2.1 Phosphorylation in development

Protein phosphorylation is critical in growth and development.⁷⁹ It is essential for the precise regulation of cell proliferation, cell cycle arrest, and differentiation into various cell types during embryonic development.⁸⁰ During early embryonic development, the metabolism of mammalian totipotent stem cells is tightly regulated by the kinases HK and PFK1. In addition, phosphorylation regulates the process of embryonic development by mediating chromosome condensation and spindle assembly.⁸¹ EGF promotes AKT1 phosphorylation through PI3K, which further stimulates the proliferation of stem cells and precursor mesenchymal cells while blocking their differentiation.⁸² Phosphorylation of the RNA-binding protein MSY2 during oocyte-to-embryo transition drives maternal mRNA degradation and converts a highly differentiated oocyte to totipotent blastomeres.⁸³ PKD stimulates the phosphorylation of MAPK for spindle organization and cofilin for actin assembly and plays an important role in meiotic maturation of porcine oocytes.⁸⁴

Growth inhibitory signaling is regulated by the Raf/MEK/ERK pathway, which plays an important role in early development and neuronal differentiation.⁸⁵ The persistent activation of ERK1/2 is a common feature of growth inhibitory signaling in the Raf/MEK/ERK pathway.⁸⁶ The target of rapamycin (TOR) is a kinase that regulates cell growth and metabolism by stimulating cell growth through anabolism and inhibiting catabolism.⁸⁷ In mammalian cells, cyclin E plays a role in the G1 and S phases of cell cycle. Cyclin E1 and cyclin E2 affect cell growth and development by activating the cyclin-dependent kinase CDK2 and then phosphorylating a series of proteins involved in cell cycle progression, male meiosis, and stem cell maintenance.⁸⁸ Deficiency of both cyclin E1 and cyclin E2 in mice is embryonic lethal.⁸⁹

2.2 Phosphorylation in aging

Aging is a process characterized by declines in both organism and organ functions, which can result in various diseases.⁹⁰ This process is characterized by cell cycle arrest,⁹¹ abnormal accumulation of senescent cells in tissues,⁹² and altered neurotransmission and response ability to external stimuli.⁹³ In quiescent cells, most protein phosphorylation does not change significantly with age.⁹⁴ However, some protein phosphorylation significantly changes with age and has crucial physiological functions. For example, αB-crystallin is a lenticular protein, and its phosphorylation can be boosted by aging, stress, and diseases.⁹⁵ αB-crystallin phosphorylation is also increased in aged muscle tissues and eye lenses.^{96, 97} Modulation of αB-crystallin phosphorylation is a potential strategy to address aging-related complications.⁹⁸

p53 is an important tumor suppressor,⁹⁹ and its ability to suppress tumors is related to the function of p53 in regulating the transcription of genes associated with cell cycle arrest and senescence.¹⁰⁰ Phosphorylation of the p53 DNA-binding domain can reduce its activity and prevent senescence.⁹⁸ p53-triggered senescence is also mediated by phosphorylation of other proteins, such as MDM2 at Ser183, which can activate p53-mediated senescence and delay tumor progression.¹⁰⁰

The brain is one of the most functionally affected organs during aging, and dysregulation of protein phosphorylation is common during brain aging.¹⁰¹ Protein phosphorylation signals in the brain are rich and diverse and mediated by kinases such as PKA, PKC, and CAM during aging.^{102, 103} The phosphorylation levels of B50/GAP-43 protein, which plays a role in long-term memory, are significantly reduced in the hippocampus of aging rats. An imbalance in protein phosphorylation, including Tau phosphorylation, acts as a key factor causing brain aging.¹⁰¹ Specifically, the accumulation of Tau phosphorylation at Ser396/404 in mitochondria is associated with cognitive dysfunction.¹⁰⁴

Sarcopenia is characterized by the loss of skeletal muscle mass and strength with age.^{105, 106} The elderly population may experience basal hyperphosphorylation of mTORC1, which could potentially contribute to insulin resistance and the age-related anabolic resistance of skeletal muscle protein metabolism in response to nutrition and exercise.¹⁰⁷ The decreased phosphorylation of myosin regulatory light chain (RLC), a critical protein involved in the modulation of muscle contractility, at Ser14/15 with age is the cause of sarcopenia-associated muscle dysfunction (Table 1).¹⁰⁸

2.3 Phosphorylation in immune regulation

Phosphorylation, a common PTM, plays a crucial role in regulating innate and acquired immunity, a process that is coregulated by kinases and phosphatases.^{109, 110} Phosphorylation and other PTMs work together to regulate the signaling networks of the immune system. For example, the MAP4K family of kinases play an important role in immune cell signaling, immune response, and inflammation; PKC is involved in regulating the important signaling pathways of innate and adaptive immunity, and plays an intermediary role in the signaling process of immune cells through immune synapses; PKA is involved in multiple processes that regulate immune activation and immune control, not only regulating lymphocyte activation, but also modulating antigen receptor-induced signaling by altering protein interactions and altering enzyme activity of substrate proteins.^111-113

Normally, phosphorylation and dephosphorylation maintain a dynamic balance in maintaining the immune homeostasis of organisms. On the one hand, protein phosphorylation is widely involved in immune regulation, for example, the receptors on immune cells trigger phosphorylation signals through the recruitment of TKs, resulting in the activation of immune cells.¹¹⁴ Shuai et al.¹¹⁵ showed that serine phosphorylation of STAT1, an important signal converter in IFN signaling, is required for the body's resistance to viral infection. On the other hand, dephosphorylation of proteins is also widely involved in immune responses and this process is mediated by phosphatases.¹¹⁰

Innate immunity is the first line of defense against pathogen invasion. Phosphorylation plays an important role in innate immunity. It has been shown that the transcription factor (TF) interferon regulatory factor 3 (IRF-3) regulates gene expression in innate immune responses, and IRF-3 activation is mediated by phosphorylation of kinase IKK/TBK1.¹¹⁶ The toll-like receptors (TLRs) are principal sensors capable of sensing multiple microbial stimuli and inducing innate immune responses through a cascade of phosphorylation signals. TLR signaling reaches its peak during the activation of nuclear factor-kappaB (NF-κB), which is mediated by phosphorylation and controls the expression of a series of inflammatory cytokine genes and further triggers the innate immune response against viruses. During viral infection, the adaptor proteins MAVS and STING are phosphorylated by the kinase IKK/TBK1 in response to stimulation, inducing type I interferons (IFNs) and other antiviral molecules.¹¹⁷ Chen et al. found that ionizing radiation leads to phosphorylation of phosphoribosyl pyrophosphate synthetase 1/2 at T228, triggering innate immune response in the body.¹¹⁸

The antiviral immune response also requires phosphorylation to mediate. STAT6 is essential for antiviral innate immunity. After viral infection, STAT6 is aggregated in the endoplasmic reticulum and phosphorylated by TBK1, which then dimerizes into the nucleus and regulates the expression of antiviral immunity genes.¹¹⁹ And phosphorylation of STAT2 at S-734 inhibits IFN-α-induced antiviral response.¹²⁰ The virus also activates the kinase IKKε, which phosphorylates YAP at Ser403, triggering degradation of YAP in lysosomes and antagonizing innate antiviral immunity.¹²¹ In addition, kinase complex mTORC2, which is involved in phosphorylation of AKT and GSK3β kinase, can maintain reactive oxygen species balance in mitochondria and maintain the lifespan of virus-specific memory CD4⁺ T cells in vivo, playing an important role in antiviral immunity.¹²²

Phosphorylation plays a key role in signal transduction during tumor immunity, mediating immune escape in a variety of tumors. PD-1 is crucial for inhibiting the activation of T cells in vitro and in vivo, and its immunosuppression process also requires phosphorylation mediated by the specific mechanism as follows: PD-1 binds to its ligand PD-L1, then aggregates with T cell receptors (TCRs) and binds briefly to phosphatase SHP2 to initiate dephosphorylation of TCR, resulting in inhibition of T cell activation.¹²³ Inhibition of CDK4/6 in vivo has been shown to inhibit cyclin D-CdK4-mediated Spoz protein phosphorylation, thereby increasing PD-L1 protein levels, and this can increase the number of tumor infiltrating lymphocytes and enhance tumor immunity.¹²⁴ Yang et al. found that phosphorylated PDHE1α (pyruvate dehydrogenase complex E1 subunit α) at S327 by ERK2 in cytoplasm can induce its transfer to mitochondria and improve NF-κB signal in the cytoplasm, which increases resistance to cytotoxic lymphocytes and promotes tumor immune escape.¹²⁵ In human glioblastoma cells, a high glucose environment promotes mitochondrial separation of hexokinase 2 (HK2), which binds to the T291 site of IκBα and phosphorylates it, subsequently mediating upregulation of PD-L1 and promoting tumor immune escape.¹²⁶

2.4 Phosphorylation in metabolic disorders

Abnormal phosphorylation may lead to the blockage of cell signaling and in turn result in metabolic disorders in the human body.⁴² Diabetes mellitus is a metabolic syndrome characterized by long-term hyperglycemia, 90% of which is T2DM. Insulin resistance is a fundamental mechanism leading to T2DM.¹²⁷ Glucose homeostasis is maintained by insulin in insulin-responsive tissues, while phosphorylation is a critical mechanism for regulating insulin secretion and insulin signaling processes.^{33, 128, 129} Over 1000 phosphorylation events are dysregulated in T2DM.¹³⁰ The effect of phosphorylation on diabetes occurs mainly through the cascade of kinases and phosphatases that regulate insulin signaling.¹²⁹ Several key molecules in the insulin pathway, such as IR, IRS1, IRS2, PDK, and mTORC1, are phosphorylated upon insulin stimulation.¹³¹ In mammals, GLP1 acts as an incretin to promote the release of insulin from pancreatic B cells. It is speculated that phosphorylation at Arg91 may inhibit processing of the glucagon precursor to GLP1 to affect blood glucose levels.¹³² In addition, phosphorylation of obesity-associated PPARγ at S273 induces insulin resistance by upregulating Gdf3 expression and inhibiting the BMP signaling pathway1.¹³³ Phosphorylation of Afadin at S1795 also promotes insulin resistance in the early stages of diet-induced obesity.¹³⁴

Obesity, a common metabolic disorder, results from the accumulation of adipose tissue caused by energy imbalances.¹³⁵ Phosphorylation plays a role in the pathogenesis of obesity by regulating adipogenesis and metabolism. For example, S6K1 participates in many key metabolic pathways, including lipid synthesis in the body, by mediating the phosphorylation of H2BS36 in obese patients. S6K1 is a potential therapeutic target for obesity.¹³⁶ Mammalian white adipose tissue (WAT) is critical for whole-body homeostasis. Smyd2 is abundant in WAT and regulates STAT2 phosphorylation to regulate adipocyte differentiation.¹³⁷ PPARγ is indispensable in the process of adipocyte differentiation, and the phosphorylation level of PPARγ at Thr166 is positively correlated with obesity status. Specifically, blocking PPARγ phosphorylation at Thr166 prevents obesity-related metabolic dysfunction (Table 1).¹³⁸

2.5 Phosphorylation in cancers

Abnormal kinase activity and expression are implicated in various types of cancers. In recent years, with the increasing development of mass spectrometry (MS) technology, the Clinical Proteomic Tumor Analysis Consortium and many other teams have conducted phosphoproteomics investigations in various cancers, such as lung cancer,^139-142 colorectal cancer (CRC),^143-146 liver cancer,¹⁴⁷ breast cancer,¹⁴⁸ prostate cancer,¹⁴⁹ gastric cancer,^{150, 151} head and neck cancer,¹⁵² esophageal cancer,¹⁵³ pancreatic cancer (PC),^{154, 155} kidney cancer,^{156, 157} melanoma,¹⁵⁸ skin cancer,¹⁵⁹ leukemia,¹⁶⁰ pancreatic ductal adenocarcinoma (PDAC),¹⁶¹ pituitary neuroendocrine tumors,¹⁶² cholangiocarcinoma,¹⁶³ and urothelial carcinoma of the bladder.¹⁶⁴

Protein phosphorylation mediates metabolic reprogramming of tumors. Studies have found that c-Src phosphorylates HK1 at Tyr732, which promotes the glycolysis rate of tumor cells and their proliferation, invasion, and metastasis abilities.⁷¹ Aerobic glycolysis can promote tumor immune escape through IκBα^T291 phosphorylation mediated by HK2.¹²⁶ Moreover, HK2 can be phosphorylated at Thr473 by the kinase PIM2, which increases its stability and enzymatic activity and promotes glycolysis and breast tumor growth, enhancing its drug resistance to paclitaxel.¹⁶⁵ AKT2 may also be an upstream kinase leading to HK2 Thr473 phosphorylation in CRC.¹⁶⁶ PFK also plays an important role in the regulation of tumor metabolism. The homologous isoform PFKP of PFK1 can be phosphorylated by AKT at Ser386, which inhibits the degradation of PFKP and promotes aerobic glycolysis in glioma cells and tumor growth.¹⁶⁷ RSK directly phosphorylates PFKFB2 to increase PFKFB2 activity and glycolysis, which accelerates the growth of BRAF-mutated melanoma.¹⁶⁸ PFKFB3 in the cytoplasm is phosphorylated and activated by AMPK. Targeted inhibition of PFKFB3 improves the sensitivity of chemotherapy drugs such as cisplatin.¹⁶⁹ Phosphorylation at Tyr105 of PKM2 is significantly increased in various tumors to mediate the transformation of the tumor cell metabolic mode to aerobic glycolysis.¹⁷⁰ Hyperphosphorylation of Ser295 and Ser314 of PDHA redirects tumor metabolism to the tricarboxylic acid (TCA) cycle by increasing PDH activity. This protects cancer cells from metabolic and oxidative stress-induced cell death and promotes tumor metastasis.¹⁷¹

Protein phosphorylation extensively regulates cancer cell proliferation, metastasis, and invasion.¹⁷² For example, DAPK3 directly phosphorylates Ser556 of ULK1, which increases the activity of ULK1 and promotes the formation of the ULK1 complex, leading to inhibition of the proliferation of gastric cancer cells. The downregulation of DAPK3 in gastric cancer patients is related to poor prognosis.¹⁷³ Phosphorylation at Ser267 of ACSS2 by CDK5 kinase inhibits the degradation of ACSS2 and promotes the growth of GBM tumor cells.¹⁷⁴ BZW1 enhances the phosphorylation of eIF2α to promote tumor progression. This process can be prevented by the PERK/eIF2α phosphorylation inhibitors GSK2606414 and ISRIB.¹⁷⁵ The kinase ERK catalyzes Drp1 phosphorylation at Ser616 to activate Drp1. Activated Drp1 changes the metabolic pathway, facilitates the oxidation of fatty acids, and promotes the proliferation of colon cancer cells.¹⁷⁶

Dysregulated phosphorylation can promote tumor metastasis. For example, AKT promotes TGF-β-driven breast cancer metastasis by mediating RNF12 phosphorylation and enhancing RNF12 stability.¹⁷⁷ KNSTRN, a component of the mitotic spindle, phosphorylates AKT at Thr308 and Ser473 to activate AKT and promote bladder cancer metastasis.¹⁷⁸ TKT, a key metabolic enzyme in the pentose phosphate pathway (PPP), interacts with GRP78 to promote glycolysis by increasing AKT phosphorylation, which promotes CRC metastasis.¹⁷⁹ PKM2 phosphorylation at Ser37 is a prominent feature of invasive breast cancer. The use of the pyruvate kinase activator TEPP-46 or the potent CDK inhibitor dinaciclib to bind to phosphorylation sites can reduce its nuclear localization and inhibit cancer cell migration and invasion (Table 1).¹⁸⁰

2.6 Phosphorylation in neurodegenerative diseases

Parkin is a tumor suppressor gene, and its overexpression can inhibit the growth of tumor cells. Parkin mutations exist in a variety of malignant tumors, such as colon cancer,¹⁸¹ PC,¹⁸² and cervical cancer.¹⁸³ However, Parkin is also a causative gene related to Parkinson's disease (PD). It has a neuroprotective effect, and mutations in Parkin lead to the loss of dopaminergic neurons in the substantia nigra.¹⁸⁴ Parkin is almost inactive in vitro, and its activation is regulated by PINK1-mediated phosphorylation.¹⁸⁵ After phosphorylation, the protein conformation, solubility, and affinity with the substrate of Parkin are changed. Parkin amplifies the PINK1-induced signaling pathway through positive feedback, which enhances mitophagy and selectively degrades defective mitochondria to maintain the stability of the intracellular environment. Abnormalities in this pathway may cause PD.¹⁸⁴ In addition, PINK1 controls XBP1s transcriptional activity by phosphorylating XBP1s at Ser61 and Thr48, which consequently enhances PINK1 transcription, and triggers a promitophagic phenotype.¹⁸⁶ Notably, functional deficiency of Parkin leads to ineffective ubiquitination and a large accumulation of cyclins. These cyclins are responsible for initiating the cell cycle in both neurons and mitotically active cells. However, due to the lack of mitogenic capacity in neurons, their inability to undergo cell division ultimately leads to apoptosis.¹⁸⁷

Tau hyperphosphorylation has an intrinsic link with neurodevelopment and degeneration, and the phosphorylation level of Tau in the AD brain is three to four times higher than that of normal peers.¹⁸⁸ Transient Tau hyperphosphorylation is protective on neurons. However, persistent accumulation of hyperphosphorylated Tau may cause neurodegeneration.¹⁸⁹ Hyperphosphorylated Tau depolymerizes normal microtubule-associated proteins after forming neuronfibrillary tangles, disrupts cellular dynamic structures, blocks intracellular material exchange and cell signaling, inhibits ubiquitin (Ub)–proteasome activity, and finally leads to neurodegenerative diseases.^{190, 191} In a cohort study of 593 elderly people with an average age of 64 years, it was found that compared with cognitively normal controls, the plasma concentrations of P-tau217 and P-tau181 are increased in clinical AD patients, suggesting that P-tau217 and P-tau181 may be useful biomarkers for AD diagnosis (Table 1).¹⁹²

2.7 Phosphorylation-associated targeted therapies

Compared with traditional cytotoxic anticancer drugs, targeted anticancer drugs have the advantages of high efficiency, low toxicity, and strong specificity.²⁰⁷ Given the important roles of protein kinases in tumor growth and metastasis, if the kinase signaling pathway is effectively blocked, the malignant progression of tumors may be prevented.²⁰⁸ To date, the United States Food and Drug Administration (US FDA) has approved 68 small molecule kinase inhibitors.²⁰⁹ These kinase inhibitors can be roughly divided into four classes according to the way they bind to protein kinases. Type I kinase inhibitors are by far the most US FDA-approved drugs, such as bosutinib, dasatinib, and crizotinib.²¹⁰ Dasatinib acts on multiple targets, such as BCR-Abl and the SRC kinase family, and is mainly used for the treatment of leukemia.²¹¹ Crizotinib has been confirmed in tumor patients with abnormal ALK, ROS kinase, and HGFR/c-MET activities.²¹² Type II kinase inhibitors, including the BCR-Abl inhibitors imatinib and nilotinib, are mainly used for the treatment of chronic myeloid leukemia (CML).^{213, 214} Another representative drug, sorafenib,²¹⁵ is a typical multitarget drug targeting TKs such as VEGFR2 and PDGFR-β, as well as the serine/threonine kinase Raf-1,²¹⁶ and can be used for the treatment of hepatocellular carcinoma (HCC) and renal cell carcinoma (RCC).^{217, 218} Allosteric kinase inhibitors are another type of kinase inhibitor.²¹⁵ Trametinib and cobimetinib are allosteric kinase inhibitors targeting MEK1/2, both of which can be used for the treatment of non-small cell lung cancer (NSCLC).²¹⁹ Allosteric kinase inhibitors do not bind to the ATP binding site, so they act together with ATP-competitive inhibitors, which makes allosteric inhibitors useful for overcoming the low selectivity, off-target effects and resistance of small molecule inhibitors.²²⁰ The fourth type of kinase inhibitors are covalent inhibitors, such as afatinib, neratinib, ibrutinib and acalabrutinib.^{215, 221} Afatinib acts on EGFR and is mainly used to NSCLC.²²² Neratinib inhibits HER2 and is used for the treatment of HER2-positive breast cancer.²²³ The BTK inhibitors ibrutinib and acalabrutinib are mainly used for the treatment of chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL).^{224, 225} Notably, acalabrutinib significantly prolonged the progression-free survival of patients with CLL (Table 2).²²¹

Tyrosine kinase inhibitors (TKIs) are currently the most widely studied. The TK EGFR is mutated or overexpressed in a variety of tumors. Abnormal expression of EGFR is closely related to the occurrence of cancer. Thus, the development of drugs targeting EGFR is a research hotspot.²²⁶ The current small-molecule inhibitors designed to target EGFR have been developed into the fourth generation.²²⁷ The first three generations of inhibitors are widely used in the clinic and have gradually become the first choice for NSCLC treatment, mainly by inhibiting the phosphorylation of the intracellular TK domain.²²⁸ First-generation EGFR-TKIs, including gefitinib and erlotinib, are reversible inhibitors.^{229, 230} Second-generation EGFR-TKIs, including dacomitinib and afatinib, are irreversible.²³⁰ Third-generation EGFR-TKIs mainly target T790M mutant EGFR and are irreversible as well. The representative drug is osimertinib.²³¹ Although TKIs, represented by third-generation EGFR-TKIs, have achieved remarkable success in the field of cancer treatment, clinical results show that there are still inevitable toxic side effects in the gastrointestinal tract, skin and other organs.²³² In addition, TKIs have also been used to treat T1DM and T2DM.²³³ For example, c-Abl²³⁴ and VEGFR2²³⁵ inhibitors have been shown to enhance β cell survival and insulin secretion, while PDGFR²³⁶ and EGFR²³⁷ inhibitors have been demonstrated to improve insulin sensitivity.

3.1 Acetylation in development

Previous studies have shown that HDACs regulate histone acetylation to affect the proliferation, differentiation, apoptosis, migration, and synapse regeneration of nerve cells.³³⁷ HDAC1 and HDAC2 are essential for cortical lamination and play a crucial role in maintaining the progenitor pool during cortical development. Deletion of both HDAC1 and HDAC2 results in a deficiency in neocortical development.³³⁸ HDACs also play roles in the development and differentiation of various immune cells.³¹² Inactivating HDAC3 during the double-negative stages of thymocyte development will cause significant damage at the CD8 immature single-positive (ISP) stage and the CD4/CD8 double-positive stage, resulting in the production of few mature CD4(+) or CD8(+) single-positive cells.³³⁹ Deletion of HDAC3 in early B-progenitor cells caused a defect in VDJ recombination and failure in B cell development.³⁴⁰ In addition, HDAC3 can also indirectly regulate the development and function of these immune cells through stromal cells or target cells interacting with immune cells. For example, HDAC3 is an important component of the Notch signaling pathway that regulates the development of medullary thymic epithelial cells (mTECs).³⁴¹ Loss of HDAC3 expression can lead to developmental arrest of mTECs with impaired T-cell negative selection. However, whether the regulation of HDAC3 on the development of neurons and immune cells depends on its deacetylation function remains to be further studied.

3.2 Acetylation in aging

As a key metabolite, acetyl-CoA is an important donor of acetylation modifications.^{342, 343} Previous studies have shown that fasting and caloric restriction (CR) reduce glucose-derived metabolic flux and cytoplasmic acetyl-CoA levels through ATP-citrate lyase,³⁴⁴ which decreases p300 activity to stimulate long-lived autophagy. However, increased nuclear acetyl-CoA can promote lifespan by increasing the levels of histone acetylation.^{343, 345, 346} Sirtuins are epigenetic enzymes that are key regulators of aging and CR.³⁴⁷ In yeast, CR prolongs lifespan by increasing the activity of Sir2.^{347, 348} In mammals, the role of SIRT1–7 in extending lifespan is also largely based on their deacetylase functions.³⁴⁹

Nicotinamide mononucleotide (NMN) supplementation not only inhibits the aging-associated increase in protein acetylation but also modulates fatty acid β-oxidation, TCA cycle, and valine degradation. Aged livers show increased acetylation compared with young livers, but NMN supplementation decreases acetylation. These results reveal the potential of NMN in combating aging and aging-related functional declines.³⁵⁰ Inflammatory aging of the brain is a hallmark of age-related neurodegenerative diseases. Integrated analysis of H3K27ac and gene expression data in human and mouse brains shows that genes upregulated and downregulated with aging are correlated with different H3K27ac modification patterns.³⁵¹ By using aging mouse models under inflammatory conditions, it has been found that the pattern recognition receptor NLRP3 is acetylated in macrophages and deacetylated by NAD⁺-dependent sirtuins. Dysregulation of the NLRP3 inflammasome acetylation switch may be the cause of aging-associated chronic inflammation.³⁵²

During the aging process of mesenchymal stem cells, histone acetylation on the promoters and enhancers of osteogenic genes, as well as the chromatin accessibility, decreases, which leads to the downregulation of osteogenic gene expression and a decrease in osteogenesis.³⁵³ Comparing the changes in sirtuins in experimental animals of three different age groups, young, middle-aged and old, it has been found that the expression of sirtuin family proteins in skeletal muscle increases during the aging process, but acetylation is not effectively reduced, which is associated with a severe reduction in NAD⁺ content.³⁵⁴

In addition, the acetyltransferase KAT7 can promote H3K14ac-related gene expression and induce cell senescence. Inactivation of KAT7 reduces H3K14ac and represses the transcription of p15INK4b, which attenuates the senescence of human peritoneal mesothelial cells.³⁵⁵ DNA damage can activate ATM and inhibit LARP7-regulated SIRT1 activity, leading to increased p53 and p65 acetylation and transcriptional activity to promote cellular senescence. Activation of this pathway exacerbates aging and atherosclerosis in ApoE-knockout mice, while inactivation of this pathway can reverse these phenotypes (Table 3).³⁵⁶

3.3 Acetylation in metabolic disorders

Diabetes and obesity are related to mutations in the acetylation sites of metabolic enzymes.^{357, 358} Persistent hyperglycemia in diabetes can increase acetyl-CoA and protein acetylation levels, which may impair protein functions.³⁵⁹ In diabetic rat models, organs with high protein acetylation are susceptible to diabetic complications.³⁶⁰ The acetylation level of NF-κB in the hearts of diabetic rats is elevated, and the expression of Nrf2-related genes and mitochondrial activity are impaired. Consequently, this results in the persistence of inflammation, impairs the functions of the heart to resist oxidative stress, and increases the risk of cardiovascular complications in diabetes.³⁶¹ HDACs and sirtuins play key roles in diabetes by affecting insulin signaling and secretion.^{362, 363} The GLUT4 gene promoter is composed of an MEF2-binding domain and domain I. Transcriptional activity is highest when MEF2 is bound to the MEF2-binding domain and the GLUT4 enhancer GEF is bound to domain I.³⁶⁴ HDACs downregulate the transcription of MEF2-related genes.³⁶⁵ HDAC2 reduces acetylation by binding to IRS1 in hepatocytes, thereby reducing insulin receptor-mediated IRS1 tyrosine phosphorylation³⁶⁶ and downregulating pancreatic insulin formation and secretion.³⁶⁷ Notably, SIRT1 can enhance glucose-induced pancreatic insulin secretion.³⁶⁸ HDACs and p300/CBP mediate STAT3 acetylation to regulate gluconeogenesis.³⁶⁹

The p300/CBP family and SIRT3/SIRT6 are involved in the process of obesity,^370-372 and the histone acetylation level is positively correlated with adipogenic differentiation.^373-375 p300/CBP in the PPARγ complex is the main enzyme that activates gene transcription, which can increase the expression of CEBPα and PPARγ and promote adipogenesis.^376-378 This process has been associated with p300/CBP-mediated H3K27ac.^{379, 380} p300/CBP double knockout mice develop severe lipodystrophy with hepatic steatosis, hyperglycemia, and hyperlipidemia.³⁸¹ In a cardiac-specific SIRT6 knockout mouse model fed a high-fat diet (HFD), loss of SIRT6 function exacerbates cardiac injury, including left ventricular hypertrophy and lipid accumulation.³⁸² Enzymes of the HDAC family are also involved in the regulation of obesity. For example, by regulating fat metabolism, HDAC3 can promote fat absorption and diet-induced obesity.³⁷² HDACs also inhibit adipogenesis by downregulating histone acetylation.^{383, 384} HDAC1 but not HDAC2 can inhibit adipogenesis by reducing CEBPα and PPARγ expression (Table 3).³⁸⁵

3.4 Acetylation in CVDs

The modulation of HDAC functions can improve CVDs such as cardiac hypertrophy, heart failure, arrhythmia, myocardial infarction, hypertension, atherosclerosis, and fibrosis.^{386, 387} Although both class I and class II HDACs have conserved HDAC domains, they have completely different functions in CVDs. Class I HDACs have procardiac hypertrophic effects, whereas class II HDACs are expressed in a relatively tissue-specific manner and have anticardiac hypertrophic effects.³⁸⁸ HDAC7 is localized in the cardiac cytoplasm, and its overexpression induces the expression of cardiac hypertrophy and heart failure-related genes such as Nppa and Nppb.³⁸⁹

Vascular endothelial dysfunction is the main cause of CVDs, and one of the characteristics of endothelial dysfunction is insufficient synthesis of nitric oxide (NO). The main enzyme responsible for the synthesis of NO in endothelial cells is endothelial nitric oxide synthase (eNOS). The interaction between SIRT1 and eNOS can activate eNOS by reducing its acetylation level, which promotes NO production and vasodilation.³⁹⁰ CKIP-1 regulates physiological cardiac hypertrophy by inhibiting HDAC4 phosphorylation.³⁹¹ In an AngII-induced mouse model of pathological cardiac hypertrophy, the pan-HDAC inhibitor (HDACI) emodin ameliorates hypertrophy by inhibiting the activity of class I, IIa, and IIb HDACs.³⁹² HDACs are also involved in myocardial fibrosis. Overexpression of class I HDACs significantly enhances the proliferation of cardiac fibroblasts and the expression of proteins associated with fibrosis. Silencing of HDAC3 upregulates miR-18a and reduces ADRB3 expression, thereby inhibiting cardiomyocyte fibrosis and hypertrophy.³⁹³ The HDAC8 inhibitor PCI34051 mediates the p38 MAPK pathway to alleviate isoproterenol-induced cardiac hypertrophy and fibrosis³⁹⁴ and attenuates myocardial fibrosis induced by transverse aortic constriction in mice through downregulation of Ace1.³⁹⁵ The sirtuin family also plays key roles in preventing cardiomyocyte fibrosis, regulating cardiomyocyte apoptosis, improving cellular energy metabolism remodeling and inflammation, and maintaining cardiac homeostasis.³⁹⁶ Stimulation of SIRT3 reduces ROS and protein kinase levels and prevents cardiac hypertrophy, which may be a mechanism to inhibit cardiac remodeling.³⁹⁷ Last, p300 is a potential therapeutic target for heart failure. Mice with p300 knockout exhibit remarkable cardiac defects and embryonic lethality (Table 3).^{398, 399}

3.5 Acetylation in neurodegenerative diseases

The imbalance between acetylation and deacetylation processes is related to neurodegenerative diseases such as AD and HD.^{400, 401} Abnormal histone acetylation in AD affects the expression of memory-related genes and dysregulates several signaling pathways, including cell differentiation, apoptosis, inflammation, and neuronal and vascular remodeling.^{402, 403} In a transgenic AD fly model, loss of Tip60 activity significantly increases the transcriptional expression of amyloid precursor protein (APP), leading to neuronal apoptosis, while overexpression of Tip60 HAT activity can potentially serve as a neuroprotective agent.⁴⁰⁴ p300/CBP is widely expressed in the nervous system. It has been proposed that inhibiting the activity of CBP/p300 acetyltransferase may affect the death of brain neurons and the long-term memory of animals.⁴⁰⁵ Among various HDACs, HDAC2 modulates chromatin plasticity to regulate the expression of learning and memory-related genes, and its dysregulation leads to the dysfunction of cholinergic nbM neurons, neurofibrillary tangle (NFT) pathology, and cognitive decline in AD.⁴⁰⁶ HDAC3 controls gene expression during the development and maintenance of neural stem cells,⁴⁰⁷ while HDAC4 may also play a role in the area of learning and memory. Selective deletion of HDAC4 in the brain leads to impaired long-term synaptic plasticity.⁴⁰⁸ HDAC6 plays a leading role in neuronal health or dysfunction. Selective inhibition of HDAC6 can promote growth cone function, synaptic plasticity, transport, and autophagosomal degradation, which can help protect neurons.⁴⁰⁹ Notably, HDAC6 is significantly elevated in the brains of AD patients. Sirtuins restore protein microenvironmental homeostasis mainly by reducing toxic protein aggregates. They also improve neural plasticity by increasing gene transcription activity, which can reduce oxidative stress, enhance mitochondrial function, and improve learning and memory abilities.⁴¹⁰ SIRT3 expression is significantly increased in the temporal cortex in AD patients.⁴¹¹ High SIRT3 expression can promote antioxidant effects in mutant HTT cells, enhance mitochondrial function, and exert neuroprotective effects in HD.⁴¹² In PD mice, a neuroprotective effect of SIRT3 has also been found.⁴¹³ SIRT3 may play a protective role in neurons by scavenging free radicals in mitochondria.⁴¹⁴ Decreased SIRT3 function increases mitochondrial oxidative stress and cell death in substantia nigra dopaminergic neurons in PD models.⁴¹⁵ The expression of SIRT3 is significantly reduced in MPTP-induced PD cell models, and overexpression of SIRT3 inhibits cell apoptosis. PGC-1α can promote the transcription of SIRT3 and inhibit the loss of dopaminergic neurons (Table 3).⁴¹⁶

3.6 Acetylation in cancers

Abnormal acetylation exists in various cancers.³¹⁰ Most histones are in a hypoacetylated state in tumor cells, and mutations in the acetyltransferases CBP and p300 are often found in tumors. An imbalance in acetylation leads to dysregulated gene expression related to cancer cell proliferation, differentiation, migration, invasion, and apoptosis.^{417, 418} For example, H4K16ac alters the chromatin state and promotes gene transcription to regulate tumorigenesis and development.⁴¹⁹ miR24-2 inhibits histone deacetylase HDAC3 through miR675 to promote histone H4K16ac, which acts on PI3K and enhances the interaction between LC3 and ATG4, consequently triggering autophagy that affects cancer cell proliferation.⁴²⁰ The acetylation of the cytoskeleton is related to tumorigenesis. The acetylation of α-tubulin, a component of the cytoskeleton, is an important indicator of microtubule stability. Tubulin is the target of many anticancer drugs.⁴²¹ Tumors are resistant to apoptosis. PDCD5, a protein related to apoptosis, can bind to Tip60 and increase p53 acetylation at K120, which affects the expression of apoptosis-related genes such as Bax.⁴²² The acetylation of the hypoxia-induced autophagy regulator PAK1 regulates the phosphorylation of ATG5 at Thr101 in GBM and is important for hypoxia-induced autophagy and tumor growth.⁴²³

Increasing evidence shows that carcinogenesis is affected by metabolism in the body. Most metabolic proteins are substrates of lysine acetylation,⁴²¹ such as ATM, ABL1, CDK9, BTK, and CDK1. PKM2 is the last rate-limiting enzyme in the glycolytic pathway responsible for the conversion of phosphoenolpyruvate to pyruvate. In a high glucose environment, PCAF acetylates PKM2 at K305, which reduces its binding to the substrate PEP, inhibits its enzymatic activity and promotes its chaperone-mediated autophagy and lysosome-dependent degradation.⁴²⁴ The acetylation of LDHA at K5 inhibits its enzymatic activity and is recognized and mediated by the heat shock protein HSC70, which downregulates the level of LDHA. The acetylation level of LDHA at K5 in early PC tissues is significantly lower than that in adjacent tissues, suggesting that acetylation of LDHA at K5 may be related to the occurrence of PC.⁴²⁵ FBP1 is the rate-limiting enzyme in gluconeogenesis and is lost in many types of cancer. Reduced FBP1 is associated with poor prognosis in HCC. HDAC-mediated repression of FBP1 expression is associated with a reduction in H3K27ac in the FBP1 enhancer.⁴²⁶ PDC is located within the mitochondria and is responsible for the irreversible conversion of pyruvate to acetyl-CoA. Phosphorylation of PDP1 at Tyr381 triggers SIRT3 to detach from the PDC center but recruits the acetyltransferase ACAT1 to the PDC center to acetylate PDP1 at K202 and PDHA at K321. This reconstructs the structure of the PDC center and inhibits PDC activity, further promoting tumor cell proliferation and growth.⁴²⁷ 6PGD is an important enzyme in the PPP. Acetylation of 6PGD at K294 promotes the formation of highly active 6PGD dimers, thereby further activating the 6PGD and PPP pathways to produce more ribulose-5-phosphate and NADPH for nucleic acid synthesis and resisting oxidative free radical damage.⁴²⁸ Fatty acid metabolism is important for tumor growth and metastasis. Acetylation of FASN in the fatty acid synthesis pathway promotes its degradation. The deacetylation process is regulated by HDAC3, which functions in the initiation and development of liver cancer.⁴²⁹ Furthermore, SIRT4 can regulate branched-chain amino acid catabolism by deacetylating BCAT2 and promote PDAC growth (Table 3).⁴³⁰

3.7 Acetylation-associated targeted therapies

Due to the important functions of acetylation in diseases, HDACIs have now shown good application prospects in the treatment of various diseases, such as heart disease, diabetes, and cancers.⁴⁴⁶ Currently, HDACIs can be divided into four classes, including short-chain fatty acids (SCFAs) predominantly inhibiting class I HDACs (e.g., butyrate, phenylbutyrate, and valproate), hydroxamic acids inhibiting class I and II HDACs (e.g., trichostatin A (TSA) and suberoylanilide hydroxamic acid (SAHA)), cyclic tetrapeptides displaying class I HDAC selectivity in vitro, and benzamides inhibiting class I HDACs (e.g., RGFP136 and MS-275).⁴⁴⁷ Restoring normal protein acetylation may be a new approach for the treatment of malignant tumors.⁴⁴⁸

Upregulation of HDAC expression is a characteristic of various malignant cancers,⁴⁴⁹ such as prostate cancer,⁴⁵⁰ gastric cancer,⁴⁵¹ breast cancer,⁴⁵² renal cancer,⁴⁵³ and Hodgkin's lymphoma.⁴⁵⁴ HDACIs can inhibit tumor cell proliferation by inducing cell differentiation, growth arrest, and apoptosis.^{455, 456} A variety of HDACIs have been approved or entered clinical trials. HDACIs not only show direct inhibitory effects on tumor cells but also overcome the resistance of tumors to other drugs, which makes the combination of HDACIs and other antitumor drugs possible.⁴⁵⁷

HDACIs have bidirectional effects on inflammation and anti-inflammatory effects. HDAC inhibition may not only increase inflammation but also attenuate the expression of specific genes to reduce infiltrating inflammatory cells, leading to a beneficial result.⁴⁵⁸ Additionally, HDACIs can reverse neuronal degeneration and aging and enhance synaptic plasticity in mouse models. On the one hand, HDACIs have a direct influence on gene transcription by remolding histone acetylation. On the other hand, HDACIs can increase the acetylation of transcriptional regulators such as HNF4a to indirectly regulate gene expression.⁴⁵⁹ HDACIs have been found to exhibit neuroprotective effects on neurological diseases such as PD, AD, amyotrophic lateral sclerosis (ALS), and HD.⁴⁶⁰ For example, the class I HDACI valproic acid may exert neuroprotective effects by regulating the BDNF/TrkB signaling axis.⁴⁶¹ In a cellular model of PD patients, selective inhibition of SIRT2 increases tubulin acetylation and improves microtubule-mediated transport.⁴⁶² Moreover, SIRT2 inhibitors such as AGK2, AK-7, and AK1 have been demonstrated to decrease neuroinflammation and cytotoxicity induced by toxins or mutant protein aggregation.⁴⁶³ Additionally, the HDAC6 inhibitor tubastatin A, which increases autophagic flux and protects neurons in HD patients, is a potential drug for the treatment of HD.⁴⁶⁴

Deacetylase inhibitors are also used to treat metabolic disorders. SAHA has been shown to target eNOS uncoupling and oxidative stress in diabetes.⁴⁶⁵ TSA can prevent ischemia-induced left ventricular remodeling by inhibiting TNF-α transcription. In addition, it also enhances AKT phosphorylation to promote angiogenesis and cardiomyocyte survival.⁴⁶⁶ However, a SIRT6 inhibitor aggravates diabetes-induced cardiomyocyte apoptosis and fibrosis in mice by increasing the levels of inflammatory factors and ROS.⁴⁶⁷

4.1 CVDs

SCFA modifications play a role in regulating the progression of CVDs through enzymatic switches and oxidative stress. Additionally, they can affect the cellular localization and PPIs of many cardioprotective proteins.⁵⁴⁵ Maintaining metabolic stability of SCFAs is essential for cardiac and vascular function.⁵⁴³ p53 Kbhb mediates the protective effect of β-hydroxybutyrate on vascular cell senescence, possibly by reducing the expression of p21 and PUMA, which leads to cell growth arrest and reduced apoptosis.⁴⁹⁹ Kpr can cause FAO disorders and impair mitochondrial functions.⁵²⁴ H3K14pr is highly expressed in promoter regions of transcriptionally activating genes, including FAO-related genes associated with CVD progression.⁴⁹¹ SIRT5 defects lead to an increase in succinyl-coA in the heart.⁵⁰⁴ Elevated levels of Ksucc can lead to hypertrophic obstructive cardiomyopathy,⁵²⁸ while oxidative stress caused by Kglu may be an important mechanism for inducing CVDs.⁵⁴⁶ A high-fat diet causes adverse effects on cardiovascular health, especially under stressful conditions. The levels of H3k9bu affected by ACADS can moderate the expression of stress-regulated genes.⁵⁴⁷

4.2 Metabolism-associated diseases

Metabolic disorders cause dysregulated SCFA modifications, which can lead to various metabolic diseases.^{17, 548} Obesity can lead to sperm DNA damage and decreased sperm quality,⁵⁴⁹ and SCFA modifications cause male reproductive dysfunction in obese men. In high-fat diet mice, acetylation and crotonylation decrease in the testes, while other metabolism-related lysine acylations, including propionylation, malonylation, succinylation, glutarylation, 2-hydroxyisobutyrylation, and benzoylation, increase.⁵⁵⁰ Obesity and metabolic syndrome accelerate the occurrence of osteoarthritis during the aging process, and SIRT5-regulated malonylation may impair chondrocyte metabolism.⁵⁵¹ The mechanism by which energy restriction improves fat metabolism may be involved in Ksucc. Acute fasting regulates Ksucc through SIRT5 to modulate lipid metabolism in adipose tissues and improve obesity.⁵⁵² In addition, specific inhibition of p300-mediated butyrylation at H4K5 by LTK-14A in adipocytes and liver improves obesity.⁴⁹⁰

Decreased SCFA modifications, such as malonylation, butyrylation, and propionylation, are found in liver histones of obese mice induced by a high-fat diet.⁵⁵³ Dapagliflozin treatment leads to elevated 3-hydroxybutyrate in the plasma and adipose tissues of obese diabetic mice, which further induces H3K9 3-hydroxybutyrylation to promote the expression of apolipoproteins in adipocytes. Apolipoproteins are anti-inflammatory and antiatherosclerotic, indicating that 3-hydroxybutyrate is protective against obesity-associated diabetes by modulating H3K9 3-hydroxybutyrylation.⁵⁵⁴ In addition, the dysregulation of Kmal in fatty acid oxidative metabolism can also lead to mitochondrial fatty acid metabolism diseases such as malonyl-CoA synthetase ACSF3 deficiency.⁵³² These studies provide additional evidence for the link between metabolic disorders and epigenetic regulation by SCFA modifications.

4.3 Kidney diseases

Kidney disease includes acute kidney injury (AKI) and chronic kidney disease (CKD), some of which progress to end-stage renal disease (ESRD). SCFA modification, as a type of epigenetic mechanism, is involved in the progression of kidney disease.⁴⁸¹ Histone lysine crotonylation was observed in mouse and human renal tubular cells, and histone crotonylation was observed to be increased in renal tissues during AKI. Crotonate supplementation can increase overall histone crotonylation and have a protective effect on the kidneys.⁵⁵⁵ Crotonyl-CoA hydratase CDYL regulates the crotonylation of histone H3K18 and affects the disease process of ADPKD.⁵⁵⁶ Diabetic kidney disease (DKD) is the main cause of ESRD, and inflammation and fibrosis are key processes in its development. Butyrate inhibits the expression of renal inflammation and fibrosis genes through p300-mediated histone Kbu and improves DKD.⁵⁵⁷ Other studies have found that crotonylation and 2-hydroxyisobutyrylation also play a significant role in ESRD. By analyzing crotonylation and 2-hydroxyisobutyrylation in PBMCs of patients with ESRD, it is speculated that crotonylation and 2-hydroxyisobutyrylation may affect immune cell numbers and induce immune senescence, which may be due to regulation of the glycolytic/gluconeogenesis pathway and protein processing.⁵⁵⁸ In STZ-induced diabetic SD rats, 3-hydroxybutyrate treatment increases H3K9 3-hydroxybutyrylation in the gene promoter to upregulate MMP-2, which reduces collagen IV content and glomerular fibrosis.⁵⁵⁹

4.4 Cancers

Metabolic reprogramming is a common feature of cancer.⁵⁶⁰ The “Warburg effect” is an important feature of tumor cell metabolism.⁵⁴⁸ Tumor cells predominantly rely on aerobic glycolysis for energy production and metabolite synthesis, resulting in extracellular acidification due to lactate accumulation, which is a hallmark of cancer.⁵⁶¹ Lactate is involved in the regulation of the tumor microenvironment, which promotes macrophage polarization into an M2-like phenotype, thereby inhibiting the immune response in the tumor microenvironment.⁴⁸⁰ Histone Kla has been proven to promote the development of tumors. Histone Kla regulates the transcription of the m6A reader YTHDF2, which can recognize the m6A modification on the 3'UTR of the tumor suppressor genes PER1 and TP53 mRNA, resulting in their degradation and further impacting the development of melanoma.⁵⁶² Kla is more abundant in gastric cancer tumor tissues than in adjacent normal tissues, indicating its potential as a prognostic indicator for gastric cancer.⁵⁶³ In PDAC, elevated tumor-mediated Kla levels can enhance the expression of cancer-associated fibroblasts (CAFs) to promote cancer cell invasiveness.⁵²²

Besides lactylation, other SCFA modifications also contribute to cancer development. In PDAC, SUCLA2-coupled regulation of GLS succinylation promotes tumor growth.⁵⁶⁴ Lower levels of the mitochondrial protein GCDH result in Kglu of the TF NRF2, leading to cell death, indicating that GCDH pathway inhibition is a potential therapeutic strategy for melanoma treatment.⁵⁶⁵ Khib is widely distributed in PC, and treatment with the TIP60 inhibitor MG149 can significantly reduce the total Khib level in PC, which leads to the inhibition of PC cell proliferation, migration, and invasion.⁴⁹⁸ Kcr is also involved in cancer regulation, with decreased levels observed in liver, gastric, and kidney cancers and increased levels in thyroid, esophageal, and PC.⁵⁶⁶ In addition, p300-mediated crotonylation enhances the expression of HNRNPA1, promoting HeLa cell malignancies.⁵⁶⁷ By knocking out HDACs or adding the HDACI TSA, the level of Kcr is increased, which inhibits the motility and proliferation of HCC cells.⁵⁶⁶ The level of Kcr in prostate cancer tissue is higher than that in adjacent tissues, and its level increases as the malignancy increases.⁵⁶⁸ Hyperpropionylation of H3K23 in the leukemia cell line U937 may serve as a stage-specific biomarker in hematopoiesis and leukemogenesis.⁵⁶⁹ Moreover, propionate can induce global Kpr, which inhibits colon cancer development by upregulating the expression of MICA/B.⁵⁷⁰ Ksucc can affect the synthesis of thyroid hormones. Radiation-induced thyroid cancer and cancer cell metastasis in apoptotic cell lines can be inhibited by Ksucc.⁵⁷¹ Downregulation of TFAM can induce Kmal of mDia2 to promote its nuclear translocation, which further induces lung metastasis of mouse liver cancer.⁵⁷²

4.5 Neurological diseases

SCFA modifications are closely related to neurological function. In cases of syndromic intellectual disability, there is an alteration in histone H3K23 propionylation.⁴⁸⁸ The level of Kbu is significantly changed in the brains of rats with vascular dementia compared to the control group.⁵⁷³ Crotonylation and succinylation levels are increased in the cerebral cortex of mice with developmental disorders of the central nervous system (CNS).⁵⁷⁴ Neural excitation can modulate Kla levels in brain cells, suggesting that protein Kla in the brain may be associated with neuropsychiatric disorders.⁵⁷⁵ CDYL mediates histone crotonylation, which regulates gene transcription and promotes the development of stress-induced depression in rodents, providing a potential therapeutic target for major depression.⁵⁷⁶ Lysine succinylation and malonylation are related to protein regulation, glycolysis, and energy metabolism. Mitochondrial dysfunction causes an imbalance in succinylation, which in turn leads to schizophrenia and other psychiatric disorders.⁵⁷⁷ Histone Kbhb is enriched in the promoter region of active genes and affects the organism by reprogramming the epigenetic map.⁵³⁶ Kbhb plays an important role in the development of neurological diseases, and 3-hydroxybutyrate can be used to treat certain neurological diseases, such as epilepsy and AD.^{578, 579} Experiments in mice have shown that 3-hydroxybutyrate may alleviate depressive behavior by increasing histone H3k9 3-hydroxybutyrylation.⁵⁸⁰

In patients with AD, the succinylation levels of various mitochondrial proteins are decreased, while the succinylation of APP is increased. This disrupts the normal proteolytic process of APP and leads to abnormal protein deposition in the brain.⁵⁸¹ In the AD mouse model, elevated levels of H4K12 lactylation in microglia activate the transcription of glycolytic genes, resulting in proinflammatory activation of microglia.⁵⁸² During mammalian development, histone crotonylation and lactylation are widely distributed in the brain and play important roles in neurodevelopmental processes by contributing to transcriptome remodeling.⁵⁸³

5.1 Palmitoylation

The regulation of protein palmitoylation balance is carried out by palmitoyl acyltransferases (PATs) and depalmitoylating enzymes (Figure 10A).⁵⁸⁷ PATs belong to the PAT family and contain a conserved DHHC (aspartic acid-histidine-histidine-cysteine) motif, hence, they are referred to as DHHC-PAT .⁵⁹³ These enzymes are also known as zinc finger-containing DHHCs (ZDHHCs) as the DHHC motifs form zinc finger domains. Palmitoylation can be categorized into three types based on the way palmitoyl groups are attached to proteins: S-type, N-type, and O-type.⁵⁹⁴ S-type palmitoylation refers to the attachment of palmitoyl groups to the cysteine residues of proteins through an unstable thioester bond. N-type palmitoylation refers to the attachment of palmitoyl groups to the amino groups of various amino acids (e.g., glycine, cysteine, lysine), while O-type palmitoylation is the attachment of a few palmitoyl groups to the hydroxyl groups of serine or threonine. S-palmitoylation, which dominates the majority of palmitoylated proteins and is a reversible process, represents typical palmitoylation. To date, 23 PATs have been discovered (Table 5).⁵⁹⁵ The thioester bond is hydrolyzed by depalmitoylases, leading to the dissociation of palmitoyl groups from cysteine residues. Five depalmitoylases have been found to date,^596-600 including PPT1, PPT2, APT1, APT2, and ABHD17. PPT1, which is located mainly in lysosomes, is a thioesterase that mediates the depalmitoylation of various palmitoylated proteins in neurodegenerative diseases.⁵⁹⁶ PPT2,which has a different crystal structure from PPT1, is essential for depalmitoylation in protein degradation.⁶⁰¹ APT1, which is mainly localized in the cytoplasm of yeast and mammalian cells, is a highly conserved α/β hydrolase containing the S-H-D catalytic triad and the G-X-S-X-G motif, with palmitoylated Ras proteins being its main substrates.⁶⁰² APT2 is highly homologous to APT1.⁶⁰³ ABHD17 is essential for N-Ras depalmitoylation and the relocalization of N-Ras to internal cellular membranes.⁶⁰⁴

Palmitoylation is essential for protein localization. The palmitoylation of Cdc42 at Cys188 plays a crucial role in its localization to the plasma membrane, which regulates gene transcription and neuronal morphology in hippocampal neurons.⁶⁷³ The precise localization of calcineurin CNAβ1 and CD36 is also regulated by palmitoylation.⁶⁷⁴ Palmitoylation enhances the hydrophobicity of CD36,⁶⁷⁵ increasing its ability to bind to the membrane and absorb fatty acids.⁶⁷⁶ In contrast, inhibiting CD36 palmitoylation reduces its hydrophobicity and localization to the cytoplasmic membrane.⁶⁷⁷ The palmitoylation of CD36 is precisely regulated by DHHC4 and DHHC5.⁶⁷⁸ In addition, the palmitoyltransferase ZDHHC5 mediates the palmitoylation of NOD1/2 to promote its membrane recruitment and immune signaling, which are extremely important for microorganisms to establish an effective immune response.⁶⁷⁹

Cellular palmitoylation maintains a dynamic balance to ensure normal life activities, and any disruptions of this balance can lead to various diseases, such as autoimmune diseases,⁶¹³ neurodegenerative diseases,⁵⁹³ T2DM,³³ and nonalcoholic fatty liver disease,⁶⁷⁷ tumors,^680-683 Friedreich ataxia, and peripheral artery disease.^{684, 685} Elevated palmitoylation of NOD2 mutants in autoinflammatory diseases leads to inflammation and inhibits autophagic degradation (Figure 11).⁶⁸⁶ In autoimmune diseases, palmitoylation activates the STING signal associated with the type I IFN response and induces the expression of inflammatory genes through recruitment of TBK1 and IRF3. Treatment with the palmitoylation inhibitor 2-bromopalmitate (2-BP) can abolish the type I IFN response by inhibiting the palmitoylation of STING.⁶⁸⁷ In major depressive disorder, deletion of ZDHHC21 reduces palmitoylation of 5-HT1AR and affects its signaling function.⁶⁶⁵ Reduced palmitoylation of Cdc42 in ZDHHC8-deficient neurons interferes with Akt/Gsk3β signaling, leading to schizophrenia.⁶⁸⁸ Increased palmitoylation in the brains of HD mice can alleviate the anxiety and depression behaviors of mice⁶⁸⁹ and reduce cytotoxicity in YAC128 neurons.³² CD36 palmitoylation on the plasma membrane in nonalcoholic steatohepatitis (NASH) is significantly increased, but inhibition of CD36 palmitoylation protects against NASH in mice.⁶⁷⁷ Aberrant protein palmitoylation mediates cell barrier disruption and leads to spermatogenesis dysfunction in spermatodysplastic patients.⁶⁹⁰ ZDHHC17 mediates palmitoylation of Oct4A in human GBM and protects it from lysosomal degradation, which maintains tumorigenicity.⁶⁹¹ Palmitoylated PCSK9 can activate the PI3K/AKT pathway, confer drug resistance in HCC cells, and promote cancer cell proliferation.⁶⁹² In addition, palmitoylated EGFR in TKI-resistant EGFR-mutant NSCLC cells positively regulates FASN and further promotes cancer cell growth through the Akt pathway.⁶⁹³

At present, various therapeutic strategies have been developed to address diseases caused by imbalanced palmitoylation. FLT3 is palmitoylated in primary human AML cells, which hinders the activation of AKT signaling and AML progression. A novel therapeutic strategy has been developed for FLT3-ITD⁺ leukemia by promoting FLT3 depalmitoylation.⁶²² The palmitoyltransferase ZDHHC3 mediates the palmitoylation of PD-L1, which further inhibits PD-L1 ubiquitination and degradation. The palmitoylation inhibitor 2-BP can lower PD-L1 palmitoylation and boost PD-L1 degradation through the lysosomal pathway, thereby increasing the immune response of T cells against tumors.⁶¹³ Furthermore, 2-BP can block STING palmitoylation and impair the type I IFN response.⁶⁸⁷ ABD957 acts as a potent and selective inhibitor of ABHD17 depalmitoylase. Specifically, it inhibits N-Ras depalmitoylation in AML cells and disrupts the balance of N-Ras palmitoylation, suggesting ABHD17 as a promising target for the treatment of N-Ras mutant tumors.⁶⁰⁴

5.2 Myristoylation

Protein myristoylation is a process mediated by NMT, with the majority of this transfer occurring at the amino group of glycine.^{585, 694} On rare occasions, it takes place at the side chain of lysine.⁶⁹⁵ Until now, no demyristoylase has been discovered, but a study has shown that a cysteine protease IpaJ expressed by Shigella bacteria can cleave myristoylglycine from the N-terminus of host proteins, serving a similar function as a hypothetical demyristoylase.⁶⁹⁶ The N-terminus of the myristoylated protein contains a conserved sequence Met-Gly-XXX-Ser/Thr (XXX could be any natual amino acid). The N-terminal amino acid methionine must be removed by methionine aminopeptidase to expose glycine before myristoylation (Figure 10B).⁶⁹⁷ There are two NMTs (NMT1 and NMT2) in humans, NMT1 and NMT2, which have 77% sequence similarity and partially overlapping substrates and biological functions.^{695, 698}

Myristoylation affects the subcellular localization of proteins and regulates PPIs and protein–membrane interactions. Myristoylated proteins located on the membrane can trigger subsequent cellular responses by sensing and transmitting signals.^{699, 700} For example, the myristoylation of the signal peptide of the virus envelope glycoproteins is essential for the fusion of the virus with the cell membrane and promotes the virus infection of host cells.⁷⁰¹ EV71 has a myristoylation modification site on the glycine residue of VP4, which improves membrane permeability. When this modification is missing, the viral genome replication is impaired.⁷⁰² ZYG11B and ZER1 are E3 ligase complexes that control the quality of N-myristoylated proteins.⁵⁸⁵

N-myristoylation endows proteins with stronger hydrophobicity, and the disruption of cellular N-myristoylation balance may lead to the occurrence of malignant tumors, CVDs, and immune diseases.^{699, 703, 704} N-myristoylation of EZH2 promotes phase separation of EZH2 with its substrate STAT3, leading to the activation of STAT3 signaling and growth of lung cancer cells, making N-myristoylation of EZH2 a potential target for lung cancer therapy.⁷⁰⁵ In rheumatoid arthritis (RA), T cell deficits in NMT1 can lead to inflammation in synovial tissue due to impaired lysosomal transfer and AMPK activation.⁷⁰⁴ During vascular lesions, N-myristoylation of LMCD1 specifically suppresses E2F1 and NFATc1, resulting in increased CDC6 and IL-33, which further affects VSMC proliferation and migration.⁷⁰⁶

A variety of treatments have also been developed for diseases caused by N-myristoylation imbalance. NMT1 is significantly upregulated in bladder cancer, and high NMT1 expression is linked to poor patient prognosis. NMT1 mediates the myristoylation of LAMTOR1 at Gly2 to increase LAMTOR1 stability and lysosomal localization, which is critical for amino acid sensing and mTORC1 activation. The inhibitor B13 can abrogate the functions of NMT1 and suppress tumor growth, suggesting that targeting NMT1 is a potential treatment for bladder cancer.⁷⁰⁷ B13 inhibits NMT1 activity by blocking Src myristoylation and reducing its cytoplasmic membrane localization, which inhibits prostate cancer cell proliferation.⁷⁰⁸

6.1 Methylation in aging

Histone methylation and methylated proteins have recently been shown to play a role in regulating lifespan and tissue aging in organisms. EZH2 is involved in the regulation of aging. Compared with young mice, more EZH2 was recruited to the SDF1 promoter region in aged mice to inhibit SDF1 expression and promote skin tissue regeneration and repair in aged mice.⁷⁴⁶ KDM2B is a regulator of mouse embryonic fibroblast (MEF) lifespan.⁷⁴⁷ KDM2B inhibits MEF senescence through demethylation of H3K36me2, resulting in cell immortalization (Table 6).⁷⁴⁸

6.2 Methylation in heart development and CVDS

Abnormal protein methylation or mutations in methyltransferases often lead to many diseases, such as CVDs, metabolic diseases, immune diseases, neurodegenerative diseases, and tumors. Mutations in multiple SETD family members have been associated with abnormal development of the cardiovascular system, mainly involving SETD2,⁷⁴⁹ SETD5,⁷⁵⁰ and SETD7.⁷⁵¹ For example, the loss of Setd2 in cardiac progenitor cells leads to obvious coronary vascular defects and ventricular noncompaction, which causes the fetus to die in mid-gestation. The mechanism may be that Setd2 deletion significantly reduces the level of H3K36me3 and affects the expression of the heart development-related genes Rspo3 and Flrt2.⁷⁴⁹ Both SETD7 and SMYD3 are H3K4 methyltransferases that are highly expressed during heart development in zebrafish. Knockout and overexpression of both Setd7 and Smyd3 induce severe defects in cardiac morphogenesis, suggesting that SETD7 and SMYD3 have a synergistic effect on heart development.⁷⁵² Moreover, abnormal expression of SETD family members is potentially related to pulmonary hypertension. SETD3 may be protective factors against hypoxic pulmonary hypertension,⁷⁵³ while SETD2, SETD8, and SETD9 may be pathogenic factors in hypoxic pulmonary hypertension or pulmonary fibrosis.^754-756 In human and mouse hypertrophic hearts, the expression of JMJD1C is increased, and the methylation level of H3K9 is decreased. Knockdown of Jmjd1c can inhibit Ang II-induced expression of hypertrophy-related genes and cardiomyocyte hypertrophy, while overexpression of JMJD1C can promote cardiomyocyte hypertrophy. Elevated JMJD1C expression induced by pathological conditions reduces the levels of H3K9me1/2/3 at the CaMKK2 promoter, which was associated with CaMKK2 gene silence. CAMKK2 could facilitate the development of metabolic dysfunction and cardiac hypertrophy.⁷⁵⁷

6.3 Methylation in metabolic disorders

Histone methylation may be responsible for diabetic complications, including diabetic neuropathy, and the phenomenon of “metabolic memory” of long-term changes, and plays a crucial role in pathways of fibrosis, inflammation, and oxidative stress.⁷⁵⁸ PRMT1 is associated with abnormal glucose tolerance by affecting hepatic glucose metabolism and insulin secretion. PRMT1 knockdown reduced the activation of insulin signaling and inhibited the expression of gluconeogenic genes in hepatocytes.⁷⁵⁹ High expression of SHP can inhibit the activity of some metabolic enzymes, which increases glucose tolerance and reduces the levels of bile acid and triglycerides. PRMT5 catalyzes Arg57 methylation of SHP to augments the SHP repression function and reduce the occurrence of metabolic syndrome.⁷⁶⁰ Increased expression of SETD7 is one of the mechanisms of vascular dysfunction in T2DM.^{761, 762} In endothelial cells, high glucose induced sustained expression of NF-κB p65 subunit and inflammatory genes by increasing H3K4me1 via SETD7 activation.^{763, 764} Moreover, high-glucose stimulation can reduce the level of H3K9me3 and increase the expression of inflammatory genes in normal human vascular smooth muscle cells (VSMCs).⁷⁵⁸ On the contrary, SETD8 is a protective factor against endothelial damage in hyperglycemic patients (Table 6).⁷⁶⁵

6.4 Methylation in immune diseases

PRMTs play a critical role in the establishment and maintenance of lymphoid and myeloid cell lines. PRMT1 is essential for lymphocyte development, proliferation, and differentiation in vivo, as well as for cytokine production by Th cells. CARM1 regulates the differentiation of early thymocyte progenitors by methylating the T cell-specific factor TARPP at R650, while PRMT5-mediated arginine methylation is crucial for the recruitment of TFs during cytokine gene expression in activated T cells. PRMTs have a role in regulating inflammation, with PRMT1 acting as a negative regulator. It interacts with and methylates the NF-κB subunit, RelA/p65, at R30 to suppress its activation by TNF-α. Asymmetric dimethylation of RelA/p65 at R30 inhibits its function as a TF.⁷⁶⁶ On the other hand, PRMT5 is a positive regulator of inflammation, as it contributes to the activation of IKK and NF-κB, and the induction of several NF-κB target genes.⁷⁶⁷ Additionally, PRMT6 and CARM1 also positively regulate inflammation.⁷⁶⁸

PRMT1 is involved in both acute and chronic asthma in epithelial cells and fibroblasts.⁷⁶⁸ The PRMT5 inhibitor C220 can reduce T cell proliferation and cytokine production, thereby alleviating acute graft-versus-host disease.⁷⁶⁹ Moreover, selectively inhibiting PRMT5 may prove to be an effective therapeutic strategy for RA and ulcerative colitis.⁷⁶⁸ PRMT7 is an essential contributor to B cell lymphomagenesis.⁷⁶⁸ FOXP3, a TF critical for Treg cell identity and immunosuppressive function, is dimethylated by PRMT5 (or PRMT1, PRMT6) at R48 and R51. This methylation reduces the expression of immunosuppressive genes, consequently leading to autoimmunity, tumor shrinkage, and associated CD8⁺ T cell infiltration.²⁷ Anti-hnRNP reactivity in RA, systemic lupus erythematosus (SLE), and mixed connective tissue diseases (MCTD) is mainly derived from arginine-methylated proteins such as hnRNP A1, A2, and K (Table 6).⁷⁷⁰

6.5 Methylation in neurogenerative diseases

Protein methylation is closely related to neurological diseases.⁷⁷¹ PRMT5 is highly expressed in mammalian neurons. In neurons, β-amyloid peptide (Aβ) deposition reduces PRMT5 expression, increasing E2F-1 expression and activating GSK-3β and NF-κB, leading to caspase-3-dependent neuronal apoptosis.⁷⁷² KMT2A can monomethylate and trimethylate H3K4 to promote neuronal gene expression. Mice heterozygous for loss-of-function mutations in the KMT2A gene exhibit learning and memory deficits.⁷⁷¹ The histone demethylase KDM2B is a candidate gene associated with intellectual disability, autism, epilepsy, and craniofacial abnormalities.⁷⁷³ KDM7B is a key factor in learning and memory. KDM7B-knockout mice show impaired learning and memory, accompanied by abnormal long-term potentiation in the hippocampus.⁷⁷⁴

The abnormal protein interactions of mutant HTT have been implicated in the pathogenesis of HD. Normal HTT stimulates PRMT5 activity in vitro. However, the presence of mutant HTT reduced the symmetrical dimethylation of arginine (sDMA) of histones H2A and H4 in primary cultured neurons and in HD brain, consistent with impaired gene transcription and RNA splicing in HD.⁷⁷⁵ In terms of PD, increased levels of α-synuclein can boost the H3K9 methylation activity of EHMT2, which may affect SNARE complex assembly and effectively vesicle fusion events.⁷⁷⁶ Methyltransferase KMT2A (MLL1) and G9a are associated with AD. KMT2A plays a protective role in AD, while G9a plays a harmful role. Inhibition of the G9a/GLP complex promotes long term potentiation and synaptic tagging/capture in the hippocampus (Table 6).⁷⁷¹

6.6 Methylation in cancers

Aberrant methylation is closely associated with the occurrence and development of cancer.⁷³⁴ Compared with normal tissues, PRMT1, PRMT4, and PRMT6 showed higher expression in lung cancer tissues.^{777, 778} Similarly, PRMT1, PRMT2, PRMT3, PRMT4, and PRMT7 are highly expressed in breast cancer tissues.⁷⁷⁹ EZH2 R342 can be methylated by PRMT1, which increases epithelial–mesenchymal transition (EMT) in breast cancer cells and predicts poor prognosis in breast cancer patients.⁷⁸⁰ Downregulation of PRMT1 expression in neuroblastoma results in reduced activity of the prosurvival factor ATF5 and inhibits tumor cell growth.⁷⁸¹ In clear cell RCC (ccRCC), a novel potent inhibitor, DCPT1061, was found to induce G1 cell cycle arrest by targeting PRMT1 activity.⁷⁸² G9a is highly expressed in diverse tumors and indicates poor prognosis. G9a can induce H3K9me2 to affect cancer cell growth and apoptosis.⁷⁸³ Set7-mediated methylation of Gli3 at K436 and K595 in the Sonic Hedgehog pathway promotes NCSLC.⁷⁸⁴ Aberrant SMYD3 expression may contribute to carcinogenesis.^785-787 In PDAC, SMYD3-catalyzed MAP3K2 methylation at lysine 260 is involved in the regulation of oncogenic Ras signaling.⁷⁸⁸ SMYD3 also exhibits a proto-oncogenic role in prostate cancer due to its methyltransferase enzymatic activity.⁷⁸⁹ Moreover, key methylation sites, such as H3K27me3, have also been found to be upregulated in many cancers including prostate cancer, breast cancer, and lymphoma, indicating their involvement in tumor progression (Table 6).⁷²⁸

6.7 Methylation-associated targeted therapies

EZH2, a key histone methyltransferase and EMT inducer, is overexpressed in diverse carcinomas. Given its role in tumorigenesis and progression, EZH2 has emerged as a potential antitumor therapeutic target.^{812, 813} It has been reported that EZH2 can enhance adhesion turnover and accelerate tumorigenesis by increasing cytoskeletal regulatory protein, Talin1 methylation and cleavage. However, this capacity is abolished by targeted disruption of the EZH2 interaction with cytoskeleton remodeling effector, VAV. The interaction of EZH2 with VAV family proteins in the cytoplasm contributes to initial tumor transformation and may maintain cancer stem cells by regulating adhesion dynamics and STAT3 signaling pathways.⁸¹⁴ Some anticancer drugs targeting mutant or wild-type EZH2, such as GSK126, were used to inhibit EZH2-mutant lymphoma cells,⁸¹⁵ and EPZ-6438 in a phase I/II clinical trial was designed for treating patients with relapsed or refractory B-cell non-Hodgkin lymphoma or advanced solid tumors.⁸¹² Many G9a inhibitors, such as diazepinquinazolin-amines and benzimidazoles, have also been developed. The current G9a inhibitors are roughly classified into three types according to their binding modes, including substrate competitive inhibitors, SAM cofactor competitive inhibitors, and inhibitors whose mechanism of inhibition remains elusive. In general, substrate-competitive inhibitors show better selectivity for G9a than SAM inhibitors.⁷⁸³ In addition, the PRMT1 inhibitor GSK3368715 has also entered phase I clinical trials,⁸¹⁶ and other inhibitors, including AMI-1, allantodapsone and furamidine, have also started preclinical studies.⁸¹⁷

7.1 Ubiquitination in development

An increasing number of studies have shown that members of various E2 and E3 families are involved in sperm capacitation, oocyte maturation, and embryonic development in mammals.⁸⁵⁶ For example, the absence of UCH-L1, an neuronal deubiquitinating enzyme, impacts the maintenance of spermatogonial stem cells homeostasis and metabolism and impacts the differentiation competence.⁸⁵⁷ Testicular macrophage USP2 promotes sperm motility, activation, and capacitation.⁸⁵⁸ Polycomb repressive complex 1 (PRC1) is known to play a crucial role in stem cell and tissue development. It has been demonstrated that PRC1, in conjunction with H2AK119ub, influences early embryonic development. High levels of expression of Polycomb repressive DUB complex (PR–DUB) in zygotes can rapidly reduce H2AK119ub levels, resulting in developmental arrest at the 4-cell stage.⁸⁵⁹ Furthermore, Cbls play a role in promoting the ubiquitination and degradation of FLT3, which results in the inhibition of FLT3 signaling and limits the development of CD8α⁺/CD103⁺ DC1 (cDC1). When Cbls are absent, activated FLT3 cannot be efficiently removed, leading to constant FLT3 signaling that favors cDC1 development and expansion.⁸⁶⁰

7.2 Ubiquitination in aging

During aging, there is a buildup of damaged and aggregated proteins, which can lead to a decline in cellular function. Ub-dependent proteolytic pathways are critical for the efficient turnover of defective proteins.⁸⁶¹ However, age-related impairment of these pathways can lead to a greater accumulation of damaged proteins, thereby exacerbating the aging process.^{861, 862} For example, in aged C. elegans, 192 proteins with low levels of ubiquitination accumulate, further contributing to the decline in cellular function.⁸⁶³ Notably, ageing causes a global loss of ubiquitination that is triggered by increased DUB activity. Parkin-mediated mitophagy is essential to ensure mitochondrial quality control in myocardium. The main mechanism of action is that the interaction between Parkin and TBKI promotes the K63 polyubiquitination of TBK1, which in turn promotes TBK1 phosphorylation to enhance mitophagy and alleviate cardiac aging.⁸⁶⁴ However, excessive or inappropriate protein ubiquitination may also shorten longevity. For example, the Ub ligase RLE-1 selectively polyubiquitinates daf-16, a key component in the insulin/IGF signaling pathway, leading to its degradation by the proteasome. As a result, inhibition of RLE-1 extends lifespan in C. elegans.⁸⁶⁵ In human fibroblasts, the degradation of BMAL1 is mediated by the E3 Ub ligase STUB1. Reduced BMAL1 can attenuate cellular senescence induced by hydrogen peroxide.⁸⁶⁶ Nrf2 is an important regulator in healthy aging, and its activity is also affected by ubiquitination. For example, p62 prevents Nrf2 from being ubiquitinated by combining with Keap1. Some studies have found that the expression of p62 decreases with age. Hrd1 is a negative regulator of Nrf2. It interacts with Nrf2 through its Neh4–5 domain to enhance its ubiquitination.^{867, 868} Stem cell dysfunction and reduced regenerative capacity are hallmarks of aging. NANOG, one of homeobox proteins, plays a crucial role in regulating self-renewal and pluripotency for embryonic stem cells (ESCs).The deubiquitinating enzyme USP21 increases NANOG levels to maintain pluripotency in ESCs by deubiquitinating NANOG to reduce NANOG proteasomal degradation (Table 7).⁸⁶⁹

7.3 Ubiquitination in immune regulation

Ubiquitination modulates the function of immune cells by regulating biological processes such as protein degradation and signal pathway transduction.⁸⁷⁰ K63- or K48-linked ubiquitination is common in these processes.⁸⁷¹ For example, the E3 Ub ligase NEURL3 promotes host antiviral immune response by catalyzing K63-linked polyubiquitination of IRF7 at K375.⁸⁷² The removal of K63 ubiquitination of TBK1 by USP15 negatively regulates TBK1 activity to suppress macrophage antiviral innate immune responses.⁸⁷³ Pellino 1 (Peli1) can mediate TLR-stimulated K63 ubiquitination of c-IAP2 in microglia to trigger the Ub ligase activity of c-IAP2, which catalyzes the K48 ubiquitination and degradation of TRAF3 and consequently induces proinflammatory cytokine production and the recruitment of autoimmune T cells in peripheral lymphoid organs to the CNS.⁸⁷⁴ Peli1 also negatively regulates T-cell activation and inhibits the development of autoimmunity through K48 ubiquitination-dependent degradation of c-Rel.⁸⁷⁵ The unanchored K48-polyubiquitin chain synthesized by the E3 ligase TRIM6 can promote the binding of DHX16 to RIG-I and mediate the production of IFN-I and the expression of IFN-stimulated genes (ISGs).⁸⁷⁶

During thymocyte development, multiple E3 ligases have been shown to play a role in T cell development. Protein ubiquitination regulates T cell development and differentiation. For example, Ub ligases, including Itch, LNX, DTX, Mib1, Mib2, Neur1, and Neur2, catalyze Notch ubiquitination, which is critical to the early stage of T cell development.⁸⁷⁷ Ubiquitination also affects the proliferation and development of B cells by regulating NF-κB signaling and participating in BCR and BAFFR signal transduction. E3 Ub ligase Hrd1 mediates the downregulation of pre-BCR through ubiquitination and promotes the maturation of pre-B cells.⁸⁷⁸

Viruses can escape from immunity by degrading target proteins. For example, the CoV nucleocapsid (N) protein of SADS-CoV interacts with RIG-I and promotes its K27-, K48-, and K63-linked ubiquitination to induce RIG-I degradation, which further inhibits the host IFN-β response.⁸⁷⁹ lncRNAs can promote influenza A virus (IAV) replication and immune evasion by restricting RIG-I K63 ubiquitination mediated by TRIM25.⁸⁸⁰ Mycobacterial PPE proteins are ubiquitinated by MKRN1, which suppresses the innate immune response.⁸⁸¹ Additionally, ubiquitination plays a role in regulating early innate immune responses triggered by human respiratory syncytial virus. RIG-I, MAVS, TRAF3/6, and NEMO are the main proteins involved in these processes.⁸⁷¹ Activation of MAVS is indispensable for antiviral immunity. Viral infection enhances the interaction between USP18 and MAVS and promotes the K63-linked ubiquitination and subsequent aggregation of MAVS to upregulate the production of IFN-I.⁸⁸² RNF115 interacts with MAVS to promote K48 ubiquitination of MAVS, and loss of RNF115 enhances antiviral signaling triggered by RNA viruses (Table 7).⁸⁸³

7.4 Ubiquitination in metabolic disorders

Dysfunction of the Ub-proteasomal system can lead to obesity-related metabolic disorders such as diabetes and fatty liver. Chronic insulin stimulation inhibits hepatocyte ubiquitination by activating USP14, which also increases the nuclear translocation of the lipogenic TF SREBP-1c to inhibit mature SREBP-1c.⁸⁸⁴ USP7 is increased in diabetic foot ulcers and human umbilical vein endothelial cells (HUVECs). USP7 inhibition can suppress AGEs-induced cell cycle arrest and cellular senescence in HUVECs by promoting p53 ubiquitination.⁸⁸⁵ E3 Ub ligase FBW7 prevents type I diabetes in nonobese diabetic mice by mediating EZH2 ubiquitination.⁸⁸⁶ MG53 acts as an E3 ligase targeting insulin receptor and IRS1 for Ub-dependent degradation. Overexpression of MG53 is sufficient to induce muscle insulin resistance and metabolic syndrome.⁸⁸⁷ Diabetic cataract is also a common complication of diabetes. The E3 Ub ligase MDM2 may promote high glucose-induced EMT and oxidative stress damage by downregulating LKB1. The EMT of lens epithelial cells is an important step in the development of diabetic cataracts.⁸⁸⁸ Pregnant women with obesity or gestational diabetes have reduced blood levels of adiponectin, which is thought to be associated with an increased risk of obesity or obesity-related insulin resistance and fetal overgrowth. Adiponectin ubiquitination is increased in the visceral fat of obese pregnant women compared to their lean counterparts, and it is a key mechanism through which obesity curtails adiponectin secretion during pregnancy (Table 7).⁸⁸⁹

7.5 Ubiquitination in cancers

Dysregulation of ubiquitination may cause a range of adverse consequences, such as abnormal activation or inactivation of signaling pathways, abnormal protein complex formation, accumulation of misfolded proteins and mislocalization of proteins,⁸⁹⁰ and even cancers.⁸³⁴ Due to the specificity of E3 in recognizing protein substrates, E3 has received increasing attention.⁸⁹¹ Some E3 ligases are carcinogenic factors, some are tumor suppressors, and some have both functions dependent on the context.⁸⁹² E3 usually participates in tumorigenesis and development by regulating the stability of oncoproteins and tumor suppressors. Members of the Cbl family of E3 ligases are involved in tumorigenesis and development by mediating lysosomal sorting and degradation of activated RTKs.⁸⁹³ Mutations and aberrant expression of C-CBL are most common in myelodysplastic syndromes.⁸⁹⁴ The tumor suppressor p53 is degraded by ubiquitination mediated by the E3 ligase MDM2, resulting in immortal cancer cell proliferation.⁸⁹⁵

DUBs also affect cancer signaling pathways by deubiquitination. The changes in DUBs may cause continuous activation or abnormal blockade of downstream signal transduction molecules such as PI3K/AKT⁸⁹⁶ and NF-κB⁸⁹⁷ to affect the progression of malignant tumors. USP7 can deubiquitinate and stabilize MDM2 (Murine double minute 2) oncoproteins, which is the major negative regulator of the p53 tumor suppressor, thereby inducing the initiation, progression, and metastasis of human cancers.⁸⁹⁸ On the other hand, USP10 can counteract the effects of MDM2-induced p53 nuclear export and degradation by deubiquitinating p53.⁸⁹⁹ Additionally, USP10 can also stabilize Smad4 by ubiquitinating it, which contributes to liver cancer metastasis.⁹⁰⁰ USP4 can interact directly with and deubiquitinates ARF-BP1, leading to the stabilization of ARF-BP1 and subsequent reduction of p53 levels.⁹⁰¹ In addition, USP4 is highly expressed in PC tumors. It stabilizes TRAF6 and activates the NF-κB signaling pathway to enhance the proliferation, migration and invasion of PC cells.⁹⁰² In cervical cancer cell lines SiHa and Caski, silencing USP18 resulted in the inhibition of cell proliferation, induction of apoptosis, and promotion of cleaved caspase-3 expression.⁹⁰³ USP14 has increased expression in cisplatin-resistant ovarian cancer cells. It inhibits ovarian cancer cell apoptosis by stabilizing the level of BCL6, which increases ovarian cancer cisplatin resistance.⁹⁰⁴ USP22 is highly expressed in human PDAC tissues. It enhances the growth and colony formation ability of cancer cells by regulating the expression of DRYK1A.⁹⁰⁵ USP32 is highly expressed in gastric cancer and is closely related to the high T-staging and poor prognosis of gastric cancer patients. Downregulation of USP32 can significantly inhibit the expression of SMAD2, thereby inhibiting the proliferation, migration, and chemoresistance to cisplatin of gastric cancer cells.⁹⁰⁶ TBLR1 plays an important role in regulating the Wnt signaling pathway. USP1 promotes the survival of liver circulating tumor cells in the bloodstream by deubiquitinating and stabilizing TBLR1.⁹⁰⁷ USP2a is highly expressed in HCC tissues and is positively correlated with poor prognosis. USP2a can deubiquitinate and stabilize RAB1A to promote HCC progression.⁹⁰⁸

In addition to impacting protein activity and degradation, ubiquitination is also implicated in cancer signaling regulation by modulating PPIs. Monoubiquitination of K-Ras at K147 can lead to enhanced GTP loading and increases its affinity for specific downstream effectors PI3K and Raf, which results in anomalous activation of the PI3K–AKT signaling pathway. This is one of the mechanisms by which the G12V-K-Ras mutant spurs malignant cell proliferation (Table 7).⁹⁰⁹

7.6 Ubiquitination in CVDS

Ubiquitination is also critical in the development of CVDs. Elevated levels of myocardial ubiquitinated proteins have been observed in most primary causes of heart failure, such as cardiac muscle loss⁹¹⁰ and cardiomyopathy.⁹¹¹ Changes in Ub protein ligases targeting myofibrillar and other cardiac proteins, such as atrogin-1 (MAFbx) and MURF-1, are associated with pathological cardiac remodeling.⁹¹² Moreover, ubiquitination is linked to atherosclerosis, with Ub–proteasome system (UPS) regulating eNOS activity and oxidative stress in the initiation and development of atherosclerosis. It also activates the NF-κB pathway, affecting adhesion molecule expression, cytokine release, and proliferation. The UPS also influences foam cell formation and maintenance, which can impact atherosclerosis progression.⁹¹² Ubiquitination also plays an important role in the development of myocardial fibrosis. By inhibiting the expression of the E3 Ub ligase Pellino1, it is possible to prevent the production of α-SMA, collagen I and collagen III, and thereby attenuate myocardial interstitial fibrosis. Pellino1 has been shown to facilitate the binding of NF-κB and AP-1 to the TGF-β promoter, which regulates the fibrogenic capability of cardiac fibroblast cells and contributes to the development of fibrosis in the heart (Table 7).⁹¹³

7.7 Ubiquitination in neurodegenerative diseases

Abnormal UPS function is closely related to the formation of protein aggregates in neurodegenerative diseases.⁹¹⁴ The accumulation of insoluble Aβ in extracellular plaques and hyperphosphorylated tau protein (P-tau) in NFTs within neuronal cytoplasm is a significant pathological factor observed in the brains of AD patients.⁹¹⁵ Reduced UPS efficiency and the inhibited autophagy-lysosomal pathway are significantly positively correlated with the abnormal accumulation of Tau at synaptic terminals.^{916, 917} Molecular misreading allows the formation of mutant proteins in the absence of gene mutations. Ubb⁺¹, a frameshift mutation product of Ub protein in the brains of AD patients, can inhibit the function of the 26S proteasome and lead to an accumulation of a large number of pathogenic proteins, such as Aβ.^{918, 919} In addition, many ubiquitination-related enzymes are abnormally expressed in AD, such as increased E2 ligase E2-25K/Hip-2⁹²⁰ and E3 ligase CHIP⁹²¹ and RNF182,⁹²² and decreased E3 ligases Parkin,⁹²³ HRD1,⁹²⁴ and the DUB UCHL1.⁹²⁵

Aβ42 can lead to hyperphosphorylation of the E3 Ub ligase Itch by abnormally activating the JNK signaling pathway. Hyperphosphorylated Itch ubiquitinates and degrades TAp73, leading to abnormal expression of important neuronal cyclins and causing neuronal apoptosis, which accelerates AD progression.⁹²⁶ The HECT family protein E6AP can activate the transcription of the ESR2 gene encoding ER-β, which reduces Aβ deposition in the hippocampus and improves learning and memory in AD rats.⁹²⁷ Loss of the E3 Ub ligase COP1 results in rapid accumulation of the TF C/EBPβ, which drives the expression of proinflammatory and neurodegeneration-related genes and accelerates the neurodegeneration of AD.⁹²⁸ However, C/EBPβ is also the main TF responsible for the transcription of the scavenger receptor CD36. The E3 Ub ligase Peli1 can directly ubiquitinate and degrade C/EBPβ, which further reduces CD36 and inhibits the phagocytosis of microglial cells, slowing the clearance of Aβ in the brains of AD mice.⁹²⁹

While the precise mechanisms of PD are not yet entirely clear, it is widely acknowledged that α-synuclein plays key pathophysiological roles as the main constituent of the cytoplasmic inclusions known as Lewy bodies. SIAH is an E3 Ub ligase that plays a key role in stress-induced cell death and α-synuclein degradation. This suggests that ubiquitination could protect against PD.⁹³⁰ However, some studies have found that SIAH can actually promote α-synuclein aggregation and enhance its toxicity,^{931, 932} leading to more inclusions in dopaminergic neurons. Inhibiting SIAH could prevent Lewy bodies formation and be a potential therapy for PD.⁹³³

The accumulation of RNA-binding protein TDP-43 in neuronal cytoplasmic and intranuclear aggregates is a defining feature of neurodegenerative disorders, including ALS and frontotemporal lobar degeneration. TDP-43 is typically modified with polyubiquitin chains that are mainly K48- or K63-linked.⁹³⁴ Insufficient degradation of abnormally aggregated TDP-43 protein leads to cell death and inflammation, which is an critical mechanism in the pathogenesis of ALS.⁹³⁵ Several studies have examined the potential therapeutic value of targeting TDP-43 ubiquitination by preventing the removal of Ub chains, with conflicting results. Inhibition of the DUB USP14 promotes TDP-43 clearance by maintaining Ub chains.⁹³⁶ However, in Drosophila, knockdown of the DUB UBPY increased TDP-43 toxicity, despite retaining Ub chains (Table 7).⁹³⁷

7.8 Ubiquitination-associated targeted therapies

An increasing number of studies have shown that ubiquitination-related enzymes are a class of important drug targets. For example, the E3 ligase inhibitors thalidomide, lenalidomide, and permadomide have been used to treat multiple myeloma.⁹⁴² These inhibitors bind to CRBN, activate the activity of CRL4^CRBN E3 Ub ligase, and induce the degradation of two important TFs, Ikaros/Aiolos, to kill cancer cells.⁹⁴³ With the continuous advancement of drug screening technology and chemical synthesis technology, an increasing number of novel small molecules targeting ubiquitination have been discovered, such as the E2 ligase UBE2 inhibitor NSC697923,⁹⁴⁴ E3 ligase MDM2 inhibitor BI-0252,⁹⁴⁵ CRL inhibitors 33-11 and KH-4-43,⁹⁴⁶ XIAP and cIAP1 inhibitor AT-IAP,⁹⁴⁷ VHL inhibitor VH298,⁹⁴⁸ DUB USP2 inhibitor 6TG,⁹⁴⁹ USP7 inhibitor FT671,⁹⁵⁰ USP9X inhibitor G9,⁹⁵¹ USP14 inhibitor IU1–47,⁹⁵² and PSMD14 inhibitor THL.⁹⁵³ For proteins that pose challenges for targeting, regulating their upstream ubiquitination-related enzymes has emerged as a novel drug development strategy.⁹⁵⁴

In 2001, the proteolysis-targeting chimera (PROTAC) was first proposed as a chemical biology tool for targeted therapies. After 20 years of development, PROTAC technology has matured.⁹⁵⁵ The structures of PROTACs are similar to a dumbbell, connecting the “ligand of target protein” and “recruitment ligand of E3 ubiquitin ligase” through a linker.^{691, 692, 956} The tagged target proteins are recognized and degraded by the intracellular 26S proteasome.⁹⁵⁵ The most significant advantage of PROTAC technology is its ability to convert potentially undruggable targets into druggable ones.⁹⁵⁷ Moreover, PROTACs overcome drug resistance.^958-960 Some PROTACs developed against cancer-associated proteins outperform traditional small-molecule inhibitors for cancer therapy. For example, oral PROTACs targeting ER and AR (ARV-110 and ARV-471) are used for the treatment of breast and prostate cancer, respectively.^961-963

8.1 Sumoylation in immune regulation

SUMOylation plays a significant role in the host immune response, as numerous SUMOylated proteins are involved in the development and activation of various immune cells.⁹⁸⁰ For example, SUMOylation of PKC-θ is required for T cell activation and formation of a mature immunological synapse.⁹⁸¹ Viruses can manipulate the process of SUMOylation through the SUMO pathway, while SUMOylation can also eliminate viral infections by regulating host antiviral immune components.⁹⁸² Additionally, SUMOylation has also been linked to autoimmune diseases, especially RA. SUMOylation bidirectionally regulates immune pathways, which can prevent hyperresponsiveness of the immune system and inhibit the development of inflammatory and autoimmune diseases.⁹⁸³ In RA patients, the expression of SUMO1 and SUMO2 is elevated in fibroblast-like synoviocytes (FLSs), especially SUMO1, and FLSs are important for promoting RA pathogenesis. SUMO-1 suppression could be protective against joint destruction in RA by inhibiting aggressive behavior of RA FLSs.⁹⁸⁴ Moreover, SUMOylation is crucial in maintaining the stability of the intestinal epithelial barrier by changing the intestinal flora, regulating immune cells, and regulating cytokines, such as IL-6, TNF-α, and IFN-γ. SUMOylation of intestinal epithelial cells (IECs) can reduce the severity of inflammatory bowel disease (IBD) by inhibiting the activity of master regulators, including the serine-threonine kinase AKT1 (Table 8).⁹⁸⁵

8.2 Sumoylation in development

The role of SUMOylation in embryonic development has been confirmed in Drosophila,⁹⁸⁶ nematodes,⁹⁸⁷ zebrafish,⁹⁸⁸ Xenopus laevis,⁹⁸⁹ silkworms,⁹⁹⁰ and various plants.⁹⁹¹ For example, an imbalance of SUMO leads to defects in embryonic patterning in Drosophila,⁹⁹² while the absence of SUMO activity disrupts multiple signaling pathways and causes neural tube and heart defects in Xenopus embryos.⁹⁸⁹ In mammals, dysregulation of SUMO leads to defects in embryonic development, craniofacial defects,⁹⁹³ and even embryonic lethality. SUMO-deficient mice suffer from severe developmental disabilities, and mice die during embryonic development. Further studies have found that only SUMO2-deficient mice die earlier in embryonic development, while SUMO1- or SUMO3-deficient mice survive and reproduce well.^{969, 994, 995}

The balance of SUMOylation in organisms is critical to the development of tissues and organs such as the heart,⁹⁹⁶ blood vessels,⁹⁹⁷ reproductive system,⁹⁹⁸ lung,⁹⁹⁹ and nervous system.¹⁰⁰⁰ Too high or too low SUMO levels may lead to organ dysfunction. SUMO1 has important and specific functions in normal heart development. Both hetero- and homozygous SUMO-1 knockout mice exhibited atrial septal defects and ventricular septal defects with high mortality rates, which were rescued by cardiac reexpression of the SUMO-1 transgene.¹⁰⁰¹ Similarly, high expression of SENP2 can enhance deSUMOylation in the mouse heart, leading to congenital heart defects and cardiac dysfunction in mice.¹⁰⁰² Angiogenesis is essential for embryonic development and tissue growth, and the NOTCH pathway is a significant negative regulator of endothelial sprouting and vascular growth. SUMOylation negatively regulates angiogenesis by targeting endothelial NOTCH signaling. Endothelial SENP1 deletion in newly generated mice significantly delayed retinal vascularization by maintaining prolonged NOTCH1 signaling.⁹⁹⁷ The dynamic SUMOylation of endothelial FGFR1 regulates the balance of the angiogenesis core pathways VEGF/VEGFR and FGF/FGFR and enables the body to complete angiogenesis in different microenvironments.¹⁰⁰³ SUMOylation also plays an important role in the generation of germ cells. SUMO1 and SUMO2/3 function at different stages of male meiosis and precisely regulate the formation of sex chromosomes.¹⁰⁰⁴ C/EBPα is a core TF that regulates cell growth and differentiation. During lung development, C/EBPα is SUMOylated and participates in C/EBPα-mediated lung growth and differentiation.¹⁰⁰⁵ Utf1 is a key SUMOylation target during neurogenesis and determines normal neurogenesis.¹⁰⁰⁶ SENP2 can regulate the calcium homeostasis of mouse neurons through the SENP2–PLCβ4 signaling axis and then regulate neurogenesis in the hippocampus.¹⁰⁰⁷ Furthermore, SUMOylation is important to maintain the development of human induced pluripotent stem cells.¹⁰⁰⁸

8.3 Sumoylation in aging

During normal aging, SUMOylation is very important.^{1009, 1010} In C. elegans, enhanced insulin/IGF signaling activity promotes SUMOylation of the germ cell protein CAR-1, resulting in shortened lifespan and impaired proteostasis.¹⁰¹¹ The degree of SUMOylation at Lys49 of UBC9 increases during aging. It has been found that the SUMOylation of UBC9 at Lys49 is conducive to its relocation to PML-NBs and promotes the translocation of target proteins into the nuclear bodies, which can transmit the antiaging phenotype. Whereas SUMOylation of proteins by the non-SUMOylated UBC9 promotes senescence.¹⁰¹² Persistent DNA damage triggers cells to undergo apoptosis or senescence to prevent replicating a damaged genome. Sp1, a protein involved in double-strand break (DSB) repair, has been linked to aging, with Sp1 levels decreasing with age. Proteasomal degradation of Sp1 in senescent cells is mediated via SUMOylation, where SUMOylation of Sp1 on lysine 16 is increased in senescent cells. Prdx6 is important in maintaining redox homeostasis and localizes to ROS-producing organelles. A gradual decrease in Prdx6 expression is associated with increased Sp1 SUMOylation and decreased Sp1 expression during aging.^{1013, 1014} SUMOylation of KLF1 at K74 regulates its transcriptional activity during erythroid differentiation. K74 SUMOylation deficiency contributes to health and longevity in mice.^{1015, 1016} SUMO can also affect lifespan by affecting the mitochondrial unfolded protein response (UPR). During mitochondrial stress, ULP4 prolongs lifespan in C. elegans by removing SUMOylated DVE1 and ATFS1 (Table 8).¹⁰¹⁷

8.4 Sumoylation in metabolic disorders

SUMOylation also plays an important role in the regulation of cellular metabolism.⁹⁶⁶ SUMOylation acts on factors related to cholesterol homeostasis, including SREBPs and members of the nuclear receptor superfamily, such as LXR, FXR, LRH1, and PPAR. These receptors are potential therapeutic targets for lipid metabolism disorders.¹⁰¹⁸ Studies have found that insufficient insulin secretion or output disorders will eventually lead to diabetes. The E3 SUMO ligase PIASy can inhibit insulin secretion by reducing the interaction between ICA512 and STAT5 through SUMOylation of ICA512.¹⁰¹⁹ Furthermore, SUMOylation can prevent stress-induced β-cell apoptosis by upregulating the levels of antioxidant genes, including Ho-1, Cat, and Nqo-1.¹⁰²⁰ ERK5 is one of the major targets of SUMOylation in diabetic hearts. ERK5 SUMOylation enhances the inhibition of ROS-mediated ERK5 transcription, which leads to the deterioration of left ventricular function after myocardial infarction in diabetic. The phenotype can be significantly reversed by inhibiting ERK5 SUMOylation.¹⁰²¹ Overexpression of SUMO4 promotes SUMOylation of IκBα, which inhibits the activation of NF-κB by external stimuli. This modification is considered to be related to type 1 diabetes (Table 8).¹⁰²²

8.5 Sumoylation in cancers

The important role of SUMOylation in human tumorigenesis has gradually emerged. SUMOylation enzymes E1, E2 and E3 are highly expressed in many types of tumors. For example, Ubc9 is associated with the occurrence and development of ovarian carcinoma, advanced melanomas, colon cancer and primary prostate cancer.⁹⁶⁸ SUMO E3 ligase PIAS1 has been implicated in the regulation of several oncogenes and tumor suppressors. In B-cell lymphoma, PIAS1-mediated SUMOylation of Myc leads to a longer half-life of the protein and increased oncogenic activity, contributing to the development of B-cell lymphoma.¹⁰²³ PIAS1 is highly expressed in prostate cancer and leads to accelerated tumor cell proliferation by inhibiting p21 expression.¹⁰²⁴ High expression of PIAS3 is very common in CRC.¹⁰²⁵

SUMOylation has an impact on cancer cell signaling and gene networks that regulate DNA damage, metabolism, inflammation and immunity, which provides link with carcinogenesis, proliferation, metastasis and apoptosis. SUMOylation is important for mammalian DNA damage response. BRCA1 participates in the DNA damage response and mutations in BRCA1 are associated with a high risk of breast and ovarian cancer. Hybrid SUMO-Ub chains are synthesized by RNF4, a SUMO-targeted Ub E3 ligase, and recognized by RAP80 to promote BRCA1 recruitment and repair of DNA DSBs. PIAS1 and PIAS4 are necessary for efficient Ub-adduct formation by RNF8, RNF168, and BRCA1 at DNA damage sites.^{1026, 968}

SUMOylation is also involved in the regulation of cancer metabolism. For example, K270 of PKM2 can be modified by SUMO1, which promotes its transformation from a tetramer to a dimer. After entering the nucleus, it binds to the SIM on RUNX1, recruits RUNX1, and regulates the differentiation process of leukemia cells.¹⁰²⁷ K315 and K492 are the SUMOylation sites of HK2. SUMOylation-deficient HK2 enhances its binding to mitochondria, which reduces mitochondrial oxidative phosphorylation and increases glycolysis and lactic acid production. This process promotes the growth of prostate cancer cells that resist chemotherapeutic drug-induced apoptosis.¹⁰²⁸

SENP3 is involved in the regulation of immune cell function, which in turn affects the progression of tumor development.¹⁰²⁹ Knockdown of SENP3 in DCs inactivates the STING-dependent type-I IFN signaling pathway and weakens the antitumor immune response. In the tumor microenvironment, SENP3 senses oxidative stimulation from DCs by deSUMOylating IFI204 and activating the STING signaling pathway.¹⁰³⁰ SENP7 can regulate the metabolic homeostasis and antitumor activity of CD8⁺ T cells in response to oxidative stress. The key deSUMOylation substrate of SENP7 in this process is PTEN.¹⁰³¹

SUMOylation plays a role in cell differentiation and carcinogenesis. Myc mutation results in the constitutive expression of Myc protein, which leads to the uncontrolled expression of various genes, including those that drive cell proliferation, ultimately leading to cancer. Loss of SAE1/2 enzymatic activity is synthetically lethal with Myc. SUMOylation-dependent Myc switchers (SMS genes) are necessary for Myc-driven tumorigenesis. Patients with breast cancer who have Myc overexpression and low expression of SAE1/2 show significantly reduced cancer cell metastasis and improved survival compared with those with high SAE1/2 expression. Similarly, in a Myc-overexpressing PDAC model, the highly selective SAE small-molecule inhibitor ML-93 significantly inhibited protein SUMOylation and tumor growth.¹⁰³² In addition, BIRC5, EG5, and TPX2 are synthetic lethal partners of Myc. The functions of these three proteins are also dependent on SUMOylation.^{1033, 1034} Moreover, SUMOylation is associated with tumor metastasis.¹⁰³⁵ hTERT is the catalytic component of human telomerase, and SUMOylation of hTERT promotes the migration and invasion of breast cancer cells.¹⁰³⁶ The SUMOylated E2-conjugating enzyme Ubc9 modifies METTL3 through SUMO1, and the SUMOylated METTL3/Snail axis is correlated with high metastatic potential of liver cancer (Table 8).¹⁰³⁷

8.6 Sumoylation in neurodegenerative diseases

Tight control of the CNS by SUMOylation is critical for maintaining neuronal cell viability, function, and connectivity.¹⁰³⁸ SUMOylation plays important roles in the repair of DNA damage in neurons, axonal mRNA transport, and the regulation of synaptic plasticity.¹⁰³⁹ Dysregulation of SUMOylation has been observed in the AD brain, with increased levels of hippocampal SUMO1 transcription possibly contributing to Aβ aggregations and impaired learning and memory abilities.¹⁰⁴⁰ In fact, several proteins involved in the physiopathological process of AD, such as BACE1, GSK3-β tau, Aβ precursor protein (AβPP), and JNK, are in fact subject to protein SUMOylation or interactions.¹⁰⁴¹ Mature Aβ is produced by hydrolysis of APP. SUMO1 modification of APP promotes the generation of Aβ plaques in AD mouse models.¹⁰⁴² In addition, melatonin can induce the SUMOylation of the APP intracellular domain (AICD). SUMOylation of AICD activates the transcription of two Aβ-degrading enzymes, which promotes Aβ degradation and delays the occurrence of AD.¹⁰⁴³ SUMO can also modify Tau. SUMOylation and phosphorylation of Tau promote each other and inhibit ubiquitination-dependent Tau degradation, thereby promoting NFT formation.¹⁰⁴⁴ α-synuclein has two SUMOylation sites, Lys96/102, which are modified by SUMO1.¹⁰⁴⁴ SUMOylation may regulate the normal and pathological functions of α-synuclein, including degradation, intracellular distribution, and PPIs and aggregation.¹⁰⁴⁵ SUMOylation promotes PD onset by preventing proteasomal degradation of α-synuclein.¹⁰⁴⁶ SUMOylation is also involved in regulating the pathogenesis of ALS.¹⁰⁴⁷ Aggregation of SOD1 is characteristic of patients with SOD1 variant-induced ALS. The modification of SOD1 by SUMO3 enhances the aggregation of familial ALS (fALS)-linked SOD1 mutants, while SENP1 decreases the number of cells exhibiting SOD1-mutant aggregation.¹⁰⁴⁸ TDP-43 can also be SUMOylated. SUMOylation promotes the formation of TDP-43 aggregates and affects the nuclear localization of TDP-43, which is involved in the pathological process of ALS (Table 8).¹⁰⁴⁹

8.7 Sumoylation in CVDS

SUMOylation is also closely related to the development, metabolism, and pathology of the heart. For example, SUMO1 is essential for normal cardiac development, and cardiac-specific overexpression of SUMO1 improves cardiac functions. SUMO1 mutant or knockout mice are more prone to congenital heart defects.¹⁰⁵⁰ Specifically, UBC9 is the sole E2 enzyme essential for GATA4's role in cardiac development and function. It boosts GATA4's transcriptional activity and affects its nuclear localization. UBC9 adds a SUMO group to GATA4 at K366, which activates particular gene expression in pluripotent cardiac cells.¹⁰⁵¹

Protein SUMOylation and its dysregulation are implicated in various CVDs, including atherosclerosis, heart failure, and ischemic cardiomyopathy. Several factors, including ERK5, NF-κB, p53, and PKC, undergo SUMOylation, which contributes to atherosclerosis progression.¹⁰⁵¹ For example, SUMO1-mediated SUMOylation of NF-κB inhibits IκBα degradation and reduces NF-κB activation.¹⁰⁵² In contrast, SUMO2/3 modification promotes IκBα detachment from NF-κB and enhances NF-κB activation, ultimately inducing atherosclerosis.^{1052, 1053} Additionally, SUMOylation of HSF2, myocardin, PARIS, PPARγ1, and SERCA2a plays crucial roles in heart failure progression.¹⁰⁵¹ The protein levels of SUMO1 and SUMOylated SERCA2a are significantly decreased in failing hearts, whereas increased SUMOylation of SERCA2a improves myocardial contractility and ventricular function in heart failure mice.¹⁰⁵⁴ Conversely, hypoxia induces SUMOylation of HIF-1α, which promotes HIF-1α degradation, whereas SENP1 deSUMOylates HIF-1α and enhances its stability.¹⁰⁵⁵ Reduced SENP1 levels exacerbate ischemia/reperfusion (I/R) injury in cardiomyocytes through the HIF-1α pathway, while HIF-1α overexpression counteracts the detrimental impact of SENP1 downregulation on cell death (Table 8).¹⁰⁵⁶

8.8 Sumoylation-associated targeted therapies

Targeted cancer therapy may be achieved by inhibiting the SUMO pathway.¹⁰⁶⁷ In 2021, Bellail's team screened the hit compound CPD1 that can specifically target and degrade SUMO1 from the drug-like compound library of the National Institutes of Health (NCI). CPD1 specifically reduces SUMO1 protein levels without affecting SUMO1 mRNA levels. Further druggability optimization identifies the first highly selective SUMO1 degrader, HB007. HB007 inhibits the proliferation of various tumor cells by selectively degrading SUMO1.¹⁰⁶⁸ The highly selective SAE inhibitor TAK-981 can significantly upregulate the expression of IFN1 and activate IFN1-dependent innate immune cells, including macrophages, NK cells, DCs, and T cells, promoting antitumor immune responses.¹⁰⁶⁹ Moreover, TAK-981 is currently in phase 1 clinical trials in patients with solid tumors and lymphomas.¹⁰⁶⁹ Although there are still many blind spots on the mechanism of SUMOylation involved in the occurrence and development of tumors, AD/PD, and CVDs at this stage, a large number of experiments have confirmed the role of SUMOylation in these diseases. Therefore, drugs targeting the SUMOylation mechanism may represent a promising treatment strategy.¹⁰⁷⁰

9.1 Glycosylation in development

During the developmental process, protein glycosylation occurs at various times and locations, and these characteristic glycans cover the surface of nearly all cells. The numerous and complex structures of glycans provide strong support for cell-to-cell communication during growth and development.^{1134, 1135} Genetic defects in glycosylation are often embryonic lethal.¹¹³⁶ Congenital disorders of glycosylation (CDGs) are diseases caused by disorders of glycoprotein synthesis, with various clinical manifestations, such as the appearance of special facial features and lesions in the organs of the body, which can be divided into Type I and Type II CDGs.^{1137, 1138} The causative factors of CDGs include abnormal activation, presentation, or transport of glycolipid precursors, abnormal expression or activity of glycosidases or glycosyltransferases, and abnormal functions of proteins that control glycosylation or maintain the Golgi apparatus.¹⁰⁸² Furthermore, during embryonic development, N-glycosylation plays a key role in the generation of hematopoietic stem cells (HSCs) from arterial endothelial cells through the process of endothelial to hematopoietic transition (EHT) and is a determinant of hematopoietic fate.¹¹³⁹ CDGs often have serious consequences, suggesting the important roles of protein glycosylation in maintaining the normal growth and development of individuals (Figure 19).

9.2 Glycosylation in aging

Protein glycosylation is a dynamic process highly sensitive to aging,¹¹⁴⁰ and acts as a potential important molecular effector in aging and age-related diseases.¹¹⁴¹ Numerous studies have shown that the glycan at Asn279 on IgG heavy chains undergoes changes during aging that are often similar to those found in inflammatory states,¹¹⁴² but the mechanism by which the glycans at Asn279 on IgG heavy chains are altered is unclear (Figure 19).^{1143, 1144} In addition, reduced expression of the glycosyltransferase B4GALT1 can prevent senescence-associated IgG glycan changes and improve the senescence phenotype.¹¹⁴⁵ The glycosyltransferase ST6GAL1 can alter the glycans of fibroblasts, leading to the transition of fibroblasts to proinflammatory cells.¹¹⁴⁶ Sialic acid-binding immunoglobulin-like lectins (Siglecs) act as inhibitory receptors on the immune cell surface.¹¹⁴⁷ The activity of Siglecs appears to correlate with longevity, possibly due to their ability to suppress aging-related inflammation.¹¹⁴⁸

9.3 Glycosylation in immunity

A large number of glycoproteins on immune cell surfaces are extensively involved in many immune processes by receiving signals from the extracellular environment.¹¹⁴⁹ Neutrophils are the most abundant innate immune cells and act as the host's first line of defense against pathogen invasion, utilizing bioactive glycoproteins assembled in the cytoplasm to fight pathogenic infections.^{1150, 1151} N-glycosylated and O-glycosylated proteins exhibit structural and functional diversity in different life stages of neutrophils during bone marrow maturation, blood circulation, and sterilization of inflammatory peripheral tissues.¹¹⁵² Numerous modified granule glycoproteins are present in neutrophils, including neutrophil elastase, myeloperoxidase, and cathepsin G.¹¹⁵³ In addition, neutrophils possess unique glycans that are not typically observed, such as hypertruncated chitobiose core- and paucimannosidic-type N-glycans and monoantennary complex-type N-glycans.^{1154, 1155} The glycoproteins major histocompatibility complex (MHC) classes I and II play key roles in adaptive immunity. They are closely associated with the presentation of cell surface antigen peptides and circulating T lymphocyte recognition and activation. Glycosylated protein antigens are critical for antigen uptake by cells, proteolysis, antigen presentation by MHC, and activation and initiation of T cells.^{1156, 1157} In the adaptive immune system, protein glycosylation also has multifaceted roles in the differentiation of B cells and T cells, cell–cell interactions and the recognition of glycosylated antigens (Figure 19).^1157-1159 Protein glycosylation has broad impacts on the function of the immune system.

9.4 Glycosylation in the gut

There are numerous glycosylation modifications on the surface proteins of IECs that form a physiological barrier to protect the intestinal tract from bacterial infection and invasion. For example, Golgi glycosyltransferases modify mucin in the gut to separate IECs from commensal microorganisms.¹¹⁶⁰ Changes in the glycosylation level of proteins on the surface of IECs can result in the destruction of the mucus layer and the occurrence of intestinal IBD (Figure 19).¹¹⁶¹ Glycosylation of IEC surface proteins can also alter the structure and function of the microbiota.¹¹⁶² IL-22-mediated glycosylation of intestinal cells facilitates the growth of succinate-consuming Bacillus in the gut microbiome, which reduces the availability of succinate, a key metabolite for Clostridium difficile growth, and prevents Clostridium difficile infection.¹¹⁶³

9.5 Glycosylation in neurodegenerative diseases

A growing number of studies have shown significant differences in the levels of N-glycosylation and O-glycosylation in brains between AD patients and healthy individuals.^1164-1168 APP is concentrated at the synapses of neurons. After being cleaved by proteases, it produces toxic Aβ protein that accumulates in AD patients.¹¹⁶⁹ N-glycosylation- and O-glycosylation-modified APP is found in the cerebrospinal fluid of AD patients.¹¹⁷⁰ The structure of N-glycans can affect APP transport and Aβ production.¹¹⁷¹ The modification of APP-linked N-glycans by sialylation may affect APP processing, resulting in increased APP secretion and Aβ production.¹¹⁷² In addition, various O-glycosylation sites have also been identified on APP in human cerebrospinal fluid,¹¹⁷³ and O-glycosylation modification can also increase nonamyloidogenic α-secretase processing, thus affecting APP processing and reducing Aβ secretion.¹¹⁷⁴ In AD patients, Tau proteins modified by N-glycosylation and O-GlcNAcylation can also be detected,^{1175, 1176} and the levels of N-glycosylation in AD patients are higher than those in healthy people,¹¹⁷⁷ which may affect the aggregation of Tau proteins.¹¹⁷⁵ Interestingly, the level of O-GlcNAcylation is reduced in AD patients.¹¹⁷⁸

TREM2 expressed on myeloid cells is a PD-related protein that has multiple ligands, including APOE and lipids. By interacting with DNAX activating protein to transduce signals, TREM2 plays an anti-inflammatory role in various diseases, including PD,¹¹⁷⁹ and can be modified by sialylated and fucosylated complex glycans. N-glycan changes alter TREM2 conformation and affect the stability and antioxidant capacity of the protein.¹¹⁸⁰ α-Synuclein can also be modified by O-GlcNAcylation.¹¹⁸¹ O-GlcNAcylation of α-synuclein affects its phosphorylation and blocks the toxicity of α-synuclein, suggesting that an increase in O-GlcNAcylation may prevent α-synuclein aggregation (Figure 19).¹¹⁸²

9.6 Glycosylation in viruses

Protein glycosylation is involved in the regulation of host–pathogen interactions and mediates the adhesion, recognition, invasion and immune evasion of pathogens in host cells, which affects pathogen virulence or host cell resistance.^1183-1188 COVID-19 caused by SARS-CoV-2 is currently a major global health problem.¹¹⁸⁹ Protein glycosylation affects the toxicity and viability of SARS-CoV-2, which utilizes its highly glycosylated modified spike (S) protein to interact with the glycosylated host receptor ACE2 and to facilitate SARS-CoV-2 invasion of host cells.¹¹²⁷

Acquired immune deficiency syndrome caused by human immunodeficiency virus (HIV) seriously endangers human health. On the HIV-1 envelope (Env), there are approximately 25 and 4 glycosites on each gp120 monomer and gp41 subunit, respectively, and 18–33 glycans on each gp120 monomer. These glycans are dominated by the high mannose type and sialylated complex type.^{1190, 1191} which can bind to the chemokine receptors CD4 and DC-SIGN of host cells or mannose receptors of macrophages and regulate HIV-1 invasion of host cells (Figure 19).^{1192, 1193} Highly glycosylated HIV binds to C-type lectin receptors (CLRs) of different antigen-presenting cell subsets, possibly regulating T cell priming and B cell activation.^{1194, 1195} N-glycosylation of gp120 in HIV-1 is critical for CD4⁺ T cell recognition.¹¹⁹⁶ HIV infection also leads to altered glycosylation of host IgG. The levels of galactosylation in HIV⁺ patients are lower than those in healthy people, and it is more pronounced in the IgG1 subtype.¹¹⁹⁷ Low sialylation of IgG is also found in HIV-infected patients.¹¹⁹⁷ During the evolution of HIV, the glycans on the HIV-1 envelope become more complicated under the guidance of natural selection, which also makes HIV more diverse.¹¹⁹⁸

9.7 Glycosylation in cancer

O-GlcNAc of the key cell cycle regulators participates in the processes of cell division, DNA repair, and cell death and is dynamically changed in a cell cycle stage-dependent manner.¹¹⁹⁹ MUC1, a transmembrane glycoprotein associated with the cell cycle,¹²⁰⁰ is overexpressed and aberrantly glycosylated in a variety of epithelial cancers and plays an important role in disease progression.¹²⁰¹ Dysregulation of glycosyltransferases such as ST6GalNAcI, C1GalT1, and ST3GalI alters the level of O-glycosylation of MUC1.^1202-1204 In addition, changes in glycosylation motifs on MUC1 also affect cancer immune surveillance. For example, the binding of sialylated MUC1 to the cell surface lectin CD169 can enhance macrophage activation and promote tumor growth.¹²⁰⁵

The growth of tumor cells is accompanied by immune escape. There are ligands of programmed death receptors on the surface of tumor cells, which can bind to programmed death receptors (such as PD1) on the surface of T cells, making T cells exhausted and unable to kill tumor cells. Aberrant glycosylation on the surface of tumor cells can alter how the immune system senses tumors and induce immunosuppressive signals. Thus, specific glycans on tumor cells represent a novel immune checkpoint. Aberrant glycosylation on the surface of tumor cells may affect antitumor responses. Changing the level of glycosylated proteins on the surface of tumor cells can enhance the killing effect of CAR-T cells on solid malignant tumors.¹²⁰⁶ Moreover, the glycosylation of tumor cell surface proteins also provides new antigen targets for tumor-specific T cells (Figure 19).¹²⁰⁷ The most common tumor-associated glycans include sialylated glycans, Tn antigen and Lewis antigen. The level of sialylation on melanoma cells correlates with the level of tumor growth in vivo, which is associated with the accumulation of Treg cells, reduction of effector T cells, and decreased activity of NK cells.¹²⁰⁸ Poor survival in patients with stage III colon cancer is associated with BRAF mutation and increased Tn antigen.¹²⁰⁹ In addition, increased Lewis antigen in the tumor microenvironment can drive innate immune suppression.¹²⁰⁷

9.8 Therapeutic glycosylated proteins

Almost all therapeutic proteins are glycosylated, such as EPO, ENPP1, and IgG antibodies. Carbohydrate components play an important role in the safety and pharmacokinetic properties of these protein-based drugs.¹²¹⁰ Rapid advances in the field of glycobiology have provided more opportunities for the development of glycoprotein therapeutics.¹²¹¹ Technologies are being developed for glycan processing of therapeutic proteins using chemical, chemoenzymatic and genetic approaches in different cell types.^{1210, 1212, 1213} In addition, engineered cells provide a more powerful tool for developing more complex protein glycan structures and improving the pharmacodynamic properties of therapeutic proteins.¹²¹¹

EPO is an endogenous glycosylated hormone that can stimulate erythropoiesis and has a variety of important physiological functions.¹²¹⁴ It can be used to treat anemia caused by CKD and cancer. The recombinant EPO used for clinical treatment can be divided into four types according to their different glycosylation levels. Sialylation and branching N-glycans on EPO can prolong its half-life in serum,^{1215, 1216} while EPO lacking sialylation exhibits neuroprotective effects in vivo.¹²¹⁷ This shows the importance of the type of glycosylation for therapeutic EPO.

Mutations in ENPP1 cause generalized arterial calcification in infancy (GACI), an extremely rare neonatal disease associated with extensive arterial calcification and narrowing. ENPP1 cleaves ATP into PPi and AMP extracellularly. Recombinant ENPP1-Fc protein has preventive and therapeutic effects on GACI animal models.¹²¹⁸ Enhancing the sialylation level of recombinant ENPP1-Fc protein has a significant effect on prolonging the protein half-life and improving drug efficacy.¹²¹⁹

Glycosylation-dependent IgGs are therapeutic antibodies for cancers.^{1220, 1221} Glycosylation of the IgG Fc region affects the safety and clinical efficacy of therapeutic antibodies. Biantennary complex oligosaccharide modification of Fc at Asn297 is essential for the effector function of antibodies. Fucose and outer arm sugars linked to the core heptasaccharide create structural heterogeneity that also exhibits unique biological activities.¹²²² Clarifying the glycosylation profile of Fc is the key to the development and quality control of therapeutic antibodies.

Glycosylated vaccines also have broad application prospects. For example, changes in the glycans of the glycoprotein gp120 expressed on the HIV-1 envelope may lead to immune escape of the virus.¹²²³ In the process of vaccine design, adding a new glycosylated epitope on recombinant gp120 is beneficial to improve the ability of neutralizing monoclonal antibodies to recognize HIV-1, thereby optimizing the design of viral vaccines.

10.1 Citrullination in immune diseases

Inflammation, chronic pain, and polyarthritis are the main features of RA.¹²⁵⁸ A growing amount of evidence shows that the autoimmune reaction to RA is driven by dysregulation of protein citrullination mediated by PAD4, as 75% of patients have anticitrullinated protein antibodies (ACPAs).¹²³⁹ ACPAs are the most specific autoantibodies in RA serum.¹²⁵⁹ Most of these autoantibodies can be detected early in the plasma, making them useful diagnostic markers for RA.^{1260, 1261} In inflammatory synovial tissues, macrophages and neutrophils express more PAD2 and PAD4, respectively. Both PAD2 and PAD4 are released into joint citrullinated proteins, such as fibrin, fibrinogen and vimentin,¹⁷⁵ which further initiate immune responses and induce autoantibodies (Figure 21).^{1262, 1263} Blood coagulation factor citrullination is also important in RA.¹²⁶⁴ Thrombin can generate inflammatory mediators that promote inflammation, resulting in excessive capillary formation and fibrin deposition in synovial tissues (Figure 21). PAD4-mediated citrullination of antithrombin inhibits thrombin activity, resulting in an increase in the coagulation rate.¹²⁶⁵ The levels of citrullinated antithrombin are significantly elevated in RA patients compared to healthy controls.¹²²⁹ Inhibiting hyperactivated PAD enzymes to reduce citrullination is a promising therapeutic strategy for RA patients.¹²⁶⁶

MS is characterized by inflammatory demyelinating lesions of the CNS. The pathogenesis of MS is complex and is currently thought to be caused by a combination of genetic and environmental factors.¹²⁶⁴ MS is primarily associated with PAD2-mediated over-citrullination of myelin basic protein and GFAP, leading to demyelination and affecting nerve signal transduction (Figure 21).^{1267, 1268} Overexpression of PAD2 in transgenic mice increases the amount of citrullinated myelin basic protein and accelerates demyelinating changes.¹²⁶⁹ Citrullinated myelin basic protein alters its processing and presentation by T cells. Despite the presence of T cells specific for citrullinated myelin peptides, no autoantibodies against citrullinated proteins have been found in the serum of MS patients.¹²⁷⁰ Furthermore, the upregulation of PAD2 and inflammatory signaling may locally increase PAD4 to further aggravate inflammatory disease. Collectively, myelin basic protein citrullination mediated by PAD2 and PAD4 promotes proteolysis, demyelination, and signaling blockade, ultimately leading to MS.

SLE is manifested by the immune system attacking healthy cells and tissues throughout the body. SLE immune system activation is characterized by B cell and T cell hyperreactivity and loss of immune tolerance to self-antigens.¹²⁷¹ Autoantigens in SLE patients mainly come from apoptosis and the formation of NETs, and citrullination is involved in these processes and affects the occurrence and development of SLE. For example, LL37 binds to self-DNA/RNA and stimulates plasmacytoid dendritic cells to produce IFN-I, leading to an autoimmune response. In the skin and kidney of SLE, citrullinated LL37 (cit-LL37) is significantly increased, and LL37-specific T cells show a significant response to cit-LL37 (Figure 21).¹²⁷² Many NETs-related proteins are posttranslationally modified, especially histones found to be methylated, acetylated, and citrullinated, indicating that NETs may be a source of self-antigens in autoimmune diseases (Figure 21).¹²⁷³ Aberrant apoptotic pathways prevent immune cell clearance, which prolongs the exposure of self-antigens and induces the production of autoantibodies.^{1264, 1274} Among the autoantibodies associated with SLE, many antigens, including nuclear DNA and proteins, can be detected in NETs.^{1283, 1293}

10.2 Citrullination in cancers

p53 is a well-known tumor suppressor and TF.¹²⁷⁵ Based on pathological studies of patient samples, PAD4 is highly expressed in a variety of tumors, including colon cancer, esophageal cancer, ovarian cancer, PC, and gastric cancer,^{1237, 1276} suggesting possible involvement of PAD4 in tumorigenesis. PAD4 expression is regulated by p53. PAD4 citrullinates the growth inhibitor ING4, which subsequently prevents the binding of p53 to ING4 to inhibit p53 expression, further inhibiting downstream p21 expression and promoting tumor growth (Figure 21). Notably, PAD4 forms negative feedback to regulate p53 through histone citrullination.¹²⁷⁷ PAD inhibitors may also prevent the expression of genes related to cancer cell invasion and metastasis.¹²⁵⁶

10.3 Citrullination in inflammatory diseases

Ulcerative colitis is a chronic inflammatory disease occurring in the colonic mucosa. Ulcerative colitis patients often suffer from serious complications, the most common of which is peripheral arthropathy.¹²⁷⁸ Immunohistochemical analysis shows increased PAD2 and PAD4 in damaged tissues in ulcerative colitis patients.¹²⁷⁹ The upregulation of PAD4 is associated with neutrophils and NETs in the colonic mucosa of ulcerative colitis patients, even in remission.¹²⁸⁰ Proteins that promote NETs formation are potential therapeutic targets to reduce inflammation in ulcerative colitis.¹²⁸¹ The severity of acute colitis in PAD4-deficient mice with considerable rectal bleeding is evidence that PAD4 is essential for maintaining colitis mucosal homeostasis and regulating rectal bleeding.¹²⁸²

11.1 Carbamylation in aging

Studies have found that various physiological and pathological processes are related to protein carbamylation, such as aging, cataracts, CKD, atherosclerosis, RA, and neurological diseases (Figure 22B).¹²⁸³ By measuring the changes in homocitrulline, a typical carbamylation derivative product (CDP), with age, carbamylation occurs throughout the life cycle and contributes to carbamylated protein accumulation in organs. The accumulation rate of CDPs is negatively correlated with lifespan, suggesting that longer-lived species may have efficient turnover, repair, or degradation systems to limit carbamylated protein accumulation in organs.¹²⁹⁶ Modifications of salivary proteins increase with age, as evidenced by decreased total thiol levels and increased carbamylated proteins in the saliva of older adults.¹²⁹⁷ Therefore, protein carbamylation may serve as a marker of mammalian aging.¹²⁸⁵ In addition, protein carbamylation in peripheral blood is associated with age-related oxidative damage.¹²⁹⁸ In elderly cataract patients, crystallins, including α-, β-, and γ-crystallin, can be modified by carbamylation, with seven lysine residues modified in α-crystallin.¹²⁹⁹ An important function of α-crystallin in the lens is to ensure the activity of its chaperones, which limit protein aggregation and thus keep the lens transparent. However, α-crystallin carbamylation significantly affects its chaperone activity.¹³⁰⁰

11.2 Carbamylation in kidney diseases

Carbamylated protein levels have been found to be significantly elevated in CKD (Figure 22B), and carbamylated albumin is considered an important biomarker for risk of death.¹³⁰¹ Carbamylated proteins may cause CKD complications such as atherosclerosis. Higher levels of carbamylated proteins are detected in the plasma of 75% of kidney-removed CKD mice.¹³⁰² C-Alb carbamylation levels are associated with higher mortality in diabetic patients with ESRD.¹³⁰³ Carbamylated HDL but not carbamylated LDL in plasma is independently related to CKD progression in T2DM patients.¹³⁰⁴ By investigating the levels of fibrinogen carbamylation in patients with renal disease and the effect of carbamylation on thrombin fibrinogen cleavage, fibrin polymerization and in vitro crosslinking, it has been found that although carbamylation itself does not affect thrombin cleavage, it alters fibrin polymerization kinetics and impairs cross-linking and clot degradation.¹²⁹⁵

11.3 Carbamylation in atherosclerosis

Protein carbamylation is closely associated with atherosclerosis (Figure 22B).¹³⁰⁵ “Uremic dyslipidemia” in CKD patients is characterized by normal low-density lipoprotein cholesterol, low high-density lipoprotein cholesterol, and high triglyceride plasma levels.¹³⁰⁶ cLDL induces the prothrombotic effects of vascular cells and platelets by activating LOX-1 receptors and promotes thrombus formation.¹³⁰⁷ An increased incidence of acute thrombosis has been observed in patients with CKD. In addition, the slight carbamylation of LDL leads to a decrease in the uptake of LDL by fibroblasts, which results in a lower clearance rate and prolongs the residence time of these particles in the blood circulation. This consequently increases the chance of further carbamylation of LDL. High cLDL leads to the accumulation of cholesteryl esters in macrophages.^{1308, 1309} Meanwhile, cLDL causes damage to vascular endothelial cells and induces abnormal proliferation of VSMCs, eventually leading to atherosclerosis.¹³¹⁰ Compared with cLDL, HDL has antiatherosclerotic properties and protective effects on the heart. However, carbamylation of HDL is involved in the occurrence and development of atherosclerotic CVD.¹³¹¹ Carbamylated HDL engages in the formation of foam cells and becomes proatherosclerotic lipoproteins.¹³¹² In addition, after carbamylation, vascular elastin fiber morphology and susceptibility to elastase degradation remain unchanged, but elastic fiber stiffness increases. These changes in the mechanical properties of the vascular wall may lead to aortic stiffness.¹³¹³

11.4 Carbamylation in immune diseases

In addition to ACPAs,¹³¹⁴ autoantibodies against carbamylated proteins (anti-CarP) have been detected in the serum of RA patients (Figure 22B).¹³¹⁵ Anti-CarP has important implications in the pathophysiology of RA and can be used to assess the risk level of RA patients. RA patients also develop autoantibodies against carbamylated NET (cNET) antigens, and the levels of these antibodies correlate with anti-CarP levels, making them a new biomarker for RA.^{1316, 1317} In addition, carbamylated histone-IgG immune complexes can promote osteoclast differentiation and enhance the matrix resorption of osteoclasts, suggesting that carbamylated proteins in NETs can increase pathogenic immune responses and bone destruction. This explains the link between anti-CarP levels and erosive arthritis in RA patients.¹³¹⁷

11.5 Carbamylation in neuropathy

Carbamylation has also been related to neuropathy (Figure 22B). The higher the level of carbamylation in the rat brain, the more severe the memory loss.¹²⁸³ Carbamylation has also been implicated in AD,¹³¹⁸ and prevention of carbamylation may protect against cyanate-induced neuropathy.¹³¹⁹ Collectively, protein carbamylation is a potential biomarker for various human diseases and has important clinical significance.

12.1 S-Nitrosylation

The formation of SNO is a reversible nonenzymatic catalyzed reaction in which NO is covalently bound to the thiols of cysteines to form SNO.¹³²⁵ In general, there are three pathways for the synthesis of SNO (Figure 23B). NO reacts directly with the thiol group of the cysteine through NO⁺,¹³²⁶ the SNO-modified small molecule or the proximal SNO-modified protein provides NO⁺,¹³²⁷ or the thiol of cysteine can be activated to form a sulfur radical (S•), which then reacts with a NO radical (NO•) to generate SNO.¹³²⁶ S-nitrosothiols are biologically stable reservoirs of NO,¹³²⁸ have selective and transient modification properties and are excellent signal sensors.¹³²⁹ For each protein, SNO can occur at a single cysteine or multiple cysteines.¹³³⁰ There are more than 3000 proteins that are regulated by SNO and are involved in the regulation of protein stability, DNA damage repair, transcriptional regulation, cell growth, differentiation, and apoptosis (Figure 23B).¹³³⁰ In addition, SNO also regulates the protein conformation of overlapping or nonoverlapping residues, PPIs, and other PTMs (e.g., phosphorylation, acetylation, ubiquitination, and disulfide linkage).¹³³¹ Dysregulation of SNO contributes to many diseases.^1332-1334

12.2 S-Glutathionylation

Glutathione is covalently bound to reactive cysteines in proteins through disulfide bonds, termed S-glutathionylation.¹³³⁵ S-Glutathionylation can occur via nucleophilic sulfur, where the thioanion (S-) reacts with oxidized glutathione (GSSG), or via the reaction between GSH and electrophilic sulfur intermediates such as sulfenic acid, S-nitrosothiol, and thiol radical (Figure 23C).¹³³⁶ S-glutathionylation is involved in redox signaling and protects the thiols of cysteines from irreversible oxidation during oxidative stress. Many enzymes are known to catalyze glutathionylation/deglutathionylation reactions, including GSTP and GRX.¹³³⁵ GSTP possesses both molecular chaperone and catalytic properties and controls the redox balance in the oxidative endoplasmic reticulum. It catalyzes the S-glutathionylation of target proteins and impacts the function of unfolded proteins.¹³³⁷ GRX plays a crucial role in removing S-glutathionylation, preserving cellular redox homeostasis by regulating the S-glutathionylation of essential proteins such as phosphatases, kinases, and TFs.¹³³⁸ Therefore, S-glutathionylation balance serves as a feature of normal cellular redox homeostasis.¹³³⁵ Moreover, S-glutathionylation also regulates the activities of mitochondrial enzymes, heat shock proteins, TFs, and cytoskeletal proteins.¹³³⁹ Hsp90 is a widely distributed molecular chaperone that interacts with a variety of proteins and regulates a variety of cellular processes. S-glutathionylation of Hsp90 results in inactivation of ATPase.¹³⁴⁰ S-glutathionylation of C/EBPβ stabilizes the protein and increases its levels, promoting 3T3l1 cell differentiation.¹³⁴¹ Additionally, S-glutathionylation plays a role in regulating apoptosis. For example, S-glutathionylation of GADPH may transmit signals to the nucleus where GADPH trans-glutathionylates nuclear proteins such as Sirt1 to trigger apoptosis. GRX removes S-glutathionylation of GAPDH and prevents its nuclear transport.¹³⁴² FASL-induced activation of airway epithelial cell apoptosis is accompanied by an increase in protein S-glutathionylation.¹³⁴³

12.3 Sulfenylation

Cysteine thiols are oxidized by hydrogen peroxide to produce sulfenic acids. Sulfenylation is a nonenzymatic modification that can also be converted from other oxidized forms of cysteine, such as SNO (Figure 23D).¹³²⁶ Sulfenic acids have long been considered transient reaction intermediates formed by cysteine thiols under oxidative stress.¹³⁴⁴ However, they have been discovered to play much more significant roles in cellular biology. These sulfenic acids serve not only as indicators of oxidation-sensitive cysteines and intermediate oxidation states, but also as key regulators of protein function, signal transduction, and initiators of disulfide bond formation.^{1345, 1346} For example, the formation of sulfenic acid is associated with hydrogen peroxide-mediated inactivation of PTPs, and the oxidation of cysteine thiol affects cellular PTP activity.¹³⁴⁴ Sulfenylation increases the kinase activity of EGFR by oxidizing the active site Cys797.¹³⁴⁷ The activity of Src is regulated by a redox-dependent mechanism, in which sulfenylation at Cys185 and Cys277 can enhance its activity.¹³⁴⁸ Platelet CD36 signaling can promote hydrogen peroxide-mediated sulfenylation of Src family kinases, which is critical for oxLDL/CD36 proaggregation and procoagulant functions.¹³⁴⁹ UCP1 is a protein required for thermogenesis in adipose tissue. Cys253 of UCP1 is sulfenylated during thermogenesis and affects sensitivity to UCP1-dependent thermogenesis.¹³⁵⁰ The redox state of a single cysteine can alter biological processes in response to changes in cellular redox homeostasis.¹³⁵¹ For example, ROS-induced sulfonylation of C663 inhibits the UPR and stimulates the antioxidant response mediated by p38 MAPK signaling.¹³⁵² Reversible sulfenylation can also regulate the enzymatic activity of transcription and transduction factors, such as Mfn2, which undergoes sulfenylation after inflammatory stimulation and negatively regulates the transcriptional activity of β-catenin.¹³⁵³ In addition, protein sulfenylation can also directly or indirectly affect many PTMs in cells, such as the oxidation of specific cysteines in PTPs, PTK, and cysteine proteases.¹³⁵⁴

12.4 Sulfinylation and sulfonylation

Cysteine sulfinic and sulfonic acids are the peroxidation products of cysteine.¹³⁴⁵ In the presence of excess oxidants, sulfenic acid can be oxidized to sulfinic acids or even sulfonic acids (Figure 23D). Sulfonic acids are the most oxidized form of cysteine. Although sulfinic acid was once thought to be an irreversible oxidative modification, the oxidation of the active site cysteine of Prx I or Prx II to sulfinic acids is reversible.¹³⁵⁵ With the discovery of sulfiredoxin,^{1356, 1357} the regulation of sulfinylation in biology has also received attention. By using electrophilic diazene probes (DiaAlk), hundreds of previously unreported protein sulfinylation sites have been identified in mammalian cells.¹³⁵⁸ Prx is an important class of human antioxidant enzymes that are present in high concentrations in human cells such as erythrocytes¹³⁵⁹ and can rapidly oxidize sulfenic to sulfinic acids.¹³⁶⁰ Under normal conditions, Prx utilizes the thiol/sulfenic acid oxidative cycle to detoxify hydrogen peroxide. However, under conditions of oxidative stress, the hydrogen peroxide concentration exceeds the reducing power of Prx, resulting in the formation of sulfinic acids. Cysteine residues in Prx sense the intracellular hydrogen peroxide concentration.¹³⁶⁰ DJ-1 is a protein associated with hereditary Parkinson's syndrome. Sulfinylation at Cys106 regulates the protective function of DJ-1.¹³⁶¹ Cysteine residues usually exist in metal-binding motifs and form coordinate bonds with metal ions, such as zinc, copper, and iron. However, sulfinylation of these proteins can lead to the release of zinc ions and changes in protein conformation, which in turn alter protein function.¹³⁶²

12.5 Disulfides

Disulfide bonds in proteins are a widespread cysteine modification that plays an important role in protein folding and stability. Disulfide bonds form intermolecularly or intramolecularly, depending on the accessibility and proximity to other cysteine groups. Disulfide bonds can be produced intracellularly through thiol-disulfide exchange, coupling between thiol radicals, and thiol reaction with nitrosylated cysteine or sulfenic acids (Figure 23E).¹³⁴⁵ Thiol-disulfide exchange can be achieved by nonenzymatic or enzymatic reactions, such as Trx, Grx, and PDI, which can accelerate disulfide bond formation.¹³⁴⁵ Disulfide bonds can introduce conformational constraints in peptides and proteins. Peptides containing disulfide bonds are expected to be used as drug leads or scaffold materials.¹³⁶³ Hinge disulfide bonds in the human IgG2 CD40 antibody regulate receptor signaling by modulating conformation and flexibility.¹³⁶⁴ Kinases are now also thought to be regulated by redox. The formation of intermolecular disulfide bonds between homodimers activates PKG1α and ATM, while intermolecular disulfide bonds between Src monomers inhibit kinase activity.¹³⁶⁵ MLL1 is a redox-regulated HMT, and peroxide-induced intramolecular disulfide bond formation results in inactivation of the HMT SET-1/MLL1.¹³⁶⁶ The protease activity of human ATG4B is also affected by reversible redox modification. A previous study found that C292 and C361 of ATG4B form a disulfide bond, which affects autophagy by regulating the activity of ATG4B.¹³⁶⁷ Disulfide bonds also affect the subcellular localization of proteins, PPIs,¹³⁶⁸ and the activity of cytokines. HMGB1 is a nuclear protein with extracellular inflammatory cytokine activity. HMGB1 requires mild oxidation to form a C23–C45 disulfide bond and unoxidized C106 to induce phosphorylation of the NF-κB p65 subunit and TNF-α production.¹³⁶⁹ Oxidative stress induces the intermolecular disulfide bond formation of TFEB/TFE3 in mammals, which enhances the activity of the TF.¹³⁷⁰ STING is a key regulator in the innate immune type I IFN pathway, and its C206 oxidation to form intermolecular disulfide bonds leads to a conformational change in the protein that prevents excessive activation of STING.¹³⁷¹ In addition, cellular structure is also dependent on cysteine disulfides, as microtubule assembly is partly mediated by disulfide bonds.¹³⁴⁵

12.6 Redox modifications in health and diseases

Redox-associated PTMs are a part of normal cell signaling and can regulate the activity of a wide variety of proteins involved in energy metabolism, protein folding and degradation, and gene transcription. Imbalances in redox homeostasis are associated with aging¹³⁷² and various diseases, such as neurodegenerative diseases,¹³⁷³ CVDs,¹³⁷⁴ and cancers (Figure 24).¹³³⁴

12.7 Redox modifications in aging

Aging-caused imbalance between RONS production and cellular antioxidant capacity may lead to oxidative stress. Proteins aberrantly modified by redox modifications become dysfunctional.¹³⁷² Protein cysteine redoxomics in various tissues of young and old mice has shown that many redox modifications in young tissues disappear in old tissues, but some new redox modifications also appear in old tissues.¹³⁷⁵ Supplementation with antioxidants can neutralize ROS, and glutathione and its precursor N-acetylcysteine (NAC) are common dietary supplements. However, long-term use of GSH and NAC inhibits skn-1-mediated gene transcription and accelerates aging.¹³⁷⁶ Endoplasmic reticulum sulfhydryl oxidase Ero1α produces SNO to reduce its activity, leading to reduced stress in the ER and compromised ER proteostasis and UPR, which in turn promotes cell senescence.¹³⁷⁷ The KEAP1–NRF2 system is a key defense mechanism to prevent oxidative stress and aging.¹³⁷⁸

Sarcopenia is a hallmark of human aging.¹³⁷⁹ Muscle wasting is accompanied by decreased oxygen consumption and increased ROS production in sarcopenia.¹³⁸⁰ The restoration of redox balance by elamipretide (SS-31) in aged mice can enhance mitochondrial function and improve skeletal muscle function. S-glutathionylation has been significantly reversed in aged mice, and the gastrocnemius muscle of SS-31-treated mice has greater fatigue resistance and mass.¹³⁸¹ S-glutathionylation is significantly increased in mitochondrial complexes I, II and V, ACO2, GAPDH, and MDH1 after rest and fatigue contraction, indicating that redox plays important roles in the control of muscle physiology, metabolism, and exercise adaptation response.¹³⁸²

12.8 Redox modifications in metabolic disorders

The production and metabolism of active substances and the recovery and removal of the antioxidant system are highly synchronized processes in normal physiological conditions. When these systems are disturbed, a series of metabolic diseases, such as obesity, metabolic syndrome, and T2DM, can occur (Figure 24).^{1383, 1384} Excessive production of ROS in vivo can lead to oxidation of proteins, leading to decreased insulin secretion and increased insulin resistance, and contributing to the development of diabetes.¹³⁸⁵ For example, ROS-induced oxidative stress can activate the IKKβ/NF-kβ pathway, leading to pancreatic β-cell dysfunction. Cys179 of IKKβ may be a vulnerable site to redox modification.¹³⁸⁶ Oxidative modification of cysteine residues in Keap1 also affects the development of diabetes by regulating the Nrf2/Keap1/ARE pathway.¹³⁸⁵

Increased intracellular NO production and reduced bioavailability are key factors leading to imbalances in redox homeostasis in metabolic diseases.¹³⁸⁷ Protein SNO is important to the entire process of insulin action, including processing and secretion by pancreatic β cells, transport by endothelial cells, signal transduction, and degradation of insulin.^{1388, 1389} For example, glucokinase SNO at Cys371 dissociates glucokinase from ISG and facilitates its transition to the active conformation to increase insulin secretion.¹³⁹⁰

Obesity is a metabolic disorder affected by oxidative stress.^{1391, 1392} SNO of IRE1α in obese mice leads to a decrease in glucose homeostasis.¹³⁹³ ROS can activate thermogenesis through direct redox modification of UCP1. UCP1 Cys253 in brown and beige adipose tissues undergoes sulfenylation during thermogenesis, and the heat generated by these adipocytes can fight obesity and diabetes.^{1394, 1395}

In addition to the influence of ROS/RNS, antioxidant enzymes also play a role in metabolic diseases. Numerous studies have demonstrated that the activity of antioxidant enzymes can be used as potential biomarkers of metabolic diseases. For example, elevated levels of glutathione S-transferase have been observed in the blood plasma of individuals with T2DM.¹³⁹⁶ The activities of enzymes such as superoxide dismutases, catalases, and glutathione peroxidases in PBMCs of obese patients are found to be significantly lower.¹³⁹⁷ In addition, the regulation of the Trx/trxR system has been identified as a potential target for treating metabolic syndrome, T2DM and hypertension.^{1398, 1399, 14}

12.9 Redox modifications in the cardiovascular system

The heart is one of the organs most severely affected by SNO, which plays a key role in regulating redox homeostasis in the stressed heart.¹⁴⁰⁰ In mouse cardiac proteins, a total of 1974 SNO sites from 761 proteins have been identified. In the cardiovascular system, NO signaling regulates vasodilation and myocardial contraction, and SNO of proteins may represent the third functional dimension of NO signaling in the cardiovascular system to ensure optimal cardiac function.¹⁴⁰¹ SNO at Cys589 of Hsp90 promotes cardiac hypertrophy.¹⁴⁰² Moreover, the SNO levels of MLP are significantly elevated in patients with hypertrophic myocardium and in spontaneously hypertensive rats and mice with transverse aortic constriction.¹⁴⁰³ Cardiac fibrosis is an irreversible pathological process, and inhibition of SNO of Hsp90 can alleviate myocardial fibrosis through the TGFβ/SMAD3 signaling pathway (Figure 24).¹⁴⁰⁴ SNO at C116/C163 of JNK accelerates cardiac fibrosis.¹⁴⁰⁵ SNO of Hsp90 affects cardiac hypertrophy and myocardial fibrosis. A recent study found that SNO of C521 of Hsp90 can inhibit the interaction between Hsp90 and AHA1, promote the recruitment of CDC37, and aggravate atherosclerosis.¹⁴⁰⁶ In addition, SNO mediates the coupling of GNAI2 to CXCR5, activating YAP-dependent endothelial inflammation to drive diabetes-accelerated atherosclerosis.¹⁴⁰⁷ SNO is also involved in calcific aortic valve diseases. USP9X SNO is reduced in calcified human aortic valves. SNO of USP9X can stabilize MIB1, which activates the NOTCH1 signaling pathway in adjacent cells to prevent calcification.¹⁴⁰⁸ In addition to SNO, protein S-glutathionylation also plays a role in CVDs. During the development of calcific aortic valve stenosis, abnormal S-glutathionylation promotes tissue phenotypic switching in the aortic valve, eventually leading to calcium deposition.¹⁴⁰⁹

12.10 Redox modifications in neurological disorders

Redox homeostasis is linked to neurological disorders. ALS is a motor neuron disease. The G93A mutation in the antioxidant enzyme SOD1 is a gain-of-function mutation that causes SOD1 aggregation and motor neuron degeneration. Aggregation of SOD1 is associated with the oxidation of cysteine residues on SOD1 and increases when Cys6 and Cys111 are oxidized (Figure 24).^{1410, 1411} The pathogenesis of AD is related to Aβ, neuronal hyperexcitability, and aging-related neuroinflammation. Excessive NO production leads to aberrant SNO of various proteins.¹⁴¹² Moreover, noncanonical transnitrosylation can lead to synaptic loss, which may be one of the pathological causes of cognitive decline in AD patients.¹⁴¹² SNO also affects the occurrence and progression of PD. HMGB1 is a DNA-binding protein that regulates gene transcription and genome stability in the nucleus. SNO in C106 of HMGB1 can promote the secretion of HMGB1 and enhance its binding force with microglial Mac1, which is one of the key mechanisms for the development of PD.¹⁴¹³ In addition, SNO of foreign α-synuclein also promotes Parkinopathy.¹⁴¹⁴

12.11 Redox modifications in cancers

ROS-induced oxidative stress is a fundamental feature of cancer. Tumor cells produce more ROS than normal cells, resulting in abnormal redox homeostasis.¹⁴¹⁵ Oxidative stress can regulate intracellular signaling pathways and promote the growth of tumor cells, while excessive oxidative stress may lead to oxidative damage and even tumor cell death.¹⁴¹⁶ Tumor cells can rely on their own powerful antioxidant systems to withstand oxidative stress with high levels of ROS at different stages. For example, in the early stages of tumors, they can adapt to oxidative stress by activating antioxidant TFs or increasing NADPH through the PPP. During tumor growth and metastasis, tumor cells can activate AMPK, PPP, and reductive glutamine to increase NADPH, allowing cells to survive under conditions of high ROS.¹⁴¹⁷ During the whole process, the redox modifications of proteins in tumors play a crucial role (Figure 24). For example, SNO at C277 of VEGFD inhibits its expression and promotes the occurrence and development of LUAD.¹⁴¹⁸ In early CRC, PTPS is highly expressed and phosphorylated at Thr58 under hypoxic conditions, which promotes binding to LTBP1 and drives LTBP1 SNO, thereby maintaining tumor cell growth under hypoxic conditions.¹⁴¹⁹ Tumor cells can resist high levels of ROS by activating the activity of antioxidant TFs such as Nrf2. Nrf2 deletion affects the redox proteome of PC and NCSLC, which has impacts on mRNA translation machinery and the glycolytic pathway.^{1420, 1421} ROS can also affect tumor cells by regulating the functions of metabolic enzymes through redox modification. For example, TPβ is a redox-sensitive protein and a key rate-limiting enzyme in the FAO process. Under the stimulation of ROS, C458 of TPβ will be oxidized and inactivated. In oxidative stress caused by glucose starvation, Nur77 can enter the mitochondria and be oxidized by ROS, protecting C458 of TPβ from oxidation, resulting in the production of FAO-mediated NADPH to relieve intracellular oxidative stress and promote melanoma cell survival and metastasis. This suggests that Nur77 may be a potential target for the treatment of melanoma.¹⁴²² In the glycolysis pathway, PFKM is one of the most important regulatory enzymes, and SNO at C351 of PFKM can stabilize the tetramer of PFKM, which contributes to the metabolic reprogramming of ovarian cancer cells.¹⁴²³ Additionally, elevated expression of Nm23-H1 is linked to a favorable prognosis in patients with breast cancer, and activation of Nm23-H1 through redox regulation can inhibit breast cancer metastasis.¹⁴²⁴ PTEN is a tumor suppressor. The oxidative modification of the cysteine of PTEN makes it inactive. The recovery of its activity mainly depends on the availability of Trx and Prx.¹⁴²⁵ Methionine is an amino acid residue prone to redox modifications that are regulated by MSRA. MSRA deletion promotes the oxidation of methionine on PKM2, which in turn promotes mitochondrial respiration and cell metastasis.¹⁴²⁶

13.1 ADP-ribosylation

ADP-ribosylation is a process in which ADP-ribosyl transferases transfer ADP-ribosyl to the target protein's ADP-ribose binding domain using NAD⁺ as the substrate.¹⁴²⁷ Currently, over 800 proteins have been found to contain the ADP-ribose binding domain.¹⁴²⁸ The structure and function of many proteins, including nuclear proteins topoisomerase I, DNA ligase II, endonuclease, histones H1, H2B and H4, DNA polymerases and cytoplasmic proteins adenyl cyclase and elongation factor eEF-2, are regulated by ADP-ribosylation.¹⁴²⁹ ADP-ribosylation is involved in numerous physiological and pathological processes, such as signal transduction, protein transport, transcription, DNA damage repair, cell cycle regulation, apoptosis, and necrosis.¹⁴²⁷ Poly-ADP-ribosylation (PAR) of proteins may decease during aging because the activity of poly-ADP-ribose polymerase (PARP) in senescent human fibroblasts is reduced with donor age and continuous passage in vitro.¹⁴²⁹ PAR modification plays an important role in DNA damage repair,¹⁴³⁰ and many DNA damage repair factors can recognize PAR signals, leading to the rapid recruitment of these factors for efficient repair.¹⁴³¹ Loss of PAR modification inhibits SSB and DSB repair.¹⁴³² Therefore, PARP inhibitors (PARPi), such as olaparib, rucaparib, niraparib, and talazoparib, have been developed as a class of targeted drugs for cancer treatment.¹⁴³³ These drugs compete with PARP1/2 for intracellular NAD⁺ and inhibit its catalytic activity to block DNA damage repair signals. Additionally, PARP1/2 can become trapped in damaged DNA, forming a PARP–DNA complex and blocking its release, causing replication fork stagnation and leading to cancer cell death.^{1434, 1435}

13.2 Benzoylation

Lysine benzoylation (Kbz) was the first discovered aromatic fatty acid modification and primarily occurs in the N-terminal tails of histones.¹⁴³⁶ Sodium benzoate (SB) is a widely used food additive and a clinical treatment for hyperammonemia.¹⁴³⁷ It can be converted into benzoyl-CoA in mammalian cells and as a precursor of Kbz.¹⁴³⁸ Moreover, benzoyl-CoA is also a central intermediate for the degradation of numerous aromatic growth substrates in bacteria and gut microbes.¹⁴³⁹ Previous studies have showed that HBO1 and the Spt-Ada-Gcn5 acetyltransferase complex are writers of histone Kbz.^{1440, 1441} In vivo studies have found that SIRT2, unlike other sirtuins or histone deacetylases (HDACs), acts as an eraser of histone Kbz.¹⁴³⁶ NAD⁺-dependent histone deacetylase Hst2 has debenzoylase activity in yeast.¹⁴⁴⁰ Human DPF and YEATS but not BRD domains are the readers for histone benzoylation.¹⁴⁴²

Histone Kbz is a mark enriched in gene promoters and is associated with gene expression.¹⁴³⁶ Furthermore, it has a different physiological relevance than histone acetylation. Kbz primarily targets gene promoters and regulates glycerophospholipid metabolism, ovarian steroid synthesis, and hydrolytic phospholipase signaling pathways.¹⁴³⁸ Excessive intake of SB increases Kbz levels, leading to motor coordination disorders and increased risk of diseases such as ADHD.¹⁴⁴³

13.3 Neddylation

Neddylation is a PTM that can conjugate the Ub-like protein NEDD8 to target proteins.¹⁴⁴⁴ Neddylation has a broad range of functions and can alter various aspects of protein function, including protein conformation, stability, subcellular localization, affinity for DNA, and binding of protein substrates.^{1445, 1446} NEDD8 is a highly conserved protein composed of 81 amino acids. Of all the Ub-like protein families, NEDD8 is the molecule with the highest sequence and structural similarity to Ub.¹⁴⁴⁴ After passing through the E1–E2–E3 cascade, NEDD8 covalently binds to the substrate and undergoes Neddylation on the lysine of the substrate protein clock, thereby regulating the biological function of the substrate.¹⁴⁴⁴

NEDD8 activating enzyme E1 is a heterodimer composed of APPBP1/UBA3.¹⁴⁴⁷ Through its ATP-dependent catalytic subunit, it catalyzes the formation of a high-energy sulfolipid bond at the C-terminal glycine of NEDD8 molecule and the cystine active site of UBA3 to activate the NEDD8 molecule.¹⁴⁴⁸ The NEDD8-loaded NEDD8-activating enzyme (NAE) is transferred to E2-conjugating enzymes UBC12/UBE2M or UBE2F via a trans-thiolation reaction. Ultimately, substrate specific E3 ligases transfer NEDD8 from E2 to lysine residues in their target proteins by covalent attachment. NEDD8 ligase E3 can be divided into RING finger and HECT types.¹⁴⁴⁹ Many neddylation E3s have been discovered, including RBX1/2, ROC1/2, SAG, c-CBL, MDM2, FBXO11, c-CBL, DCNL1–5, IAPs, RNF111, TFB3, and TRIM40.^{1448, 1449} Neddylation is a reversible process, and under the action of the de-neddylation enzyme, NEDD8 can be dissociated from the substrate protein.¹⁴⁵⁰

Similar to ubiquitination, the neddylation process also affects cell growth in multiple aspects such as proliferation, apoptosis, and migration in tumor cells. Many recent studies have shown that NEDD8 or enzymes related to the neddylation pathway are overexpressed in various cancers, including lung cancer,¹⁴⁵¹ liver cancer,¹⁴⁵² CRC,¹⁴⁴⁹ and esophageal squamous cell carcinoma.¹⁴⁵³ At present, the primary approach to studying the neddylation pathway involves interfering with the expression of particular genes through siRNA or inhibiting the activation enzyme NAE using small molecule inhibitors such as MLN4924 which deactivate the entire neddylation pathway.¹⁴⁵⁴ The formation of a stable covalent bond with NEDD8 and competitive inhibition of NAE's activation of NEDD8 leads to inhibition of the neddylation pathway and partially blocks the ubiquitination pathway dependent on CRL. This inhibition results in the accumulation of substrates, triggering various cellular responses, such as cell cycle arrest, apoptosis, aging, and autophagy. By utilizing this mechanism, it is possible to achieve a therapeutic effect for treating tumors.¹⁴⁵⁵ In addition, the dysregulation of neddylation is involved in the development of various diseases such as neurodegenerative diseases,¹⁴⁵⁶ inflammation,¹⁴⁵⁷ and CVDs,¹⁴⁴⁵ suggesting that it can be a potential target for disease treatment. In fact, the NAE inhibitor MLN4924 not only has significant antitumor activity, but also has activities such as antiviral¹⁴⁵⁸ and anti-inflammatory effects.¹⁴⁵⁹ Collectively, neddylation is a versatile PTM that can modulate a wide range of protein functions, with implications for many physiological and pathological processes. The neddylation pathway and its inhibitors hold significant potential for research into various disease treatments.