The role of protein palmitoylation in disease pathogenesis and therapeutic innovation.

In immunology, TCR signaling requires the assistance of CD4 or CD8 co-receptors [ 121 ]. Studies have shown that palmitoylation regulates CD4 signaling, with CD4 being palmitoylated at Cys396 and Cys399 [ 122 , 123 ]. This modification facilitates its accumulation in lipid rafts, enhancing the receptor’s tyrosine phosphorylation ability [ 122 ]. Recent research revealed that ZDHHC21 induces palmitoylation of T cell signaling proteins, activating immature CD4+ T cells in response to TCR [ 124 ]. ZDHHC21 is a key enzyme in palmitoylation of T cell signaling proteins, linked to early signaling events and expression of activation markers. Downregulation of ZDHHC21 prevents activation and differentiation of immature T cells into effector T cells [ 125 ]. STAT3 is a key transcription factor for TH17 cell differentiation [ 126 ]. ZDHHC7 catalyzes the palmitoylation of STAT3, promoting its membrane accumulation and phosphorylation. APT2 depalmitoylates p-STAT3, enabling its translocation to the nucleus. This palmitoylation-depalmitoylation cycle enhances STAT3 activation and promotes TH17 cell differentiation. Disrupting this cycle alleviates inflammation in mouse models [ 12 ]. Furthermore, ZDHHC3 is identified as the palmitoyltransferase for PD-L1. Inhibiting PD-L1 palmitoylation activates T cell immunity in MC38 tumor-bearing mice [ 12 , 127 ]. PLCβ is a common inflammatory factor in the human body, and ZDHHC21 regulates inflammatory signaling in endothelial cells by mediating the palmitoylation of PLCβ, promoting inflammation [ 128 ]. Endothelial nitric oxide synthase (eNOS) is the rate-limiting enzyme for nitric oxide (NO) production [ 129 ]. ZDHHC21 palmitoylates eNOS, promoting its membrane localization and NO production [ 128 ], while APT1 catalyzes eNOS depalmitoylation. O-palmitoylation mediated by SPTLC1 is essential for vascular development [ 130 ]. SPT enzymes are associated with the synthesis of lipid rafts in endothelial cells [ 20 , 131 ], and damage to lipid rafts triggers cardiovascular diseases [ 132 ]. This suggests that the O-palmitoylation site serine can be a target of action for some cardiovascular and cerebrovascular diseases. In drug development, simvastatin can modulate the palmitoylation of the estrogen receptor (ER-α) in endothelial cells, affecting estrogen signaling in uterine smooth muscle tumors [ 133–135 ]. Palmitoylation is closely associated with neurodegenerative diseases. Gephyrin, an anchoring protein at GABA(Gamma-aminobutyric acid)ergic synapses, is palmitoylated by ZDHHC12, enhancing GABA(Gamma-aminobutyric acid) synaptic transmission [ 136 ]. In Huntington’s disease, reduced PSD-95 leads to the loss of excitatory synapses in the thalamostriatal region, worsening symptoms [ 137 ]. In neurons, SYT11 is a synaptic binding protein with transport and cytosolic and cytotrophic effects on intracellular vesicles [ 138 ], and its palmitoylation enhances α-synuclein binding to intracellular membranes [ 139 ]. In Alzheimer’s disease, ZDHHC21 abnormally induces palmitoylation of APP, increasing Aβ production and exacerbating pathological conditions [ 140 ]. In vitro experiments have shown that palmitoylation of the γENaC subunit of the renal tubular epithelial sodium channel (ENaC) regulates extracellular fluid volume [ 141 , 142 ]. Abnormal activation can induce hereditary hypertension [ 143 ]. In intestinal epithelial cells, ZDHHC21 mediates heat-induced damage to increase intestinal epithelial permeability. Inhibiting this enzyme may offer a new approach to treating burn-induced intestinal barrier dysfunction [ 144 ]. Rhinovirus is associated with a higher risk of childhood asthma, and ORM1L3 is linked to its replication [ 145 ]. Inhibition of O-palmitoyltransferase SPT is required to support epithelial cell O-palmitoylation for viral replication [ 146 ]. Activating SPT may inhibit rhinovirus replication. In breast epithelial cells, Smad7 undergoes palmitoylation by ZDHHC17, enhancing its inhibition of the TGF-β/Smad signaling pathway and suppressing TGF-β1-dependent breast cancer cell migration [ 147 ].

Palmitoylation research dates back to the 1970s, and around 1990, scholars proposed that it could alter the physical and biological properties of proteins [ 1–4 ]. Protein palmitoylation is a post-translational modification that has a profound impact on protein structure and function. It involves the addition of palmitic acid to cysteine residues, forming palmitoylcysteine, which enhances protein stability, alters subcellular localization, and modulates function and activity [ 5 ]. Palmitoylated proteins can influence the onset and progression of a large number of diseases. For instance, in tumors, palmitoylation regulates the tumor immune microenvironment, and aberrant palmitoylation of some proteins can also activate classical pathways of tumourigenesis [ 6 , 7 ]. In the immune-inflammatory response, palmitoylation of some inflammatory vesicles can promote inflammation, such as NOD2 and Gasdermin D [ 8 , 9 ], promote inflammation upon palmitoylation, while others, like NLRP3 [ 10 , 11 ], suppress it. In colitis, the immune inducer STAT3 requires a palmitoylation-depalmitoylation cycle to be activated, which promotes TH17 cell differentiation and exacerbates inflammation [ 12 ]. This review provides an overview of S- palmitoylation, O-palmitoylation, and N-palmitoylation, introduces palmitoyl acyltransferases and acyl-protein thioesterases, and discusses the role of palmitoylation in diseases. Additionally, it explores the therapeutic potential of targeting palmitoylation and offers perspectives on future treatment strategies and drug development.

The site of protein palmitoylation modification is the cysteine residue of the protein. Depending on the type of linkage, palmitoylation can be classified into S-palmitoylation, N-palmitoylation, and O-palmitoylation. N-palmitoylation occurs when the palmitoyl group is linked to the N-terminal glycine, lysine, or cysteine residues of the protein via an amide bond, making this process irreversible due to the stability of the amide bond [ 13 ]. Sonic Hedgehog (Shh), a critical signaling pathway in the human body, undergoes N-palmitoylation through the action of its palmitoyltransferase(PAT), Hhat, catalyzing the modification of both the precursor and mature Shh proteins [ 14 ]. In pancreatic cancer, Shh signaling can be blocked by knocking down Hhat or using Hhat inhibitors, which inhibits pancreatic cancer cell growth [ 5 , 15 ]. In breast cancer, abnormal overexpression of Shh in initial breast tumor cells and invasive ductal carcinoma leads to increased metastasis and death. Thus, inhibiting the palmitoylation of Shh precursor genes or knocking down Hhat may offer effective therapeutic strategies for breast cancer [ 16 ]. O-palmitoylation involves the linking of the palmitoyl group to serine/threonine residues through ester bonds. SPT (serine palmitoyltransferase) is the classic O-palmitoyltransferase. Recent studies have shown that SPT catalyzes palmitoylation at specific sites, altering membrane sphingolipid diversity, which in turn limits tumor growth [ 17 , 18 ]. Its specific palmitoylation blocker, myristoyl-CoA, has been used as a drug therapy to hinder tumor growth [ 18 ]. In addition, SPT has been implicated in the development of some neurodegenerative and neurodevelopmental disorders (amyotrophic lateral sclerosis, hereditary spastic paraplegia) [ 19 ]. Hereditary and non-hereditary retinal diseases have been linked to O-palmitoylation [ 20 ], with SPT inhibiting de novo ceramide synthesis and disrupting ceramide metabolism in the retina [ 21 , 22 ]. Moreover, Porcn (membrane-bound O-acyltransferase) can catalyze palmitoylation of certain proteins. PORCN catalyzes the palmitoylation of Wnt ligands, activating Wnt signaling [ 23 , 24 ]. Inhibition of Porcn expression by drugs can effectively suppress Wnt-dependent tumor growth [ 25 ]. Overexpression of PORCN due to O-palmitoylation alters Wnt signaling, promoting liver cancer progression [ 26 ]. S-palmitoylation is a post-translational modification where specific cysteine residues are linked to palmitoyl groups via thioester bonds, and this process is reversible [ 27 ]. Among the various types of palmitoylation, S-palmitoylation and depalmitoylation are the most extensively studied, with dysregulation linked to numerous diseases [ 28 , 29 ]. In diabetes, depalmitoylation enzyme APT1 catalyzes the depalmitoylation of the transmembrane vesicle protein Scamp1, which is associated with insulin secretion. Impaired S-depalmitoylation of Scamp1 leads to increased insulin secretion, resulting in β-cell failure [ 28 ]. In septic myocarditis and colitis, S-palmitoylation modulates the activity of NLRP3 inflammasomes, influencing inflammation [ 30 ]. In chronic kidney disease, S-palmitoylation alleviates renal fibrosis by inhibiting Wnt/β-catenin signaling [ 12 ]. Therefore, S-palmitoylation may be the most influential palmitoylation modification affecting cellular homeostasis and human diseases. In a broader sense, protein S-acylation is encompassed within the category of palmitoylation, which refers to the covalent attachment of long-chain fatty acids to cysteine residues. Among these modifications, S-palmitoylation represents the most prevalent form. Because the present review focuses on zDHHC family–mediated modifications and their clinical relevance, the term “palmitoylation” is used throughout this manuscript to specifically denote S-palmitoylation.

Palmitoylation does not occur spontaneously but is governed by a tightly regulated enzymatic system that determines both the extent and dynamics of this modification. To understand how palmitoylation achieves homeostatic balance in vivo , it is essential to delineate the key enzyme classes responsible for catalyzing palmitoylation and de-palmitoylation, as well as their underlying mechanisms of action. The S-palmitoylation of proteins is reversible. The forward reaction, known as palmitoylation, is catalyzed by ZDHHC-type palmitoyltransferases (PATs), while the reverse reaction, depalmitoylation, is catalyzed by enzymes like APT1/2, PPT1/2, or ABHD17A/B/C [ 46 , 53 ]. S-palmitoylation requires enzymatic catalysis, and the enzymes involved belong to the family of protein acyltransferases (PATs). PATs, which are classified as ZDHHC enzymes, have a conserved zinc-finger DHHC motif located within a cysteine-rich domain (CRD) [ 54 ]. Humans have at least 23 types of ZDHHC enzymes [ 55 ]. These ZDHHC enzymes are capable of auto-palmitoylation both in vivo and in vitro without consuming energy [ 56–58 ]. The auto-palmitoylation process is complex: the palmitoyl group first binds to the cysteine residue in the ZDHHC structural domain, forming an enzyme-active intermediate, which then binds to the target substrate [ 59 ]. The S-palmitoylation reaction is reversible. In mammalian cells, the removal of S-acyl groups is mediated by a family of serine hydrolases [ 60 ]. APT1 is located in mitochondria and acts as a redox-sensing enzyme with bidirectional regulatory functions [ 61 , 62 ]. APT1 is activated under oxidative conditions and maintains orrestores its monomeric state under reducing conditions, thus regulating cellular redox homeostasis [ 62 ]. In humans, APT1-mediated depalmitoylation affects glutamatergic synaptic plasticity and may serve as a potential target for learning and memory disorders. Additionally, APT1 is regulated in human islets, and its deficiency leads to excessive insulin secretion and β-cell failure [ 28 , 63 ], which is associated with the development of type 2 diabetes. These findings point to the possibility that APT1 could be used as a target to treat complications of metabolic diseases. APT2 is a cytosolic enzyme that selectively inhibits the palmitoylation of cytosolic proteins [ 28 , 64 ]. For instance, APT2 can disrupt the palmitoylation equilibrium of the Scribble protein, inhibiting its membrane relocalization and growth attenuation [ 64 ]. High estrogen levels may induce uterine adenomyosis through this mechanism [ 65 ]. In lung adenocarcinoma patients, the infiltration of immune cells and the expression of immune checkpoint genes like CD276 are closely correlated with APT2 levels [ 66 ], suggesting that APT2 may serve as a prognostic biomarker for lung adenocarcinoma. In most cases, APT1 and APT2 work together to promote depalmitoylation in the cell [ 67–69 ], although they do not significantly affect the palmitoylation of key cancer-related proteins [ 64 , 70 ]. Other novel depalmitoylation enzymes include ABHD17A, B, and C, which regulate the palmitoylation status of cancer-related proteins [ 71 , 72 ]. Additionally, ABHD10 regulates redox homeostasis by modulating the depalmitoylation of PRDX5 [ 73 ]. ABHD16A catalyzes the depalmitoylation of IFITM proteins to exert antiviral effects [ 74 ]. ABHD7 catalyzes the depalmitoylation of lamina-associated protein A, promoting myogenic differentiation [ 75 ]. A single substrate protein can often be palmitoylated by multiple zDHHC enzymes, and such functional redundancy provides robust safeguards for essential cellular processes. During epithelial cell polarity establishment, palmitoylation of Ankyrin-G at Cys70 specifically requires the coordinated activity of zDHHC5 and zDHHC8. This redundant modification occurs within defined plasma membrane microdomains and is critical for lateral membrane assembly and polarity formation [ 76 ]. In immune responses, palmitoylation of the pyroptosis effector gasdermin D (GSDMD) is primarily mediated by zDHHC7; however, zDHHC5 and zDHHC9 provide compensatory palmitoylation that ensures inflammasome signaling can still be executed when the dominant enzyme is absent or under stress conditions, thereby maintaining pathological responsiveness through modulation of GSDMD activity [ 33 ]. Collectively, the interplay between redundancy and specificity among zDHHC enzymes constitutes a spatiotemporal “navigation system” for intracellular protein localization and function. Although this complexity increases the difficulty of developing inhibitors against single enzymes, it offers important insights into the molecular logic underlying complex disease pathogenesis. Notably, palmitoylation-related enzymes with high functional specificity display distinct expression patterns across different human tissues and organs. By precisely regulating defined substrate proteins in a cell-type–dependent manner, these enzymes contribute to the molecular basis of diverse tissue-specific physiological functions.

Abnormal palmitoylation modifications can disrupt glucose metabolism, contributing to diabetes. Palmitoylation of GLUT4 is crucial for its translocation to the plasma membrane, facilitating insulin metabolism. Enhancing palmitoylation of GSV or GLUT4 can address metabolic syndrome caused by insulin resistance. In regenerative medicine, human mesenchymal stem cells can activate the PI3K/Akt pathway to improve type 2 diabetes symptoms through the regulation of AS160 palmitoylation [ 169 , 170 ]. New drug developments include palmitoylated anorexigenic prolactin-releasing peptide analogs with anti-obesity and hypoglycemic properties [ 171 , 172 ]. GIP and GLP-1 are gut hormones that stimulate insulin secretion. GLP-1 receptor (GLP-1R) agonists are effective in the treatment of type 2 diabetes and obesity and improve insulin control of blood glucose in humans [ 171 , 173–175 ]. GIP palmitoylation analogs, used as GIPR antagonists in combination with GLP-1R agonists, enhance GLP-1R activity to lower blood glucose and control weight [ 176 ]. Inverse depalmitoylation, primarily by APT1, also affects diabetes. Hyperglycemia in type 2 diabetes reduces APT1 activity, leading to β-cell dysfunction [ 28 ]. Although research on depalmitoylation therapies is nascent, targeting APT1 substrates might hold potential for treating diabetic complications [ 63 ]. Palmitoylation influences not only glucose metabolism in diabetes but also late-stage inflammation, such as diabetic foot. Phenylalanine metabolism disorder in diabetes disrupts de-palmitoylase PPT1, increasing palmitoylation and activation of the NLRP3 inflammasome, leading to inflammation [ 177 , 178 ]. Targeting PPT1 to enhance its activity can be a strategy to treat DM-related conditions [ 29 ]. Diabetic cardiomyopathy is a leading cause of death in diabetic patients, characterized by lipid accumulation and impaired heart function [ 179 ]. The bile acid receptor TGR5, when absent, increases palmitoylation of CD36 via zDHHC4, hastening myocardial lipid uptake and diabetic cardiomyopathy [ 180 , 181 ]. Targeting TGR5 might be promising for treating this condition In cardiac electrophysiology, palmitoylation regulates lipids [ 182 ]. NCX1 controls cytoplasmic Ca2+ extrusion, affecting heart relaxation [ 30 , 183 , 185 , 186 ]. Insulin-induced zDHHC5 palmitoylation alters NCX1 conformation and deactivation, offering a target for drugs to enhance heart function by mimicking insulin [ 187 ]. APT1 regulates NCX1 depalmitoylation and transport in the body [ 188 ]. HCN4 protein palmitoylation is essential for sinoatrial node function, and defects are linked to dysfunction [ 189 ]. Recent studies have found that the initial human HCN4 protein is in a palmitoylated state and the palmitoylation site of its terminal cysteine residue plays a key role in the sinus node [ 190 ]. Septic myocarditis is the most common acute non-cardiovascular disease in cardiac intensive care units with extremely high lethality.Flavonoid VAC inhibits NLRP3 inflammasome activity, promoting palmitoylation and reducing sepsis-related heart damage [ 191 ]. The cyclic guanosine monophosphate adenosine synthase-interferon gene-stimulating factor axis (cGAS-STING) is usually associated with human immunity, but in recent years it has been found that the cGAS-STING signalling axis is also active after myocardial infarction [ 184 ]. STING palmitoylation inhibitors can mitigate ischemic heart failure progression after myocardial infarction [ 192 , 193 ]. This study has pharmacological implications for the prognostic treatment of myocardial ischaemia-reperfusion injury. Non-alcoholic steatohepatitis (NASH) can lead to liver failure, cirrhosis, and cancer. CD36 and inactive IRHOM2 are involved in NASH. zDHHC5, zDHHC4, and APT1 regulate CD36 palmitoylation [ 160 , 194 ]. Recent studies have found that inhibition of CD36 palmitoylation can also enhance hepatic fatty acid beta-oxidation [ 195 ]. Inhibition of CD36 palmitoylation is one of the feasible ways to treat NAFLD. Inactive rhodopsin 2 (IRHOM2, also known as RHBDF2) has been identified as an important regulator involved in innate immune responses and inflammation-related diseases [ 196 , 197 ]. Inhibiting CD36 palmitoylation reduces fatty acid absorption and balances metabolism, reversing liver damage. IRHOM2 stability is altered by palmitoylation, accelerating NASH progression when palmitoylated by zDHHC3 [84]. Targeting zDHHC3 might reduce NASH advancement and related complications. This rare disease involves limited palmitoylation research. DGKε is a key factor. Recent studies have shown that the palmitoylation modification site of DGKε is Cys38/40 (mDGKε/hDGKε). DGKε can be palmitoylated by zDHHC6/16, zDHHC7, and zDHHC17S-palmitoylated and this modification down-regulates DGKε activity [ 198 ]. Chronic kidney disease leads to nephron loss and kidney failure. The Wnt/beta-catenin pathway is important in kidney development and repair, with reactivation leading to fibrosis [ 199 ]. In CKD, reduced zDHHC9 expression lowers beta-catenin palmitoylation, activating signaling pathways that exacerbate fibrosis [ 114 ]. Acyl-protein thioesterase 1, which depalmitoylates beta-catenin, promotes fibrosis [ 114 ], suggesting new therapeutic strategies for CKD. In hypertension research, ADM and ADM2 palmitoylation analogs activate CLR/RAMP1 and 2 receptors, improving endothelial dysfunction [ 200–203 ]. Subcutaneous gels lower blood pressure and prolong vasodilation, indicating potential for treating resistant hypertension [ 204 ]. Endothelial knockout of acyl protein thioesterase 1 (APT1) in mice impaired recovery from chronic hindlimb ischaemia (a model of peripheral arterial disease). APT1 deficiency increases R-Ras palmitoylation, hindering vascular maturation and marking chronic occlusive diseases [ 152 ], providing further avenues for therapeutic intervention. IBD, including Crohn’s disease and ulcerative colitis, involves immune dysregulation [ 205 , 206 ]. STAT3 is key in T-cell differentiation [ 126 ]. zDHHC7-mediated palmitoylation enhances its activation. Inhibiting zDHHC7 can alleviate colitis, offering therapeutic potential [ 12 ]. The cytoplasmic pattern recognition receptor (PRR) nucleotide oligomeric structural domain 1 (NOD1) and NOD2 have been linked to the development of inflammation [ 207 ]. It was found that zDHHC5 is a palmitoyl catalase for NOD1 and NOD2 and is implicated in the development of colitis [ 208 ]. And inhibition of ABHD17 promotes the development of colitis. APT2 promotes nuclear translocation of STAT3, where inhibition can enhance therapeutic palmitoylation levels [ 12 ]. It was shown that application of the APT2-specific inhibitor ML349 significantly upregulated palmitoylation-modified STAT3 [ 209 ]. Under physiological conditions, palmitoylation supports learning and memory by finely regulating the membrane domain distribution of postsynaptic scaffold proteins. PSD-95 is a core scaffold protein within the excitatory postsynaptic density. PSD-95 undergoes palmitoylation catalyzed by zDHHC2 [ 210 ] and de-palmitoylation mediated by ABHD17 [ 211 ]. Studies have shown that palmitoylation of PSD-95 drives the ordered assembly of postsynaptic nanodomains by altering its protein conformation [ 212 ]. Recent evidence further indicates that the palmitoylation cycle and the APT1/PSD-95/AMPAR axis, which governs selective surface stabilization of glutamatergic receptors, are essential for memory formation [ 213 ]. Although APT1 does not directly participate in the palmitoylation cycle of PSD-95, its de-palmitoylation of AMPARs indirectly reduces PSD-95 expression levels. In the pathological context of AD, disruption of palmitoylation homeostasis is closely associated with β-amyloid plaque formation [ 214 ]. Recent studies have demonstrated that zDHHC21 is aberrantly upregulated in the brains of AD patients. zDHHC7 directly catalyzes palmitoylation of amyloid precursor protein (APP) at Cys186 and Cys187 [ 215 ]. This modification preferentially activates the amyloidogenic processing pathway rather than the non-amyloidogenic pathway, thereby accelerating neurodegenerative progression [ 216 ]. Encouragingly, the study by Kim Dore et al. showed that increasing PSD-95 levels protects synapses from β-amyloid toxicity. Pharmacological inhibition of PSD-95 de-palmitoylation increased synaptic PSD-95 and rescued β-amyloid–induced synaptic defects, suggesting a potential therapeutic strategy for AD [ 46 ]. In the brains of HD patients, mutant huntingtin (mHTT) exhibits abnormal binding to the palmitoyltransferase zDHHC17 (HIP14), thereby suppressing its catalytic activity toward substrates such as PSD-95 and SNAP-25. Experimental evidence indicates that reduced palmitoylation of these substrates directly leads to the loss of excitatory synapses in striatal neurons [ 217 ]. From a therapeutic perspective, recent studies have demonstrated that treatment of lymphocytes derived from HD patients with acyl-protein thioesterase (APT) inhibitors can normalize mHTT palmitoylation levels [ 218 ]. Neuronal ceroid lipofuscinosis (NCL) is a group of neurodegenerative lysosomal storage disorders characterized by progressive neuronal loss and the accumulation of autofluorescent lipopigments. Among the different subtypes, infantile neuronal ceroid lipofuscinosis (INCL) is considered the most severe form and is caused by mutations in the PPT1 gene, typically presenting during early infancy [ 219 ]. Loss of PPT1 disrupts the lysosomal degradation of proteins carrying hydrophobic lipid modifications. As a consequence, partially degraded lipid-modified protein fragments accumulate within lysosomes, forming pathological ceroid lipofuscin deposits. Experimental knockdown of PPT1 has been shown to promote the development and progression of INCL [ 220 ]. Recent studies further demonstrated that the synaptic protein cysteine-string protein-α (CSPα) undergoes depalmitoylation mediated by PPT1 at presynaptic terminals. Dysregulation of this process leads to impaired turnover of synaptic proteins and contributes to the pathogenesis of INCL [ 221 ]. Enzyme replacement therapy targeting PPT1 activity has been explored as a therapeutic strategy, and several related approaches have shown encouraging progress in clinical studies [ 222 ]. Palmitoylation does not exert a unidirectional tumor-promoting role during cancer initiation and progression. In certain physiological and pathological contexts, palmitoylation drives malignant phenotypes, whereas in other settings it serves as a protective mechanism that maintains cellular homeostasis and suppresses tumor development. In many malignant tumors, aberrantly activated palmitoylation promotes oncogenesis by enhancing oncoprotein stability or activating proliferative signaling pathways. Programmed death-ligand 1 (PD-L1) is a tumor-promoting factor involved in immune evasion. zDHHC3-mediated palmitoylation of PD-L1 promotes breast tumor growth [ 223 ]. Inhibition of this modification induces PD-L1 degradation and activates T cell–mediated antitumor immunity [ 224 ]. Recent pharmacological studies have identified benzbromarone derivative C as a zDHHC3-targeting compound that induces lysosomal degradation of PD-L1, thereby enhancing antitumor immune responses [ 225 ]. In addition, palmitoylation participates in oncogenic signal transduction. zDHHC20-mediated palmitoylation of EGFR promotes receptor localization at the plasma membrane and enhances its kinase activity, thereby activating downstream proliferative pathways such as PI3K–AKT and driving malignant progression of KRAS-mutant lung cancer [ 158 ]. In drug resistance, S-palmitoylation of PCSK9 activates the PI3K/AKT pathway and induces sorafenib resistance in hepatocellular carcinoma. zDHHC16-catalyzed palmitoylation of PCSK9 markedly enhances proliferation of advanced hepatocellular carcinoma cells by activating the AKT signaling axis [ 102 ]. Conversely, palmitoylation of specific proteins is essential for maintaining the activity of tumor suppressors. In breast cancer, zDHHC17-mediated palmitoylation of Smad7 enhances its inhibitory effect on the TGF-β signaling pathway, thereby effectively blocking TGF-β1–induced tumor cell migration [ 226 ]. Palmitoylation of the tumor suppressor Scribble is a prerequisite for its correct localization at the plasma membrane and is required to restrain abnormal tumor cell proliferation. zDHHC7 catalyzes palmitoylation of Scribble at Cys4 and Cys10 [ 227 ]. Once this modification cycle is disrupted by the de-palmitoylating enzyme APT2, Scribble dissociates from the membrane and loses its tumor-suppressive function [ 64 ]. This bidirectional role of palmitoylation reflects the intrinsic complexity of post-translational modifications, whereby the same enzyme may exert opposite effects depending on the substrate or tissue context. Therefore, palmitoylation should not be regarded as uniformly “deleterious” in cancer. Future therapeutic strategies must adopt substrate- and tumor-type–specific targeting approaches.

The functional diversity of palmitoylation-related enzymes across different cell types fundamentally arises from their precise regulation of core intracellular signaling pathways. By dynamically modifying key signaling proteins, these enzymes act as molecular “switches” that turn specific signaling cascades on or off, thereby determining cellular proliferation, metabolic activity, and stress responses. R-Ras is involved in vascular maturation, integrin transport, and the integrity of adhesion junctions [ 148–151 ]. The loss of the depalmitoylating enzyme APT1 affects R-Ras signaling [ 152 ]. The palmitoylation cycle of R-Ras drives vesicular transport along the Golgi network and regulates membrane division to maintain vascular homeostasis [ 153 ]. APT1 enzyme deficiency impedes R-Ras membrane trafficking, impairing signaling and hindering recovery from chronic hindlimb ischemia [ 152 ]. zDHHC19 catalyzes the palmitoylation of R-Ras, amplifying signaling by enhancing its association with the membrane and lipid rafts [ 154 ]. Co-expression of ZDHHC19 enhances cell vitality through R-Ras palmitoylation. Overexpression of EGFR is linked to various cancers, especially head and neck tumors, cervical cancer, and bladder cancer [ 155–157 ]. Epidermal growth factor receptor is localized at the cell membrane by zDHHC13 palmitoylation, and upon binding to EGF protein, EGFR is phosphorylated, thereby activating the PI3K-AKT pathway. Knockdown of ZDHHC20 expression can block EGFR palmitoylation, reducing PI3K kinase activity and decreasing the tumorigenic ability of KRAS mutant mice [ 158 ]. ZDHHC13 is a key enzyme for EGFR palmitoylation, helping EGFR localize to the plasma membrane and activate the PI3K-AKT signaling pathway [ 159 ]. Inhibition of zDHHC13 activity prevents palmitoylation of EGFR, which provides new ideas for managing EGFR-dependent tumours [ 160 ]. Additionally, the metabolic environment of non-alcoholic fatty liver disease promotes the production of palmitoyl lipids in colon cancer cells, increasing EGFR palmitoylation and facilitating liver metastasis of colon cancer cells [ 161 ]. The PI3K/Akt pathway is crucial for regulating the cell cycle, cell proliferation, cancer, and longevity [ 162 ]. ZDHHC16 promotes the palmitoylation of the PCSK9 protein at Cys600, inducing AKT-S473 phosphorylation and stimulating proliferation of advanced liver cancer cells [ 102 ]. ZDHHC17/24 also activates AKT by promoting PCSK9 palmitoylation [ 163 ]. High-fat diets increase palmitoyl lipids, and ZDHHC17/24 catalyze palmitoylation of PI3K proteins, activating the AKT signaling pathway. Controlling diet and targeting AKT palmitoylation may provide a viable approach for cancer treatment [ 163 ]. Medications like metformin can inhibit fatty acid synthase reduction, preventing AKT palmitoylation to intervene in inflammation [ 164 ]. Tyrosine kinase inhibitors (TKIs) are targeted drugs that suppress tumor angiogenesis and the VEGF signaling pathway [ 165 ]. ZDHHC2 catalyzes the S-palmitoylation of AGK, activating the PI3K-AKT-mTOR pathway and affecting the sensitivity of renal cell carcinoma to the TKI drug sunitinib [ 79 ]. ZDHHC12 is the palmitoyltransferase for NLRP3, promoting its degradation through the molecular chaperone-mediated autophagy pathway to suppress inflammation. Mice lacking zDHHC12 show enhanced peritonitis and lethality [ 10 , 121 ], indicating its role in terminating NLRP3 activation. On the other hand, ZDHHC7 palmitoylates NLRP3 at Cys126, activating the NLRP3 inflammasome on the Golgi apparatus, which stabilizes the complex and recruits ASC to cleave GSDMD and secrete GSDMD-NT small molecules to promote inflammation [ 8 , 33 ]. Knockout of ZDHHC7 disrupts NLRP3 palmitoylation at Cys126, leading to partial inactivation of NLRP3 in macrophages [ 166 ]. Both ZDHHC12 and ZDHHC7 are involved in NLRP3 palmitoylation, but they have opposing effects. Both zDHHC7 and zDHHC9 can catalyze palmitoylation of glucose transporters. zDHHC7 is the palmitoyltransferase for GLUT4, and knockout of this enzyme in mice results in defective GLUT4 vesicle translocation in adipocytes. As a result, GLUT4 cannot anchor to the plasma membrane, leading to insulin resistance and glucose intolerance [ 36 ]. Upon insulin receptor activation, the PI3K-AKT pathway phosphorylates AS160 protein, leading to the palmitoylation of GSV proteins. This allows GSVs to anchor to the cell membrane and fix GLUT4 as a glucose transporter, accelerating glucose uptake [ 167 ]. Studies show that knockout of ZDHHC9 eliminates GLUT1 palmitoylation and membrane distribution, impairing glycolysis and reducing glioblastoma development [ 41 , 168 ].

Biochemically, palmitoylation significantly enhances the hydrophobicity of certain regions of proteins, influencing various aspects of their characteristics and functions. Cellular pyroptosis, mediated by the GSDMD protein, plays a role in immune responses [ 31 , 32 ]. It has been found that GSDMD is activated by palmitoylation at the zDHHC7 site, which activates Caspase cleavage [ 33 ], releasing the GSDMD-NT domain. This domain oligomerizes at the cell membrane, forming pores and promoting cell death [ 34 ]. APT2, a depalmitoylase, can prevent this oligomerization [ 35 ]. Therefore, palmitoylation and depalmitoylation of GSDMD can act as a switch in the cellular pyroptosis pathway, which has an important regulatory role in the inflammatory immune response [ 8 ]. GLUT1 and GLUT4 are glucose transporters in the GLUT family. During glucose uptake, ZDHHC7 catalyzes palmitoylation at Cys223 of GLUT4 [ 36 , 37 ], while VAMP2 and IRAP on glucose storage vesicles (GSV) also undergo palmitoylation [ 38 , 39 ]. This facilitates the translocation of GSVs to the cell membrane, where VAMP2 interacts with palmitoylated syntaxin 4, anchoring GLUT4 at the membrane and accelerating glucose uptake [ 40 ]. Similarly, ZDHHC9 mediates palmitoylation at Cys207 of GLUT1, also affecting glucose uptake [ 41 ], though the intracellular palmitoylation of GLUT1 is not yet fully understood. Palmitoylation alters the physical properties of palmitoyl-binding proteins. SynDIG1, a type II transmembrane protein, moves and anchors at the cell membrane after its cysteine residues undergo palmitoylation [ 42 ]. Inhibition of SynDIG1 palmitoylation prevents its binding to the AMPA receptor and alleviates symptoms of depression [ 43 ]. FLT3 internal tandem duplication (FLT3-ITD) is a common mutation in acute myeloid leukemia (AML) [ 44 ]. After palmitoylation by ZDHHC6, FLT3-ITD is retained longer in the endoplasmic reticulum. Inhibition of palmitoylation on FLT3-ITD suppresses the growth of FLT3-ITD-mutated AML cells [ 45 ]. PSD-95 is an excitatory postsynaptic density scaffold protein whose palmitoylation affects neuronal signaling [ 46 ]. Inhibition of PSD-95 depalmitoylation increases its abundance at the cell membrane. The levels of amyloid-beta (aβ) peptides are closely associated with Alzheimer’s disease [ 47 , 48 ]. An increase in PSD-95 levels can promote synaptic protein expression and reduce issues caused by amyloid-beta, providing new therapeutic approaches for combating Alzheimer’s disease [ 46 ]. Palmitoylation can alter protein structure and enhance stability. In tumor research, McClellan et al. found that palmitoylation of Flotillin-1 in triple-negative breast cancer inhibited protein degradation and promoted invasion and metastasis [ 49 ]. Zhang et al. demonstrated that palmitoylation inhibits autophagic degradation of YTHDF3 in pancreatic cancer, leading to abnormal MYC accumulation and promoting cancer progression [ 50 ]. In immune inflammation, Zhou et al. found that palmitoylation stabilizes NOD2, promoting excessive inflammation [ 9 ]. Lu et al. showed that palmitoylation protects T-cell regulator protein CD80 from ubiquitination-mediated degradation, ensuring the proper localization of CD80 [ 51 ]. In neuronal cells, Piguel et al. discovered that in bipolar disorder, palmitoylation stabilizes AnkG-190 at the spine head and dendritic membrane nanodomains, influencing mood fluctuations in patients [ 52 ].

This review systematically summarizes the molecular mechanisms of protein palmitoylation and its critical roles in the pathogenesis of diverse diseases. Protein palmitoylation frequently interacts with other post-translational modifications (PTMs), including phosphorylation and ubiquitination, forming complex regulatory networks. Due to the reversible nature of S-palmitoylation, it is widely regarded as a dynamic “molecular switch” that regulates protein subcellular localization, stability, and signal transduction [ 228 ]. For example, in the case of the immune checkpoint protein PD-L1, palmitoylation at Cys272 mediated by zDHHC3 has been shown to enhance protein stability by masking nearby phosphorylation sites and creating steric hindrance that limits ubiquitination [ 229 ]. This modification prolongs PD-L1 residence at the plasma membrane and consequently strengthens immune evasion in tumor cells. These findings illustrate that palmitoylation participates in PTM cross-talk through mechanisms such as signaling shielding and structural modulation, thereby contributing to the maintenance of protein functional homeostasis. Despite significant progress in the detection of protein palmitoylation, the development of high-throughput and highly specific analytical techniques remains a major challenge in the field. The classical Acyl-Biotin Exchange (ABE) method relies on blocking free cysteine residues with N-ethylmaleimide (NEM), followed by hydroxylamine-mediated cleavage of thioester bonds and biotin labeling to enrich palmitoylated proteins. However, this technique is highly sensitive to incomplete cysteine blocking, which may lead to false-positive signals. To reduce background interference, an improved low-background ABE (LB-ABE) strategy has been developed, introducing a sequential blocking step using 2,2′-dipyridyl disulfide to eliminate residual thiols [ 230 , 231 ]. Nevertheless, the subsequent biotinylation and multiple precipitation steps often lead to substantial protein loss, which has gradually limited the use of conventional ABE approaches. To overcome these limitations, Acyl-Resin-Assisted Capture (Acyl-RAC) was developed as an alternative strategy. This method captures hydroxylamine-released proteins directly using thiol-reactive resins, eliminating the need for biotin labeling and repeated precipitation steps, thereby improving protein recovery and experimental reproducibility [ 232 ]. In contrast, metabolic labeling combined with click chemistry enables the incorporation of palmitic acid analogs containing alkyne or azide groups (such as 17-ODYA or 15-YNE) into proteins during live-cell culture. Subsequent click reactions with biotin or fluorescent probes allow not only the identification of palmitoylated proteins but also dynamic monitoring of palmitoylation turnover [ 233 ]. In addition, emerging nanopore sensing technologies provide a potential approach for single-molecule detection of protein modifications by directly reading electrical signals associated with modified residues, enabling the simultaneous detection of multiple PTMs [ 234 ]. However, these approaches remain at an early stage of development. Collectively, these methods should be viewed as complementary rather than mutually exclusive, with ABE-based approaches suited for static site identification and metabolic labeling techniques more appropriate for studying dynamic palmitoylation processes. Current therapeutic development targeting palmitoylation is largely constrained by the high structural homology and functional redundancy within the zDHHC palmitoyltransferase family. Traditional broad-spectrum inhibitors such as 2-bromopalmitate (2-BP) exhibit substantial off-target toxicity, which significantly limits their clinical applicability. Future drug discovery efforts will likely rely on advances in computer-aided drug design (CADD) and high-throughput screening to identify allosteric inhibitors or peptide-based competitive inhibitors targeting specific zDHHC domains. Meanwhile, the therapeutic potential of depalmitoylation processes should not be overlooked. Compared with the large zDHHC enzyme family, depalmitoylating enzymes such as APT1/2, PPT1, and members of the ABHD family are fewer in number and possess relatively more restricted substrate spectra. The successful application of the APT1/2 inhibitor Palmostatin B in melanoma models suggests that modulating the activity of these “eraser” enzymes may provide an effective strategy to disrupt pathological palmitoylation-dependent signaling. Another important area requiring further investigation is the mechanism by which high-fat diets (HFDs) promote palmitoylation dysregulation. Epidemiological and experimental studies have consistently identified HFD as a critical environmental factor that exacerbates various palmitoylation-associated diseases. Mechanistically, HFD may influence palmitoylation through two principal pathways. First, elevated dietary palmitate increases intracellular palmitoyl-CoA availability, thereby enhancing the substrate pool for palmitoylation reactions. Second, lipid-rich environments may modulate the expression of specific zDHHC enzymes, indirectly affecting palmitoylation kinetics. For instance, exogenous palmitate has been reported to increase intracellular palmitoyl-CoA levels and upregulate zDHHC17 and zDHHC24 expression, thereby promoting AKT palmitoylation and membrane recruitment. This mechanism suggests that lipid metabolic disorders may contribute to oncogenic signaling through palmitoylation-dependent pathways in metabolic syndrome–associated cancers such as hepatocellular carcinoma. Furthermore, CD36-mediated lipid uptake has been shown to drive immune evasion by activating the TLR4–p38/JNK signaling pathway and promoting an immunosuppressive tumor microenvironment. Targeting CD36 palmitoylation may therefore not only disrupt tumor metabolic support but also enhance the efficacy of immune checkpoint inhibitors by remodeling the immune microenvironment. Drug development targeting specific zDHHC enzymes remains challenging due to the conserved nature of their catalytic domains. Among the 23 human zDHHC isoforms, several have emerged as promising therapeutic targets based on structural and functional studies. For example, zDHHC20 functions as an important regulator of EGFR signaling and plays a crucial role in the progression of multiple cancers, including pancreatic, breast, and lung cancers. Structural analyses have revealed a well-defined transmembrane hydrophobic cavity that provides a potential platform for rational inhibitor design [ 235 ]. zDHHC9, the primary palmitoyltransferase for Ras family proteins, has also attracted considerable attention because Ras mutations represent one of the most common oncogenic drivers in human cancers. Structural modeling and recent cryo-electron microscopy studies have demonstrated that zDHHC9 activity depends on its interaction with the accessory protein GOLGA7, revealing potential opportunities for disrupting this interface therapeutically [ 236 ]. In contrast, zDHHC5 is one of the few palmitoyltransferases predominantly localized to the plasma membrane and plays important roles in neuronal development, synaptic plasticity, and cardiovascular diseases. Its unusually long C-terminal cytoplasmic domain contains multiple regulatory PTM sites, suggesting a complex regulatory mechanism that may also present opportunities for therapeutic intervention [ 237 ]. Although the development of highly selective zDHHC inhibitors remains in its early stages, several innovative strategies have begun to emerge. For example, PROTAC-based degraders targeting zDHHC5 and zDHHC20 have been designed to induce proteasomal degradation of these enzymes, thereby reducing the palmitoylation levels of substrates such as PD-L1 and IFITM3 [239]. This strategy offers improved specificity and may circumvent compensatory enzymatic upregulation. In addition, the lipid-lowering drug lomitapide has recently been identified as a potential zDHHC5 inhibitor capable of suppressing pancreatic tumor progression, highlighting the promise of drug repurposing strategies. Historically, most functional studies of palmitoyltransferases have relied on the broad-spectrum inhibitor 2-BP [ 238 , 239 ]. However, because of its structural similarity to palmitoyl groups, 2-BP inhibits not only zDHHC enzymes but also fatty acid metabolism–related enzymes such as ACSLs and thioesterases including APTs. Moreover, intracellular metabolism of 2-BP into 2-BP-CoA can cause nonspecific protein alkylation and cytotoxicity [ 240 ]. These limitations, together with the high conservation of the DHHC cysteine-rich domain and the ability of multiple zDHHC isoforms to modify the same substrates, highlight the challenges associated with achieving isoform-specific inhibition. Consequently, future therapeutic strategies will likely integrate structural biology, computational drug design, and targeted protein degradation technologies to overcome these barriers. In the context of precision medicine, palmitoylation states may also serve as potential diagnostic and prognostic biomarkers. Emerging evidence suggests that the palmitoylation status of specific proteins may more accurately reflect disease-associated signaling activity than total protein abundance. For example, the palmitoylation level of PD-L1 has been linked to its stability and immunosuppressive function, potentially influencing tumor responsiveness to immune checkpoint blockade. In neurodegenerative diseases, alterations in the palmitoylation of synaptic proteins such as PSD-95 have been proposed as indicators of synaptic dysfunction. With the continued advancement of mass spectrometry and palmitoyl-proteomics technologies, systematic profiling of disease-associated palmitoylation signatures may provide valuable insights for disease stratification and personalized therapeutic strategies. In summary, research on protein palmitoylation is currently transitioning from mechanistic exploration toward translational and therapeutic applications. Future anticancer strategies may no longer rely on single-target interventions but instead integrate metabolic modulation (such as dietary lipid restriction), enzymatic regulation (including zDHHC-targeting PROTACs or APT inhibitors), and immunotherapeutic approaches that destabilize PD-L1. By combining drug repurposing strategies with emerging proteomic technologies, it may become possible to overcome the long-standing challenges associated with targeting this major post-translational modification and ultimately provide new therapeutic opportunities for patients.

Based on the above discussion, although the development of specific palmitoylation inhibitors and a deeper understanding of the exact molecular mechanisms of palmitoylation in certain pathways are still pending, the current understanding of palmitoylation’s properties and functions provides a solid foundation for emerging research technologies and clinical innovations. This could have profound implications for the development of clinical drug therapies and the innovation of clinical treatment strategies. Three types of palmitoylation: N-palmitoylation, O-palmitoylation, and S-palmitoylation, and all of them can affect signal transduction, protein subcellular localization, and protein stability. The impact of palmitoylation on the metabolism of diverse cell types. Signaling pathways regulated by palmitoylation. PI3K/Akt palmitoylation regulates the cell cycle and is involved in cell proliferation, cancer development, and cell lifespan; NLRP3 palmitoylation is mainly involved in inflammatory response and apoptosis; GLUTs palmitoylation is involved in glucose metabolism, and is associated with glucose metabolism diseases. Enzymes of the zDHHC family. Diabetes and its complications. Palmitoylation-related diseases.