Extracellular vesicles (EVs) originating from testicular somatic cells act as pivotal intermediaries in cell signaling crosstalk between spermatogenic cells and the testicular microenvironment. The intricate balance between palmitoylation and depalmitoylation governs the positioning of protein cargos on the membrane, thereby influencing cellular activities by concentrating these proteins in EVs for delivery to recipient cells. Here, we reveal that GNA13 undergoes specific S-palmitoylation at Cys14 and Cys18 residues in Sertoli cells (SCs), a modification essential for its localization to the plasma membrane. We identify DHHC13, a member of the zinc finger DHHC-type palmitoyltransferase family that catalyzes protein S-palmitoylation, as the enzyme responsible for this critical post-translational modification. Additionally, GNA13 palmitoylation is indispensable for its selective enrichment in EVs emanating from SCs. Intriguingly, we discovered the presence of palmitoylated GNA13 in SC-derived EVs significantly downregulates autophagy levels in spermatogonial stem cells (SSCs), and the inhibition of GNA13 palmitoylation attenuates its interaction with ARHGEF12 which leads to diminished RhoA activity and consequent elevation of autophagy in SSCs. Our results illuminate the crucial role of DHHC13-mediated GNA13 S-palmitoylation in modulating autophagy levels in SSCs through SCs-derived EVs, suggesting that PM-GNA13-EV may serve as a potential candidate for further exploration in addressing fertility-related challenges during spermatogenesis.

The dynamic addition and removal of long-chain fatty acids modulate protein function and localization. The α/β hydrolase domain-containing (ABHD) 17 enzymes remove acyl chains from membrane-localized proteins such as the oncoprotein NRas, but how the ABHD17 proteins are regulated is unknown. Here, we used cell-based studies and molecular dynamics simulations to show that ABHD17 activity is controlled by two mobile elements-an S-acylated N-terminal helix and a loop-that flank the putative substrate-binding pocket. Multiple S-acylation events anchor the N-terminal helix in the membrane, enabling hydrophobic residues in the loop to engage with the bilayer. This stabilizes the conformation of both helix and loop, alters the conformation of the binding pocket, and optimally positions the enzyme for substrate engagement. S-acylation may be a general feature of acyl-protein thioesterases. By providing a mechanistic understanding of how the lipid modification of a lipid-removing enzyme promotes its enzymatic activity, this work contributes to our understanding of cellular S-acylation cycles.

NLRP3 inflammasome is activated by diverse stimuli including infections, intracellular and environmental irritants. How NLRP3 senses these unrelated stimuli and what activates NLRP3 remain unknown. Here we report that signal-dependent NLRP3 phase separation initiated its activation, in which the palmitoyltransferase ZDHHC7-mediated tonic NLRP3 palmitoylation and an IDR region in the FISNA domain of NLRP3 play important roles. Moreover, three conserved hydrophobic residues in the IDR critically mediate multivalent weak interactions. NLRP3-activating stimuli including K+ efflux and NLRP3-interacting molecules imiquimod, palmitate, and cardiolipin all cause NLRP3 conformational change and induce its phase separation and activation in cells and/or in vitro. Surprisingly, amphiphilic molecules like di-alcohols used to inhibit biomolecular phase separation and chemotherapeutic drugs doxorubicin and paclitaxel activate NLRP3 independently of ZDHHC7 by directly inducing NLRP3 phase separation. Mechanistically, amphiphilic molecules decrease the solubility of both palmitoylated and non-palmitoylated NLRP3 to directly induce its phase separation and activation while NLRP3 palmitoylation reduces its solubility to some extent without activation. Therefore, ZDHHC7-mediated NLRP3 palmitoylation in resting cells licenses its activation by lowering the threshold for NLRP3 phase separation in response to any of the diverse stimuli whereas NLRP3 solubility-reducing molecules like di-alcohols and chemotherapeutic drugs activate NLRP3 directly. The signal-induced NLRP3 phase separation likely provides the simplest and most direct mechanistic basis for NLRP3 activation.

Protein post-translational modification (PTM) serves as an important mechanism for regulating protein function. Accurate assay of PTM stoichiometry, or PTM occupancy, which refers to the proportion of proteins that contain specific modifications, is important for understanding the function of PTMs. We previously developed a novel chemoproteomic strategy "STO-MS" to quantify the PTM stoichiometry in complex biological samples, which employs a resolvable polymer mass tag to differentiate modified proteins and utilizes liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) techniques to measure PTM stoichiometry. However, the resolution of STO-MS is constrained by the relatively low molecular weight of the mass tag, and the incorporation of isotopic labels not only complicates the sample preparation but also restricts the measurement throughput. To address these challenges, we herein developed "STO-MS+", an enhanced workflow, that incorporates an optimized DNA mass tag and employs a label-free quantitative data analysis approach. We applied STO-MS+ to measure stoichiometry of three distinct PTMs, including endogenous carbonylation induced by arachidonic acid (AA), itaconation, and endogenous O-GlcNAcylation. Our work marks a notable improvement in chemoproteomic methodologies for quantifying post-translational modifications and provides a powerful analytical tool for PTM research.

Elevated uptake of saturated fatty acid palmitic acid (PA) is associated with tumor metastasis; however, the precise mechanisms remain partially understood, hindering the development of therapy for PA-driven tumor metastasis. The Hippo-Yes-associated protein (Hippo/YAP) pathway is implicated in cancer progression. Here it is shown that a high-palm oil diet potentiates tumor metastasis in murine xenografts in part through YAP. It is found that the palmitoyltransferase ZDHHC15 is a YAP-regulated gene that forms a feedback loop with YAP. Notably, PA drives the ZDHHC15-YAP feedback loop, thus enforces YAP signaling, and hence promotes tumor metastasis in murine xenografts. In addition, it is shown that ZDHHC15 associates with Kidney and brain protein (KIBRA, also known as WW- and C2 domain-containing protein 1, WWC1), an upstream component of Hippo signaling, and mediates its palmitoylation. KIBRA palmitoylation leads to its degradation and regulates its subcellular localization and activity toward the Hippo/YAP pathway. Moreover, PA enhances KIBRA palmitoylation and degradation. It is further shown that combinatorial targeting of YAP and fatty acid synthesis exhibits augmented effects against metastasis formation in mice fed with a Palm diet. Collectively, these findings uncover a ZDHHC15-YAP feedback loop as a previously unrecognized mechanism underlying PA-promoted tumor metastasis and support targeting YAP and fatty acid synthesis as potential therapeutic targets in PA-driven tumor metastasis.

Interferon-inducible transmembrane (IFITM) proteins are broad-spectrum antiviral factors that confer cellular resistance to virus invasion. α/β-Hydrolase domain-containing 16A (ABHD16A) has recently been identified as a novel depalmitoylase that can inhibit the antiviral activity of IFITM proteins by catalyzing the depalmitoyl reaction; this pattern may be crucial for the host to avoid damage caused by excessive immune response. However, it remains largely elusive about how host cells regulate the activity of ABHD16A. In the present study, we performed the AlphaFold2-based protein-protein interaction prediction and identified swine E3 ubiquitin ligase ring finger protein 5 (sRNF5) as a sABHD16A-interacting protein and negatively regulated the stability of sABHD16A. Using immunofluorescence and co-immunoprecipitation techniques, we uncovered that sRNF5 targeted sABHD16A for ubiquitination and degradation via the proteasomal pathway at residues K3 and K452. Furthermore, sABHD16A catalyzed the depalmitoylation of sIFITM1, which obstructed the antiviral function of sIFITM1, while sRNF5 caused ubiquitination of sABHD16A, which attenuated the depalmitoylation effect on sIFITM1, and consequently restored the antiviral activity of sIFITM1. Collectively, our findings demonstrate for the first time that sRNF5 positively regulates the antiviral function of sIFITM1 by mediating the degradation of sABHD16A, which expands the biological functions of RNF5 and ABHD16A in immune regulation. Moreover, our work highlights the well-designed interplay between RNF5, ABHD16A, and IFITM, which balances antiviral immune responses to avoid the disorders induced by excessive immune response.

Interferon and interferon-stimulated genes play significant and protective roles in the host's defense against viral infection. IFITM family proteins, which can be strongly induced by interferon, have been identified as the first line of defense to prevent invasion of various viruses. Further analysis reveals the antiviral activity of IFITMs depends on palmitoylation/depalmitoylation. Recently, we reported that ABHD16A, as the first depalmitoylase of IFITMs, negatively regulated the antiviral activity of IFITMs. However, these raise crucial questions: how ABHD16A is regulated and remained in a balanced manner? Here, we show that swine RNF5 attenuates the negative regulation of sIFITM1 against virus invasion by modifying sABHD16A through ubiquitination and guiding sABHD16A for degradation. Thus, sRNF5-sABHD16A interplay plays an indispensable role in regulating immune response and avoiding the disorders induced by elevated interferon levels. Overall, our findings extend the upstream subtle regulatory molecular mechanism of IFITMs and provide potential targets for viral disease therapy.

Calcineurin, the Ca 2+ /calmodulin-activated protein phosphatase, recognizes substrates and regulators via short linear motifs, PxIxIT and LxVP, which dock to distinct sites on calcineurin and determine calcineurin distribution and catalysis, respectively. Calcimembrin/C16orf74 (CLMB), an intrinsically disordered microprotein whose expression correlates with poor cancer outcomes, targets calcineurin to membranes where it may promote oncogenesis by shaping calcineurin signaling. We show that CLMB associates with membranes via lipidation, i.e. N-myristoylation and reversible S-acylation. Furthermore, CLMB contains an unusual composite 'LxVPxIxIT' motif, that binds the PxIxIT-docking site on calcineurin with extraordinarily high affinity when phosphorylated, 33 LxVPxIxITxx(p)T 44 . Calcineurin dephosphorylates CLMB to decrease this affinity, but Thr44 is protected from dephosphorylation when PxIxIT-bound. We propose that CLMB is dephosphorylated in multimeric complexes, where one PxIxIT-bound CLMB recruits calcineurin to membranes, allowing a second CLMB to engage via its LxVP motif to be dephosphorylated. In vivo and in vitro data, including nuclear magnetic resonance (NMR) analyses of CLMB-calcineurin complexes, supports this model. Thus, the CLMB composite motif imposes unique properties to calcineurin signaling at membranes including sensitivity to CLMB:calcineurin ratios, CLMB phosphorylation and dynamic S-acylation.

Protein S-acyl transferases (PATs) are a family of enzymes that catalyze protein S-acylation, a post-translational lipid modification involved in protein membrane targeting, trafficking, stability, and protein-protein interaction. S-acylation plays important roles in plant growth, development, and stress responses. Here, we report the genome-wide analysis of the PAT family genes in the woodland strawberry (Fragaria vesca), a model plant for studying the economically important Rosaceae family. In total, 21 'Asp-His-His-Cys' Cys Rich Domain (DHHC-CRD)-containing sequences were identified, named here as FvPAT1-21. Expression profiling by reverse transcription quantitative PCR (RT-qPCR) showed that all the 21 FvPATs were expressed ubiquitously in seedlings and different tissues from adult plants, with notably high levels present in vegetative tissues and young fruits. Treating seedlings with hormones indole-3-acetic acid (IAA), abscisic acid (ABA), and salicylic acid (SA) rapidly increased the transcription of most FvPATs. A complementation assay in yeast PAT mutant akr1 and auto-S-acylation assay of one FvPAT (FvPAT19) confirmed its enzyme activity where the Cys in the DHHC motif was required. An AlphaFold prediction of the DHHC and the mutated DHHC155S of FvPAT19 provided further proof of the importance of C155 in fatty acid binding. Together, our data clearly demonstrated that S-acylation catalyzed by FvPATs plays important roles in growth, development, and stress signaling in strawberries. These preliminary results could contribute to further research to understand S-acylation in strawberries and plants in general.

Protein S-palmitoylation is a reversible form of protein lipidation in which the formation of a thioester bond occurs between a cysteine (Cys) residue of a protein and a 16-carbon fatty acid chain. This modification is catalyzed by a family of palmitoyl acyl transferases, the DHHC enzymes, so called because of their Asp-His-His-Cys (DHHC) catalytic motif. Deregulation of DHHC enzymes has been linked to various diseases, including cancer and infections. Cancer, a major cause of global mortality, is characterized by features like uncontrolled cell growth, resistance to cell death, angiogenesis, invasion, and metastasis. Several of these processes are controlled by DHHC-mediated S-palmitoylation of oncogenes or tumor suppressors, including growth factor receptors (e.g., EGFR), kinases (e.g., AKT), and transcription factors (e.g., β-catenin). Dynamic regulation of S-palmitoylation is also governed by protein depalmitoylases. These enzymes balance the cycling of palmitoylation and regulate cellular signaling, cell growth, and its organization. Given the significance of S-palmitoylation in cancer, the DHHCs and protein depalmitoylases are promising targets for cancer therapy. Here we summarize the catalytic mechanisms of DHHC enzymes and depalmitoylases, their role in cancer progression and prevention, as well as the crosstalk of palmitoylation with other post-translational modifications. Additionally, we discuss the methods to detect S-palmitoylation, the limitations of available DHHC-targeting inhibitors, and ongoing research efforts to address these obstacles.

Protein lipidation dramatically affects protein structure, localization, and trafficking via remodeling protein-membrane and protein-protein interactions through hydrophobic lipid moieties. Understanding the biosynthesis of lipidated proteins, whether natural ones or mimetics, is crucial for reconstructing, validating, and studying the molecular mechanisms and biological functions of protein lipidation. In this Perspective, we first provide an overview of the natural enzymatic biosynthetic pathways of protein lipidation in mammalian cells, focusing on the enzymatic machineries and their chemical linkages. We then discuss strategies to biosynthesize protein lipidation in mammalian cells by engineering modification machineries and substrates. Additionally, we explore site-specific protein lipidation biosynthesis in vitro via enzyme-mediated ligations and in vivo primarily through genetic code expansion strategies. We also discuss the use of small molecule tools to modulate the process of protein lipidation biosynthesis. Finally, we provide concluding remarks and discuss future directions for the biosynthesis and applications of protein lipidation.

The 23 human zinc finger Asp-His-His-Cys motif-containing (ZDHHC) S-acyltransferases catalyze long-chain S-acylation at cysteine residues across an extensive network of hundreds of proteins important for normal physiology or dysregulated in disease. Here we present a technology to directly map the protein substrates of a specific ZDHHC at the whole-proteome level, in intact cells. Structure-guided engineering of paired ZDHHC 'hole' mutants and 'bumped' chemically tagged fatty acid probes enabled probe transfer to specific protein substrates with excellent selectivity over wild-type ZDHHCs. Chemical-genetic systems were exemplified for five human ZDHHCs (3, 7, 11, 15 and 20) and applied to generate de novo ZDHHC substrate profiles, identifying >300 substrates and S-acylation sites for new functionally diverse proteins across multiple cell lines. We expect that this platform will elucidate S-acylation biology for a wide range of models and organisms.

S-palmitoylation is a reversible and dynamic process that involves the addition of long-chain fatty acids to proteins. This protein modification regulates various aspects of protein function, including subcellular localization, stability, conformation, and biomolecular interactions. The zinc finger DHHC (ZDHHC) domain-containing protein family is the main group of enzymes responsible for catalyzing protein S-palmitoylation, and 23 members have been identified in mammalian cells. Many proteins that undergo S-palmitoylation have been linked to disease pathogenesis and progression, suggesting that the development of effective inhibitors is a promising therapeutic strategy. Reducing the protein S-palmitoylation level can target either the PATs directly or their substrates. However, there are rare clinically effective S-palmitoylation inhibitors. This review aims to provide an overview of the S-palmitoylation field, including the catalytic mechanism of ZDHHC, S-palmitoylation detection methods, and the functional impact of protein S-palmitoylation. Additionally, this review focuses on current strategies for expanding the chemical toolbox to develop novel and effective inhibitors that can reduce the level of S-palmitoylation of the target protein.

Here, we present an optimized acyl-PEGyl exchange gel shift (APEGS) assay to monitor palmitoylation of high-molecular-weight proteins from primary neuronal cultures. We describe steps for culturing cortical neurons from rat embryos and expressing proteins of interest. We then detail procedures for employing a fatty acyl exchange technique wherein hydroxylamine is used to cleave palmitic acid from the palmitoyl-thioester bond, exposing cysteine residues that are subsequently labeled with methoxy polyethylene glycol maleimide (mPEG-MAL-10k). For complete details on the use and execution of this protocol, please refer to Yucel et al.1.

Autophagy is a conserved catabolic recycling pathway that can eliminate cytosolic materials to maintain homeostasis and organelle functions. Many studies over the past few decades have demonstrated that abnormal autophagy is associated with a variety of diseases. Protein lipidation plays an important role in the regulation of autophagy by affecting protein trafficking, localization, stability, interactions and signal transduction. Here, we review recent advances in the understanding of the role of lipidation in autophagy, including S-palmitoylation, N-myristoylation, S-prenylation, glycosylphosphatidylinositol (GPI) anchor modification and cholesterylation. We comprehensively review the enzymes and catalytic mechanisms of lipidation and discuss the relationship between lipidation and autophagy, aiming to deepen the understanding of lipidation and promote the discovery of drug targets for the treatment of autophagy-related diseases.

Rap2b, a proto-oncogene upregulated in colorectal cancer (CRC), undergoes protein S-palmitoylation at specific C-terminus sites (C176/C177). These palmitoylation sites are crucial for Rap2b localization on the plasma membrane (PM), as mutation of C176 or C177 results in cytosolic relocation of Rap2b. Our study demonstrates that Rap2b influences cell migration and invasion in CRC cells, independent of proliferation, and this activity relies on its palmitoylation. We identify ABHD17a as the depalmitoylating enzyme for Rap2b, altering PM localization and inhibiting cell migration and invasion. EGFR/PI3K signaling regulates Rap2b palmitoylation, with PI3K phosphorylating ABHD17a to modulate its activity. These findings highlight the potential of targeting Rap2b palmitoylation as an intervention strategy. Blocking the C176/C177 sites using an interacting peptide attenuates Rap2b palmitoylation, disrupting PM localization, and suppressing CRC metastasis. This study offers insights into therapeutic approaches targeting Rap2b palmitoylation for the treatment of metastatic CRC, presenting opportunities to improve patient outcomes.

Proteins undergo reversible S-acylation via a thioester linkage in vivo. S-palmitoylation, modification by C16:0 fatty acid, is a common S-acylation that mediates critical protein-membrane and protein-protein interactions. The most widely used S-acylation assays, including acyl-biotin exchange and acyl resin-assisted capture, utilize blocking of free Cys thiols, hydroxylamine-dependent cleavage of the thioester and subsequent labeling of nascent thiol. These assays generally require >500 μg of protein input material per sample and numerous reagent removal and washing steps, making them laborious and ill-suited for high throughput and low input applications. To overcome these limitations, we devised "Acyl-Trap", a suspension trap-based assay that utilizes a thiol-reactive quartz to enable buffer exchange and hydroxylamine-mediated S-acyl enrichment. We show that the method is compatible with protein-level detection of S-acylated proteins (e.g., H-Ras) as well as S-acyl site identification and quantification using "on trap" isobaric labeling and LC-MS/MS from as little as 20 μg of protein input. In mouse brain, Acyl-Trap identified 279 reported sites of S-acylation and 1298 previously unreported putative sites. Also described are conditions for long-term hydroxylamine storage, which streamline the assay. More generally, Acyl-Trap serves as a proof-of-concept for PTM-tailored suspension traps suitable for both traditional protein detection and chemoproteomic workflows.

Defects in planar cell polarity (PCP) have been implicated in diverse human pathologies. Vangl2 is one of the core PCP components crucial for PCP signaling. Dysregulation of Vangl2 has been associated with severe neural tube defects and cancers. However, how Vangl2 protein is regulated at the posttranslational level has not been well understood. Using chemical reporters of fatty acylation and biochemical validation, here we present that Vangl2 subcellular localization is regulated by a reversible S-stearoylation cycle. The dynamic process is mainly regulated by acyltransferase ZDHHC9 and deacylase acyl-protein thioesterase 1 (APT1). The stearoylation-deficient mutant of Vangl2 shows decreased plasma membrane localization, resulting in disruption of PCP establishment during cell migration. Genetically or pharmacologically inhibiting ZDHHC9 phenocopies the effects of the stearoylation loss of Vangl2. In addition, loss of Vangl2 stearoylation enhances the activation of oncogenic Yes-associated protein 1 (YAP), serine-threonine kinase AKT, and extracellular signal-regulated protein kinase (ERK) signaling and promotes breast cancer cell growth and HRas G12V mutant (HRasV12)-induced oncogenic transformation. Our results reveal a regulation mechanism of Vangl2, and provide mechanistic insight into how fatty acid metabolism and protein fatty acylation regulate PCP signaling and tumorigenesis by core PCP protein lipidation.

Proteins undergo reversible S-acylation via a thioester linkage in vivo. S-palmitoylation, modification by C16:0 fatty acid, is a common S-acylation that mediates critical protein-membrane and protein-protein interactions. The most widely used S-acylation assays, including acyl-biotin exchange and acyl resin-assisted capture, utilize blocking of free Cys thiols, hydroxylamine-dependent cleavage of the thioester and subsequent labeling of nascent thiol. These assays generally require >500 micrograms of protein input material per sample and numerous reagent removal and washing steps, making them laborious and ill-suited for high throughput and low input applications. To overcome these limitations, we devised "Acyl-Trap", a suspension trap-based assay that utilizes a thiol-reactive quartz to enable buffer exchange and hydroxylamine-mediated S-acyl enrichment. We show that the method is compatible with protein-level detection of S-acylated proteins (e.g. H-Ras) as well as S-acyl site identification and quantification using "on trap" isobaric labeling and LC-MS/MS from as little as 20 micrograms of protein input. In mouse brain, Acyl-Trap identified 279 reported sites of S-acylation and 1298 previously unreported putative sites. Also described are conditions for long-term hydroxylamine storage, which streamlines the assay. More generally, Acyl-Trap serves as a proof-of-concept for PTM-tailored suspension traps suitable for both traditional protein detection and chemoproteomic workflows.

Here, we show that insulin induces palmitoylation turnover of Caveolin-2 (Cav-2) in adipocytes. Acyl protein thioesterases-1 (APT1) catalyzes Cav-2 depalmitoylation, and zinc finger DHHC domain-containing protein palmitoyltransferase 21 (ZDHHC21) repalmitoylation of the depalmitoylated Cav-2 for the turnover, thereby controlling insulin receptor (IR)-Cav-2-insulin receptor substrate-1 (IRS-1)-Akt-driven signaling. Insulin-induced palmitoylation turnover of Cav-2 facilitated glucose uptake and fat storage through induction of lipogenic genes. Cav-2-, APT1-, and ZDHHC21-deficient adipocytes, however, showed increased induction of lipolytic genes and glycerol release. In addition, white adipose tissues from insulin sensitive and resistant obese patients exhibited augmented expression of LYPLA1 (APT1) and ZDHHC20 (ZDHHC20). Our study identifies the specific enzymes regulating Cav-2 palmitoylation turnover, and reveals a new mechanism by which insulin-mediated lipid metabolism is controlled in adipocytes.

The global impact of emerging viral infections emphasizes the urgent need for effective broad-spectrum antivirals. The cellular organelle, lipid droplet (LD), is utilized by many types of viruses for replication, but its reduction does not affect cell survival. Therefore, LD is a potential target for developing broad-spectrum antivirals. In this study, we found that 2-bromopalmitate (2 BP), a previously defined palmitoylation inhibitor, depletes LD across all studied cell lines and exerts remarkable antiviral effects on different coronaviruses. We comprehensively utilized 2 BP, alongside other palmitoylation inhibitors such as cerulenin and 2-fluoro palmitic acid (2-FPA), as well as the enhancer palmostatin B and evaluated their impact on LD and the replication of human coronaviruses (hCoV-229E, hCoV-Oc43) and murine hepatitis virus (MHV-A59) at non-cytotoxic concentrations. While cerulenin and 2-FPA exhibited moderate inhibition of viral replication, 2 BP exhibited a much stronger suppressive effect on MHV-A59 replication, although they share similar inhibitory effects on palmitoylation. As expected, palmostatin B significantly enhanced viral replication, it failed to rescue the inhibitory effects of 2 BP, whereas it effectively counteracted the effects of cerulenin and 2-FPA. This suggests that the mechanism that 2 BP used to inhibit viral replication is beyond palmitoylation inhibition. Further investigations unveil that 2 BP uniquely depletes LDs, a phenomenon not exhibited by 2-FPA and cerulenin. Importantly, the depletion of LDs was closely associated with the inhibition of viral replication because the addition of oleic acid to 2 BP significantly rescued LD depletion and its inhibitory effects on MHV-A59. Our findings indicate that the inhibitory effects of 2 BP on viral replication primarily stem from LD disruption rather than palmitoylation inhibition. Intriguingly, fatty acid (FA) assays demonstrated that 2 BP reduces the FA level in mitochondria while concurrently increasing FA levels in the cytoplasm. These results highlight the crucial role of LDs in viral replication and uncover a novel biological activity of 2 BP. These insights contribute to the development of broad-spectrum antiviral strategies.

In our study, we conducted a comparative investigation into the antiviral effects of palmitoylation inhibitors including 2-bromopalmitate (2-BP), 2-fluoro palmitic acid (2-FPA), and cerulenin. Surprisingly, we discovered that 2-BP has superior inhibitory effects on viral replication compared to 2-FPA and cerulenin. However, their inhibitory effects on palmitoylation were the same. Intrigued by this finding, we delved deeper into the underlying mechanism of 2-BP's potent antiviral activity, and we unveiled a novel biological activity of 2-BP: depletion of lipid droplets (LDs). Importantly, we also highlighted the crucial role of LDs in viral replication. Our insights shed new light on the antiviral mechanism of LD depletion paving the way for the development of broad-spectrum antiviral strategies by targeting LDs.

Caveolae are small flask-shaped invaginations of the surface membrane which are proposed to recruit and co-localize signaling molecules. The distinctive caveolar shape is achieved by the oligomeric structural protein caveolin, of which three isoforms exist. Aside from the finding that caveolin-3 is specifically expressed in muscle, functional differences between the caveolin isoforms have not been rigorously investigated. Caveolin-3 is relatively cysteine-rich compared to caveolins 1 and 2, so we investigated its cysteine post-translational modifications. We find that caveolin-3 is palmitoylated at 6 cysteines and becomes glutathiolated following redox stress. We map the caveolin-3 palmitoylation sites to a cluster of cysteines in its C terminal membrane domain, and the glutathiolation site to an N terminal cysteine close to the region of caveolin-3 proposed to engage in protein interactions. Glutathiolation abolishes caveolin-3 interaction with heterotrimeric G protein alpha subunits. Our results indicate that a caveolin-3 oligomer contains up to 66 palmitates, compared to up to 33 for caveolin-1. The additional palmitoylation sites in caveolin-3 therefore provide a mechanistic basis by which caveolae in smooth and striated muscle can possess unique phospholipid and protein cargoes. These unique adaptations of the muscle-specific caveolin isoform have important implications for caveolar assembly and signaling.

Abnormal activation of FAK is associated with tumor development and metastasis. Through interactions with other intracellular signalling molecules, FAK influences cytoskeletal remodelling, modulation of adhesion signalling, and activation of transcription factors, promoting migration and invasion of tumor cells. However, the exact mechanism that regulates these processes remains unresolved. Herein, our findings indicate that the S-palmitoylation of FAK is crucial for both its membrane localization and activation.

The palmitoylation of FAK in U251 and T98G cells was assessed by an acyl-PEG exchange (APE) assay and a metabolic incorporation assay. Cellular palmitoylation was inhibited using 2-bromopalmitate, and the palmitoylation status and cellular localization of FAK were determined. A metabolic incorporation assay was used to identify the potential palmitoyl acyltransferase and the palmitoylation site of FAK. Cell Counting Kit-8 (CCK8) assays, colony formation assays, and Transwell assays were conducted to assess the impact of ZDHHC5 in GBM. Additionally, intracranial GBM xenografts were utilized to investigate the effects of genetically silencing ZDHHC5 on tumor growth.

Inhibiting FAK palmitoylation leads to its redistribution from the membrane to the cytoplasm and a decrease in its phosphorylation. Moreover, ZDHHC5, a protein-acyl-transferase (PAT), catalyzes this key modification of FAK at C456. Knockdown of ZDHHC5 abrogates the S-palmitoylation and membrane distribution of FAK and impairs cell proliferation, invasion, and epithelial-mesenchymal transition (EMT). Taken together, our research reveals the crucial role of ZDHHC5 as a PAT responsible for FAK S-palmitoylation, membrane localization, and activation.

These results imply that targeting the ZDHHC5/FAK axis has the potential to be a promising strategy for therapeutic interventions for glioblastoma (GBM). Video Abstract.

Diacylglycerol kinase-ε (DGKε) catalyzes phosphorylation of diacylglycerol to phosphatidic acid with a unique specificity toward 1-stearoyl-2-arachidonoyl-sn-glycerol, which is a backbone of phosphatidylinositol (PI). Owing to this specificity, DGKε is involved in the PI cycle maintaining the cellular level of phosphorylated PI derivatives of signaling activity and was also found crucial for lipid metabolism. DGKε dysfunction is linked with the development of atypical hemolytic uremic syndrome (aHUS) and possibly other human diseases. Despite the DGKε significance, data on its regulation by cotranslational and/or post-translational modifications are scarce. Here, we report that DGKε is S-palmitoylated at Cys38/40 (mouse/human DGKε) located in the cytoplasmic end of its N-terminal putative transmembrane fragment. The S-palmitoylation of DGKε was revealed by metabolic labeling of cells with a palmitic acid analogue followed by click chemistry and with acyl-biotin and acyl-polyethylene glycol exchange assays. The S-acyltransferases zDHHC7 (zinc finger DHHC domain containing) and zDHHC17 and the zDHHC6/16 tandem were found to catalyze DGKε S-palmitoylation, which also increased the DGKε abundance. Mouse DGKε-Myc ectopically expressed in human embryonic kidney 293 cells localized to the endoplasmic reticulum where zDHHC6/16 reside and in small amounts also to the Golgi apparatus where zDHHC7 and zDHHC17 are present. The Cys38Ala substitution upregulated, whereas hyperpalmitoylation of wild-type DGKε reduced the kinase activity, indicating an inhibitory effect of the Cys38 S-palmitoylation. In addition, the substitution of neighboring Pro31 with Ala also diminished the activity of DGKε. Taken together, our data indicate that S-palmitoylation can fine-tune DGKε activity in distinct cellular compartments, possibly by affecting the distance between the kinase and its substrate in a membrane.

Septin proteins are involved in diverse physiological functions, including the formation of specialized cytoskeletal structures. Septin 8 (Sept8) is implicated in spine morphogenesis and dendritic branching through palmitoylation. We explored the role and regulation of a Sept8 variant in human neural-like cells and in the mouse brain. We identified Sept8-204 as a brain-specific variant of Sept8 that was abundant in neurons and modified by palmitoylation, specifically at Cys469, Cys470, and Cys472. Sept8-204 palmitoylation was mediated by the palmitoyltransferase ZDHHC7 and was removed by the depalmitoylase PPT1. Palmitoylation of Sept8-204 bound to F-actin and induced cytoskeletal dynamics to promote the outgrowth of filopodia in N2a cells and the arborization of neurites in hippocampal neurons. In contrast, a Sept8-204 variant that could not be palmitoylated because of mutation of all three Cys residues (Sept8-204-3CA) lost its ability to bind F-actin, and expression of this mutant did not promote morphological changes. Genetic deletion of Sept8, Sept8-204, or Zdhhc7 caused deficits in learning and memory and promoted anxiety-like behaviors in mice. Our findings provide greater insight into the regulation of Sept8-204 by palmitoylation and its role in neuronal morphology and function in relation to cognition.

Interferon-induced transmembrane proteins (IFITM1, 2 and 3) are important host antiviral defense factors. They are active against viruses like the influenza A virus (IAV), dengue virus (DENV), Ebola virus (EBOV), Zika virus (ZIKV) and severe acute respiratory syndrome coronavirus (SARS-CoV). In this review, we focus on IFITM3 S-palmitoylation, a reversible lipid modification, and describe its role in modulating IFITM3 antiviral activity. Our laboratory discovered S-palmitoylation of IFITMs using chemical proteomics and demonstrated the importance of highly conserved fatty acid-modified Cys residues in IFITM3 antiviral activity. Further studies showed that site-specific S-palmitoylation at Cys72 is important for IFITM3 trafficking to restricted viruses (IAV and EBOV) and membrane-sterol interactions. Thus, site-specific lipid modification of IFITM3 directly regulates its antiviral activity, cellular trafficking, and membrane-lipid interactions.

palmitoylation, a reversible post-translational modification, is initiated by the DHHC family of palmitoyltransferases and reversed by several acyl protein thioesterases. However, the role and mechanisms for protein palmitoylation in renal fibrosis have not been elucidated. Here we show protein palmitoylation and DHHC9 were downregulated in the fibrotic kidneys of mouse models and chronic kidney disease (CKD) patients. Ablating DHHC9 in tubular cells aggravated, while inducing DHHC9 overexpression with adeno-DHHC9 transfection or iproniazid treatment protected against kidney fibrosis in male mouse models. Mechanistically, DHHC9 palmitoylated β-catenin, thereby promoted its ubiquitination and degradation. Additionally, acyl protein thioesterase 1 (APT1) was induced in the fibrotic kidneys, which depalmitoylated β-catenin, increased its abundance and nuclear translocation. Ablating tubular APT1 or inhibiting APT1 with ML348 markedly protected against unilateral ureter obstruction (UUO) or ischemia/reperfusion injury (IRI)-induced kidney fibrosis in male mice. This study reveals the regulatory mechanism of protein palmitoylation in kidney fibrosis.

Kainate receptors (KARs) are key regulators of neuronal excitability and synaptic transmission. KAR surface expression is tightly controlled in part by post-translational modifications (PTMs) of the GluK2 subunit. We have shown previously that agonist activation of GluK2-containing KARs leads to phosphorylation of GluK2 at S868, which promotes subsequent SUMOylation at K886 and receptor endocytosis. Furthermore, GluK2 has been shown to be palmitoylated. However, how the interplay between palmitoylation, phosphorylation and SUMOylation orchestrate KAR trafficking remains unclear. Here, we used a library of site-specific GluK2 mutants to investigate the interrelationship between GluK2 PTMs, and their impact on KAR surface expression. We show that GluK2 is basally palmitoylated and that this is decreased by kainate (KA) stimulation. Moreover, a non-palmitoylatable GluK2 mutant (C858/C871A) shows enhanced S868 phosphorylation and K886 SUMOylation under basal conditions and is insensitive to KA-induced internalisation. These results indicate that GluK2 palmitoylation contributes to stabilising KAR surface expression and that dynamic depalmitoylation promotes downstream phosphorylation and SUMOylation to mediate activity-dependent KAR endocytosis.

Protein S-palmitoylation, a type of post-translational modification, refers to the reversible process of attachment of a fatty acyl chain-a 16-carbon palmitate acid-to the specific cysteine residues on target proteins. By adding the lipid chain to proteins, it increases the hydrophobicity of proteins and modulates protein stability, interaction with effector proteins, subcellular localization, and membrane trafficking. Palmitoylation is catalyzed by a group of zinc finger DHHC-containing proteins (ZDHHCs), whereas depalmitoylation is catalyzed by a family of acyl-protein thioesterases. Increasing numbers of oncoproteins and tumor suppressors have been identified to be palmitoylated, and palmitoylation is essential for their functions. Understanding how palmitoylation influences the function of individual proteins, the physiological roles of palmitoylation, and how dysregulated palmitoylation leads to pathological consequences are important drivers of current research in this research field. Further, due to the critical roles in modifying functions of oncoproteins and tumor suppressors, targeting palmitoylation has been used as a candidate therapeutic strategy for cancer treatment. Here, based on recent literatures, we discuss the progress of investigating roles of palmitoylation in regulating cancer progression, immune responses against cancer, and cancer stem cell properties.

S-acylation is a reversible posttranslational protein modification consisting of attachment of a fatty acid to a cysteine via a thioester bond. Research over the last few years has shown that a variety of different fatty acids, such as palmitic acid (C16:0), stearate (C18:0), or oleate (C18:1), are used in cells to S-acylate proteins. We recently showed that GNAI proteins can be acylated on a single residue, Cys3, with either C16:0 or C18:1, and that the relative proportion of acylation with these fatty acids depends on the level of the respective fatty acid in the cell's environment. This has functional consequences for GNAI proteins, with the identity of the acylating fatty acid affecting the subcellular localization of GNAIs. Unclear is whether this competitive acylation is specific to GNAI proteins or a more general phenomenon in the proteome. We perform here a proteome screen to identify proteins acylated with different fatty acids. We identify 218 proteins acylated with C16:0 and 308 proteins acylated with C18-lipids, thereby uncovering novel targets of acylation. We find that most proteins that can be acylated by C16:0 can also be acylated with C18-fatty acids. For proteins with more than one acylation site, we find that this competitive acylation occurs on each individual cysteine residue. This raises the possibility that the function of many different proteins can be regulated by the lipid environment via differential S-acylation.

Oral squamous cell carcinoma (OSCC) is the most prevalent subtype of head and neck tumors, highly prone to lymph node metastasis. This study aims to examine the expression pattern of Ras-related protein Rab-27A (RAB27A) and explore its potential implications in OSCC. The expression of RAB27A was assessed through immunohistochemical analysis utilizing tissue microarrays. In vitro experiments were conducted using RAB27A-knockdown cells to investigate its impact on OSCC tumor cells. Additionally, transcriptome sequencing was performed to elucidate potential underlying mechanisms. RAB27A was significantly overexpressed in OSCC, and particularly in metastatic lymph nodes. It was positively correlated with the clinical progression and poor survival prognosis. Silencing RAB27A notably decreased the proliferation, migration, and invasion abilities of OSCC cells in vitro. A Gene Ontology (GO) enrichment analysis indicated a strong association between RAB27A and the epidermal growth factor receptor (EGFR) signaling pathway. Further investigations revealed that RAB27A regulated the palmitoylation of EGFR via zinc finger DHHC-type containing 13 (ZDHHC13). These findings provide insights into OSCC progression and highlight RAB27A as a potential therapeutic target for combating this aggressive cancer.

Neovascular inflammatory vitreoretinopathy (NIV) is a rare eye disease that ultimately leads to complete blindness and is caused by mutations in the gene encoding calpain-5 (CAPN5), with six pathogenic mutations identified. In transfected SH-SY5Y cells, five of the mutations resulted in decreased membrane association, diminished S-acylation, and reduced calcium-induced autoproteolysis of CAPN5. CAPN5 proteolysis of the autoimmune regulator AIRE was impacted by several NIV mutations. R243, L244, K250 and the adjacent V249 are on β-strands in the protease core 2 domain. Conformational changes induced by Ca2+binding result in these β-strands forming a β-sheet and a hydrophobic pocket which docks W286 side chain away from the catalytic cleft, enabling calpain activation based on comparison with the Ca2+-bound CAPN1 protease core. The pathologic variants R243L, L244P, K250N, and R289W are predicted to disrupt the β-strands, β-sheet, and hydrophobic pocket, impairing calpain activation. The mechanism by which these variants impair membrane association is unclear. G376S impacts a conserved residue in the CBSW domain and is predicted to disrupt a loop containing acidic residues which may contribute to membrane binding. G267S did not impair membrane association and resulted in a slight but significant increase in autoproteolytic and proteolytic activity. However, G267S is also identified in individuals without NIV. Combined with the autosomal dominant pattern of NIV inheritance and evidence that CAPN5 may dimerize, the results are consistent with a dominant negative mechanism for the five pathogenic variants which resulted in impaired CAPN5 activity and membrane association and a gain-of-function for the G267S variant.

RAS mutations are among the most prevalent oncogenic drivers in cancers. RAS proteins propagate signals only when associated with cellular membranes as a consequence of lipid modifications that impact their trafficking. Here, we discovered that RAB27B, a RAB family small GTPase, controlled NRAS palmitoylation and trafficking to the plasma membrane, a localization required for activation. Our proteomic studies revealed RAB27B upregulation in CBL- or JAK2-mutated myeloid malignancies, and its expression correlated with poor prognosis in acute myeloid leukemias (AMLs). RAB27B depletion inhibited the growth of CBL-deficient or NRAS-mutant cell lines. Strikingly, Rab27b deficiency in mice abrogated mutant but not WT NRAS-mediated progenitor cell growth, ERK signaling, and NRAS palmitoylation. Further, Rab27b deficiency significantly reduced myelomonocytic leukemia development in vivo. Mechanistically, RAB27B interacted with ZDHHC9, a palmitoyl acyltransferase that modifies NRAS. By regulating palmitoylation, RAB27B controlled c-RAF/MEK/ERK signaling and affected leukemia development. Importantly, RAB27B depletion in primary human AMLs inhibited oncogenic NRAS signaling and leukemic growth. We further revealed a significant correlation between RAB27B expression and sensitivity to MEK inhibitors in AMLs. Thus, our studies presented a link between RAB proteins and fundamental aspects of RAS posttranslational modification and trafficking, highlighting future therapeutic strategies for RAS-driven cancers.

Regulator of protein signaling (RGS20) is a member of the RGS protein superfamily, which serve as key negative regulators of G protein-mediated signal transduction. Through their GTPase accelerating protein (GAP) activity, RGS proteins deactivate α-subunits of heterotrimeric G proteins. In addition, the majority of RGS proteins also have the ability to act through other, non-GAP related, functions. RGS20 is one of three members of the RZ subfamily, which all show selective GAP activity towards Gα_z, however emerging data suggest that RGS20 can also regulate Gi/o-mediated signaling. While increased RGS20 expression is associated with the progression of multiple cancers, a large gap still exists relating to the mechanisms of RGS20 regulation and function. RGS20 contains a poly-cysteine string motif and a conserved cysteine in RGS domain, which are assumed to be palmitoylated. Palmitoylation, an important post-translational modification, plays an important role in cells by changing cellular functions of proteins. Consequently, the aim of this study was to confirm that RGS20 is palmitoylated and determine how palmitoylation affects its inhibition of Gα_o-mediated signaling. We found a significant positive correlation between RGS20 palmitoylation and its association with active Gα_o. We also showed that a conserved cysteine residue in the RGS domain is a critical site for its palmitoylation, with large impact on its association with Gα_o. Palmitoylation on this site did not affect its GAP activity, however, it increased the inhibition of Gα_o-mediated cAMP signaling. Altogether these data suggest that palmitoylation is a regulatory mechanism controlling RGS20 function, and that RGS20 can inhibit Gα_o signaling through both GAP activity and non-GAP mechanisms.

Mammalian voltage-activated L-type Ca2+ channels, such as Ca(v)1.2, control transmembrane Ca2+ fluxes in numerous excitable tissues. Here, we report that the pore-forming α1C subunit of Ca(v)1.2 is reversibly palmitoylated in rat, rabbit, and human ventricular myocytes. We map the palmitoylation sites to two regions of the channel: The N terminus and the linker between domains I and II. Whole-cell voltage clamping revealed a rightward shift of the Ca(v)1.2 current-voltage relationship when α1C was not palmitoylated. To examine function, we expressed dihydropyridine-resistant α1C in human induced pluripotent stem cell-derived cardiomyocytes and measured Ca2+ transients in the presence of nifedipine to block the endogenous channels. The transients generated by unpalmitoylatable channels displayed a similar activation time course but significantly reduced amplitude compared to those generated by wild-type channels. We thus conclude that palmitoylation controls the voltage sensitivity of Ca(v)1.2. Given that the identified Ca(v)1.2 palmitoylation sites are also conserved in most Ca(v)1 isoforms, we propose that palmitoylation of the pore-forming α1C subunit provides a means to regulate the voltage sensitivity of voltage-activated Ca2+ channels in excitable cells.

Many CAAX proteins, such as Ras GTPase, undergo a series of posttranslational modifications at their carboxyl terminus (i.e., cysteine prenylation, endoproteolysis of AAX, and carboxylmethylation). Some CAAX proteins, however, undergo prenylation-only modification, such as Saccharomyces cerevisiae Hsp40 Ydj1. We previously observed that altering the CAAX motif of Ydj1 from prenylation-only to canonical resulted in altered Ydj1 function and localization. Here, we investigated the effects of a reciprocal change that altered the well-characterized canonical CAAX motif of S. cerevisiae Ras2 to prenylation-only. We observed that the type of CAAX motif impacted Ras2 protein levels, localization, and function. Moreover, we observed that using a prenylation-only sequence to stage hyperactive Ras2-G19V as a farnesylated and nonproteolyzed intermediate resulted in a different phenotype relative to staging by a genetic RCE1 deletion strategy that simultaneously affected many CAAX proteins. These findings suggested that a prenylation-only CAAX motif is useful for probing the specific impact of CAAX proteolysis on Ras2 under conditions where other CAAX proteins are normally modified. We propose that our strategy could be easily applied to a wide range of CAAX proteins for examining the specific impact of CAAX proteolysis on their functions. IMPORTANCE CAAX proteins are subject to multiple posttranslational modifications: cysteine prenylation, CAAX proteolysis, and carboxylmethylation. For investigations of CAAX proteolysis, this study took the novel approach of using a proteolysis-resistant CAAX sequence to stage Saccharomyces cerevisiae Ras2 GTPase in a farnesylated and nonproteolyzed state. Our approach specifically limited the effects of disrupting CAAX proteolysis to Ras2. This represented an improvement over previous methods where CAAX proteolysis was inhibited by gene knockout, small interfering RNA knockdown, or biochemical inhibition of the Rce1 CAAX protease, which can lead to pleiotropic and unclear attribution of effects due to the action of Rce1 on multiple CAAX proteins. Our approach yielded results that demonstrated specific impacts of CAAX proteolysis on the function, localization, and other properties of Ras2, highlighting the utility of this approach for investigating the impact of CAAX proteolysis in other protein contexts.

Infections caused by enveloped viruses require fusion with cellular membranes for viral genome entry. Viral entry occurs following an interaction of viral and cellular membranes allowing the formation of fusion pores, by which the virus accesses the cytoplasm. Here, we focus on interferon-induced transmembrane protein 3 (IFITM3) and its antiviral activity. IFITM3 is predicted to block or stall viral fusion at an intermediate state, causing viral propagation to fail. After introducing IFITM3, we describe the generalized lipid membrane fusion pathway and how it can be stalled, particularly with respect to IFITM3, and current questions regarding IFITM3's topology, with specific emphasis on IFITM3's amphipathic α-helix (AAH) ₅₉V-₆₈M, which is necessary for the antiviral activity. We report new hydrophobicity and hydrophobic moment calculations for this peptide and a variety of active site peptides from known membrane-remodeling proteins. Finally, we discuss the effects of posttranslational modifications and localization, how IFITM3's AAH may block viral fusion, and possible ramifications of membrane composition.

Frizzled (Fzd) proteins are Wnt receptors and play essential roles in development, homeostasis, and oncogenesis. How Wnt/Fzd signaling is coupled to physiological regulation remains unknown. Cholesterol is reported as a signaling molecule regulating morphogen such as Hedgehog signaling. Despite the elusiveness of the in-depth mechanism, it is well-established that pancreatic cancer specially requires abnormal cholesterol metabolism levels for growth. In this study, it is unexpectedly found that among ten Fzds, Fzd5 has a unique capacity to bind cholesterol specifically through its conserved extracellular linker region. Cholesterol-binding enables Fzd5 palmitoylation, which is indispensable for receptor maturation and trafficking to the plasma membrane. In Wnt-addicted pancreatic ductal adenocarcinoma (PDAC), cholesterol stimulates tumor growth via Fzd5-mediated Wnt/β-catenin signaling. A natural oxysterol, 25-hydroxylsterol competes with cholesterol and inhibits Fzd5 maturation and Wnt signaling, thereby alleviating PDAC growth. This cholesterol-receptor interaction and ensuing receptor lipidation uncover a novel mechanism by which Fzd5 acts as a cholesterol sensor and pivotal connection coupling lipid metabolism to morphogen signaling. These findings further suggest that cholesterol-targeting may provide new therapeutic opportunities for treating Wnt-dependent cancers.

Calpain-5 (CAPN5) is a member of the calpain family of calcium-activated neutral thiol proteases. CAPN5 is partly membrane associated, despite its lack of a transmembrane domain. Unlike classical calpains, CAPN5 contains a C-terminal C2 domain. C2 domains often have affinity to lipids, mediating membrane association. We recently reported that the C2 domain of CAPN5 was essential for its membrane association and the activation of its autolytic activity. However, despite the removal of the C2 domain by autolysis, the N-terminal fragment of CAPN5 remained membrane associated. S-acylation, also referred to as S-palmitoylation, is a reversible post-translational lipid modification of cysteine residues that promotes membrane association of soluble proteins. In the present study several S-acylated cysteine residues were identified in CAPN5 with the acyl-PEG exchange method. Data reported here demonstrate that CAPN5 is S-acylated on up to three cysteine residues including Cys-4 and Cys-512, and likely Cys-507. The D589N mutation in a potential calcium binding loop within the C2 domain interfered with the S-acylation of CAPN5, likely preventing initial membrane association. Mutating specific cysteine residues of CAPN5 interfered with both its membrane association and the activation of CAPN5 autolysis. Taken together, our results suggest that the S-acylation of CAPN5 is critical for its membrane localization which appears to favor its enzymatic activity.

Interferon-induced transmembrane proteins (IFITM1, 2, and 3) are important antiviral proteins that are active against many viruses, including influenza A virus (IAV), dengue virus (DENV), Ebola virus (EBOV), Zika virus (ZIKV), and severe acute respiratory syndrome coronavirus (SARS-CoV). IFITM proteins exhibit specificity in activity, but their distinct mechanisms of action and regulation are unclear. Since S-palmitoylation and cholesterol homeostasis are crucial for viral infections, we investigated IFITM interactions with cholesterol by photoaffinity cross-linking in mammalian cells along with molecular dynamic simulations and nuclear magnetic resonance analysis in vitro. These studies suggest that cholesterol can directly interact with S-palmitoylated IFITMs in cells and alter the conformation of IFITMs in membrane bilayers. Notably, we discovered that the S-palmitoylation levels regulate differential IFITM protein interactions with cholesterol in mammalian cells and specificity of antiviral activity toward IAV, SARS-CoV-2, and EBOV. Our studies suggest that modulation of IFITM S-palmitoylation levels and cholesterol interaction influence host susceptibility to different viruses.