Mechanisms and functions of protein S-acylation.

Over the past two decades, protein S-acylation (often referred to as S-palmitoylation) has emerged as an important regulator of vital signalling pathways. S-Acylation is a reversible post-translational modification that involves the attachment of a fatty acid to a protein. Maintenance of the equilibrium between protein S-acylation and deacylation has demonstrated profound effects on various cellular processes, including innate immunity, inflammation, glucose metabolism and fat metabolism, as well as on brain and heart function. This Review provides an overview of current understanding of S-acylation and deacylation enzymes, their spatiotemporal regulation by sophisticated multilayered mechanisms, and their influence on protein function, cellular processes and physiological pathways. Furthermore, we examine how disruptions in protein S-acylation are associated with a broad spectrum of diseases from cancer to autoinflammatory disorders and neurological conditions.

MAVS Cys508 palmitoylation promotes its aggregation on the mitochondrial outer membrane and antiviral innate immunity.

Cysteine palmitoylation or S-palmitoylation catalyzed by the ZDHHC family of acyltransferases regulates the biological function of numerous mammalian proteins as well as viral proteins. However, understanding of the role of S-palmitoylation in antiviral immunity against RNA viruses remains very limited. The adaptor protein MAVS forms functionally essential prion-like aggregates upon activation by viral RNA-sensing RIG-I-like receptors. Here, we identify that MAVS, a C-terminal tail-anchored mitochondrial outer membrane protein, is S-palmitoylated by ZDHHC7 at Cys508, a residue adjacent to the tail-anchor transmembrane helix. Using superresolution microscopy and other biochemical techniques, we found that the mitochondrial localization of MAVS at resting state mainly depends on its transmembrane tail-anchor, without regulation by Cys508 S-palmitoylation. However, upon viral infection, MAVS S-palmitoylation stabilizes its aggregation on the mitochondrial outer membrane and thus promotes subsequent propagation of antiviral signaling. We further show that inhibition of MAVS S-palmitoylation increases the host susceptibility to RNA virus infection, highlighting the importance of S-palmitoylation in the antiviral innate immunity. Also, our results indicate ZDHHC7 as a potential therapeutic target for MAVS-related autoimmune diseases.

A STAT3 palmitoylation cycle promotes TH17 differentiation and colitis.

Cysteine palmitoylation (S-palmitoylation) is a reversible post-translational modification that is installed by the DHHC family of palmitoyltransferases and is reversed by several acyl protein thioesterases1,2. Although thousands of human proteins are known to undergo S-palmitoylation, how this modification is regulated to modulate specific biological functions is poorly understood. Here we report that the key T helper 17 (TH17) cell differentiation stimulator, STAT33,4, is subject to reversible S-palmitoylation on cysteine 108. DHHC7 palmitoylates STAT3 and promotes its membrane recruitment and phosphorylation. Acyl protein thioesterase 2 (APT2, also known as LYPLA2) depalmitoylates phosphorylated STAT3 (p-STAT3) and enables it to translocate to the nucleus. This palmitoylation-depalmitoylation cycle enhances STAT3 activation and promotes TH17 cell differentiation; perturbation of either palmitoylation or depalmitoylation negatively affects TH17 cell differentiation. Overactivation of TH17 cells is associated with several inflammatory diseases, including inflammatory bowel disease (IBD). In a mouse model, pharmacological inhibition of APT2 or knockout of Zdhhc7-which encodes DHHC7-relieves the symptoms of IBD. Our study reveals not only a potential therapeutic strategy for the treatment of IBD but also a model through which S-palmitoylation regulates cell signalling, which might be broadly applicable for understanding the signalling functions of numerous S-palmitoylation events.

Target deconvolution of HDAC pharmacopoeia reveals MBLAC2 as common off-target.

Drugs that target histone deacetylase (HDAC) entered the pharmacopoeia in the 2000s. However, some enigmatic phenotypes suggest off-target engagement. Here, we developed a quantitative chemical proteomics assay using immobilized HDAC inhibitors and mass spectrometry that we deployed to establish the target landscape of 53 drugs. The assay covers 9 of the 11 human zinc-dependent HDACs, questions the reported selectivity of some widely-used molecules (notably for HDAC6) and delineates how the composition of HDAC complexes influences drug potency. Unexpectedly, metallo-ß-lactamase domain-containing protein 2 (MBLAC2) featured as a frequent off-target of hydroxamate drugs. This poorly characterized palmitoyl-CoA hydrolase is inhibited by 24 HDAC inhibitors at low nanomolar potency. MBLAC2 enzymatic inhibition and knockdown led to the accumulation of extracellular vesicles. Given the importance of extracellular vesicle biology in neurological diseases and cancer, this HDAC-independent drug effect may qualify MBLAC2 as a target for drug discovery.

Metallo-ß-lactamase domain-containing protein 2 is S-palmitoylated and exhibits acyl-CoA hydrolase activity.

Members of the metallo-ß-lactamase (MBL) superfamily of enzymes harbor a highly conserved αßßα MBL-fold domain and were first described as inactivators of common ß-lactam antibiotics. In humans, these enzymes have been shown to exhibit diverse functions, including hydrolase activity toward amides, esters, and thioesters. An uncharacterized member of the human MBL family, MBLAC2, was detected in multiple palmitoylproteomes, identified as a zDHHC20 S-acyltransferase interactor, and annotated as a potential thioesterase. In this study, we confirmed that MBLAC2 is palmitoylated and identified the likely S-palmitoylation site as Cys254. S-palmitoylation of MBLAC2 is increased in cells when expressed with zDHHC20, and MBLAC2 is a substrate for purified zDHHC20 in vitro. To determine its biochemical function, we tested the ability of MBLAC2 to hydrolyze a variety of small molecules and acylprotein substrates. MBLAC2 has acyl-CoA thioesterase activity with kinetic parameters and acyl-CoA selectivity comparable with acyl-CoA thioesterase 1 (ACOT1). Two predicted zinc-binding residues, Asp87 and His88, are required for MBLAC2 hydrolase activity. Consistent with a role in fatty acid metabolism in cells, MBLAC2 was cross-linked to a photoactivatable fatty acid in a manner that was independent of its S-fatty acylation at Cys254. Our study adds to previous investigations demonstrating the versatility of the MBL-fold domain in supporting a variety of enzymatic reactions.

FKBP12 binds to acylated H-ras and promotes depalmitoylation.

A cycle of palmitoylation/depalmitoylation of H-Ras mediates bidirectional trafficking between the Golgi apparatus and the plasma membrane, but nothing is known about how this cycle is regulated. We show that the prolyl isomerase (PI) FKBP12 binds to H-Ras in a palmitoylation-dependent fashion and promotes depalmitoylation. A variety of inhibitors of the PI activity of FKBP12, including FK506, rapamycin, and cycloheximide, increase steady-state palmitoylation. FK506 inhibits retrograde trafficking of H-Ras from the plasma membrane to the Golgi in a proline 179-dependent fashion, augments early GTP loading of Ras in response to growth factors, and promotes H-Ras-dependent neurite outgrowth from PC12 cells. These data demonstrate that FKBP12 regulates H-Ras trafficking by promoting depalmitoylation through cis-trans isomerization of a peptidyl-prolyl bond in proximity to the palmitoylated cysteines.

Structure and function of DHHC protein S-acyltransferases.

It has been estimated that 10% of the human genome encodes proteins that are fatty acylated at cysteine residues. The vast majority of these proteins are modified by members of the DHHC protein family, which carry out their enzymatic function on the cytoplasmic face of cell membranes. The biomedical importance of DHHC proteins is underscored by their association with human disease; unique and essential roles for DHHC proteins have been uncovered using DHHC-deficient mouse models. Accordingly, there is great interest in elucidating the molecular mechanisms that underlie DHHC protein function. In this review, we present recent insights into the structure and function of DHHC enzymes.

The Cysteine-rich Domain of the DHHC3 Palmitoyltransferase Is Palmitoylated and Contains Tightly Bound Zinc.

DHHC palmitoyltransferases catalyze the addition of the fatty acid palmitate to proteins on the cytoplasmic leaflet of cell membranes. There are 23 members of the highly diverse mammalian DHHC protein family, all of which contain a conserved catalytic domain called the cysteine-rich domain (CRD). DHHC proteins transfer palmitate via a two-step catalytic mechanism in which the enzyme first modifies itself with palmitate in a process termed autoacylation. The enzyme then transfers palmitate from itself onto substrate proteins. The number and location of palmitoylated cysteines in the autoacylated intermediate is unknown. In this study, we present evidence using mass spectrometry that DHHC3 is palmitoylated at the cysteine in the DHHC motif. Mutation of highly conserved CRD cysteines outside the DHHC motif resulted in activity deficits and a structural perturbation revealed by limited proteolysis. Treatment of DHHC3 with chelating agents in vitro replicated both the specific structural perturbations and activity deficits observed in conserved cysteine mutants, suggesting metal ion-binding in the CRD. Using the fluorescent indicator mag-fura-2, the metal released from DHHC3 was identified as zinc. The stoichiometry of zinc binding was measured as 2 mol of zinc/mol of DHHC3 protein. Taken together, our data demonstrate that coordination of zinc ions by cysteine residues within the CRD is required for the structural integrity of DHHC proteins.

Protein S-palmitoylation and cancer.

Protein S-palmitoylation is a reversible posttranslational modification of proteins with fatty acids, an enzymatic process driven by a recently discovered family of protein acyltransferases (PATs) that are defined by a conserved catalytic domain characterized by a DHHC sequence motif. Protein S-palmitoylation has a prominent role in regulating protein location, trafficking and function. Recent studies of DHHC PATs and their functional effects have demonstrated that their dysregulation is associated with human diseases, including schizophrenia, X-linked mental retardation, and Huntington's Disease. A growing number of reports indicate an important role for DHHC proteins and their substrates in tumorigenesis. Whereas DHHC PATs comprise a family of 23 enzymes in humans, a smaller number of enzymes that remove palmitate have been identified and characterized as potential therapeutic targets. Here we review current knowledge of the enzymes that mediate reversible palmitoylation and their cancer-associated substrates and discuss potential therapeutic applications.

Oligomerization of DHHC protein S-acyltransferases.

The formation of dimers or higher-order oligomers is a property of numerous integral membrane proteins, including ion channels, transporters, and receptors. In this study, we examined whether members of the DHHC-S-acyltransferase family oligomerize in intact cells and in vitro. DHHC-S-acyltransferases are integral membrane proteins that catalyze the addition of palmitate to cysteine residues on proteins at the cytoplasmic face of cell membranes. Bioluminescence resonance energy transfer (BRET) experiments revealed that DHHC2 or DHHC3 (Golgi-specific DHHC zinc finger protein (GODZ)) self-associate when expressed in HEK-293 cells. Homomultimer formation was confirmed by coimmunoprecipitation. Purified DHHC3 resolved predominately as a monomer and dimer on blue native polyacrylamide gels. In intact cells and in vitro, catalytically inactive DHHC proteins displayed a greater propensity to form dimers. BRET signals were higher for the catalytically inactive DHHC2 or DHHC3 than their wild-type counterparts. DHHC3 BRET in cell membranes was decreased by the addition of its lipid substrate palmitoyl-CoA, a treatment that results in autoacylation of the enzyme. Enzyme activity of a covalently linked DHHC3 dimer was less than that of the monomeric form, suggesting that enzyme activity may be modulated by the oligomerization status of the protein.

NLRP3 Cys126 palmitoylation by ZDHHC7 promotes inflammasome activation.

Nucleotide oligomerization domain (NOD)-like receptor protein 3 (NLRP3) inflammasome hyperactivation contributes to many human chronic inflammatory diseases, and understanding how NLRP3 inflammasome is regulated can provide strategies to treat inflammatory diseases. Here, we demonstrate that NLRP3 Cys126 is palmitoylated by zinc finger DHHC-type palmitoyl transferase 7 (ZDHHC7), which is critical for NLRP3-mediated inflammasome activation. Perturbing NLRP3 Cys126 palmitoylation by ZDHHC7 knockout, pharmacological inhibition, or modification site mutation diminishes NLRP3 activation in macrophages. Furthermore, Cys126 palmitoylation is vital for inflammasome activation in vivo. Mechanistically, ZDHHC7-mediated NLRP3 Cys126 palmitoylation promotes resting NLRP3 localizing on the trans-Golgi network (TGN) and activated NLRP3 on the dispersed TGN, which is indispensable for recruitment and oligomerization of the adaptor ASC (apoptosis-associated speck-like protein containing a CARD). The activation of NLRP3 by ZDHHC7 is different from the termination effect mediated by ZDHHC12, highlighting versatile regulatory roles of S-palmitoylation. Our study identifies an important regulatory mechanism of NLRP3 activation that suggests targeting ZDHHC7 or the NLRP3 Cys126 residue as a potential therapeutic strategy to treat NLRP3-related human disorders.

DHHC protein S-acyltransferases use similar ping-pong kinetic mechanisms but display different acyl-CoA specificities.

DHHC proteins catalyze the reversible S-acylation of proteins at cysteine residues, a modification important for regulating protein localization, stability, and activity. However, little is known about the kinetic mechanism of DHHC proteins. A high-performance liquid chromatography (HPLC), fluorescent peptide-based assay for protein S-acylation activity was developed to characterize mammalian DHHC2 and DHHC3. Time courses and substrate saturation curves allowed the determination of V(max) and K(m) values for both the peptide N-myristoylated-GCG and palmitoyl-coenzyme A. DHHC proteins acylate themselves upon incubation with palmitoyl-CoA, which is hypothesized to reflect a transient acyl enzyme transfer intermediate. Single turnover assays with DHHC2 and DHHC3 demonstrated that a radiolabeled acyl group on the enzyme transferred to the protein substrate, consistent with a two-step ping-pong mechanism. Enzyme autoacylation and acyltransfer to substrate displayed the same acyl-CoA specificities, further supporting a two-step mechanism. Interestingly, DHHC2 efficiently transferred acyl chains 14 carbons and longer, whereas DHHC3 activity was greatly reduced by acyl-CoAs with chain lengths longer than 16 carbons. The rate and extent of autoacylation of DHHC3, as well as the rate of acyl chain transfer to protein substrate, were reduced with stearoyl-CoA when compared with palmitoyl-CoA. This is the first observation of lipid substrate specificity among DHHC proteins and may account for the differential S-acylation of proteins observed in cells.

Mechanism and function of DHHC S-acyltransferases.

Protein S-palmitoylation is a reversible post-translational modification of proteins with fatty acids. In the last 5 years, improved proteomic methods have increased the number of proteins identified as substrates for palmitoylation from tens to hundreds. Palmitoylation regulates protein membrane interactions, activity, trafficking and stability and can be constitutive or regulated by signalling inputs. A family of PATs (protein acyltransferases) is responsible for modifying proteins with palmitate or other long-chain fatty acids on the cytoplasmic face of cellular membranes. PATs share a signature DHHC (Asp-His-His-Cys) cysteine-rich domain that is the catalytic centre of the enzyme. The biomedical importance of members of this family is underscored by their association with intellectual disability, Huntington's disease and cancer in humans, and raises the possibility of DHHC PATs as targets for therapeutic intervention. In the present paper, we discuss recent progress in understanding enzyme mechanism, regulation and substrate specificity.

GCP16 stabilizes the DHHC9 subfamily of protein acyltransferases through a conserved C-terminal cysteine motif.

Protein S-acylation is a reversible lipid post-translational modification that allows dynamic regulation of processes such as protein stability, membrane association, and localization. Palmitoyltransferase ZDHHC9 (DHHC9) is one of the 23 human DHHC acyltransferases that catalyze protein S-acylation. Dysregulation of DHHC9 is associated with X-linked intellectual disability and increased epilepsy risk. Interestingly, activation of DHHC9 requires an accessory protein-GCP16. However, the exact role of GCP16 and the prevalence of a requirement for accessory proteins among other DHHC proteins remain unclear. Here, we report that one role of GCP16 is to stabilize DHHC9 by preventing its aggregation through formation of a protein complex. Using a combination of size-exclusion chromatography and palmitoyl acyltransferase assays, we demonstrate that only properly folded DHHC9-GCP16 complex is enzymatically active in vitro. Additionally, the ZDHHC9 mutations linked to X-linked intellectual disability result in reduced protein stability and DHHC9-GCP16 complex formation. Notably, we discovered that the C-terminal cysteine motif (CCM) that is conserved among the DHHC9 subfamily (DHHC14, -18, -5, and -8) is required for DHHC9 and GCP16 complex formation and activity in vitro. Co-expression of GCP16 with DHHCs containing the CCM improves DHHC protein stability. Like DHHC9, DHHC14 and DHHC18 require GCP16 for their enzymatic activity. Furthermore, GOLGA7B, an accessory protein with 75% sequence identity to GCP16, improves protein stability of DHHC5 and DHHC8, but not the other members of the DHHC9 subfamily, suggesting selectivity in accessory protein interactions. Our study supports a broader role for GCP16 and GOLGA7B in the function of human DHHCs.

Gi/o signaling and the palmitoyltransferase DHHC2 regulate palmitate cycling and shuttling of RGS7 family-binding protein.

R7BP (RGS7 family-binding protein) has been proposed to function in neurons as a palmitoylation-regulated protein that shuttles heterodimeric, G(i/o)α-specific GTPase-activating protein (GAP) complexes composed of Gß5 and RGS7 (R7) isoforms between the plasma membrane and nucleus. To test this hypothesis we studied R7BP palmitoylation and localization in neuronal cells. We report that R7BP undergoes dynamic, signal-regulated palmitate turnover; the palmitoyltransferase DHHC2 mediates de novo and turnover palmitoylation of R7BP; DHHC2 silencing redistributes R7BP from the plasma membrane to the nucleus; and G(i/o) signaling inhibits R7BP depalmitoylation whereas G(i/o) inactivation induces nuclear accumulation of R7BP. In concert with previous evidence, our findings suggest that agonist-induced changes in palmitoylation state facilitate GAP action by (i) promoting Giα depalmitoylation to create optimal GAP substrates, and (ii) inhibiting R7BP depalmitoylation to stabilize membrane association of R7-Gß5 GAP complexes. Regulated palmitate turnover may also enable R7BP-bound GAPs to shuttle between sites of low and high G(i/o) activity or the plasma membrane and nucleus, potentially providing spatio-temporal control of signaling by G(i/o)-coupled receptors.

Plasma membrane association of p63 Rho guanine nucleotide exchange factor (p63RhoGEF) is mediated by palmitoylation and is required for basal activity in cells.

Activation of G protein-coupled receptors at the cell surface leads to the activation or inhibition of intracellular effector enzymes, which include various Rho guanine nucleotide exchange factors (RhoGEFs). RhoGEFs activate small molecular weight GTPases at the plasma membrane (PM). Many of the known G protein-coupled receptor-regulated RhoGEFs are found in the cytoplasm of unstimulated cells, and PM recruitment is a critical aspect of their regulation. In contrast, p63RhoGEF, a Gα(q)-regulated RhoGEF, appears to be constitutively localized to the PM. The objective of this study was to determine the molecular basis for the localization of p63RhoGEF and the impact of its subcellular localization on its regulation by Gα(q). Herein, we show that the pleckstrin homology domain of p63RhoGEF is not involved in its PM targeting. Instead, a conserved string of cysteines (Cys-23/25/26) at the N terminus of the enzyme is palmitoylated and required for membrane localization and full basal activity in cells. Conversion of these residues to serine relocates p63RhoGEF from the PM to the cytoplasm, diminishes its basal activity, and eliminates palmitoylation. The activity of palmitoylation-deficient p63RhoGEF can be rescued by targeting to the PM by fusion with tandem phospholipase C-δ1 pleckstrin homology domains or by co-expression with wild-type Gα(q) but not with palmitoylation-deficient Gα(q). Our data suggest that p63RhoGEF is regulated chiefly through allosteric control by Gα(q), as opposed to other known Gα-regulated RhoGEFs, which are instead sequestered in the cytoplasm, perhaps because of their high basal activity.

Substrate recruitment by zDHHC protein acyltransferases.

Protein palmitoylation is the post-translational attachment of fatty acids, most commonly palmitate (C16 : 0), onto a cysteine residue of a protein. This reaction is catalysed by a family of integral membrane proteins, the zDHHC protein acyltransferases (PATs), so-called due to the presence of an invariant Asp-His-His-Cys (DHHC) cysteine-rich domain harbouring the catalytic centre of the enzyme. Conserved throughout eukaryotes, the zDHHC PATs are encoded by multigene families and mediate palmitoylation of thousands of protein substrates. In humans, a number of zDHHC proteins are associated with human diseases, including intellectual disability, Huntington's disease, schizophrenia and cancer. Key to understanding the physiological and pathophysiological importance of individual zDHHC proteins is the identification of their protein substrates. Here, we will describe the approaches and challenges in assigning substrates for individual zDHHCs, highlighting key mechanisms that underlie substrate recruitment.

High-Throughput Enzyme Assay for Screening Inhibitors of the ZDHHC3/7/20 Acyltransferases.

As enzymes that mediate the attachment of long-chain fatty acids to cysteine residues, ZDHHC proteins have been reported to be promising therapeutic targets for treating cancer and autoimmune diseases. Yet, due to the lack of potent selective inhibitors, scrutiny of the biological functions of ZDHHCs has been limited. The main hindrance for developing ZDHHC inhibitors is the lack of a facile high-throughput assay. Here, we developed a ZDHHC3/7/20 high-throughput assay based on the acylation-coupled lipophilic induction of polarization (Acyl-cLIP) method and screened several potential ZDHHC inhibitors. Furthermore, we demonstrated that in vitro results from the Acyl-cLIP assay are supported by the results from cell-based assays. We envision that this new ZDHHC3/7/20 Acyl-cLIP assay will accelerate the high-throughput screening of large compound libraries for improved ZDHHC inhibitors and provide therapeutic benefits for cancer and autoimmune diseases.

The vacuolar DHHC-CRD protein Pfa3p is a protein acyltransferase for Vac8p.

Palmitoylation of the vacuolar membrane protein Vac8p is essential for vacuole fusion in yeast (Veit, M., R. Laage, L. Dietrich, L. Wang, and C. Ungermann. 2001. EMBO J. 20:3145-3155; Wang, Y.X., E.J. Kauffman, J.E. Duex, and L.S. Weisman. 2001. J. Biol. Chem. 276:35133-35140). Proteins that contain an Asp-His-His-Cys (DHHC)-cysteine rich domain (CRD) are emerging as a family of protein acyltransferases, and are therefore candidates for mediators of Vac8p palmitoylation. Here we demonstrate that the DHHC-CRD proteins Pfa3p (protein fatty acyltransferase 3, encoded by YNL326c) and Swf1p are important for vacuole fusion. Cells lacking Pfa3p had fragmented vacuoles when stressed, and cells lacking both Pfa3p and Swf1p had fragmented vacuoles under normal growth conditions. Pfa3p promoted Vac8p membrane association and palmitoylation in vivo and partially purified Pfa3p palmitoylated Vac8p in vitro, establishing Vac8p as a substrate for palmitoylation by Pfa3p. Vac8p is the first N-myristoylated, palmitoylated protein identified as a substrate for a DHHC-CRD protein.