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.

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.

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.

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.

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.

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.

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.

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.

Purification of Recombinant DHHC Proteins Using an Insect Cell Expression System.

DHHC enzymes are a family of integral membrane proteins that catalyze the posttranslational addition of palmitate, a 16-carbon fatty acid, onto a cysteine residue of a protein. While the library of identified palmitoylated proteins has grown tremendously over the years, biochemical and mechanistic studies on DHHC proteins are challenged by the innate difficulty of purifying the enzyme in large amounts. Here we describe our protocol for preparing recombinant DHHC proteins tagged with a hexahistidine sequence and a FLAG epitope that aid in the purification. This procedure has been tested successfully in purifying several members of the enzyme family; DHHC3 and its catalytically inactive cysteine mutant, DHHS3 are used as examples. The recombinant protein is extracted from whole cell lysates using the detergent dodecylmaltoside (DDM) and is subjected to a two-column purification. Homogeneity and monodispersity of the purified protein are checked by size exclusion chromatography (SEC). A preparation from a 400-mL infection of Sf9 insect cell culture typically yields 0.5 mg of DHHC3 and 1.0 mg of catalytically inactive DHHS3. Both forms appear monodisperse up to a concentration of 1 mg/mL by SEC.

Monitoring RhoGDI Extraction of Lipid-Modified Rho GTPases from Membranes Using Click Chemistry.

The posttranslational lipid modification of Rho GTPases is important for their proper subcellular localization and signal transduction. Rho GTPases terminate in a CaaX motif, in which the cysteine residue is modified with either a farnesyl or geranylgeranyl isoprenoid. RhoGDI renders Rho GTPases soluble by masking their lipid moieties. We recently identified that the brain-specific splice variant of Cdc42 (bCdc42) containing a noncanonical CCaX motif harbors a dual prenyl-palmitoyl modification that prevents its binding to RhoGDI. This chapter describes a method to analyze RhoGDI extraction of Rho GTPases containing different lipid modifications from membranes using a liposome reconstitution assay and click chemistry.

SIRT2 and lysine fatty acylation regulate the transforming activity of K-Ras4a.

Ras proteins play vital roles in numerous biological processes and Ras mutations are found in many human tumors. Understanding how Ras proteins are regulated is important for elucidating cell signaling pathways and identifying new targets for treating human diseases. Here we report that one of the K-Ras splice variants, K-Ras4a, is subject to lysine fatty acylation, a previously under-studied protein post-translational modification. Sirtuin 2 (SIRT2), one of the mammalian nicotinamide adenine dinucleotide (NAD)-dependent lysine deacylases, catalyzes the removal of fatty acylation from K-Ras4a. We further demonstrate that SIRT2-mediated lysine defatty-acylation promotes endomembrane localization of K-Ras4a, enhances its interaction with A-Raf, and thus promotes cellular transformation. Our study identifies lysine fatty acylation as a previously unknown regulatory mechanism for the Ras family of GTPases that is distinct from cysteine fatty acylation. These findings highlight the biological significance of lysine fatty acylation and sirtuin-catalyzed protein lysine defatty-acylation.

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.

Massive endocytosis triggered by surface membrane palmitoylation under mitochondrial control in BHK fibroblasts.

Large Ca transients cause massive endocytosis (MEND) in BHK fibroblasts by nonclassical mechanisms. We present evidence that MEND depends on mitochondrial permeability transition pore (PTP) openings, followed by coenzyme A (CoA) release, acyl CoA synthesis, and membrane protein palmitoylation. MEND is blocked by inhibiting mitochondrial Ca uptake or PTP openings, depleting fatty acids, blocking acyl CoA synthesis, metabolizing CoA, or inhibiting palmitoylation. It is triggered by depolarizing mitochondria or promoting PTP openings. After mitochondrial MEND blockade, MEND is restored by cytoplasmic acyl CoA or CoA. MEND is blocked by siRNA knockdown of the plasmalemmal acyl transferase, DHHC5. When acyl CoA is abundant, transient H2O2 oxidative stress or PKC activation initiates MEND, but the immediate presence of H2O2 prevents MEND. The PTP inhibitor, NIM811, significantly increases plasmalemma in normally growing cells. Thus, the MEND pathway may contribute to constitutive as well as pathological plasmalemma turnover in dependence on mitochondrial stress signaling. DOI: http://dx.doi.org/10.7554/eLife.01293.001.

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.

Identification of a novel prenyl and palmitoyl modification at the CaaX motif of Cdc42 that regulates RhoGDI binding.

Membrane localization of Rho GTPases is essential for their biological functions and is dictated in part by a series of posttranslational modifications at a carboxyl-terminal CaaX motif: prenylation at cysteine, proteolysis of the aaX tripeptide, and carboxymethylation. The fidelity and variability of these CaaX processing steps are uncertain. The brain-specific splice variant of Cdc42 (bCdc42) terminates in a CCIF sequence. Here we show that brain Cdc42 undergoes two different types of posttranslational modification: classical CaaX processing or novel tandem prenylation and palmitoylation at the CCaX cysteines. In the dual lipidation pathway, bCdc42 was prenylated, but it bypassed proteolysis and carboxymethylation to undergo modification with palmitate at the second cysteine. The alternative postprenylation processing fates were conserved in the GTPases RalA and RalB and the phosphatase PRL-3, proteins terminating in a CCaX motif. The differentially modified forms of bCdc42 displayed functional differences. Prenylated and palmitoylated brain Cdc42 did not interact with RhoGDIα and was enriched in the plasma membrane relative to the classically processed form. The alternative processing of prenylated CCaX motif proteins by palmitoylation or by endoproteolysis and methylation expands the diversity of signaling GTPases and enables another level of regulation through reversible modification with palmitate.

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.