Quantitative analysis of protein lipidation and acyl-CoAs reveals substrate preferences of the S-acylation machinery.

Protein palmitoylation or S-acylation has emerged as a key regulator of cellular processes. Increasing evidence shows that this modification is not restricted to palmitate but it can include additional fatty acids, raising the possibility that differential S-acylation contributes to the fine-tuning of protein activity. However, methods to profile the acyl moieties attached to proteins are scarce. Herein, we report a method for the identification and quantification of lipids bound to proteins that relies on hydroxylamine treatment and mass spectrometry analysis of fatty acid hydroxamates. This method has enabled unprecedented and extensive profiling of the S-acylome in different cell lines and tissues and has shed light on the substrate specificity of some S-acylating enzymes. Moreover, we could extend it to quantify also the acyl-CoAs, which are thioesters formed between a fatty acid and a coenzyme A, overcoming many of the previously described challenges for the detection of such species. Importantly, the simultaneous analysis of the lipid fraction and the proteome allowed us to establish, for the first time, a direct correlation between the endogenous levels of acyl-CoAs and the S-acylation profile of its proteome.

The endoplasmic reticulum-localized enzyme zDHHC6 mediates S-acylation of short transmembrane constructs from multiple type I and II membrane proteins.

In this study, we investigated the S-acylation of two host cell proteins important for viral infection: TMPRSS2 (transmembrane serine protease 2), which cleaves severe acute respiratory syndrome coronavirus 2 spike to facilitate viral entry, and bone marrow stromal antigen 2, a general viral restriction factor. We found that both proteins were S-acylated by zDHHC6, an S-acyltransferase enzyme localized at the endoplasmic reticulum, in coexpression experiments. Mutagenic analysis revealed that zDHHC6 modifies a single cysteine in each protein, which are in proximity to the transmembrane domains (TMDs). For TMPRSS2, the modified cysteine is positioned two residues into the TMD, whereas the modified cysteine in bone marrow stromal antigen 2 has a cytosolic location two amino acids upstream of the TMD. Cysteine swapping revealed that repositioning the target cysteine of TMPRSS2 further into the TMD substantially reduced S-acylation by zDHHC6. Interestingly, zDHHC6 efficiently S-acylated truncated forms of these proteins that contained only the TMDs and short juxtamembrane regions. The ability of zDHHC6 to modify short TMD sequences was also seen for the transferrin receptor (another type II membrane protein) and for five different type I membrane protein constructs, including cluster of differentiation 4. Collectively, the results of this study show that zDHHC6 can modify diverse membrane proteins (type I and II) and requires only the presence of the TMD and target cysteine for efficient S-acylation. Thus, zDHHC6 may be a broad specificity S-acyltransferase specialized for the modification of a diverse set of transmembrane proteins at the endoplasmic reticulum.

S-acylation of Sprouty and SPRED proteins by the S-acyltransferase zDHHC17 involves a novel mode of enzyme-substrate interaction.

S-acylation is an essential post-translational modification, which is mediated by a family of 23 zDHHC enzymes in humans. Several thousand proteins are modified by S-acylation; however, we lack a detailed understanding of how enzyme-substrate recognition and specificity is achieved. Previous work showed that the ankyrin repeat domain of zDHHC17 (ANK17) recognizes a short linear motif, known as the zDHHC ANK binding motif (zDABM) in substrate protein SNAP25, as a mechanism of substrate recruitment prior to S-acylation. Here, we investigated the S-acylation of the Sprouty and SPRED family of proteins by zDHHC17. Interestingly, although Sprouty-2 (Spry2) contains a zDABM that interacts with ANK17, this mode of binding is dispensable for S-acylation, and indeed removal of the zDABM does not completely ablate binding to zDHHC17. Furthermore, the related SPRED3 protein interacts with and is efficiently S-acylated by zDHHC17, despite lacking a zDABM. We undertook mutational analysis of SPRED3 to better understand the basis of its zDABM-independent interaction with zDHHC17. This analysis found that the cysteine-rich SPR domain of SPRED3, which is the defining feature of all Sprouty and SPRED proteins, interacts with zDHHC17. Surprisingly, the interaction with SPRED3 was independent of ANK17. Our mutational analysis of Spry2 was consistent with the SPR domain of this protein containing a zDHHC17-binding site, and Spry2 also showed detectable binding to a zDHHC17 mutant lacking the ANK domain. Thus, zDHHC17 can recognize its substrates through zDABM-dependent and/or zDABM-independent mechanisms, and some substrates display more than one mode of binding to this enzyme.

Development of a novel high-throughput screen for the identification of new inhibitors of protein S-acylation.

Protein S-acylation is a reversible post-translational modification that modulates the localization and function of many cellular proteins. S-acylation is mediated by a family of zinc finger DHHC (Asp-His-His-Cys) domain-containing (zDHHC) proteins encoded by 23 distinct ZDHHC genes in the human genome. These enzymes catalyze S-acylation in a two-step process involving "autoacylation" of the cysteine residue in the catalytic DHHC motif followed by transfer of the acyl chain to a substrate cysteine. S-acylation is essential for many fundamental physiological processes, and there is growing interest in zDHHC enzymes as novel drug targets for a range of disorders. However, there is currently a lack of chemical modulators of S-acylation either for use as tool compounds or for potential development for therapeutic purposes. Here, we developed and implemented a novel FRET-based high-throughput assay for the discovery of compounds that interfere with autoacylation of zDHHC2, an enzyme that is implicated in neuronal S-acylation pathways. Our screen of >350,000 compounds identified two related tetrazole-containing compounds (TTZ-1 and TTZ-2) that inhibited both zDHHC2 autoacylation and substrate S-acylation in cell-free systems. These compounds were also active in human embryonic kidney 293T cells, where they inhibited the S-acylation of two substrates (SNAP25 and PSD95 [postsynaptic density protein 95]) mediated by different zDHHC enzymes, with some apparent isoform selectivity. Furthermore, we confirmed activity of the hit compounds through resynthesis, which provided sufficient quantities of material for further investigations. The assays developed provide novel strategies to screen for zDHHC inhibitors, and the identified compounds add to the chemical toolbox for interrogating cellular activities of zDHHC enzymes in S-acylation.

Regulatory effects of protein S-acylation on insulin secretion and insulin action.

Post-translational modifications (PTMs) such as phosphorylation and ubiquitination are well-studied events with a recognized importance in all aspects of cellular function. By contrast, protein S-acylation, although a widespread PTM with important functions in most physiological systems, has received far less attention. Perturbations in S-acylation are linked to various disorders, including intellectual disability, cancer and diabetes, suggesting that this less-studied modification is likely to be of considerable biological importance. As an exemplar, in this review, we focus on the newly emerging links between S-acylation and the hormone insulin. Specifically, we examine how S-acylation regulates key components of the insulin secretion and insulin response pathways. The proteins discussed highlight the diverse array of proteins that are modified by S-acylation, including channels, transporters, receptors and trafficking proteins and also illustrate the diverse effects that S-acylation has on these proteins, from membrane binding and micro-localization to regulation of protein sorting and protein interactions.

Accessory proteins of the zDHHC family of S-acylation enzymes.

Almost two decades have passed since seminal work in Saccharomyces cerevisiae identified zinc finger DHHC domain-containing (zDHHC) enzymes as S-acyltransferases. These enzymes are ubiquitous in the eukarya domain, with 23 distinct zDHHC-encoding genes in the human genome. zDHHC enzymes mediate the bulk of S-acylation (also known as palmitoylation) reactions in cells, transferring acyl chains to cysteine thiolates, and in so-doing affecting the stability, localisation and function of several thousand proteins. Studies using purified components have shown that the minimal requirements for S-acylation are an appropriate zDHHC enzyme-substrate pair and fatty acyl-CoA. However, additional proteins including GCP16 (also known as Golga7), Golga7b, huntingtin and selenoprotein K, have been suggested to regulate the activity, stability and trafficking of certain zDHHC enzymes. In this Review, we discuss the role of these accessory proteins as essential components of the cellular S-acylation system.

Identification of key features required for efficient S-acylation and plasma membrane targeting of sprouty-2.

Sprouty-2 is an important regulator of growth factor signalling and a tumour suppressor protein. The defining feature of this protein is a cysteine-rich domain (CRD) that contains twenty-six cysteine residues and is modified by S-acylation. In this study, we show that the CRD of sprouty-2 is differentially modified by S-acyltransferase enzymes. The high specificity/low activity zDHHC17 enzyme mediated restricted S-acylation of sprouty-2, and cysteine-265 and -268 were identified as key targets of this enzyme. In contrast, the low specificity/high activity zDHHC3 and zDHHC7 enzymes mediated more expansive modification of the sprouty-2 CRD. Nevertheless, S-acylation by all enzymes enhanced sprouty-2 expression, suggesting that S-acylation stabilises this protein. In addition, we identified two charged residues (aspartate-214 and lysine-223), present on opposite faces of a predicted α-helix in the CRD, which are essential for S-acylation of sprouty-2. Interestingly, mutations that perturbed S-acylation also led to a loss of plasma membrane localisation of sprouty-2 in PC12 cells. This study provides insight into the mechanisms and outcomes of sprouty-2 S-acylation, and highlights distinct patterns of S-acylation mediated by different classes of zDHHC enzymes.

Regulatory effects of post-translational modifications on zDHHC S-acyltransferases.

The human zDHHC S-acyltransferase family comprises 23 enzymes that mediate the S-acylation of a multitude of cellular proteins, including channels, receptors, transporters, signaling molecules, scaffolds, and chaperones. This reversible post-transitional modification (PTM) involves the attachment of a fatty acyl chain, usually derived from palmitoyl-CoA, to specific cysteine residues on target proteins, which affects their stability, localization, and function. These outcomes are essential to control many processes, including synaptic transmission and plasticity, cell growth and differentiation, and infectivity of viruses and other pathogens. Given the physiological importance of S-acylation, it is unsurprising that perturbations in this process, including mutations in ZDHHC genes, have been linked to different neurological pathologies and cancers, and there is growing interest in zDHHC enzymes as novel drug targets. Although zDHHC enzymes control a diverse array of cellular processes and are associated with major disorders, our understanding of these enzymes is surprisingly incomplete, particularly with regard to the regulatory mechanisms controlling these enzymes. However, there is growing evidence highlighting the role of different PTMs in this process. In this review, we discuss how PTMs, including phosphorylation, S-acylation, and ubiquitination, affect the stability, localization, and function of zDHHC enzymes and speculate on possible effects of PTMs that have emerged from larger screening studies. Developing a better understanding of the regulatory effects of PTMs on zDHHC enzymes will provide new insight into the intracellular dynamics of S-acylation and may also highlight novel approaches to modulate S-acylation for clinical gain.

The linker domain of the SNARE protein SNAP25 acts as a flexible molecular spacer that ensures efficient S-acylation.

S-Acylation of the SNARE protein SNAP25 (synaptosome-associated protein of 25 kDa) is mediated by a subset of Golgi zinc finger DHHC-type palmitoyltransferase (zDHHC) enzymes, particularly zDHHC17. The ankyrin repeat domain of zDHHC17 interacts with a short linear motif known as the zDHHC ankyrin repeat-binding motif (zDABM) in SNAP25 (112VVASQP117), which is downstream of its S-acylated, cysteine-rich domain (85CGLCVCPC92). Here, we investigated the importance of a flexible linker region (amino acids 93-111, referred to hereafter as the "mini-linker" region) that separates the zDABM and S-acylated cysteines in SNAP25. Shortening the mini-linker did not affect the SNAP25-zDHHC17 interaction but blocked S-acylation. Insertion of additional flexible glycine-serine repeats had no effect on S-acylation, but extended and rigid alanine-proline repeats perturbed it. A SNAP25 mutant in which the mini-linker region was substituted with a flexible glycine-serine linker of the same length underwent efficient S-acylation. Furthermore, this mutant displayed the same intracellular localization as WT SNAP25, indicating that the amino acid composition of the mini-linker is not important for SNAP25 localization. Using the results of previous peptide array experiments, we generated a SNAP25 mutant predicted to have a higher-affinity zDABM. This mutant interacted with zDHHC17 more strongly but was S-acylated with reduced efficiency in HEK293T cells, implying that a lower-affinity interaction of the SNAP25 zDABM with zDHHC17 is optimal for S-acylation efficiency. These results show that amino acids 93-111 in SNAP25 act as a flexible molecular spacer that ensures efficient coupling of the SNAP25-zDHHC17 interaction and S-acylation of SNAP25.

S-acylation regulates the trafficking and stability of the unconventional Q-SNARE STX19.

STX19 is an unusual Qa-SNARE as it lacks a C-terminal transmembrane domain. However, it is efficiently targeted to post-Golgi membranes. Here, we set out to determine the intracellular localisation of endogenous STX19 and elucidate the mechanism by which it is targeted to membranes. We have found that a pool of STX19 is localised to tubular recycling endosomes where it colocalises with MICAL-L1 and Rab8 (which has Rab8a and Rab8b forms). Using a combination of genetic, biochemical and cell-based approaches, we have identified that STX19 is S-acylated at its C-terminus and is a substrate for several Golgi-localised S-acyltransferases, suggesting that STX19 is initially S-acylated at the Golgi before trafficking to the plasma membrane and endosomes. Surprisingly, we have found that S-acylation is a key determinant in targeting STX19 to tubular recycling endosomes, suggesting that S-acylation may play a general role in directing proteins to this compartment. In addition, S-acylation also protects STX19 from proteosomal degradation, indicating that S-acylation regulates the function of STX19 at multiple levels.This article has an associated First Person interview with the first author of the paper.

Fam49/CYRI interacts with Rac1 and locally suppresses protrusions.

Actin-based protrusions are reinforced through positive feedback, but it is unclear what restricts their size, or limits positive signals when they retract or split. We identify an evolutionarily conserved regulator of actin-based protrusion: CYRI (CYFIP-related Rac interactor) also known as Fam49 (family of unknown function 49). CYRI binds activated Rac1 via a domain of unknown function (DUF1394) shared with CYFIP, defining DUF1394 as a Rac1-binding module. CYRI-depleted cells have broad lamellipodia enriched in Scar/WAVE, but reduced protrusion-retraction dynamics. Pseudopods induced by optogenetic Rac1 activation in CYRI-depleted cells are larger and longer lived. Conversely, CYRI overexpression suppresses recruitment of active Scar/WAVE to the cell edge, resulting in short-lived, unproductive protrusions. CYRI thus focuses protrusion signals and regulates pseudopod complexity by inhibiting Scar/WAVE-induced actin polymerization. It thus behaves like a 'local inhibitor' as predicted in widely accepted mathematical models, but not previously identified in cells. CYRI therefore regulates chemotaxis, cell migration and epithelial polarization by controlling the polarity and plasticity of protrusions.

LIF-dependent survival of embryonic stem cells is regulated by a novel palmitoylated Gab1 signalling protein.

The cytokine leukaemia inhibitory factor (LIF) promotes self-renewal of mouse embryonic stem cells (ESCs) through activation of the transcription factor Stat3. However, the contribution of other ancillary pathways stimulated by LIF in ESCs, such as the MAPK and PI3K pathways, is less well understood. We show here that naive-type mouse ESCs express high levels of a novel effector of the MAPK and PI3K pathways. This effector is an isoform of the Gab1 (Grb2-associated binder protein 1) adaptor protein that lacks the N-terminal pleckstrin homology (PH) membrane-binding domain. Although not essential for rapid unrestricted growth of ESCs under optimal conditions, the novel Gab1 variant (Gab1ß) is required for LIF-mediated cell survival under conditions of limited nutrient availability. This enhanced survival is absolutely dependent upon a latent palmitoylation site that targets Gab1ß directly to ESC membranes. These results show that constitutive association of Gab1 with membranes through a novel mechanism promotes LIF-dependent survival of murine ESCs in nutrient-poor conditions.

Disruption of the Zdhhc9 intellectual disability gene leads to behavioural abnormalities in a mouse model.

Protein S-acylation is a widespread post-translational modification that regulates the trafficking and function of a diverse array of proteins. This modification is catalysed by a family of twenty-three zDHHC enzymes that exhibit both specific and overlapping substrate interactions. Mutations in the gene encoding zDHHC9 cause mild-to-moderate intellectual disability, seizures, speech and language impairment, hypoplasia of the corpus callosum and reduced volume of sub-cortical structures. In this study, we have undertaken behavioural phenotyping, magnetic resonance imaging (MRI) and isolation of S-acylated proteins to investigate the effect of disruption of the Zdhhc9 gene in mice in a C57BL/6 genetic background. Zdhhc9 mutant male mice exhibit a range of abnormalities compared with their wild-type littermates: altered behaviour in the open-field test, elevated plus maze and acoustic startle test that is consistent with a reduced anxiety level; a reduced hang time in the hanging wire test that suggests underlying hypotonia but which may also be linked to reduced anxiety; deficits in the Morris water maze test of hippocampal-dependent spatial learning and memory; and a 36% reduction in corpus callosum volume revealed by MRI. Surprisingly, membrane association and S-acylation of H-Ras was not disrupted in either whole brain or hippocampus of Zdhhc9 mutant mice, suggesting that other substrates of this enzyme are linked to the observed changes. Overall, this study highlights a key role for zDHHC9 in brain development and behaviour, and supports the utility of the Zdhhc9 mutant mouse line to investigate molecular and cellular changes linked to intellectual disability and other deficits in the human population.

Toll-like receptor 3 activation impairs excitability and synaptic activity via TRIF signalling in immature rat and human neurons.

Toll like receptor 3 (TLR3) belongs to a family of pattern recognition receptors that recognise molecules found on pathogens referred to as pathogen associated molecular patterns (PAMPs). Its involvement in innate immunity is well known but despite its presence in the central nervous system (CNS), our knowledge of its function is limited. Here, we have investigated whether TLR3 activation modulates synaptic activity in primary hippocampal cultures and induced pluripotent stem cell (iPSC)-derived neurons. Synaptically driven spontaneous action potential (AP) firing was significantly reduced by the TLR3 specific activator, poly I:C, in a concentration-dependent manner following both short (5 min) and long exposures (1h) in rat hippocampal cultures. Notably, the consequence of TLR3 activation on neuronal function was reproduced in iPSC-derived cortical neurons, with poly I:C (25 µg/ml, 1h) significantly inhibiting sAP firing. We examined the mechanisms underlying these effects, with poly I:C significantly reducing peak sodium current, an effect dependent on the MyD88-independent TRIF dependent pathway. Furthermore, poly I:C (25 µg/ml, 1h) resulted in a significant reduction in miniature excitatory postsynaptic potential (mEPSC) frequency and amplitude and significantly reduced surface AMPAR expression. These novel findings reveal that TLR3 activation inhibits neuronal excitability and synaptic activity through multiple mechanisms, with this being observed in both rat and human iPSC-derived neurons. These data might provide further insight into how TLR3 activation may contribute to neurodevelopmental disorders following maternal infection and in patients with increased susceptibility to herpes simplex encephalitis.

Tracking intracellular uptake and localisation of alkyne tagged fatty acids using Raman spectroscopy.

Intracellular uptake, distribution and metabolism of lipids are tightly regulated characteristics in healthy cells. An analytical technique capable of understanding these characteristics with a high level of species specificity in a minimally invasive manner is highly desirable in order to understand better how these become disrupted during disease. In this study, the uptake and distribution of three different alkyne tagged fatty acids in single cells were monitored and compared, highlighting the ability of Raman spectroscopy combined with alkyne tags for better understanding of the fine details with regard to uptake, distribution and metabolism of very chemically specific lipid species. This indicates the promise of using Raman spectroscopy directly with alkyne tagged lipids for cellular studies as opposed to subsequently clicking of a fluorophore onto the alkyne for fluorescence imaging.

Peptide array-based screening reveals a large number of proteins interacting with the ankyrin-repeat domain of the zDHHC17 S-acyltransferase.

zDHHC S-acyltransferases are enzymes catalyzing protein S-acylation, a common post-translational modification on proteins frequently affecting their membrane targeting and trafficking. The ankyrin repeat (AR) domain of zDHHC17 (HIP14) and zDHHC13 (HIP14L) S-acyltransferases, which is involved in both substrate recruitment and S-acylation-independent functions, was recently shown to bind at least six proteins, by specific recognition of a consensus sequence in them. To further refine the rules governing binding to the AR of zDHHC17, we employed peptide arrays based on zDHHC AR-binding motif (zDABM) sequences of synaptosomal-associated protein 25 (SNAP25) and cysteine string protein α (CSPα). Quantitative comparisons of the binding preferences of 400 peptides allowed us to construct a position-specific scoring matrix (PSSM) for zDHHC17 AR binding, with which we predicted and subsequently validated many putative zDHHC17 interactors. We identified 95 human zDABM sequences with unexpected versatility in amino acid usage; these sequences were distributed among 90 proteins, of which 62 have not been previously implicated in zDHHC17/13 binding. These zDABM-containing proteins included all family members of the SNAP25, sprouty, cornifelin, ankyrin, and SLAIN-motif containing families; seven endogenous Gag polyproteins sharing the same binding sequence; and several proteins involved in cytoskeletal organization, cell communication, and regulation of signaling. A dozen of the zDABM-containing proteins had more than one zDABM sequence, whereas isoform-specific binding to the AR of zDHHC17 was identified for the Ena/VASP-like protein. The large number of zDABM sequences within the human proteome suggests that zDHHC17 may be an interaction hub regulating many cellular processes.

The C-terminal domain of zDHHC2 contains distinct sorting signals that regulate intracellular localisation in neurons and neuroendocrine cells.

The S-acyltransferase zDHHC2 mediates dynamic S-acylation of PSD95 and AKAP79/150, which impacts synaptic targeting of AMPA receptors. zDHHC2 is responsive to synaptic activity and catalyses the increased S-acylation of PSD95 that occurs following action potential blockade or application of ionotropic glutamate receptor antagonists. These treatments have been proposed to increase plasma membrane delivery of zDHHC2 via an endosomal cycling pathway, enhancing substrate accessibility. To generate an improved understanding of zDHHC2 trafficking and how this might be regulated by neuronal activity, we searched for intramolecular signals that regulate enzyme localisation. Two signals were mapped to the C-terminal tail of zDHHC2: a non-canonical dileucine motif [SxxxLL] and a downstream NP motif. Mutation of these signals enhanced plasma membrane accumulation of zDHHC2 in both neuroendocrine PC12 cells and rat hippocampal neurons, consistent with reduced endocytic retrieval. Furthermore, mutation of these signals also increased accumulation of the enzyme in neurites. Interestingly, several threonine and serine residues are adjacent to these sorting motifs and analysis of phospho-mimetic mutants highlighted a potential role for phosphorylation in regulating the efficacy of these signals. This study offers new molecular insight into the signals that determine zDHHC2 localisation and highlights a potential mechanism to regulate these trafficking signals.

Substrate selectivity in the zDHHC family of S-acyltransferases.

S-acylation is a reversible lipid modification occurring on cysteine residues mediated by a family of membrane-bound 'zDHHC' enzymes. S-acylation predominantly results in anchoring of soluble proteins to membrane compartments or in the trafficking of membrane proteins to different compartments. Recent work has shown that although S-acylation of some proteins may involve very weak interactions with zDHHC enzymes, a pool of zDHHC enzymes exhibit strong and specific interactions with substrates, thereby recruiting them for S-acylation. For example, the ankyrin-repeat domains of zDHHC17 and zDHHC13 interact specifically with unstructured consensus sequences present in some proteins, thus contributing to substrate specificity of these enzymes. In addition to this new information on zDHHC enzyme protein substrate specificity, recent work has also identified marked differences in selectivity of zDHHC enzymes for acyl-CoA substrates and has started to unravel the underlying molecular basis for this lipid selectivity. This review will focus on the protein and acyl-CoA selectivity of zDHHC enzymes.

Molecular basis of fatty acid selectivity in the zDHHC family of S-acyltransferases revealed by click chemistry.

S-acylation is a major posttranslational modification, catalyzed by the zinc finger DHHC domain containing (zDHHC) enzyme family. S-acylated proteins can be modified by different fatty acids; however, very little is known about how zDHHC enzymes contribute to acyl chain heterogeneity. Here, we used fatty acid-azide/alkyne labeling of mammalian cells, showing their transformation into acyl-CoAs and subsequent click chemistry-based detection, to demonstrate that zDHHC enzymes have marked differences in their fatty acid selectivity. This difference in selectivity was apparent even for highly related enzymes, such as zDHHC3 and zDHHC7, which displayed a marked difference in their ability to use C18:0 acyl-CoA as a substrate. Furthermore, we identified isoleucine-182 in transmembrane domain 3 of zDHHC3 as a key determinant in limiting the use of longer chain acyl-CoAs by this enzyme. This study uncovered differences in the fatty acid selectivity profiles of cellular zDHHC enzymes and mapped molecular determinants governing this selectivity.