Littérature scientifique sur le sujet « S-Fatty acylation »

Fatty acylation of cysteine residues provides spatial and temporal control of protein function in cells and regulates important biological pathways in eukaryotes. Although recent methods have improved the detection and proteomic analysis of cysteine fatty (S-fatty) acylated proteins, understanding how specific sites and quantitative levels of this posttranslational modification modulate cellular pathways are still challenging. To analyze the endogenous levels of protein S-fatty acylation in cells, we developed a mass-tag labeling method based on hydroxylamine-sensitivity of thioesters and selective maleimide-modification of cysteines, termed acyl-PEG exchange (APE). We demonstrate that APE enables sensitive detection of protein S-acylation levels and is broadly applicable to different classes of S-palmitoylated membrane proteins. Using APE, we show that endogenous interferon-induced transmembrane protein 3 is S-fatty acylated on three cysteine residues and site-specific modification of highly conserved cysteines are crucial for the antiviral activity of this IFN-stimulated immune effector. APE therefore provides a general and sensitive method for analyzing the endogenous levels of protein S-fatty acylation and should facilitate quantitative studies of this regulated and dynamic lipid modification in biological systems.

Post-translational modifications often regulate protein functioning. Covalent attachment of long chain fatty acids to cysteine residues via a thioester linkage (known as protein palmitoylation or S-acylation) affects protein trafficking, protein-protein and protein-membrane interactions. This post-translational modification is coupled to membrane fusion or virus assembly and may affect viral replication in vitro and thus also virus pathogenesis in vivo. In this review we outline modern methods to study S-acylation of viral proteins and to characterize palmitoylproteomes of virus infected cells. The palmitoylation site predictor CSS-palm is critically tested against the Class I enveloped virus proteins. We further focus on identifying the S-acylation sites directly within acyl-peptides and the specific fatty acid (e.g, palmitate, stearate) bound to them using MALDI-TOF MS-based approaches. The fatty acid heterogeneity/ selectivity issue attracts now more attention since the recently published 3D-structures of two DHHC-acyl-transferases gave a hint how this might be achieved.

S-acylation is a common yet poorly understood fatty acid-based post-translational modification of proteins in all eukaryotes, including plants. While exact roles for S-acylation in protein function are largely unknown the reversibility of S-acylation indicates that it is likely able to play a regulatory role. As more studies reveal the roles of S-acylation within the cell it is becoming apparent that how S-acylation affects proteins is conceptually different from other reversible modifications such as phosphorylation or ubiquitination; a new mind-set is therefore required to fully integrate these data into our knowledge of plant biology. This review aims to highlight recent advances made in the function and enzymology of S-acylation in plants, highlights current and emerging technologies for its study and suggests future avenues for investigation.

Protein lipidation is a collection of important post-translational modifications that modulate protein localization and stability. Protein lipidation affects protein function by facilitating interactions with cellular membranes, changing the local environment of protein interactions. Among these modifications, S-acylation has emerged as a key regulator of various cellular processes, including different forms of cell death. In this mini-review, we highlight the role of S-acylation in apoptosis and its emerging contributions to necroptosis and pyroptosis. While traditionally associated with the incorporation of palmitic acid (palmitoylation), recent findings indicate that other fatty acids can also participate in S-acylation, expanding its functional repertoire. In apoptosis, S-acylation influences the localization and function of key regulators such as Bcl-2-associated X protein and other proteins modulating their role in mitochondrial permeabilization and death receptor signaling. Similarly, in necroptosis, S-acylation of mixed lineage kinase domain-like protein (MLKL) with palmitic acid and very long-chain fatty acids enhances membrane binding and membrane permeabilization, contributing to cell death and inflammatory responses. Recent studies also highlight the role of S-acylation in pyroptosis, where S-acylated gasdermin D facilitates membrane localization and pore assembly upon inflammasome activation. Blocking palmitoylation has shown to suppress pyroptosis and cytokine release, reducing inflammatory activity and tissue damage in septic models. Collectively, these findings underscore S-acylation as a shared and important regulatory mechanism across cell death pathways affecting membrane association of key signaling proteins and membrane dynamics, and offer insights into the spatial and temporal control of protein function.

The ileal apical sodium-dependent bile acid transporter (ASBT) is crucial for the enterohepatic circulation of bile acids. ASBT function is rapidly regulated by several posttranslational modifications. One reversible posttranslational modification is S-acylation, involving the covalent attachment of fatty acids to cysteine residues in proteins. However, whether S-acylation affects ASBT function and membrane expression has not been determined. Using the acyl resin-assisted capture method, we found that the majority of ASBT (∼80%) was S-acylated in ileal brush border membrane vesicles from human organ donors, as well as in HEK293 cells stably transfected with ASBT (2BT cells). Metabolic labeling with alkyne–palmitic acid (100 μm for 15 h) also showed that ASBT is S-acylated in 2BT cells. Incubation with the acyltransferase inhibitor 2-bromopalmitate (25 μm for 15 h) significantly reduced ASBT S-acylation, function, and levels on the plasma membrane. Treatment of 2BT cells with saturated palmitic acid (100 μm for 15 h) increased ASBT function, whereas treatment with unsaturated oleic acid significantly reduced ASBT function. Metabolic labeling with alkyne–oleic acid (100 μm for 15 h) revealed that oleic acid attaches to ASBT, suggesting that unsaturated fatty acids may decrease ASBT's function via a direct covalent interaction with ASBT. We also identified Cys-314 as a potential S-acylation site. In conclusion, these results provide evidence that S-acylation is involved in the modulation of ASBT function. These findings underscore the potential for unsaturated fatty acids to reduce ASBT function, which may be useful in disorders in which bile acid toxicity is implicated.

Defects in cilia have been associated with an expanding human disease spectrum known as ciliopathies. Regulatory Factor X 3 (RFX3) is one of the major transcription factors required for ciliogenesis and cilia functions. In addition, RFX3 regulates pancreatic islet cell differentiation and mature β-cell functions. However, how RFX3 protein is regulated at the posttranslational level remains poorly understood. Using chemical reporters of protein fatty acylation and mass spectrometry analysis, here we show that RFX3 transcriptional activity is regulated by S-fatty acylation at a highly conserved cysteine residue in the dimerization domain. Surprisingly, RFX3 undergoes enzyme-independent, “self-catalyzed” auto-fatty acylation and displays preferences for 18-carbon stearic acid and oleic acid. The fatty acylation-deficient mutant of RFX3 shows decreased homodimerization; fails to promote ciliary gene expression, ciliogenesis, and elongation; and impairs Hedgehog signaling. Our findings reveal a regulation of RFX3 transcription factor and link fatty acid metabolism and protein lipidation to the regulation of ciliogenesis.

S-acylation, the attachment of fatty acids onto cysteine residues, regulates protein trafficking and function and is mediated by a family of zDHHC enzymes. The S-acylation of peripheral membrane proteins has been proposed to occur at the Golgi, catalyzed by an S-acylation machinery that displays little substrate specificity. To advance understanding of how S-acylation of peripheral membrane proteins is handled by Golgi zDHHC enzymes, we investigated interactions between a subset of four Golgi zDHHC enzymes and two S-acylated proteins—synaptosomal-associated protein 25 (SNAP25) and cysteine-string protein (CSP). Our results uncover major differences in substrate recognition and S-acylation by these zDHHC enzymes. The ankyrin-repeat domains of zDHHC17 and zDHHC13 mediated strong and selective interactions with SNAP25/CSP, whereas binding of zDHHC3 and zDHHC7 to these proteins was barely detectable. Despite this, zDHHC3/zDHHC7 could S-acylate SNAP25/CSP more efficiently than zDHHC17, whereas zDHHC13 lacked S-acylation activity toward these proteins. Overall the results of this study support a model in which dynamic intracellular localization of peripheral membrane proteins is achieved by highly selective recruitment by a subset of zDHHC enzymes at the Golgi, combined with highly efficient S-acylation by other Golgi zDHHC enzymes.

Protein S-acylation, the reversible covalent fatty-acid modification of cysteine residues, has emerged as a dynamic posttranslational modification (PTM) that controls the diversity, life cycle, and physiological function of numerous ligand- and voltage-gated ion channels. S-acylation is enzymatically mediated by a diverse family of acyltransferases (zDHHCs) and is reversed by acylthioesterases. However, for most ion channels, the dynamics and subcellular localization at which S-acylation and deacylation cycles occur are not known. S-acylation can control the two fundamental determinants of ion channel function: (1) the number of channels resident in a membrane and (2) the activity of the channel at the membrane. It controls the former by regulating channel trafficking and the latter by controlling channel kinetics and modulation by other PTMs. Ion channel function may be modulated by S-acylation of both pore-forming and regulatory subunits as well as through control of adapter, signaling, and scaffolding proteins in ion channel complexes. Importantly, cross-talk of S-acylation with other PTMs of both cysteine residues by themselves and neighboring sites of phosphorylation is an emerging concept in the control of ion channel physiology. In this review, I discuss the fundamentals of protein S-acylation and the tools available to investigate ion channel S-acylation. The mechanisms and role of S-acylation in controlling diverse stages of the ion channel life cycle and its effect on ion channel function are highlighted. Finally, I discuss future goals and challenges for the field to understand both the mechanistic basis for S-acylation control of ion channels and the functional consequence and implications for understanding the physiological function of ion channel S-acylation in health and disease.

La S-acylation est une modification post-traductionnelle des protéines qui correspond à la formation d’une liaison covalente entre un acide gras et un résidu cystéine. L'ajout de ce groupement hydrophobe peut modifier la localisation des protéines et affecter leur structure et/ou leur stabilité. La réversibilité et le dynamisme de cette modification enzymatique lui confèrent un rôle de régulateur dans les cellules, avec une implication observée dans de nombreux processus biologiques importants. Des aberrations dans les cycles de S-acylation ont été associées à différentes maladies humaines, telles que des cancers ou des maladies neurodégénératives. Les méthodes de protéomique existantes reposent principalement sur la quantification relative de la S-acylation des protéines. Notamment, il n’existe pas de méthodes permettant de quantifier précisément les variations des niveaux de S-acylation de chaque cystéine. Ce projet de thèse s'est concentré sur le développement d'une méthode de quantification de la S-acylation des cystéines dans un seul échantillon de protéome. Nous avons choisi de baser la méthode sur le marquage séquentiel et différentiel des cystéines libres et des cystéines S-acylées à l'aide d'une paire de sondes chimiques marquées isotopiquement. Les sondes ont des structures identiques, composées d'un électrophile pour alkyler les cystéines, d'une fonction alcyne, et d’isotopes légers et lourds permettant d’introduire une différence de masse (sonde « légère » (12C, 14N) et sonde « lourde » (13C, 15N)). Les cystéines marquées sont ensuite couplées par réaction click à un agent de capture portant un motif biotine et un azoture. Après digestion par la trypsine, les peptides contenant des cystéines sont enrichis à l'aide de billes de NeutrAvidin. Suite à leur élution des billes, les peptides marqués sont analysés par LC-MS/MS. L'analyse MS fournit un ratio de marquage lourd/léger pour les cystéines marquées dans un seul échantillon de protéome, révélant le pourcentage de S-acylation de ces cystéines. Les outils chimiques (sondes et agents de capture) ont été synthétisés et le pipeline a été développé, avec la sélection des différents paramètres des étapes de traitement des protéines à l'aide d'analyses qualitatives (Western blot). Des analyses par protéomique ont ensuite permis d'étudier plus en détail des variables, telles que les couples de sondes et d’agents de capture, les paramètres d'analyse LC-MS/MS et le traitement des données pour calculer les pourcentages de S-acylation. La méthode a été appliquée pour détecter les changements des niveaux de S-acylation suite à un stimulus externe, permettant l’acquisition de nouvelles informations sur certaines voies de signalisation et des processus biologiques associés. Les optimisations de la méthode seront poursuivies, notamment avec la comparaison de sondes possédant de nouvelles structures et une automatisation du pipeline. Nous prévoyons une large application de la méthode développée à l'étude des niveaux de S-acylation des cystéines des protéines et de leur lien avec différentes pathologies, ce qui pourrait révéler des traitements innovants
S-acylation is a post-translational modification of proteins involving the covalent attachment of a fatty acid to cysteine residues. This addition of a hydrophobic moiety can alter the protein localisation, and can also affect their structure and/or stability. The reversibility and dynamism of this enzymatic modification enable it to play a regulatory role in cellular processes, with involvement in various biological functions. Aberrant S-acylation has been linked to a variety of human diseases, including cancers or neurodegenerative diseases. Existing proteomics methods are mostly based on relative quantification of the S-acylation of proteins. Tools to precisely quantify changes in S-acylation levels of each cysteine residue are noticeably lacking. This thesis project focuses on the development of a method to quantify the S-acylation levels of cysteine residues in a single proteome sample. The method relies on the sequential and differential labelling of free cysteines and S-acylated cysteines using a pair of isotopically-tagged chemical probes. The probes have identical structures, each composed of a cysteine-reactive electrophile and an alkyne handle, with a mass difference introduced by light and heavy isotopes (“light” probe (12C, 14N) and “heavy” probe (13C, 15N)). The labelled cysteines are then coupled by click reaction to an azide-capture reagent bearing a biotin moiety. Following tryptic digestion, cysteine-containing peptides are enriched using NeutrAvidin beads, and the eluted labelled peptides are analysed by LC-MS/MS. MS analysis provides heavy-to-light ratios for each labelled cysteine within a single proteome sample, which reveals the percentage of S-acylation of the cysteines. The chemical tools were synthesised and the workflow was developed with the selection of protein treatments’ parameters using qualitative analysis approaches (e.g., Western blotting). Proteomics analyses allowed further refinement of key variables, including the combination of probes and capture reagents, the LC-MS/MS analysis parameters, and the downstream data processing to calculate the S-acylation percentages. The method was successfully applied to detect changes in S-acylation levels upon an external stimulus, providing new insights into associated signalling pathways and biological processes. Optimisations of the workflow will be pursued, notably by comparing new probes and automating the pipeline. We anticipate that the developed method will have broad applications for studying the S-acylation levels of cysteine residues and their role in different pathologies, potentially revealing innovative treatments

We have previously shown that the main source of arachidonate in thrombin-stimulated human platelets is 1-acyl-2-arachidonoyl (AA) glycerophosphocholine (GPC) and release of 3H-AA from this phospholipid also was correlated with increased 3H-AA in ether phospholipid. This ATP independent transfer of 3H-AA from 1,2 diacyl GPC to ether phospholipid (transacylation) also occurs in resting cells. Human platelets in 1/10 volume of plasma (ACD anticoagulant, pH 6.5) were radiolabelled with 3H-AA for 60 min at 37°C and then exogenous 3H-AA was removed by gel filtration into Tyrode's buffer, pH 7.4, 0.2% albumin. These radiolabelled cells were incubated in the absence of exogenous 3H-AA for four hours followed by Bligh and Dyer extraction and thin layer chromatography purification of phospholipids. 3H-AA in 1,2 diacyl GPC was found to decrease by over 20% and increase substantially in 1-0-alkyl-2-acyl GPC and 1-0-alk-1'-enyl-2-acyl glycerophospho ethanolamine (GPE), In this same time interval the mass of AA released by thrombin (5 U/ml, 10 min, 37°C, no stirring)in the presence of BIT 775C and measured by GLC, stayed the same (30 nmoles/109 cells), however, the specific activity decreased. Using reverse phase HPLC to resolve diradylglycerobenzoate derivatives of phospholipids: acylation, deacylation, and transacylation were observed for individual AA-containing molecular species of phospholipid, including those with an unsaturated fatty acid at sn-1. In particular the radiolabellinq of the 1-unsaturate-2-arachidonoyl GPC correlated with the specific activity of the 3H-AA released by stimulation with thrombin. Furthermore, 1-arachidonoyl-2-3H-arachidonoyl GPC was completely deacylated while 50 % of its mass remained. This contrasted with 16:0, and 18:0-2-arachidonoyl GPC in which the specific activity remained the same before and after deacylation. We conclude that deacylation of AA-containing molecular species of 1,2 diacyl GPC in stimulated cells includes molecular species which are also a source of arachidonic acid for transacylation reactions.