Protein palmitoylation and cancer

Protein S‐palmitoylation is a reversible post‐translational modification that alters the localization, stability, and function of hundreds of proteins in the cell. S‐palmitoylation is essential for the function of both oncogenes (e.g., NRAS and EGFR) and tumor suppressors (e.g., SCRIB, melanocortin 1 receptor). In mammalian cells, the thioesterification of palmitate to internal cysteine residues is catalyzed by 23 Asp‐His‐His‐Cys (DHHC)‐family palmitoyl S‐acyltransferases while the removal of palmitate is catalyzed by serine hydrolases, including acyl‐protein thioesterases (APTs). These enzymes modulate the function of important oncogenes and tumor suppressors and often display altered expression patterns in cancer. Targeting S‐palmitoylation or the enzymes responsible for palmitoylation dynamics may therefore represent a candidate therapeutic strategy for certain cancers.     

Swf1-dependent palmitoylation of the SNARE Tlg1 prevents its ubiquitination and degradation

Protein palmitoylation is a post-translational modification that affects a great number of proteins. In most cases, the enzymes responsible for this modification have not been identified. Some proteins use palmitoylation to attach themselves to membranes; however, palmitoylation also occurs in transmembrane proteins, and the function of this palmitoylation is less clear. Here we identify Swf1, a member of the DHHC-CDR family of palmitoyltransferases, as the protein responsible for modifying the yeast SNAREs Snc1, Syn8 and Tlg1, at cysteine residues close to the cytoplasmic end of their single transmembrane domains (TMDs). In an swf1Delta mutant, Tlg1 is mis-sorted to the vacuole. This occurs because unpalmitoylated Tlg1 is recognised by the ubiquitin ligase Tul1, resulting in its targeting to the multivesicular body pathway. Our results suggest that one role of palmitoylation is to protect TMDs from the cellular quality control machinery, and that Swf1 may be the enzyme responsible for most, if not all, TMD-associated palmitoylation in yeast.     

Global analysis of protein palmitoylation in yeast

Protein palmitoylation is a reversible lipid modification that regulates membrane tethering for key proteins in cell signaling, cancer, neuronal transmission, and membrane trafficking. Palmitoylation has proven to be a difficult study: Specifying consensuses for predicting palmitoylation remain unavailable, and first-example palmitoylation enzymes--i.e., protein acyltransferases (PATs)--were identified only recently. Here, we use a new proteomic methodology that purifies and identifies palmitoylated proteins to characterize the palmitoyl proteome of the yeast Saccharomyces cerevisiae. Thirty-five new palmitoyl proteins are identified, including many SNARE proteins and amino acid permeases as well as many other participants in cellular signaling and membrane trafficking. Analysis of mutant yeast strains defective for members of the DHHC protein family, a putative PAT family, allows a matching of substrate palmitoyl proteins to modifying PATs and reveals the DHHC family to be a family of diverse PAT specificities responsible for most of the palmitoylation within the cell.     

蛋白质的棕榈酰化修饰

棕榈酰化修饰是蛋白质翻译后脂质修饰的重要形式,是调控蛋白质的转运、稳定、定位和功能的重要机制,同时,棕榈酰化修饰还参与多种细胞生物学进程,与许多疾病的发生发展密切相关。本文主要就蛋白质棕榈酰化及其修饰酶与蛋白质功能、相关疾病的关系做一综述。     

Regulation by S-nitrosylation of protein post-translational modification

Protein post-translational modification by S-nitrosylation conveys a ubiquitous influence of nitric oxide on signal transduction in eukaryotic cells. The wide functional purview of S-nitrosylation reflects in part the regulation by S-nitrosylation of the principal protein post-translational modifications that play a role in cell signaling, including phosphorylation, acetylation, ubiquitylation and related modifications, palmitoylation, and alternative Cys-based redox modifications. In this minireview, we discuss the mechanisms through which S-nitrosylation exerts its broad pleiotropic influence on protein post-translational modification.     

Probing protein palmitoylation at the yeast vacuole

A protein's function depends on its localization to the right cellular compartment. A number of proteins require lipidation to associate with membranes. Protein palmitoylation is a reversible lipid modification and has been shown to mediate both membrane localization and control protein function. At the yeast vacuole, several palmitoylated proteins have been identified that are required for vacuole biogenesis, including the fusion factor Vac8, the SNARE Ykt6 and the casein kinase Yck3. Moreover, both the DHHC-CRD acyltransferase Pfa3 and Ykt6 are involved in palmitoylation at the vacuole Here, we present and discuss methods to probe for protein palmitoylation at vacuoles.     

Altered localization of H-Ras in caveolin-1-null cells is palmitoylation-independent

Caveolin-1 is a palmitoylated protein involved in the formation of plasma membrane subdomains termed caveolae, intracellular cholesterol transport, and assembly and regulation of signaling molecules in caveolae. Caveolin-1 interacts via a consensus binding motif with several signaling proteins, including H-Ras. Ras oncogene products function as molecular switches in several signal transduction pathways regulating cell growth and differentiation. Post-translational modifications, including palmitoylation, are critical for the membrane targeting and function of H-Ras. Subcellular localization regulates the signaling pathways engaged by H-Ras activation. We show here that H-Ras is localized at the plasma membrane in caveolin-1-expressing cells but not in caveolin-1-deficient cells. Since palmitoylation is required for trafficking of H-Ras from the endomembrane system to the plasma membrane, we tested whether the altered localization of H-Ras in caveolin-1-null cells is due to decreased H-Ras palmitoylation. Although the palmitoylation profiles of cultured embryo fibroblasts isolated from wild type and caveolin-1 gene-disrupted mice differed, suggesting that caveolin-1, or caveolae, play a role in the palmitate incorporation of a subset of palmitoylated proteins, the palmitoylation of H-Ras was not decreased in caveolin-1-null cells. We conclude that the altered localization of H-Ras in caveolin-1-deficient cells is palmitoylation-independent. This article shows two important new mechanisms by which loss of caveolin-1 expression may perturb intracellular signaling, namely the mislocalization of signaling proteins and alterations in protein palmitoylation.     

Altered localization of H-Ras in caveolin-1-null cells is palmitoylation-independent

Caveolin-1 is a palmitoylated protein involved in the formation of plasma membrane subdomains termed caveolae, intracellular cholesterol transport, and assembly and regulation of signaling molecules in caveolae. Caveolin-1 interacts via a consensus binding motif with several signaling proteins, including H-Ras. Ras oncogene products function as molecular switches in several signal transduction pathways regulating cell growth and differentiation. Post-translational modifications, including palmitoylation, are critical for the membrane targeting and function of H-Ras. Subcellular localization regulates the signaling pathways engaged by H-Ras activation. We show here that H-Ras is localized at the plasma membrane in caveolin-1-expressing cells but not in caveolin-1-deficient cells. Since palmitoylation is required for trafficking of H-Ras from the endomembrane system to the plasma membrane, we tested whether the altered localization of H-Ras in caveolin-1-null cells is due to decreased H-Ras palmitoylation. Although the palmitoylation profiles of cultured embryo fibroblasts isolated from wild type and caveolin-1 gene-disrupted mice differed, suggesting that caveolin-1, or caveolae, play a role in the palmitate incorporation of a subset of palmitoylated proteins, the palmitoylation of H-Ras was not decreased in caveolin-1-null cells. We conclude that the altered localization of H-Ras in caveolin-1-deficient cells is palmitoylation-independent. This article shows two important new mechanisms by which loss of caveolin-1 expression may perturb intracellular signaling, namely the mislocalization of signaling proteins and alterations in protein palmitoylation.     

Protein palmitoylation and subcellular trafficking

Protein S-palmitoylation, the covalent lipid modification of the side chain of Cys residues with the 16-carbon fatty acid palmitate, is the most common acylation of proteins in eukaryotic cells. This post-translational modification provides an important mechanism for regulating protein subcellular localization, stability, trafficking, translocation to lipid rafts, aggregation, interaction with effectors and other aspects of protein function. In addition, N-terminal myristoylation and C-terminal prenylation, two well-studied post-translational modifications, frequently precede protein S-palmitoylation at a nearby spot of the polypeptide chain. Whereas N-myristoylation and prenylation are considered essentially irreversible attachments, S-palmitoylation is a tightly regulated, reversible modification. In addition, the unique reversibility of protein palmitoylation also allows proteins to rapidly shuttle between intracellular membrane compartments in a process controlled, in some cases, by the DHHC family of palmitoyl transferases. Recent cotransfection experiments using the DHHC family of protein palmitoyl transferases as well as RNA interference results have revealed that these enzymes, frequently localized to the Golgi apparatus, tightly control subcellular trafficking of acylated proteins. In this article we will give an overview of how protein palmitoylation regulates protein trafficking and subcellular localization.     

Palmitoylation of caveolin-1 in endothelial cells is post-translational but irreversible

Caveolin-1 is a palmitoylated protein involved in assembly of signaling molecules in plasma membrane subdomains termed caveolae and in intracellular cholesterol transport. Three cysteine residues in the C terminus of caveolin-1 are subject to palmitoylation, which is not necessary for caveolar targeting of caveolin-1. Protein palmitoylation is a post-translational and reversible modification that may be regulated and that in turn may regulate conformation, membrane association, protein-protein interactions, and intracellular localization of the target protein. We have undertaken a detailed analysis of [(3)H]palmitate incorporation into caveolin-1 in aortic endothelial cells. The linkage of palmitate to caveolin-1 was hydroxylamine-sensitive and thus presumably a thioester bond. However, contrary to expectations, palmitate incorporation was blocked completely by the protein synthesis inhibitors cycloheximide and puromycin. In parallel experiments to show specificity, palmitoylation of aortic endothelial cell-specific nitric-oxide synthase was unaffected by these reagents. Inhibitors of protein trafficking, brefeldin A and monensin, blocked caveolin-1 palmitoylation, indicating that the modification was not cotranslational but rather required caveolin-1 transport from the endoplasmic reticulum and Golgi to the plasma membrane. In addition, immunophilin chaperones that form complexes with caveolin-1, i.e. FK506-binding protein 52, cyclophilin A, and cyclophilin 40, were not necessary for caveolin-1 palmitoylation because agents that bind immunophilins did not inhibit palmitoylation. Pulse-chase experiments showed that caveolin-1 palmitoylation is essentially irreversible because the release of [(3)H]palmitate was not significant even after 24 h. These results show that [(3)H]palmitate incorporation is limited to newly synthesized caveolin-1, not because incorporation only occurs during synthesis but because the continuous presence of palmitate on caveolin-1 prevents subsequent repalmitoylation.     

Subcellular Localization Directs Signaling Specificity of the Cryptococcus neoformans Ras1 Protein

In the human fungal pathogen Cryptococcus neoformans, Ras signaling mediates sexual differentiation, morphogenesis, and pathogenesis. By studying Ras prenylation and palmitoylation in this organism, we have found that the subcellular localization of this protein dictates its downstream signaling specificity. Inhibiting C. neoformans Ras1 prenylation results in the defective general membrane targeting of this protein and the loss of all Ras function. In contrast, palmitoylation mediates localization of Ras1 to the plasma membrane and is required for normal morphogenesis and survival at high temperatures. However, palmitoylation and plasma membrane localization are not required for Ras-dependent sexual differentiation. Likely as a result of its effect on thermotolerance, Ras1 palmitoylation is also required for the pathogenesis of C. neoformans. These data support an emerging paradigm of compartmentalized Ras signaling. However, our studies also demonstrate fundamental differences between the Ras pathways in different organisms that emphasize the functional flexibility of conserved signaling cascades.     

Assays of protein palmitoylation

Protein palmitoylation plays an important role in the structure and function of a wide array of proteins. Unlike other lipid modifications, protein palmitoylation is highly dynamic and cycles of palmitoylation and depalmitoylation can regulate protein function and localization. The dynamic nature of palmitoylation is poorly resolved because of limitations in assay methods. Here, we discuss various methods that can be used to measure protein palmitoylation and identify sites of palmitoylation. We describe new methodology based on "fatty acyl exchange labeling" in which palmitate is removed via hydroxylamine-mediated cleavage of the palmitoyl-thioester bond and then exchanged with a sulfhydryl-specific labeling compound. The techniques are highly sensitive and allow for quantitative estimates of palmitoylation. Unlike other techniques used to assay posttranslational modifications, the techniques we have developed can label all sites of modification with a variety of probes, radiolabeled or non-radioactive, and can be used to assay the palmitoylation of proteins from tissue samples.     

Use of analogs and inhibitors to study the functional significance of protein palmitoylation

Covalent attachment of palmitate to proteins is a post-translational modification that exerts diverse effects on protein localization and function. The three key technical approaches required for an investigator to determine the role of palmitoylation of your favorite palmitoylated protein (YFPP) are methods to: (1) detect YFPP palmitoylation; (2) alter or inhibit palmitoylation of YFPP; (3) determine the functional significance of altered YFPP palmitoylation. Here, I describe experimental methods to address these three issues. Both radioactive (radiolabeling with [(3)H]palmitate or (125)I-IC16 palmitate) and non-radioactive (chemical labeling and mass spectrometry) methods to detect palmitoylated proteins are presented. Next, techniques to inhibit protein palmitoylation are described. These include site specific mutagenesis, and treatment of cells with inhibitors of protein palmitoylation, including 2-bromopalmitate, cerulenin, and tunicamycin. Alternative methods to replace palmitate with other fatty acids are also presented. Finally, general approaches to determining the effect of altered palmitoylation status on YFPP association with membranes and lipid rafts, as well as signal transduction, are described.     

DHHC5 Mediates β-Adrenergic Signaling in Cardiomyocytes by Targeting Gα Proteins

S-palmitoylation is a reversible posttranslational modification that plays an important role in regulating protein localization, trafficking, and stability. Recent studies have shown that some proteins undergo extremely rapid palmitoylation/depalmitoylation cycles after cellular stimulation supporting a direct signaling role for this posttranslational modification. Here, we investigated whether β-adrenergic stimulation of cardiomyocytes led to stimulus-dependent palmitoylation of downstream signaling proteins. We found that β-adrenergic stimulation led to rapidly increased Gαs and Gαi palmitoylation. The kinetics of palmitoylation was temporally consistent with the downstream production of cAMP and contractile responses. We identified the plasma membrane-localized palmitoyl acyltransferase DHHC5 as an important mediator of the stimulus-dependent palmitoylation in cardiomyocytes. Knockdown of DHHC5 showed that this enzyme is necessary for palmitoylation of Gαs, Gαi, and functional responses downstream of β-adrenergic stimulation. A palmitoylation assay with purified components revealed that Gαs and Gαi are direct substrates of DHHC5. Finally, we provided evidence that the C-terminal tail of DHHC5 can be palmitoylated in response to stimulation and such modification is important for its dynamic localization and function in the plasma membrane. Our results reveal that DHHC5 is a central regulator of signaling downstream of β-adrenergic receptors in cardiomyocytes.     

The importance of N-terminal polycysteine and polybasic sequences for G14alpha and G16alpha palmitoylation, plasma membrane localization, and signaling function

Plasma membrane targeting of G protein alpha (Galpha) subunits is essential for competent receptor-to-G protein signaling. Many Galpha are tethered to the plasma membrane by covalent lipid modifications at their N terminus. Additionally, it is hypothesized that Gq family members (Gqalpha,G11alpha,G14alpha, and G16alpha) in particular utilize a polybasic sequence of amino acids in their N terminus to promote membrane attachment and protein palmitoylation. However, this hypothesis has not been tested, and nothing is known about other mechanisms that control subcellular localization and signaling properties of G14alpha and G16alpha. Here we report critical biochemical factors that mediate membrane attachment and signaling function of G14alpha and G16alpha. We find that G14alpha and G16alpha are palmitoylated at distinct polycysteine sequences in their N termini and that the polycysteine sequence along with the adjacent polybasic region are both important for G16alpha-mediated signaling at the plasma membrane. Surprisingly, the isolated N termini of G14alpha and G16alpha expressed as peptides fused to enhanced green fluorescent protein each exhibit differential requirements for palmitoylation and membrane targeting; individual cysteine residues, but not the polybasic regions, determine lipid modification and subcellular localization. However, full-length G16alpha, more so than G14alpha, displays a functional dependence on single cysteines for membrane localization and activity, and its full signaling potential depends on the integrity of the polybasic sequence. Together, these findings indicate that G14alpha and G16alpha are palmitoylated at distinct polycysteine sequences, and that the adjacent polybasic domain is not required for Galpha palmitoylation but is important for localization and functional activity of heterotrimeric G proteins.     

Purification and characterization of recombinant protein acyltransferases

Palmitoylation enhances membrane association and plays a role in the subcellular trafficking and signaling function of proteins. Unlike other forms of protein lipidation, such as prenylation and myristoylation, palmitoylation is reversible and can therefore play a regulatory role. Enzyme activities have recently been described in mammals and yeast that carry out the palmitoylation of protein substrates. Protein acyltransferases (PATs) transfer a palmitoyl moiety derived from palmitoyl-CoA to a free thiol of a substrate protein to create a labile thioester linkage. Biochemical characterization and kinetic analysis of this new family of enzymes requires methods to purify PATs and their substrates, as well as methods to assay PAT activity. We describe a series of methods using yeast and bacterial expression systems to study protein acyltransferases.     

Lipid raft localization and palmitoylation: identification of two requirements for cell death induction by the tumor suppressors UNC5H

UNC5H receptors (UNC5H1, UNC5H2, UNC5H3) are putative tumor suppressors whose expression is lost in numerous cancers. These receptors have been shown to belong to the so-called family of dependence receptors. Such receptors induce apoptosis when their ligand netrin-1 is absent, thus conferring a state of cellular dependence towards ligand presence. Along this line, these receptors may limit tumor progression because they induce the death of tumor cells that grow in settings of ligand unavailability. We show here that UNC5H receptors are localized to cholesterol-and sphingolipid-enriched membrane domains called lipid rafts. We then demonstrate that the lipid raft localization of UNC5H2 is required for the pro-apoptotic activity of unbound UNC5H2. We also propose that this lipid raft localization is probably mediated via the recruitment of adaptor protein(s) within the death domain of UNC5H2 but is not dependent on the post-translational modification by palmitoylation of UNC5H2 even though this palmitoylation is required for UNC5H2 pro-apoptotic activity. Moreover we show that the interaction of UNC5H2 with the downstream pro-apoptotic serine threonine kinase DAPk is dependent on both UNC5H2 lipid raft localization and palmitoylation. Thus, we propose that the UNC5H dependence receptors require lipid raft localization and palmitoylation to trigger apoptosis.     

In vitro reconstitution of substrate S-acylation by the zDHHC family of protein acyltransferases

Protein S-acylation, more commonly known as protein palmitoylation, is a biological process defined by the covalent attachment of long chain fatty acids onto cysteine residues of a protein, effectively altering the local hydrophobicity and influencing its stability, localization and overall function. Observed ubiquitously in all eukaryotes, this post translational modification is mediated by the 23-member family of zDHHC protein acyltransferases in mammals. There are thousands of proteins that are S-acylated and multiple zDHHC enzymes can potentially act on a single substrate. Since its discovery, numerous methods have been developed for the identification of zDHHC substrates and the individual members of the family that catalyse their acylation. Despite these recent advances in assay development, there is a persistent gap in knowledge relating to zDHHC substrate specificity and recognition, that can only be thoroughly addressed through in vitro reconstitution. Herein, we will review the various methods currently available for reconstitution of protein S-acylation for the purposes of identifying enzyme–substrate pairs with a particular emphasis on the advantages and disadvantages of each approach.     

A role for aberrant protein palmitoylation in FFA-induced ER stress and β-cell death

Exposure of insulin-producing cells to elevated levels of the free fatty acid (FFA) palmitate results in the loss of β-cell function and induction of apoptosis. The induction of endoplasmic reticulum (ER) stress is one mechanism proposed to be responsible for the loss of β-cell viability in response to palmitate treatment; however, the pathways responsible for the induction of ER stress by palmitate have yet to be determined. Protein palmitoylation is a major posttranslational modification that regulates protein localization, stability, and activity. Defects in, or dysregulation of, protein palmitoylation could be one mechanism by which palmitate may induce ER stress in β-cells. The purpose of this study was to evaluate the hypothesis that palmitate-induced ER stress and β-cell toxicity are mediated by excess or aberrant protein palmitoylation. In a concentration-dependent fashion, palmitate treatment of RINm5F cells results in a loss of viability. Similar to palmitate, stearate also induces a concentration-related loss of RINm5F cell viability, while the monounsaturated fatty acids, such as palmoleate and oleate, are not toxic to RINm5F cells. 2-Bromopalmitate (2BrP), a classical inhibitor of protein palmitoylation that has been extensively used as an inhibitor of G protein-coupled receptor signaling, attenuates palmitate-induced RINm5F cell death in a concentration-dependent manner. The protective effects of 2BrP are associated with the inhibition of [(3)H]palmitate incorporation into RINm5F cell protein. Furthermore, 2BrP does not inhibit, but appears to enhance, the oxidation of palmitate. The induction of ER stress in response to palmitate treatment and the activation of caspase activity are attenuated by 2BrP. Consistent with protective effects on insulinoma cells, 2BrP also attenuates the inhibitory actions of prolonged palmitate treatment on insulin secretion by isolated rat islets. These studies support a role for aberrant protein palmitoylation as a mechanism by which palmitate enhances ER stress activation and causes the loss of insulinoma cell viability.