Regulation of NCX1 by palmitoylation

Palmitoylation (S-acylation) is the reversible conjugation of a fatty acid (usually C16 palmitate) to intracellular cysteine residues of proteins via a thioester linkage. Palmitoylation anchors intracellular regions of proteins to membranes because the palmitoylated cysteine is recruited to the lipid bilayer. NCX1 is palmitoylated at a single cysteine in its large regulatory intracellular loop. The presence of an amphipathic α-helix immediately adjacent to the NCX1 palmitoylation site is required for NCX1 palmitoylation. The NCX1 palmitoylation site is conserved through most metazoan phlya. Although palmitoylation does not regulate the normal forward or reverse ion transport modes of NCX1, NCX1 palmitoylation is required for its inactivation: sodium-dependent inactivation and inactivation by PIP2 depletion are significantly impaired for unpalmitoylatable NCX1. Here we review the role of palmitoylation in regulating NCX1 activity, and highlight future questions that must be addressed to fully understand the importance of this regulatory mechanism for sodium and calcium transport in cardiac muscle.     

Palmitoylation: A new mechanism for control of NCX1 function

A paper by Gök et al., identified Zinc Finger DHHC-Type Palmitoyltransferase 5 (zDHHC5) and Acyl-Protein Transferase 1 (APT1) as the enzymes responsible for the dynamic palmitoylation of NCX1. Palmitoylation occurs at the cell surface and increases the affinity of NCX1 for lipid rafts. Additionally, they discovered that palmitoylation controls the affinity of NCX1 for exchange inhibitory protein (XIP) and regulates intracellular calcium concentration. These findings provide new insights into endogenous control of NCX1 function and will drive future investigations directed at understanding its full potential.     

棕榈酰化修饰对G蛋白偶联受体功能的调节作用

G蛋白偶联受体(G-protein-coupled receptors,GPCRs)作为跨膜蛋白,其结构和功能同时受相互作用的蛋白质和脂质分子调控.S-棕榈酰化(S-palmitoylation)能够影响GPCRs与信号蛋白及膜脂分子的相互作用,在GPCRs相关的多项生理进程中发挥重要调节作用.棕榈酸与GPCRs的半胱氨酸间形成不稳定的硫酯键,其修饰动力学过程受棕榈酰转移酶(protein acly transferases,PATs)与硫酯酶(thioesterases)之间的可逆性双重调控,与受体活性及生理状态密切相关.棕榈酰化修饰多发生在GPCRs的C末端,通过棕榈酸侧链插入到质膜内侧而形成第4和/或第5个胞内环,从而影响GPCRs的构象,促进其正确折叠与成熟,并对GPCRs胞内转运、分选、下游信号转导、失敏、内化、寡聚化等活动产生影响.此外,棕榈酰化还与磷酸化、泛素化及亚硝基化等多种翻译后修饰机制相互作用,共同参与调节GPCRs的功能.GPCRs的棕榈酰化修饰酶学机制以及GPCRs蛋白复合体棕榈酰化修饰胞内动力学过程将是未来的研究热点.     

Detection of Protein Palmitoylation in Cultured Hippocampal Neurons by Immunoprecipitation and Acyl-Biotin Exchange (ABE)

Palmitoylation is a post-translational lipid modification involving the attachment of a 16-carbon saturated fatty acid, palmitate, to cysteine residues of substrate proteins through a labile thioester bond [reviewed in¹]. Palmitoylation of a substrate protein increases its hydrophobicity, and typically facilitates its trafficking toward cellular membranes. Recent studies have shown palmitoylation to be one of the most common lipid modifications in neurons^{1, 2}, suggesting that palmitate turnover is an important mechanism by which these cells regulate the targeting and trafficking of proteins. The identification and detection of palmitoylated substrates can therefore better our understanding of protein trafficking in neurons.Detection of protein palmitoylation in the past has been technically hindered due to the lack of a consensus sequence among substrate proteins, and the reliance on metabolic labeling of palmitoyl-proteins with ³H-palmitate, a time-consuming biochemical assay with low sensitivity. Development of the Acyl-Biotin Exchange (ABE) assay enables more rapid and high sensitivity detection of palmitoylated proteins^2-4, and is optimal for measuring the dynamic turnover of palmitate on neuronal proteins. The ABE assay is comprised of three biochemical steps (Figure 1): 1) irreversible blockade of unmodified cysteine thiol groups using N-ethylmaliemide (NEM), 2) specific cleavage and unmasking of the palmitoylated cysteine''s thiol group by hydroxylamine (HAM), and 3) selective labeling of the palmitoylated cysteine using a thiol-reactive biotinylation reagent, biotin-BMCC. Purification of the thiol-biotinylated proteins following the ABE steps has differed, depending on the overall goal of the experiment.Here, we describe a method to purify a palmitoylated protein of interest in primary hippocampal neurons by an initial immunoprecipitation (IP) step using an antibody directed against the protein, followed by the ABE assay and western blotting to directly measure palmitoylation levels of that protein, which is termed the IP-ABE assay. Low-density cultures of embryonic rat hippocampal neurons have been widely used to study the localization, function, and trafficking of neuronal proteins, making them ideally suited for studying neuronal protein palmitoylation using the IP-ABE assay. The IP-ABE assay mainly requires standard IP and western blotting reagents, and is only limited by the availability of antibodies against the target substrate. This assay can easily be adapted for the purification and detection of transfected palmitoylated proteins in heterologous cell cultures, primary neuronal cultures derived from various brain tissues of both mouse and rat, and even primary brain tissue itself.     

Palmitoylation of virus proteins

The article summarises the results of more than 30 years of research on palmitoylation (S-acylation) of viral proteins, the post-translational attachment of fatty acids to cysteine residues of integral and peripheral membrane proteins. Analysing viral proteins is not only important to characterise the cellular pathogens but also instrumental to decipher the palmitoylation machinery of cells. This comprehensive review describes methods to identify S-acylated proteins and covers the fundamental biochemistry of palmitoylation: the location of palmitoylation sites in viral proteins, the fatty acid species found in S-acylated proteins, the intracellular site of palmitoylation and the enzymology of the reaction. Finally, the functional consequences of palmitoylation are discussed regarding binding of proteins to membranes or membrane rafts, entry of enveloped viruses into target cells by spike-mediated membrane fusion as well as assembly and release of virus particles from infected cells. The topics are described mainly for palmitoylated proteins of influenza virus, but proteins of other important pathogens, such as the causative agents of AIDS and severe acute respiratory syndrome, and of model viruses are discussed.     

A highly conserved cytoplasmic cysteine residue in the α4 nicotinic acetylcholine receptor is palmitoylated and regulates protein expression

Nicotinic acetylcholine receptor (nAChR) cell surface expression levels are modulated during nicotine dependence and multiple disorders of the nervous system, but the mechanisms underlying nAChR trafficking remain unclear. To determine the role of cysteine residues, including their palmitoylation, on neuronal α4 nAChR subunit maturation and cell surface trafficking, the cysteines in the two intracellular regions of the receptor were replaced with serines using site-directed mutagenesis. Palmitoylation is a post-translational modification that regulates membrane receptor trafficking and function. Metabolic labeling with [(3)H]palmitate determined that the cysteine in the cytoplasmic loop between transmembrane domains 1 and 2 (M1-M2) is palmitoylated. When this cysteine is mutated to a serine, producing a depalmitoylated α4 nAChR, total protein expression decreases, but surface expression increases compared with wild-type α4 levels, as determined by Western blotting and enzyme-linked immunoassays, respectively. The cysteines in the M3-M4 cytoplasmic loop do not appear to be palmitoylated, but replacing all of the cysteines in the loop with serines increases total and cell surface expression. When all of the intracellular cysteines in both loops are mutated to serines, there is no change in total expression, but there is an increase in surface expression. Calcium accumulation assays and high affinity binding for [(3)H]epibatidine determined that all mutants retain functional activity. Thus, our results identify a novel palmitoylation site on cysteine 273 in the M1-M2 loop of the α4 nAChR and determine that cysteines in both intracellular loops are regulatory factors in total and cell surface protein expression of the α4β2 nAChR.     

Dual role of the cysteine-string domain in membrane binding and palmitoylation-dependent sorting of the molecular chaperone cysteine-string protein

S-palmitoylation occurs on intracellular membranes and, therefore, membrane anchoring of proteins must precede palmitate transfer. However, a number of palmitoylated proteins lack any obvious membrane targeting motifs and it is unclear how this class of proteins become membrane associated before palmitoylation. Cysteine-string protein (CSP), which is extensively palmitoylated on a "string" of 14 cysteine residues, is an example of such a protein. In this study, we have investigated the mechanisms that govern initial membrane targeting, palmitoylation, and membrane trafficking of CSP. We identified a hydrophobic 31 amino acid domain, which includes the cysteine-string, as a membrane-targeting motif that associates predominantly with endoplasmic reticulum (ER) membranes. Cysteine residues in this domain are not merely sites for the addition of palmitate groups, but play an essential role in membrane recognition before palmitoylation. Membrane association of the cysteine-string domain is not sufficient to trigger palmitoylation, which requires additional downstream residues that may regulate the membrane orientation of the cysteine-string domain. CSP palmitoylation-deficient mutants remain "trapped" in the ER, suggesting that palmitoylation may regulate ER exit and correct intracellular sorting of CSP. These results reveal a dual function of the cysteine-string domain: initial membrane binding and palmitoylation-dependent sorting.     

Physicochemical sequence characteristics that influence S-palmitoylation propensity

Over the past 30 years, several hundred eukaryotic proteins spanning from yeast to man have been shown to be S-palmitoylated. This post-translational modification involves the reversible addition of a 16-carbon saturated fatty acyl chain onto the cysteine residue of a protein where it regulates protein membrane association and distribution, conformation, and stability. However, the large-scale proteome-wide discovery of new palmitoylated proteins has been hindered by the difficulty of identifying a palmitoylation consensus sequence. Using a bioinformatics approach, we show that the enrichment of hydrophobic and basic residues, the cellular context of the protein, and the structural features of the residues surrounding the palmitoylated cysteine all influence the likelihood of palmitoylation. We developed a new palmitoylation predictor that incorporates these identified features, and this predictor achieves a Matthews Correlation Coefficient of .74 using 10-fold cross validation, and significantly outperforms existing predictors on unbiased testing sets. This demonstrates that palmitoylation sites can be predicted with accuracy by taking into account not only physiochemical properties of the modified cysteine and its surrounding residues, but also structural parameters and the subcellular localization of the modified cysteine. This will allow for improved predictions of palmitoylated residues in uncharacterized proteins. A web-based version of this predictor is currently under development.     

Palmitoylation of CCR5 is critical for receptor trafficking and efficient activation of intracellular signaling pathways

CCR5 is a CC chemokine receptor expressed on memory lymphocytes, macrophages, and dendritic cells and also constitutes the main coreceptor for macrophage-tropic (or R5) strains of human immunodeficiency viruses. In the present study, we investigated whether CCR5 was palmitoylated in its carboxyl-terminal domain by generating alanine substitution mutants for the three cysteine residues present in this region, individually or in combination. We found that wild-type CCR5 was palmitoylated, but a mutant lacking all three Cys residues was not. Through the use of green fluorescent fusion proteins and immunofluorescence studies, we found that the absence of receptor palmitoylation resulted in sequestration of CCR5 in intracellular biosynthetic compartments. By using the fluorescence recovery after photobleaching technique, we showed that the non-palmitoylated mutant had impaired diffusion properties within the endoplasmic reticulum. We next studied the ability of the mutants to bind and signal in response to chemokines. Chemokines binding and activation of G(i)-mediated signaling pathways, such as calcium mobilization and inhibition of adenylate cyclase, were not affected. However, the duration of the functional response, as measured by a microphysiometer, and the ability to increase [(35)S]guanosine 5'-3-O-(thio)triphosphate binding to membranes were severely affected for the non-palmitoylated mutant. The ability of RANTES (regulated on activation normal T cell expressed and secreted) and aminooxypentane-RANTES to promote CCR5 endocytosis was not altered by cysteine replacements. Finally, we found that the absence of receptor palmitoylation reduced the human immunodeficiency viruses coreceptor function of CCR5, but this effect was secondary to the reduction in surface expression. In conclusion, we found that palmitoylated cysteines play an important role in the intracellular trafficking of CCR5 and are likely necessary for efficient coupling of the receptor to part of its repertoire of signaling cascades.     

Fluorescence-based methods to image palmitoylated proteins

A well known function of palmitoylation is to promote protein binding to cell membranes. Until recently, it was unclear what additional roles, if any, palmitoylation has in controlling protein localization in cells. Recent studies of palmitoylated forms of the small GTPase Ras have now revealed that palmitoylation plays multiple roles in the regulation of protein trafficking, including targeting proteins into the secretory pathway and recycling proteins between the plasma membrane and Golgi complex. We here describe how quantitative fluorescence microscopy and photobleaching approaches can be used to study the intracellular targeting and trafficking of GFP-tagged palmitoylated proteins in living cells. We discuss (1) general considerations for fluorescence recovery after photobleaching (FRAP) measurements of GFP-tagged proteins; (2) FRAP-based assays to test the strength of binding of palmitoylated proteins to cell membranes; (3) methods to establish the kinetics and mechanisms of recycling of palmitoylated proteins between the Golgi complex and the plasma membrane; (4) the use of the palmitoylation inhibitor 2-bromo-palmitate as a tool to study the dynamic regulation of protein targeting and trafficking by palmitate turnover.     

CD81 associates with 14-3-3 in a redox-regulated palmitoylation-dependent manner

As a member of the tetraspanin superfamily of proteins, CD81 has been linked to a number of biologic functions including cellular proliferation, differentiation, activation, and degranulation. As a co-receptor for hepatitis C virus, and a requirement for hepatocytes for infectivity of human Plasmodium falciparum and rodent P. yoelii sporozoite infectivity, CD81 may also play a vital role in pathology. Despite the importance of CD81 in multiple cellular functions, the molecular mechanism of action of CD81 in these processes has remained elusive. Here we report an association between CD81 and the epsilon isoform of 14-3-3, a serine/threonine-binding intracellular signaling protein. Furthermore, we provide evidence that in human, this association is influenced by the palmitoylation state of the CD81 cytoplasmic tails. We have generated a series of CD81 cysteine mutants to identify palmitoylated intracellular motifs of CD81, and reveal palmitoylation on the N- and C-terminal tails as well as the intracellular loop between transmembrane domains 2 and 3. One of these mutants lacks all five of its intracellular cysteines and therefore cannot be palmitoylated. This unpalmitoylated version of CD81 shows constitutive association with 14-3-3. Interestingly, we find that under oxidative conditions, CD81 palmitoylation is inhibited and that condition correlates with the association of CD81 and 14-3-3. These finding suggest that CD81 signaling events could be mediated by 14-3-3 adapter proteins, and these signals may be dependent on cellular redox.     

Chemical and genetic probes for analysis of protein palmitoylation

Reversible protein palmitoylation is one of the most important posttranslational modifications that has been implicated in the regulation of protein signaling, trafficking, localizing and enzymatic activities in cells and tissues. In order to achieve a precise understanding of mechanisms and functions of protein palmitoylation as well as its roles in physiological processes and disease progression, it is necessary to develop techniques that can qualitatively and quantitatively monitor the dynamic protein palmitoylation in vivo and in vitro. This review will highlight recent advances in both chemical and genetic encoded probes that have been developed for accurate analysis of protein palmitoylation, including identification and quantification of acyl moieties and palmitoylated proteins, localization of amino acid residues on which acyl moieties are attached, and imaging of cellular distributions of palmitoylated proteins. The role of major techniques of fluorescence microscopy and mass spectrometry in facilitating the analysis of protein palmitoylation will also be explored.     

Reversible Cysteine Acylation Regulates the Activity of Human Palmitoyl-Protein Thioesterase 1 (PPT1)

Mutations in the depalmitoylating enzyme gene, PPT1, cause the infantile form of Neuronal Ceroid Lipofuscinosis (NCL), an early onset neurodegenerative disease. During recent years there have been different therapeutic attempts including enzyme replacement. Here we show that PPT1 is palmitoylated in vivo and is a substrate for two palmitoylating enzymes, DHHC3 and DHHC7. The palmitoylated protein is detected in both cell lysates and medium. The presence of PPT1 with palmitoylated signal peptide in the cell medium suggests that a subset of the protein is secreted by a nonconventional mechanism. Using a mutant form of PPT1, C6S, which was not palmitoylated, we further demonstrate that palmitoylation does not affect intracellular localization but rather that the unpalmitoylated form enhanced the depalmitoylation activity of the protein. The calculated Vmax of the enzyme was significantly affected by the palmitoylation, suggesting that the addition of a palmitate group is reminiscent of adding a noncompetitive inhibitor. Thus, we reveal the existence of a positive feedback loop, where palmitoylation of PPT1 results in decreased activity and subsequent elevation in the amount of palmitoylated proteins. This positive feedback loop is likely to initiate a vicious cycle, which will enhance disease progression. The understanding of this process may facilitate enzyme replacement strategies.     

Palmitoylation of the S0-S1 Linker Regulates Cell Surface Expression of Voltage- and Calcium-activated Potassium (BK) Channels

S-Palmitoylation is rapidly emerging as an important post-translational mechanism to regulate ion channels. We have previously demonstrated that large conductance calcium- and voltage-activated potassium (BK) channels are palmitoylated within an alternatively spliced (STREX) insert. However, these studies also revealed that additional site(s) for palmitoylation must exist outside of the STREX insert, although the identity or the functional significance of these palmitoylated cysteine residues are unknown. Here, we demonstrate that BK channels are palmitoylated at a cluster of evolutionary conserved cysteine residues (Cys-53, Cys-54, and Cys-56) within the intracellular linker between the S0 and S1 transmembrane domains. Mutation of Cys-53, Cys-54, and Cys-56 completely abolished palmitoylation of BK channels lacking the STREX insert (ZERO variant). Palmitoylation allows the S0-S1 linker to associate with the plasma membrane but has no effect on single channel conductance or the calcium/voltage sensitivity. Rather, S0-S1 linker palmitoylation is a critical determinant of cell surface expression of BK channels, as steady state surface expression levels are reduced by ∼55% in the C53:54:56A mutant. STREX variant channels that could not be palmitoylated in the S0-S1 linker also displayed significantly reduced cell surface expression even though STREX insert palmitoylation was unaffected. Thus our work reveals the functional independence of two distinct palmitoylation-dependent membrane interaction domains within the same channel protein and demonstrates the critical role of S0-S1 linker palmitoylation in the control of BK channel cell surface expression.     

The inhibitory effect of phospholemman on the sodium pump requires its palmitoylation

Phospholemman (PLM), the principal sarcolemmal substrate for protein kinases A and C in the heart, regulates the cardiac sodium pump. We investigated post-translational modifications of PLM additional to phosphorylation in adult rat ventricular myocytes (ARVM). LC-MS/MS of tryptically digested PLM immunoprecipitated from ARVM identified cysteine 40 as palmitoylated in some peptides, but no information was obtained regarding the palmitoylation status of cysteine 42. PLM palmitoylation was confirmed by immunoprecipitating PLM from ARVM loaded with [(3)H]palmitic acid and immunoblotting following streptavidin affinity purification from ARVM lysates subjected to fatty acyl biotin exchange. Mutagenesis identified both Cys-40 and Cys-42 of PLM as palmitoylated. Phosphorylation of PLM at serine 68 by PKA in ARVM or transiently transfected HEK cells increased its palmitoylation, but PKA activation did not increase the palmitoylation of S68A PLM-YFP in HEK cells. Wild type and unpalmitoylatable PLM-YFP were all correctly targeted to the cell surface membrane, but the half-life of unpalmitoylatable PLM was reduced compared with wild type. In cells stably expressing inducible PLM, PLM expression inhibited the sodium pump, but PLM did not inhibit the sodium pump when palmitoylation was inhibited. Hence, palmitoylation of PLM controls its turnover, and palmitoylated PLM inhibits the sodium pump. Surprisingly, phosphorylation of PLM enhances its palmitoylation, probably through the enhanced mobility of the phosphorylated intracellular domain increasing the accessibility of cysteines for the palmitoylating enzyme, with interesting theoretical implications. All FXYD proteins have conserved intracellular cysteines, so FXYD protein palmitoylation may be a universal means to regulate the sodium pump.     

Use of micellar electrokinetic chromatography to measure palmitoylation of a peptide

Palmitoylation is the thioester linkage of the fatty acid, palmitate (C16:0), to cysteine residues on a protein or peptide. This dynamic and reversible post-translational modification increases the hydrophobicity of proteins/peptides, facilitating protein-membrane interactions, protein-protein interactions and intracellular trafficking of proteins. Manipulation of palmitoylation provides a new mechanism for control over protein location and function, which may lead to better understanding of cell signaling disorders, such as cancer. Unfortunately, few methods exist to quantitatively monitor protein or peptide palmitoylation. In this study, a capillary electrophoresis-based assay was developed, using MEKC, to measure palmitoylation of a fluorescently-labeled peptide in vitro. A fluorescently-labeled peptide derived from the growth-associated protein, GAP-43, was palmitoylated in vitro using palmitoyl coenzyme A. Formation of a doubly palmitoylated GAP-peptide product was confirmed by mass spectrometry. The GAP-peptide substrate was separated from the palmitoylated peptide product in less than 7 min by MEKC. The rate of in vitro palmitoylation with respect to reaction time, GAP-peptide concentration, pH, and inhibitor concentration were also examined. This capillary electrophoresis-based assay for monitoring palmitoylation has applications in biochemical studies of acyltransferases and thioesterases as well as in the screening of acyltransferase and thioesterase inhibitors for drug development.     

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.     

Analysis of protein palmitoylation reveals a pervasive role in Plasmodium development and pathogenesis

Asexual stage Plasmodium falciparum replicates and undergoes a tightly regulated developmental process in human erythrocytes. One mechanism involved in the regulation of this process is posttranslational modification (PTM) of parasite proteins. Palmitoylation is a PTM in which cysteine residues undergo a reversible lipid modification, which can regulate target proteins in diverse ways. Using complementary palmitoyl protein purification approaches and quantitative mass spectrometry, we examined protein palmitoylation in asexual-stage P.?falciparum parasites and identified over 400 palmitoylated proteins, including those involved in cytoadherence, drug resistance, signaling, development, and invasion. Consistent with the prevalence of palmitoylated proteins, palmitoylation is essential for P.?falciparum asexual development and influences erythrocyte invasion by directly regulating the stability of components of the actin-myosin invasion motor. Furthermore, P.?falciparum uses palmitoylation in diverse ways, stably modifying some proteins while dynamically palmitoylating others. Palmitoylation therefore plays a central role in regulating P.?falciparum blood stage development.     

Chemoproteomics reveals Toll-like receptor fatty acylation

Background

Palmitoylation is a 16-carbon lipid post-translational modification that increases protein hydrophobicity. This form of protein fatty acylation is emerging as a critical regulatory modification for multiple aspects of cellular interactions and signaling. Despite recent advances in the development of chemical tools for the rapid identification and visualization of palmitoylated proteins, the palmitoyl proteome has not been fully defined. Here we sought to identify and compare the palmitoylated proteins in murine fibroblasts and dendritic cells.

Results

A total of 563 putative palmitoylation substrates were identified, more than 200 of which have not been previously suggested to be palmitoylated in past proteomic studies. Here we validate the palmitoylation of several new proteins including Toll-like receptors (TLRs) 2, 5 and 10, CD80, CD86, and NEDD4. Palmitoylation of TLR2, which was uniquely identified in dendritic cells, was mapped to a transmembrane domain-proximal cysteine. Inhibition of TLR2 S-palmitoylation pharmacologically or by cysteine mutagenesis led to decreased cell surface expression and a decreased inflammatory response to microbial ligands.

Conclusions

This work identifies many fatty acylated proteins involved in fundamental cellular processes as well as cell type-specific functions, highlighting the value of examining the palmitoyl proteomes of multiple cell types. S-palmitoylation of TLR2 is a previously unknown immunoregulatory mechanism that represents an entirely novel avenue for modulation of TLR2 inflammatory activity.