DHHC palmitoyl transferases: substrate interactions and (patho)physiology

S-palmitoylation is a reversible post-translational modification that occurs on diverse cellular proteins. Palmitoylation can affect proteins in many different ways, including regulating membrane attachment, intracellular trafficking, and membrane micro-localisation. Intracellular palmitoylation reactions are mediated by a family of recently identified aspartate-histidine-histidine-cysteine (DHHC) palmitoyl transferases. More than 20 DHHC proteins are encoded by mammalian genomes, and there is now a major effort to identify DHHC-substrate pairings and to determine how interaction specificity is encoded. Recent studies have highlighted how DHHC proteins regulate cell function and influence physiology and pathophysiology.     

Protein lipidation

Proteins are covalently modified with a variety of lipids, including fatty acids, isoprenoids, and cholesterol. Lipid modifications play important roles in the localization and function of proteins. The focus of this review is S-palmitoylation, the reversible addition of palmitate and other long-chain fatty acids to proteins at cysteine residues in a variety of sequence contexts. The functional consequences of palmitoylation are diverse. Palmitoylation facilitates the association of proteins with membranes, mediates protein trafficking, and more recently has been appreciated as a regulator of protein stability. Members of a family of integral membrane proteins that harbor a DHHC cysteine-rich domain mediate most cellular palmitoylation events. Here we focus on DHHC proteins that modify Ras proteins in yeast and mammalian cells.     

Identification of Giardia lamblia DHHC Proteins and the Role of Protein S-palmitoylation in the Encystation Process

Protein S-palmitoylation, a hydrophobic post-translational modification, is performed by protein acyltransferases that have a common DHHC Cys-rich domain (DHHC proteins), and provides a regulatory switch for protein membrane association. In this work, we analyzed the presence of DHHC proteins in the protozoa parasite Giardia lamblia and the function of the reversible S-palmitoylation of proteins during parasite differentiation into cyst. Two specific events were observed: encysting cells displayed a larger amount of palmitoylated proteins, and parasites treated with palmitoylation inhibitors produced a reduced number of mature cysts. With bioinformatics tools, we found nine DHHC proteins, potential protein acyltransferases, in the Giardia proteome. These proteins displayed a conserved structure when compared to different organisms and are distributed in different monophyletic clades. Although all Giardia DHHC proteins were found to be present in trophozoites and encysting cells, these proteins showed a different intracellular localization in trophozoites and seemed to be differently involved in the encystation process when they were overexpressed. dhhc transgenic parasites showed a different pattern of cyst wall protein expression and yielded different amounts of mature cysts when they were induced to encyst. Our findings disclosed some important issues regarding the role of DHHC proteins and palmitoylation during Giardia encystation.     

棕榈酰化蛋白及蛋白质的棕榈酰化研究进展

蛋白质S-棕榈酰化是最常见的具有16碳脂肪酸棕榈酸酯的脂质修饰形式,调节蛋白质的运输和功能。文中主要概括从植物到哺乳动物中发现的具有棕榈酰基转移酶活性的保守DHHC蛋白家族,并介绍蛋白质棕榈酰化的研究方法,及检测棕榈酰化蛋白质的位点预测方法(CSS-Palm、NBA-Palm、TermiNator2)、放射性标记法(用3H棕榈酸酯或125I-IC16棕榈酸酯)和非放射性标记法(化学标记和质谱法),总结蛋白棕榈酰化的抑制技术以及抑制剂类型(包括2-溴棕榈酸酯、浅蓝菌素和衣霉素)。同时概括蛋白棕榈酰化在植物胁迫中的响应,展望其在植物抗逆中的应用前景。     

Systematic screening for palmitoyl transferase activity of the DHHC protein family in mammalian cells

Posttranslational modifications, including phosphorylation, ubiquitination and lipid modifications, provide proteins with additional functions and regulation beyond genomic information. Palmitoylation is a reversible lipid modification with palmitic acid that plays critical roles in protein trafficking and function. However, the enzymes that mediate palmitoyl acyl transferase (PAT) have been elusive. Recent genetic analysis in yeast revealed that members of cysteine-rich DHHC domain containing proteins (DHHC proteins) mediate palmitoylation. In mammalian genomes, 23 DHHC proteins are predicted raising the possibility of a large family of PAT enzymes. Here, we describe a systematic method to examine which of the DHHC family members is responsible for palmitoylation of a substrate.     

Dynamic protein palmitoylation in cellular signaling

Protein S-palmitoylation, the most common lipid modification with the 16-carbon fatty acid palmitate, provides an important mechanism for regulating protein trafficking and function. The unique reversibility of protein palmitoylation allows proteins to rapidly shuttle between intracellular membrane compartments. Importantly, this palmitate cycling can be regulated by some physiological stimuli, contributing to cellular homeostasis and plasticity. Although the enzyme responsible for protein palmitoylation had been long elusive, DHHC family proteins, conserved from plants to mammals, have recently emerged as palmitoyl acyl transferases. Integrated approaches including advanced proteomics, live-cell imaging, and molecular genetics are beginning to clarify the molecular machinery for palmitoylation reaction in diverse aspects of cellular functions.     

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.     

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.     

The fat controller: roles of palmitoylation in intracellular protein trafficking and targeting to membrane microdomains (Review)

The attachment of palmitic acid to the amino acid cysteine via thioester linkage (S-palmitoylation) is a common post-translational modification of eukaryotic proteins. In this review, we discuss the role of palmitoylation as a versatile protein sorting signal, regulating protein trafficking between distinct intracellular compartments and the micro-localization of proteins within membranes.     

Plasmodium falciparum: characterization of a late asexual stage golgi protein containing both ankyrin and DHHC domains

Proteins containing the DHHC motif have been shown to function as palmitoyl transferases. The palmitoylation of proteins has been shown to play an important role in the trafficking of proteins to the proper subcellular location. Herein, we describe a protein containing both ankyrin domains and a DHHC domain that is present in the Golgi of late schizonts of P. falciparum. The timing of expression as well as the location of this protein suggests that it may play an important role in the sorting of proteins to the apical organelles during the development of the asexual stage of the parasite.     

Protein palmitoylation by a family of DHHC protein S-acyltransferases

Protein palmitoylation refers to the posttranslational addition of a 16 carbon fatty acid to the side chain of cysteine, forming a thioester linkage. This acyl modification is readily reversible, providing a potential regulatory mechanism to mediate protein-membrane interactions and subcellular trafficking of proteins. The mechanism that underlies the transfer of palmitate or other long-chain fatty acids to protein was uncovered through genetic screens in yeast. Two related S-palmitoyltransferases were discovered. Erf2 palmitoylates yeast Ras proteins, whereas Akr1 modifies the yeast casein kinase, Yck2. Erf2 and Akr1 share a common sequence referred to as a DHHC (aspartate-histidine-histidine-cysteine) domain. Numerous genes encoding DHHC domain proteins are found in all eukaryotic genome databases. Mounting evidence is consistent with this signature motif playing a direct role in protein acyltransferase (PAT) reactions, although many questions remain. This review presents the genetic and biochemical evidence for the PAT activity of DHHC proteins and discusses the mechanism of protein-mediated palmitoylation.     

Identification of PSD-95 palmitoylating enzymes

Palmitoylation is a lipid modification that plays a critical role in protein trafficking and function throughout the nervous system. Palmitoylation of PSD-95 is essential for its regulation of AMPA receptors and synaptic plasticity. The enzymes that mediate palmitoyl acyl transfer to PSD-95 have not yet been identified; however, proteins containing a DHHC cysteine-rich domain mediate palmitoyl acyl transferase activity in yeast. Here, we isolated 23 mammalian DHHC proteins and found that a subset specifically palmitoylated PSD-95 in vitro and in vivo. These PSD-95 palmitoyl transferases (P-PATs) showed substrate specificity, as they did not all enhance palmitoylation of Lck, SNAP-25b, Galpha(s), or H-Ras in cultured cells. Inhibition of P-PAT activity in neurons reduced palmitoylation and synaptic clustering of PSD-95 and diminished AMPA receptor-mediated neurotransmission. This study suggests that P-PATs regulate synaptic function through PSD-95 palmitoylation.     

Subcellular Golgi localization of stathmin family proteins is promoted by a specific set of DHHC palmitoyl transferases

Protein palmitoylation is a reversible lipid modification that plays critical roles in protein sorting and targeting to specific cellular compartments. The neuronal microtubule-regulatory phosphoproteins of the stathmin family (SCG10/stathmin 2, SCLIP/stathmin 3, and RB3/stathmin 4) are peripheral proteins that fulfill specific and complementary roles in the formation and maturation of the nervous system. All neuronal stathmins are localized at the Golgi complex and at vesicles along axons and dendrites. Their membrane anchoring results from palmitoylation of two close cysteine residues present within their homologous N-terminal targeting domains. By preventing palmitoylation with 2-bromopalmitate or disrupting the integrity of the Golgi with brefeldin A, we were able to show that palmitoylation of stathmins 2 and 3 likely occurs at the Golgi and is crucial for their specific subcellular localization and trafficking. In addition, this membrane binding is promoted by a specific set of palmitoyl transferases that localize with stathmins 2 and 3 at the Golgi, directly interact with them, and enhance their membrane association. The subcellular membrane-associated microtubule-regulatory activity of stathmins might then be fine-tuned by extracellular stimuli controlling their reversible palmitoylation, which can be viewed as a crucial regulatory process for specific and local functions of stathmins in neurons.     

TRPC5 channel instability induced by depalmitoylation protects striatal neurons against oxidative stress in Huntington's disease

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, and it enhances the membrane stability of ion channels. This post-translational modification (PTM) determines a functional mechanism of ion channel life cycle from maturation and membrane trafficking to localization. Especially, neurodevelopment is regulated by balancing the level of synaptic protein palmitoylation/depalmitoylation. Recently, we revealed the pathological role of the transient receptor potential canonical type 5 (TRPC5) channel in striatal neuronal loss during Huntington's disease (HD), which is abnormally activated by oxidative stress. Here, we report a mechanism of TRPC5 palmitoylation at a conserved cysteine residue, that is critical for intrinsic channel activity. Furthermore, we identified the therapeutic effect of TRPC5 depalmitoylation by enhancing the TRPC5 membrane instability on HD striatal cells in order to lower TRPC5 toxicity.Collectively, these findings suggest that controlling S-palmitoylation of the TRPC5 channel as a potential risk factor can modulate TRPC5 channel expression and activity, providing new insights into a therapeutic strategy for neurodegenerative diseases.     

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.     

The palmitoyl transferase DHHC2 targets a dynamic membrane cycling pathway: regulation by a C-terminal domain

Intracellular palmitoylation dynamics are regulated by a large family of DHHC (Asp-His-His-Cys) palmitoyl transferases. The majority of DHHC proteins associate with endoplasmic reticulum (ER) or Golgi membranes, but an interesting exception is DHHC2, which localizes to dendritic vesicles of unknown origin in neurons, where it regulates dynamic palmitoylation of PSD95. Dendritic targeting of newly synthesized PSD95 is likely preceded by palmitoylation on Golgi membranes by DHHC3 and/or DHHC15. The precise intracellular distribution of DHHC2 is presently unclear, and there is very little known in general about how DHHC proteins achieve their respective localizations. In this study, membrane targeting of DHHC2 in live and fixed neuroendocrine cells was investigated and mutational analysis employed to define regions of DHHC2 that regulate targeting. We report that DHHC2 associates with the plasma membrane, Rab11-positive recycling endosomes, and vesicular structures. Plasma membrane integration of DHHC2 was confirmed by labeling of an extrafacial HA epitope in nonpermeabilized cells. Antibody-uptake experiments suggested that DHHC2 traffics between the plasma membrane and intracellular membranes. This dynamic localization was confirmed using fluorescence recovery after photo-bleaching analysis, which revealed constitutive refilling of the recycling endosome (RE) pool of DHHC2. The cytoplasmic C-terminus of DHHC2 regulates membrane targeting and a mutant lacking this domain was associated with the ER. Although DHHC2 is closely related to DHHC15, these proteins populate distinct membrane compartments. Construction of chimeric DHHC2/DHHC15 proteins revealed that this difference in localization is a consequence of divergent sequences within their C-terminal tails. This study is the first to highlight dynamic cycling of a mammalian DHHC protein between clearly defined membrane compartments, and to identify domains that specify membrane targeting of this protein family.     

Global Analysis of Apicomplexan Protein S‐Acyl Transferases Reveals an Enzyme Essential for Invasion

The advent of techniques to study palmitoylation on a whole proteome scale has revealed that it is an important reversible modification that plays a role in regulating multiple biological processes. Palmitoylation can control the affinity of a protein for lipid membranes, which allows it to impact protein trafficking, stability, folding, signalling and interactions. The publication of the palmitome of the schizont stage of Plasmodium falciparum implicated a role for palmitoylation in host cell invasion, protein export and organelle biogenesis. However, nothing is known so far about the repertoire of protein S‐acyl transferases (PATs) that catalyse this modification in Apicomplexa. We undertook a comprehensive analysis of the repertoire of Asp‐His‐His‐Cys cysteine‐rich domain (DHHC‐CRD) PAT family in Toxoplasma gondii and Plasmodium berghei by assessing their localization and essentiality. Unlike functional redundancies reported in other eukaryotes, some apicomplexan‐specific DHHCs are essential for parasite growth, and several are targeted to organelles unique to this phylum. Of particular interest is DHHC7, which localizes to rhoptry organelles in all parasites tested, including the major human pathogen P. falciparum. TgDHHC7 interferes with the localization of the rhoptry palmitoylated protein TgARO and affects the apical positioning of the rhoptry organelles. This PAT has a major impact on T. gondii host cell invasion, but not on the parasite's ability to egress.     

Emerging roles for protein S-palmitoylation in Toxoplasma biology

Post-translational modifications are refined, rapidly responsive and powerful ways to modulate protein function. Among post-translational modifications, acylation is now emerging as a widespread modification exploited by eukaryotes, bacteria and viruses to control biological processes. Protein palmitoylation involves the attachment of palmitic acid, also known as hexadecanoic acid, to cysteine residues of integral and peripheral membrane proteins and increases their affinity for membranes. Importantly, similar to phosphorylation, palmitoylation is reversible and is becoming recognised as instrumental for the regulation of protein function by modulating protein interactions, stability, folding, trafficking and signalling. Palmitoylation appears to play a central role in the biology of the Apicomplexa, regulating critical processes such as host cell invasion which is vital for parasite survival and dissemination. The recent identification of over 400 palmitoylated proteins in Plasmodium falciparum erythrocytic stages illustrates the broad spread and impact of this modification on parasite biology. The main enzymes responsible for protein palmitoylation are multi-membrane protein S-acyl transferases harbouring a catalytic Asp-His-His-Cys (DHHC) motif. A global functional analysis of the repertoire of protein S-acyl transferases in Toxoplasma gondii and Plasmodium berghei has recently been performed. The essential nature of some of these enzymes illustrates the key roles played by this post-translational modification in the corresponding substrates implicated in fundamental processes such as parasite motility and organelle biogenesis. Toward a better understanding of the depalmitoylation event, a protein with palmitoyl protein thioesterase activity has been identified in T. gondii. TgPPT1/TgASH1 is the main target of specific acyl protein thioesterase inhibitors but is dispensable for parasite survival, suggesting the implication of other genes in depalmitoylation. Palmitoylation/depalmitoylation cycles are now emerging as potential novel regulatory networks and T. gondii represents a superb model organism in which to explore their significance.     

Modulation of neuronal protein trafficking and function by palmitoylation

Modification of proteins with the lipid palmitate regulates targeting to specific vesicular compartments and synaptic membranes. Mounting evidence indicates that this lipid modification modulates diverse aspects of neuronal development and synaptic transmission. In particular, palmitoylation regulates the function of proteins that control neuronal differentiation, axonal pathfinding and filopodia formation. In addition, trafficking of numerous proteins associated with synaptic vesicle release machinery requires protein palmitoylation. Remarkably, reversible palmitoylation of specific scaffolding proteins and signaling molecules dynamically regulates ion channel clustering and synaptic strength. The recent discovery of enzymes that palmitoylate specific subsets of synaptic proteins suggests that this process is tightly controlled in neurons.     

Palmitoylation and membrane interactions of the neuroprotective chaperone cysteine-string protein

Cysteine-string protein (CSP) is an extensively palmitoylated DnaJ-family chaperone, which exerts an important neuroprotective function. Palmitoylation is required for the intracellular sorting and function of CSP, and thus it is important to understand how this essential modification of CSP is regulated. Recent work identified 23 putative palmitoyl transferases containing a conserved DHHC domain in mammalian cells, and here we show that palmitoylation of CSP is enhanced specifically by co-expression of the Golgi-localized palmitoyl transferases DHHC3, DHHC7, DHHC15, or DHHC17. Indeed, these DHHC proteins promote stable membrane attachment of CSP, which is otherwise cytosolic. An inverse correlation was identified between membrane affinity of unpalmitoylated CSP mutants and subsequent palmitoylation: mutants with an increased membrane affinity localize to the endoplasmic reticulum (ER) and are physically separated from the Golgi-localized DHHC proteins. Palmitoylation of an ER-localized mutant could be rescued by brefeldin A treatment, which promotes the mixing of ER and Golgi membranes. Interestingly though, the palmitoylated mutant remained at the ER following brefeldin A washout and did not traffic to more distal membrane compartments. We propose that CSP has a weak membrane affinity that allows the protein to locate its partner Golgi-localized DHHC proteins directly by membrane "sampling." Mutations that enhance membrane association prevent sampling and lead to accumulation of CSP on cellular membranes such as the ER. The coupling of CSP palmitoylation to Golgi membranes may thus be an important requirement for subsequent sorting.