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.     

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.     

DHHC protein S-acyltransferases use similar ping-pong kinetic mechanisms but display different acyl-CoA specificities

DHHC proteins catalyze the reversible S-acylation of proteins at cysteine residues, a modification important for regulating protein localization, stability, and activity. However, little is known about the kinetic mechanism of DHHC proteins. A high-performance liquid chromatography (HPLC), fluorescent peptide-based assay for protein S-acylation activity was developed to characterize mammalian DHHC2 and DHHC3. Time courses and substrate saturation curves allowed the determination of V(max) and K(m) values for both the peptide N-myristoylated-GCG and palmitoyl-coenzyme A. DHHC proteins acylate themselves upon incubation with palmitoyl-CoA, which is hypothesized to reflect a transient acyl enzyme transfer intermediate. Single turnover assays with DHHC2 and DHHC3 demonstrated that a radiolabeled acyl group on the enzyme transferred to the protein substrate, consistent with a two-step ping-pong mechanism. Enzyme autoacylation and acyltransfer to substrate displayed the same acyl-CoA specificities, further supporting a two-step mechanism. Interestingly, DHHC2 efficiently transferred acyl chains 14 carbons and longer, whereas DHHC3 activity was greatly reduced by acyl-CoAs with chain lengths longer than 16 carbons. The rate and extent of autoacylation of DHHC3, as well as the rate of acyl chain transfer to protein substrate, were reduced with stearoyl-CoA when compared with palmitoyl-CoA. This is the first observation of lipid substrate specificity among DHHC proteins and may account for the differential S-acylation of proteins observed in cells.     

Extent of N-terminal modifications in cytosolic proteins from eukaryotes

Most proteins in all organisms undergo crucial N-terminal modifications involving N-terminal methionine excision, N-alpha-acetylation or N-myristoylation (N-Myr), or S-palmitoylation. We investigated the occurrence of these poorly annotated but essential modifications in proteomes, focusing on eukaryotes. Experimental data for the N-terminal sequences of animal, fungi, and archaeal proteins, were used to build dedicated predictive modules in a new software. In vitro N-Myr experiments were performed with both plant and animal N-myristoyltransferases, for accurate prediction of the modification. N-terminal modifications from the fully sequenced genome of Arabidopsis thaliana were determined by MS. We identified 105 new modified protein N-termini, which were used to check the accuracy of predictive data. An accuracy of more than 95% was achieved, demonstrating (i) overall conservation of the specificity of the modification machinery in higher eukaryotes and (ii) robustness of the prediction tool. Predictions were made for various proteomes. Proteins that had undergone both N-terminal methionine (Met) cleavage and N-acetylation were found to be strongly overrepresented among the most abundant proteins, in contrast to those retaining their genuine unblocked Met. Here we propose that the nature of the second residue of an ORF is a key marker of the abundance of the mature protein in eukaryotes.     

Thematic review series: lipid posttranslational modifications. Lysosomal metabolism of lipid-modified proteins

Much is now understood concerning the synthesis of prenylated and palmitoylated proteins, but what is known of their metabolic fate? This review details metabolic pathways for the lysosomal degradation of S-fatty acylated and prenylated proteins. Central to these pathways are two lysosomal enzymes, palmitoyl-protein thioesterase (PPT1) and prenylcysteine lyase (PCL). PPT1 is a soluble lipase that cleaves fatty acids from cysteine residues in proteins during lysosomal protein degradation. Notably, deficiency in the enzyme causes a neurodegenerative lysosomal storage disorder, infantile neuronal ceroid lipofuscinosis. PCL is a membrane-associated flavin-containing lysosomal monooxygenase that metabolizes prenylcysteine to prenyl aldehyde through a completely novel mechanism. The eventual metabolic fates of other lipidated proteins (such as glycosylphosphatidylinositol-anchored and N-myristoylated proteins) are poorly understood, suggesting directions for future research.     

Intracellular localization and tissue-specific distribution of human and yeast DHHC cysteine-rich domain-containing proteins

Increasing evidence indicates that DHHC cysteine-rich domain-containing proteins (DHHC proteins) are protein acyltransferases. Although multiple DHHC proteins are found in eukaryotes, characterization has been examined for only a few. Here, we have cloned all the yeast and human DHHC genes and investigated their intracellular localization and tissue-specific expression. Most DHHC proteins are localized in the ER and/or Golgi, with a few localized in the plasma membrane and one in the yeast vacuole. Human DHHC mRNAs also differ in their tissue-specific expression. These results may provide clues to aid in discovering the specific function(s) of each DHHC protein.     

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.     

Regulation of apoptosis by Bcl-2 family proteins

For multicellular organisms, the rigorous control of programmed cell death is as important as that of cell proliferation. The mechanisms involved in the regulation of cell death are not yet understood, but a key component is the family of caspases which are activated in a cascade and are responsible for the apoptotic-specific changes and disassembly of the cell. Although the caspases represent a central point in apoptosis, their activation is regulated by a variety of other factors. Among these, Bcl-2 family plays a pivotal role in caspases activation, by this deciding whether a cell will live or die. Bcl-2 family members are known to focus much of their response to the mitochondria level, upstream the irreversible cellular damage, but their functions are not yet well defined. This review summarizes the recent data regarding the Bcl-2 proteins and the ways they regulate the apoptosis.     

鹅掌楸DHHC型锌指蛋白家族基因的克隆及表达分析

棕榈酰化修饰是一种最普遍且唯一可逆的翻译后脂质修饰方式,赋予蛋白质多样化的生理功能。DHHC( Asp-His-His-Cys)蛋白家族是一类与棕榈酰化修饰相关的蛋白,多数DHHC蛋白家族成员具有蛋白质酰基转移酶( protein S-acyltransferase,PAT)活性。该研究以鹅掌楸叶芽为材料,采用RT-PCR和RACE技术,克隆获得了3个鹅掌楸DHHC蛋白家族基因cDNA全长,命名为LcPAT7、LcPAT22、LcPAT23。序列分析结果表明：LcPAT7、LcPAT22、LcPAT23基因全长分别为1933、2592、2217 bp,各包含1332、1839、1662 bp的开放阅读框( Open Reading Frame,ORF),编码433、612、533个氨基酸,预测蛋白分子量分别为40.04、67.3、60.57 kDa,理论等电点为9.15、9.03、7.29。3个基因编码的蛋白均有4个跨膜区,并且都在跨膜域( transmembrane domain, TM) TM2和 TM3之间存在一个 DHHC 蛋白家族典型的 DHHC-CRD 结构域。同源性分析表明：鹅掌楸LcPAT7、LcPAT22、LcPAT23编码的氨基酸序列与其他植物中预测的PAT具有较高的相似性。利用荧光定量PCR技术检测3个基因在鹅掌楸不同组织中的表达特性,发现3个基因在不同组织中均有表达,但表达量具有明显区别。同一家族基因表达模式的变化表明其功能非冗余。该研究结果将为鹅掌楸生长发育与形态建成,以及逆境响应信号传导等相关基因的调控研究提供了参考。     

Stilbenecarboxylate biosynthesis: a new function in the family of chalcone synthase-related proteins

Chalcone (CHS), stilbene (STS) synthases, and related proteins are key enzymes in the biosynthesis of many secondary plant products. Precursor feeding studies and mechanistic rationalization suggest that stilbenecarboxylates might also be synthesized by plant type III polyketide synthases; however, the enzyme activity leading to retention of the carboxyl moiety in a stilbene backbone has not yet been demonstrated. Hydrangea macrophylla L. (Garden Hortensia) contains stilbenecarboxylates (hydrangeic acid and lunularic acid) that are derived from 4-coumaroyl and dihydro-4-coumaroyl starter residues, respectively. We used homology-based techniques to clone CHS-related sequences, and the enzyme functions were investigated with recombinant proteins. Sequences for two proteins were obtained. One was identified as CHS. The other shared 65-70% identity with CHSs and other family members. The purified recombinant protein had stilbenecarboxylate synthase (STCS) activity with dihydro-4-coumaroyl-CoA, but not with 4-coumaroyl-CoA or other substrates. We propose that the enzyme is involved in the biosynthesis of lunularic acid. It is the first example of a STS-type reaction that does not lose the terminal carboxyl group during the ring folding to the end product. Comparisons with CHS, STS, and a pyrone synthase showed that it is the only enzyme exerting a tight control over decarboxylation reactions. The protein contains unusual residues in positions highly conserved in other CHS-related proteins, and mutagenesis studies suggest that they are important for the structure or/and the catalytic activity. The formation of the natural products in vivo requires a reducing step, and we discuss the possibility that the absence of a reductase in the in vitro reactions may be responsible for the failure to obtain stilbenecarboxylates from substrates like 4-coumaroyl-CoA.     

Identification of dually acylated proteins from complementary DNA resources by cell-free and cellular metabolic labeling

To establish a strategy to identify dually fatty acylated proteins from cDNA resources, seven N-myristoylated proteins with cysteine (Cys) residues within the 10 N-terminal residues were selected as potential candidates among 27 N-myristoylated proteins identified from a model human cDNA resource. Seven proteins C-terminally tagged with FLAG tag or EGFP were generated and their susceptibility to protein N-myristoylation and S-palmitoylation were evaluated by metabolic labeling with [³H]myristic acid or [³H]palmitic acid either in an insect cell-free protein synthesis system or in transfected mammalian cells. As a result, EEPD1, one of five proteins (RFTN1, EEPD1, GNAI1, PDE2A, RNF11) found to be dually acylated, was shown to be a novel dually fatty acylated protein. Metabolic labeling experiments using G2A and C7S mutants of EEPD1-EGFP revealed that the palmitoylation site of EEPD1 is Cys at position 7. Analysis of the intracellular localization of EEPD1 C-terminally tagged with FLAG tag or EGFP and its G2A and C7S mutants revealed that the dual acylation directs EEPD1 to localize to the plasma membrane. Thus, dually fatty acylated proteins can be identified from cDNA resources by cell-free and cellular metabolic labeling of N-myristoylated proteins with Cys residue(s) close to the N-myristoylated N-terminus.     

Oligomerization of DHHC Protein S-Acyltransferases

The formation of dimers or higher-order oligomers is a property of numerous integral membrane proteins, including ion channels, transporters, and receptors. In this study, we examined whether members of the DHHC-S-acyltransferase family oligomerize in intact cells and in vitro. DHHC-S-acyltransferases are integral membrane proteins that catalyze the addition of palmitate to cysteine residues on proteins at the cytoplasmic face of cell membranes. Bioluminescence resonance energy transfer (BRET) experiments revealed that DHHC2 or DHHC3 (Golgi-specific DHHC zinc finger protein (GODZ)) self-associate when expressed in HEK-293 cells. Homomultimer formation was confirmed by coimmunoprecipitation. Purified DHHC3 resolved predominately as a monomer and dimer on blue native polyacrylamide gels. In intact cells and in vitro, catalytically inactive DHHC proteins displayed a greater propensity to form dimers. BRET signals were higher for the catalytically inactive DHHC2 or DHHC3 than their wild-type counterparts. DHHC3 BRET in cell membranes was decreased by the addition of its lipid substrate palmitoyl-CoA, a treatment that results in autoacylation of the enzyme. Enzyme activity of a covalently linked DHHC3 dimer was less than that of the monomeric form, suggesting that enzyme activity may be modulated by the oligomerization status of the protein.     

Establishing cell polarity by the Lgl family proteins

The lethal giant larvae (lgl) gene was first identified more than 30 years ago in Drosophila and characterized as a tumor suppressor gene. Studies in budding yeast, flies and mammals all indicate that the evolutionarily conserved Lgl family proteins play an important role in cell polarity. Sro7/77, the yeast Lgl homologues, are important for the establishment and reinforcement of cell polarity through their localized interaction and kinetic activation of the post-Golgi secretion machinery. As for higher eukaryotes, both in epithelial polarity and asymmetric cell division, the role of Lgl protein is deployed by localizing proteins to the membrane in a polarized fashion. In addition, Lgl is transiently required during the establishment phase of polarity, implicating that Lgl functions at strategic time points for proliferation control. Studies in cancer biology provide direct connections between malfunction of Lgl and formation, progression and metastasis of various cancers. Here, we review recent advances in the field, focusing on the function of the Lgl family in cellular polarization.     

DHHC protein-dependent palmitoylation protects regulator of G-protein signaling 4 from proteasome degradation

Regulator of G-protein signaling 4 (RGS4), an intracellular modulator of G-protein coupled receptor (GPCR)-mediated signaling, is regulated by multiple processes including palmitoylation and proteasome degradation. We found that co-expression of DHHC acyltransferases (DHHC3 or DHHC7), but not their acyltransferase-inactive mutants, increased expression levels of RGS4 but not its Cys2 to Ser mutant (RGS4C2S). DHHC3 interacts with and palmitoylates RGS4 but not RGS4C2S in vivo. Palmitoylation prolongs the half-life of RGS4 by over 8-fold and palmitoylated RGS4 blocked α_1A-adrenergic receptor-stimulated intracellular Ca²⁺ mobilization. Together, our findings revealed that DHHC proteins could regulate GPCR-mediated signaling by increasing RGS4 stability.

Structured summary

MINT-8049215: Rgs4 (uniprotkb:P49799) physically interacts (MI:0915) with DHHC3 (uniprotkb:Q8R173) by anti-tag coimmunoprecipitation (MI:0007)     

Bcl2 family proteins in carcinogenesis and the treatment of cancer

Deregulation of Bcl2 family members is a frequent feature of human malignant diseases and causal for therapy resistance. A number of studies have recently shed light onto the role of pro- and anti-apoptotic Bcl2 family members in tumour-pathogenesis and in mediating the effects of classical as well as novel front-line anticancer agents, allowing the development of more efficient and more precisely targeted treatment regimens. Most excitingly, recent progress in our understanding of how Bcl2-like proteins maintain or perturb mitochondrial integrity has finally enabled the development of rational-design based anticancer therapies that directly target Bcl2 regulated events at the level of mitochondria. This review aims to give an overview on the most recent findings on the role of the Bcl2 family in tumour development in model systems of cancer, to relate these findings with observations made in human pathologies and drug-action.     

Bcl-2 family of proteins: life-or-death switch in mitochondria

An increase in the permeability of outer mitochondrial membrane is central to apoptotic cell death, and results in the release of several apoptogenic factors such as cytochrome c into the cytoplasm to activate downstream destructive programs. The voltage-dependent anion channel (VDAC or mitochondrial porin) plays an essential role in disrupting the mitochondrial membrane barrier and is regulated directly by members of the Bcl-2 family proteins. Anti-apoptotic Bcl-2 family members interact with and close the VDAC, whereas some, but not all, proapoptotic members interact with VDAC to open protein-conducting pore through which apoptogenic factors pass. Although the VDAC is involved directly in breaking the mitochondrial membrane barrier and is a known component of the permeability transition pore complex, VDAC-dependent increase in outer membrane permeability can be independent of the permeability transition event such as mitochondrial swelling followed by rupture of the outer mitochondrial membrane. VDAC interacts not only with Bcl-2 family members but also with proteins such as gelsolin, an actin regulatory protein, and appears to be a convergence point for a variety of cell survival and cell death signals.     

Regulation of ceramide channels by Bcl-2 family proteins

Mitochondrial outer membrane permeabilization to proteins, an irreversible step in apoptosis by which critical proteins are released, is tightly regulated by Bcl-2 family proteins. The exact nature of the release pathway is still undefined. Ceramide is an important sphingolipid, involved in various cellular processes including apoptosis. Here we describe the structural properties of ceramide channels and their regulation by the anti-apoptotic and pro-apoptotic proteins of the Bcl-2 family. The evolutionarily conserved regulation of ceramide channels by Bcl-2 family proteins, consistent with their role in apoptosis, lends credibility to the notion that ceramide channels constitute the protein release pathway.