Confocal microscopy, flow cytometry and cell surface biotinylation analyses demonstrated that palmitoylation is required for efficient cell surface expression of PAR2. We also show that receptor palmitoylation occurs within the Golgi apparatus and is required for efficient agonist-induced rab11a-mediated trafficking of PAR2 to the cell surface. These data provide new insights on the life cycle of PAR2 and demonstrate that palmitoylation is critical for efficient signalling, trafficking, cell surface localization and degradation of this receptor.
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Competing interests: The authors have declared that no competing interests exist. Unlike other GPCRs which are activated by reversible binding of soluble ligand, these receptors are irreversibly activated by proteases; almost exclusively members of the trypsin-fold serine protease family. Proteolytic cleavage within the PAR extracellular amino terminal domain exposes a new amino terminus, or tethered ligand, which binds intramolecularly to induce intracellular signal transduction [1] , [2].
The second PAR discovered, PAR2, is widely expressed and is thought to contribute to a range of normal and disease processes including embryogenesis, pain and nociception, acute and chronic inflammation, arthritis and cancer [3] , [4] , [5] , [6] , [7] , [8].
PAR2 is activated by numerous trypsin-like serine proteases including trypsin, mast cell tryptase, tissue factor complexed with factor VIIa and factor Xa, and kallikrein 4, 5, 6 and 14 [9] , [10] , [11] , [12] , [13]. Due to the irreversible nature of PAR2 activation, rapid mechanisms are required to prevent sustained and excessive receptor signalling.
PAR2 is trafficked via the early and late endosomes and degraded within lysosomes [17] , [18]. A consequence of irreversible activation and rapid desensitisation and degradation, is that large intracellular PAR2 stores are required to rapidly replenish the cell surface with nascent receptors thereby re-establishing the ability of cells to sense proteolytic activity.
Although the mechanisms controlling this process are poorly characterised, it is clear that the GTPase rab11a participates in intracellular trafficking of PAR2 within the Golgi apparatus and toward the plasma membrane [19]. Post translational modifications such as glycosylation, phosphorylation and ubiquitination of PAR2 are critical regulators of PAR2 function [15] , [16] , [20]. Recently, Botham and colleagues have also shown that PAR2 is modified by the post-translation addition of palmitate to cysteine C [21].
Palmitoylation is often dynamic and reversible and occurs commonly for GPCRs on one or more carboxyl terminal cysteines found 10 to 14 residues following the seventh transmembrane domain [22]. In addition to these thioester linkages so called S-palmitoylation , there are a small number of examples where palmitate addition to cysteine is followed by structural rearrangement leading to palmitate modification of an amide N-palmitoylation [23]. It is also proposed to regulate GPCR signalling by modifying the conformation of the carboxyl terminus thereby impacting on G protein coupling [26] , [27] , [28].
Here we report on the role of palmitoylation in regulating cell surface expression of endogenous PAR2 and use stably and transiently expressing cells to explore the function of this modification in receptor trafficking, signalling and degradation. In contrast with Botham and co-workers, who show that palmitoylation deficient PAR2 stably expressed by CHO-Pro5 cells is more highly expressed on the cell surface than wildtype receptor [21] , we demonstrate that palmitoylation of PAR2 is required for efficient plasma membrane receptor expression in 3 different endogenously expressing cell lines.
Our data suggest that S-palmitoylation is required for optimal PAR2 signalling, cellular trafficking and plasma membrane localization. Protease inhibitor cocktail was purchased from Roche Castle Hill, Australia. Protein concentrations were determined using a bicinchoninic acid assay BCA kit from Pierce. A construct encoding PAR2 tagged at the carboxyl terminal with green fluorescent protein GFP was described previously [12].
Lucia, Australia. Cell culture and transfections All cell culture media and reagents were from Invitrogen except for fetal calf serum which was from Sigma Aldrich. All cells were passaged using 0. Cell membrane preparation Crude cell membrane extracts were collected as previously described [32] with some alterations. Swollen cells were mechanically resuspended in membrane buffer 5 mM Tris, pH 7.
Analysis of palmitoylation by acyl-biotinyl exchange chemistry PAR2 palmitoylation was assessed by an acyl-biotinyl exchange approach ABE; [33] described previously [34]. To prevent protein palmitoylation, cells were incubated with 2-bromopalmitate for 16 h prior to collection of crude membrane.
Among these, N-palmitoylation can occur at both the protein N-terminus and the epsilon amino group of specific lysine residues with the palmitate group connected to the protein via an amide bond. S-palmitoylation occurs on cysteine residues, involves a thioester linkage and is a reversible modification. O-palmitoylation involves the attachment of palmitate to serine and threonine residues via an ester bond 7 — Lipid modifications can promote membrane-association to otherwise soluble proteins However, palmitoylation is more than just a simple membrane anchor.
Palmitoylation significantly increases the hydrophobicity of proteins, leading to changes in their conformation, stability, intracellular transport, localization and binding affinity to cofactors 3 , Many proteins require palmitoylation for their correct folding and proper structure.
Mutation of the palmitoylation site may lead to erroneous protein synthesis and ubiquitination-mediated protein degradation Trafficking of membrane-associated proteins from the early secretory site to the appropriate cellular destination is, in many cases, dependent on palmitoylation 14 — Palmitoylation also plays a critical role in regulating protein-protein interactions, which ensures proper signal transduction 17 — Most lipidation events, including N-myristoylation, N- and O-palmitoylation, are essentially irreversible.
In contrast, protein S-palmitoylation is a reversible and dynamically regulated post-translational modification. The labile thioester bond allows proteins to cycle between palmitoylated and de-palmitoylated forms in a time frame of seconds to hours 21 — The reversible nature of S-palmitoylation makes it a significant lipid modification in terms of controlling protein function.
For other excellent recent reviews of S-palmitoylation in immune signaling, the reader is referred to refs. In this review, we describe the process of S-palmitoylation, introduce palmitoyl acyltransferases, acyl protein thioesterases and elaborate on the role of S-palmitoylation in immune regulation. Moreover, the therapeutic potential of targeting S-palmitoylation in immunologic diseases is discussed. Protein Palmitoylation and Depalmitoylation S-palmitoylation refers to the addition of palmitate to cysteine residues.
However, palmitoleate, stearate, and oleate, as well as long-chain polyunsaturated fatty acids such as arachidonate and eicosapentaenoate can also be post-translationally linked to one or more cysteine residues via thioester S-acyl bonds. In this review, palmitoylation refers to the attachment of palmitate and other long-chain fatty acids to cysteine residues via thioester. It is estimated that more than proteins of commonly studied organisms such as human and mice can undergo palmitoylation, and more than palmitoylation has been discovered Dynamic palmitoylation is under the tight control of two opposing types of enzymes.
Palmitoyl acyltransferases PATs catalyze the attachment of palmitoyl groups, while the removal of thioester-linked long-chain fatty acids from cysteine residues is mediated by acyl protein thioesterases APTs 28 , Effector of Ras function 2 Erf2 was the first enzyme identified to catalyze protein palmitoylation. Erf2 is essential for the palmitoylation and proper subcellular localization of Ras proteins.
Deletion of ERF2 resulted in a decrease in Ras2 palmitoylation and plasma membrane association. Six other DHHC-containing proteins, Akr1, Akr2, Swf1, Pfa3, Pfa4, and Pfa5, were then discovered by screening the yeast genome, and most of them catalyze S-palmitoylation 31 — 35 Although the enzymes that catalyze protein palmitoylation were discovered in 30 , the molecular basis of their catalytic activity was not determined for another decade.
In , Mitchell and colleagues examined the molecular mechanism of palmitate transfer of the yeast Ras PAT by combining genetic and biochemical methods, and found that the palmitoylation reaction occurs in a two-step manner consisting of autopalmitoylation of the DHHC containing enzyme to create a palmitoyl-Erf2 intermediate, followed by transfer of the palmitoyl group to Ras2 Figure 1A. Palmitoyl-coenzymeA CoA was found to be the palmitate donor.
It is worth noting that the first step of the catalytic reaction takes place in a matter of seconds. This finding was subsequently confirmed in a series of experiments carried out by Jennings and co-workers who developed a high-performance liquid chromatography HPLC based method for studying the activity of mammalian DHHC2 and 3.
In these experiments, it was shown that Zinc finger DHHC domain-containing protein zDHHC -PATs first acylate themselves in the presence of palmitoyl CoA before the fatty acid group is transferred to a substrate protein, which is consistent with a two-step ping-pong kinetic transfer mechanism A Palmitate from palmitoyl-coenzyme A CoA can be attached to substrate proteins via a thioester linkage by Zinc finger aspartic acid-histidine-histidine-cysteine domain-containing protein zDHHC -palmitoyl acyltransferases PATs.
Following hydrolysis of the thioester bond, the fatty acid is released from the substrate and diffuses into the membrane. Palmitate is the approximate height of one leaflet of the lipid membrane, here it is enlarged to highlight. The palmitoyl transfer reaction occurs on the cysteine residue of the DHHC motif 41 , This cysteine is also involved in coordinating two zinc atoms, which do not play a catalytic role but are responsible for proper folding and stability of zDHHC-PATs 43 , The four TM helices of zDHHC20 adopt a tepee-like organization in the membrane, and the residues lining this cavity determine the acyl-CoA chain length selectivity of the enzyme.
However, as no consensus palmitoylation sequence motifs have yet been identified, the mechanism of zDHHC-PAT substrate specificity is only poorly understood at present This said, the cytoplasmic facing ankyrin-repeat domains of zDHHC13 and 17 are involved in determining the substrate specificity of these enzymes 53 , Some zDHHC-PATs require accessory proteins for their activity, and are essential for protein stability, plasma membrane stabilization and substrate selectivity 55 — For some substrate proteins, an earlier initial lipidation event is required before they can be palmitoylated e.
PPT1 is mainly localized to lysosomes and is critical for the depalmitoylation of proteins during the process of protein degradation In conventional type 1 dendritic cells cDC1s , PPT1 is highly expressed and acts as a protective molecular rheostat in anti-viral immunity. After cDC1s activation, PPT1 is immediately downregulated to promote efficient cross-presentation, costimulatory molecules and inflammatory cytokine production PPT2 is another lysosomal thioesterase that shares a degree of amino acid sequence similarity with PPT1 However, compared to PPT1, PPT2 has a smaller lipid-binding pocket, which contributes to differences in their substrate specificity.
PPT1 preferentially catalyzes the removal of thioester-linked long-chain fatty acids from palmitoylated substrates whereas PPT2 prefers substrate proteins to which palmitoyl-CoA is attached More recently, APT1 has been reported as having thioesterase activity APT1 can self-depalmitoylate at Cys2, and contains a hydrophobic pocket within which it can accept the fatty acids that are to be cleaved off the acylated protein, potentially resulting in its own membrane-cytosol shuttling and steady-state regulatory 71 Figure 1B.
Although APT2 can also catalyze self-depalmitoylation at Cys2, APT1 and 2 have distinct substrate specificity although why this should be is not yet understood 66 , 71 — Although soluble APT2 is vulnerable to proteasomal degradation, membrane binding can protect APT2 from this as well as provide it with sufficient time to encounter its membrane-associated target substrates. Mechanistically, APT2 can associate with membranes in three different ways: through electrostatic attraction, insertion of a hydrophobic loop and through palmitoylation by either zDHHC3 or 7.
Once bound to the membrane, APT2 is predicted to deform the lipid bilayer and trigger extraction of the acyl chain to capture it in its hydrophobic pocket In addition, Kathayat et al. Somewhat surprisingly, the proteomic profiling of dynamic protein palmitoylation revealed that the majority of proteins are not dynamically regulated by APTs at all.
The enzymatically regulated dynamic palmitoylation events exhibit markedly different turnover rates and are targeted to specific proteins with annotated roles in particular in cell growth, migration, and cancer, indicating that serine hydrolase-mediated turnover of protein palmitoylation is not a general phenomenon The ABHD17 family of enzymes have been shown to have potent thioesterase activity as well.
ABHD17 enzymes are mainly found in the plasma membrane and contribute to N-Ras and synaptic protein depalmitoylation It is thought that ABHD17 enzymes are palmitoylated at their N-terminal cysteine cluster, and that this is required for their association with the plasma membrane and interactions with substrates Additionally, ABHD10, a mitochondrial resident protein, was recently shown to have thioesterase activity. Research into the functional properties and depalmitoylation mechanisms of ABHD10 is in its infancy In short, protein depalmitoylation is carried out by an ever increasing diverse range of thioesterase enzymes that have different catalytic properties and substrate specificities.
PATs and APTs interconnect through complex regulatory networks, having crucial implications for the regulation of substrates. Emerging Roles for Palmitoylation in Innate Immunity As the first line of host defense against invading pathogens, innate immunity represents an evolutionary response that enables host survival.
The strategy for pathogen recognition is based on host germline-encoded pattern recognition receptors PRRs identifying unique pathogen-associated molecular patterns PAMPs. Endolysosomes and the cytosol are the two main sites for PAMP detection. These sensors ensure a rapid immune response to low-level stimuli In the last few years, many advances have been made in our knowledge about the role palmitoylation plays in innate immunity.
Here, we provide an overview of recent research progress related to the function of palmitoylation in innate immunity. Palmitoylation and the TLR Signaling Pathway TLRs, the first PRRs to be identified and the main integral membrane PRRs, play a central role in recognition of components derived from a wide range of pathogens and initiation of innate immune responses Furthermore, palmitoylation promotes cell surface localization of TLR2, and likely affects interactions with its ligands.
Whether and how palmitoylation regulates TLR expression, localization and function remains to be addressed. Of note, although the bacterial lipopolysaccharide LPS receptor TLR4 is not palmitoylated, palmitic acid, which binds directly to the TLR4 accessory myeloid differentiation protein 2 MD2 is required to trigger its pro-inflammatory signaling 82 , Further studies are needed to reveal the mechanism and impact of palmitoylation of TLRs on recognizing and resisting pathogens.
Although TLR4 is not palmitoylated, palmitic acid binds to its accessory protein myeloid differentiation protein 2 MD2 to promote pro-inflammatory signaling. TIR domain-containing adapters, such as the myeloid differentiation primary response protein MyD88 , modulate TLR signaling pathways These findings suggest that palmitoylation of TLRs and their adapters is a potent factor regulating the TLR-mediated pro-inflammatory responses.
Palmitoylation and the NLR Signaling Pathway Pathogens that escape endosomal detection and invade the cytosol can be detected by cytosolic sensors. NLR proteins are an important class of cytoplasmic PRRs that play a key role in microbial sensing, thereby triggering antimicrobial immune responses Biologists have been working to establish an accurate model to describe how NOD proteins are recruited to intracellular membranes and mediate ligand sensing.
However, the exact mechanism of zDHHC5 recruitment to the site of bacterial entry is not yet known. This paper elegantly demonstrates the relationship between dynamic palmitoylation and regulation of NLRs in antimicrobial responses. Function of Palmitoylation in Sensing DNA and RNA Since all viruses possess genomes that differ from host nucleic acids and generate additional nucleic acid intermediates during replication, innate immune detection of viruses relies on the nucleic acid sensing system Thus, translocation of STING appears to be critical for its activation but the underlying mechanism is not yet known.
Given that palmitoylation often localizes target proteins to lipid rafts 10 , the authors speculated that STING functions in a similar manner. These findings suggested that palmitoylation is of great importance in the innate immune system response against DNA viruses. Palmitoylation and Interferon-Stimulated Genes Activation of anti-virus PRRs triggers signaling cascades that stimulate the production of IFN-Is which induces the expression of a myriad of interferon-stimulated genes which leads to activation of the adaptive immune system and ultimately clearance of the infection However, there is a paradox here in that although 2-BP treatment severely inhibits IFNAR1 endocytosis, the palmitoylation-deficient IFNAR1 mutant does not show any obvious endocytosis or intracellular distribution abnormalities Interferon-induced transmembrane proteins IFITMs are interferon inducible proteins that restrict viral infection and modulate membrane fluidity , IFITM3 is located on the endosome that fuses with virus particles and enhances the trafficking of this pathogenic cargo to lysosomes.
The membrane proximal cysteine of IFITM3 can be palmitoylated and the palmitoylation site is highly conserved in vertebrates It was initially thought that IFITM3 lysosomal localization and its antiviral activity were differentially regulated by palmitoylation and lysine ubiquitination. However, it is now known that IFITM3 activity is negatively regulated by at least three PTMs, including lysine ubiquitination, lysine methylation, and tyrosine phosphorylation.
Up to now, only palmitoylation has been shown to have a positive effect on the antiviral activity of IFITM3 TNFR1 also undergoes palmitoylation, which is required for localization within the plasma membrane. Palmitoylation and Adaptive Immunity The innate immune system provides rapid sensing and critical elimination of pathogens and leads to activation of the adaptive immune system, which has evolved to provide a broader and more finely tuned repertoire for recognizing and clearing infections.
The adaptive immune system involves tightly regulated interplay between T and B lymphocytes and multiple adaptive immune effectors Below, we discuss the current evidence for the involvement of palmitoylation in adaptive immunity.
Palmitoylation Contributes to the Function of T Cell Co-Receptors Pathogen-derived peptides associated with major histocompatibility complex MHC proteins are recognized by T cell receptors TCRs , which perform essential functions in the initiation of the intracellular signals required for T cell activation and development. TCR engagement triggers recruitment of the co-stimulatory signaling receptors CD8 or CD4, which are transmembrane glycoproteins that increase T cell sensitivity to antigens and bind to the tyrosine kinase Lck as well as conserved regions in the MHC class I or II complexes Despite the structures of CD4 and CD8 being quite distinct, they both contain palmitoylation sites , Two cysteine residues, Cys and Cys, at the juxtamembrane regions of CD4 can be palmitoylated Palmitoylation of CD4 and binding to Lck contributes to its localization to lipid rafts, which is required for CD4-induced lipid raft aggregation, and contributes to the ability of CD4 to enhance receptor tyrosine phosphorylation and CD3 signaling Figure 5.
Similarly, the localization of CD8 to lipid rafts is essential for its co-receptor function. Moreover, unlike what was described for mouse CD8, palmitoylation of human CD8 is not required for targeting to lipid rafts Thus, there is only scarce knowledge about the significance and impact of CD4 and CD8 palmitoylation on T cell signaling. Additionally, the costimulatory molecules CD80 and CD86, which provide a second signal for full T-cell activation, are also palmitoylated in DCs However, whether palmitoylation of these molecules is involved in their trafficking to the DC surface, their subsequent clustering at the immunological synapse and impact on immune function remains to be elucidated.
Obtaining a clearer picture of the possible engagement of these proteins in acquired immune responses remains a challenge for the future. Palmitoylation of CD4 contributes to its partitioning in lipid rafts.
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AION CRYPTO REVIEW 2018
Post-translational modifications Abstract Although palmitoylation regulates numerous cellular processes, as yet efforts to manipulate this post-translational modification for therapeutic gain have proved unsuccessful. Our results suggest that by manipulating the recruitment of specific substrates to particular zDHHC-palmitoyl acyl transferases, the palmitoylation status of individual proteins can be selectively altered, thus opening the door to the development of molecular modulators of protein palmitoylation for the treatment of disease.
Introduction Palmitoylation is the reversible attachment of the fatty acid palmitate to cysteine thiols via a thioester bond 1. Many different classes of protein are palmitoylated, including G-proteins 2 , 3 , 4 , ion channels, transporters and their regulators 5 , 6 , 7 , receptors 8 , as well as kinases 9 , Palmitoylation induces substantial changes in the secondary structure and therefore function of intracellular regions of target proteins through their recruitment to the surface of a membrane bilayer via the palmitoylated cysteine.
Since palmitoylation dynamically and directly regulates the localization, interactions, turnover, and activity of numerous classes of protein 7 , 11 , 12 , 13 , 14 , and can have a profound impact on the landscape of the plasma membrane 15 , 16 , manipulation of protein palmitoylation with, for example, small molecules has the potential to exert substantial changes to cellular function.
Protein palmitoylation is catalyzed by a family of zinc finger and DHHC motif containing palmitoyl acyltransferase enzymes zDHHC-PATs , reversed by protein thioesterases, and occurs dynamically and reversibly throughout the secretory pathway in a manner analogous to phosphorylation They typically have four transmembrane TM domains, with a conserved ca.
In contrast, the intracellular amino and carboxyl termini are poorly conserved, and contribute to zDHHC isoform substrate selectivity The acyltransferase zDHHC5 is primarily located at the cell surface 19 where it regulates a diverse range of physiological processes.
Unlike kinases, for which there are an ever-increasing number of inhibitors approved for use in the clinic 26 , 27 , no therapeutically useful molecular modulators of protein palmitoylation have been developed thus far. This, in no small part, is due to a fundamental lack of knowledge regarding the molecular basis of both enzyme catalysis and substrate recruitment by zDHHC-PATs.
However, the palmitoylation status of specific proteins may also be changed by selectively manipulating their recruitment to a cognate zDHHC-PAT s. The ubiquitous Na-K ATPase Na-pump is a P-type ATPase and is located at the surface of eukaryotic cells where it carries out the active export of 3 sodium Na and import of 2 potassium K ions per catalytic cycle across the plasma membrane.
The SERCA 2a regulators phospholamban 33 and sarcolipin 34 are both palmitoylated within their transmembrane domains suggesting that palmitoylation may play an important general role in regulating P-type ATPase activity. In short, we show that the palmitoylation status of a specific substrate protein can be modified through controlling its interaction with a partner zDHHC-PAT.
The amphipathic helix is flanked at its N-terminus by two cysteine residues C, C Fig. Surprisingly, the position of the Na-pump binding site on zDHHC5 was to be found a considerable distance from the enzyme active site Fig. Each lysate was split in half and two separate purifications performed using the NBSP or an antibody specific to PLM phosphorylated at serine Full size image To establish which Na-pump sub-unit zDHHC5 interacts with, the NBSP was used to purify the Na-pump from whole rat heart lysates either as an intact complex or as individual subunits following dissociation of the pump using harsh detergents.
Biotinylated NBSP was added to one half of each lysate and binding partners captured with streptavidin sepharose beads. An antibody specific to PLM phosphorylated at serine 68 which captures PLM as part of the Na-pump complex 38 was added to the other half of each lysate, and interacting proteins purified using protein G sepharose beads.
Mutation of cysteine was essentially without effect, suggesting that this residue is not palmitoylated. The catalytically inactive enzyme had essentially identical palmitoylation in both cell lines, suggesting that the zDHHC5 dicysteine motif is palmitoylated by an enzyme other than zDHHC5 Fig. This employs a mutant RG form of the bacterial biotin ligase BirA that releases a highly reactive, labile reaction intermediate which biotinylates primary amines on nearby interacting proteins Next, we wanted to ascertain whether or not PLM palmitoylation could be reduced with a pharmacological tool by blocking the interaction between zDHHC5 and the Na-pump.
Following both 3 and 24 h treatments, PLM palmitoylation was significantly reduced in cells treated with stearate-tagged NBSP compared to an equivalent scrambled control Fig. In contrast, the disruptor peptide had no effect on H-ras palmitoylation, which was expected as H-ras is a substrate for zDHHC9 The disruptor peptide also reduced the palmitoylation of endogenous PLM but not Caveolin-3 in rat ventricular myocytes Fig.
In short, our results suggest that PLM palmitoylation can be selectively reduced using a pharmacological tool that prevents the Na-pump from interacting with zDHHC5. Full size image Discussion Although protein S-palmitoylation is a critical regulator of cellular function, the molecular mechanism by which substrates are recognized and enzyme catalysis occurs is only poorly understood.
Hence we demonstrate the selective pharmacological targeting of a palmitoylated protein by interfering with its recruitment to a zDHHC-PAT. When we carried out a detailed proteomic analysis of the proteins in cardiac muscle that interact with NBSP, however, neither Golga7 nor Golga7b were identified Therefore, if an adapter protein is required to mediate the interaction between zDHHC5 and the Na-pump in the heart, it is unlikely to be either Golga7 or 7b but rather an as yet unidentified protein of novel function.
A major finding of our work is the observation that covalent modification both palmitoylation and GlcNAcylation of residues proximal to a substrate-binding site on a zDHHC-PAT affected both the recruitment and palmitoylation of that substrate—a phenomenon that will likely apply to many other zDHHC-PATs and enzyme-substrate combinations. Regulation of substrate recruitment by palmitoylation of cysteine residues in the zDHHC5 C-tail has previously been postulated by Yang and co-workers 37 , and is now confirmed here.
Palmitoylation of the ubiquitin ligase GobX by zDHHC20 is also dependent on an amphipathic helix proximal to the palmitoylated cysteine In contrast, the zDHHC20 palmitoylation sites in the epidermal growth factor receptor lie in an unstructured part of its C tail Based on the cross-validation performance of each feature, the single feature with the best performance was incorporated along with other single features to enhance predictive power.
Detection of substrate motifs by maximal dependence decomposition Previous studies [ 1 , 15 ] have reported that S-palmitoylation can be catalyzed by palmitoyltransferases PATs , which are composed of 23 PAT enzymes defined by the presence of an aspartate-histidine-histidine-cysteine DHHC motif.
Although DHHC enzymes display substrate specificity, a substrate can be palmitoylated by one or more enzymes; e. As the number of experimentally identified S-palmitoylation peptides has increased, the investigation of substrate motifs to facilitate the study of protein S-palmitoylation is becoming imperative. Although a number of tools for predicting S-palmitoylation sites have been developed, their ability to identify S-palmitoylated sites and their corresponding substrate motifs is limited.
Thus, the aim of this study was to explore motif signatures of protein S-palmitoylation based on the amino acids surrounding substrate sites. Maximal dependence decomposition MDD [ 44 ] was utilized to cluster all fragment sequences into subgroups in order to detect the statistically conserved motifs among large-scale sequence data.
The clustering method was performed using MDDLogo [ 23 ], which demonstrated the effectiveness of dividing a group of protein sequences into smaller subgroups before the computational identification of PTM sites [ 31 , 45 — 55 ]. Based on the biochemical properties of amino acids, the 20 amino acids were categorized into 5 groups: the polar, acidic, basic, hydrophobic, and aromatic groups S1 Table.
A contingency table describes the frequency of existence of twenty amino acids in positions Ai and Aj. If a strong dependence was discovered described as a X2 value greater than After the recursive chi-square test, MDDLogo divides a group of aligned sequences into subsets that capture the most significant dependencies of positions on each other. When applying MDDLogo, a parameter, i.
If the size of a subgroup is less than the specified value of maximum cluster size, the subgroup will not be divided any further. MDDLogo will be terminated when all the subgroup sizes are less than the specified value of maximum cluster size [ 23 ]. Construction of predictive model In this study, we employed a support vector machine to build predictive models for discriminating S-palmitoylation sites and non-S-palmitoylation sites in a training dataset.
Based on a binary classification, a kernel function transformed the input samples into a higher dimensional space and then found a hyper-plane to discriminate between the two classes with maximal margin and minimal error. Two supporting factors that enhance performance are gamma and cost.
The RBF kernel is determined by the gamma parameter, while the cost parameter controls the hyper-plane softness.
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