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Luke H Chamberlain - One of the best experts on this subject based on the ideXlab platform.
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the linker domain of the snare protein SNAP25 acts as a flexible molecular spacer that ensures efficient s acylation
Journal of Biological Chemistry, 2020Co-Authors: Christine Salaun, Jennifer Greaves, Nicholas C O Tomkinson, Luke H ChamberlainAbstract:S-Acylation of the SNARE protein SNAP25 (synaptosome-associated protein of 25 kDa) is mediated by a subset of Golgi zinc finger DHHC-type palmitoyltransferase (zDHHC) enzymes, particularly zDHHC17. The ankyrin repeat domain of zDHHC17 interacts with a short linear motif known as the zDHHC ankyrin repeat-binding motif (zDABM) in SNAP25 (112VVASQP117), which is downstream of its S-acylated, cysteine-rich domain (85CGLCVCPC92). Here, we investigated the importance of a flexible linker region (amino acids 93-111, referred to hereafter as the "mini-linker" region) that separates the zDABM and S-acylated cysteines in SNAP25. Shortening the mini-linker did not affect the SNAP25-zDHHC17 interaction but blocked S-acylation. Insertion of additional flexible glycine-serine repeats had no effect on S-acylation, but extended and rigid alanine-proline repeats perturbed it. A SNAP25 mutant in which the mini-linker region was substituted with a flexible glycine-serine linker of the same length underwent efficient S-acylation. Furthermore, this mutant displayed the same intracellular localization as WT SNAP25, indicating that the amino acid composition of the mini-linker is not important for SNAP25 localization. Using the results of previous peptide array experiments, we generated a SNAP25 mutant predicted to have a higher-affinity zDABM. This mutant interacted with zDHHC17 more strongly but was S-acylated with reduced efficiency in HEK293T cells, implying that a lower-affinity interaction of the SNAP25 zDABM with zDHHC17 is optimal for S-acylation efficiency. These results show that amino acids 93-111 in SNAP25 act as a flexible molecular spacer that ensures efficient coupling of the SNAP25-zDHHC17 interaction and S-acylation of SNAP25.
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the linker domain of SNAP25 acts as a flexible molecular spacer to ensure efficient s acylation
bioRxiv, 2020Co-Authors: Christine Salaun, Jennifer Greaves, Nicholas C O Tomkinson, Luke H ChamberlainAbstract:ABSTRACT S-Acylation of the SNARE protein SNAP25 is mediated by a subset of Golgi zDHHC enzymes, in particular zDHHC17. The ankyrin repeat (ANK) domain of this enzyme interacts with a short linear motif known as the zDHHC ANK binding motif (zDABM) in SNAP25 (112-VVASQP-117), which is downstream of the S-acylated cysteine-rich domain (85-CGLCVCPC-92). In this study, we have investigated the importance of the flexible linker (amino acids 93-111; referred to as the “mini-linker” region) that separates the zDABM and S-acylated cysteines. Shortening the mini-linker had no effect of zDHHC17 interaction but blocked S-acylation. Insertion of additional flexible glycine-serine repeats had no effect on S-acylation, whereas extended and rigid alanine-proline repeats perturbed this process. Indeed, a SNAP25 mutant in which the mini-linker region was substituted with a flexible glycine-serine linker of the same length underwent efficient S-acylation. Furthermore, this mutant displayed the same intracellular localisation as wild-type SNAP25, showing that the sequence of the mini-linker is not important in this context. By using the results of previous peptide array experiments, we generated a SNAP25 mutant predicted to have a higher affinity zDABM, and this mutant showed enhanced interaction with zDHHC17 in cells. Interestingly, this mutant was S-acylated with reduced efficiency, implying that a lower affinity interaction of the SNAP25 zDABM with zDHHC17 is optimal for S-acylation efficiency. Overall, the results of this study show that amino acids 93-111 in SNAP25 act as a flexible molecular spacer to ensure efficient coupling of enzyme-substrate interaction and S-acylation.
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identification of a novel sequence motif recognized by the ankyrin repeat domain of zdhhc17 13 s acyltransferases
Journal of Biological Chemistry, 2015Co-Authors: Kimon Lemonidis, Maria C Sanchezperez, Luke H ChamberlainAbstract:S-Acylation is a major post-translational modification affecting several cellular processes. It is particularly important for neuronal functions. This modification is catalyzed by a family of transmembrane S-acyltransferases that contain a conserved zinc finger DHHC (zDHHC) domain. Typically, eukaryote genomes encode for 7–24 distinct zDHHC enzymes, with two members also harboring an ankyrin repeat (AR) domain at their cytosolic N termini. The AR domain of zDHHC enzymes is predicted to engage in numerous interactions and facilitates both substrate recruitment and S-acylation-independent functions; however, the sequence/structural features recognized by this module remain unknown. The two mammalian AR-containing S-acyltransferases are the Golgi-localized zDHHC17 and zDHHC13, also known as Huntingtin-interacting proteins 14 and 14-like, respectively; they are highly expressed in brain, and their loss in mice leads to neuropathological deficits that are reminiscent of Huntington's disease. Here, we report that zDHHC17 and zDHHC13 recognize, via their AR domain, evolutionary conserved and closely related sequences of a [VIAP][VIT]XXQP consensus in SNAP25, SNAP23, cysteine string protein, Huntingtin, cytoplasmic linker protein 3, and microtubule-associated protein 6. This novel AR-binding sequence motif is found in regions predicted to be unstructured and is present in a number of zDHHC17 substrates and zDHHC17/13-interacting S-acylated proteins. This is the first study to identify a motif recognized by AR-containing zDHHCs.
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regional and developmental brain expression patterns of SNAP25 splice variants
BMC Neuroscience, 2011Co-Authors: Gerald R Prescott, Luke H ChamberlainAbstract:Background: SNAP25 is an essential SNARE protein for regulated exocytosis in neuronal cells. Differential splicing of the SNAP25 gene results in the expression of two transcripts, SNAP25a and SNAP25b. These splice variants differ by only 9 amino acids, and studies of their expression to date have been limited to analysis of the corresponding mRNAs. Although these studies have been highly informative, it is possible that factors such as differential turnover of the SNAP25 proteins could complicate interpretations based entirely on mRNA expression profiles. Results: We report the generation and characterization of antibodies that distinguish between SNAP25a and SNAP25b isoforms, and their use to investigate the expression profile of these proteins in rat and human brain. In rat brain, SNAP25b protein expression increased dramatically during post-natal development, whereas the increase in SNAP25a expression was more modest and variable. The extent of this up-regulation in SNAP25b expression was similar across cortex, cerebellum and hippocampus. The SNAP25 isoforms also displayed distinct regional expression patterns, with SNAP25a very weakly expressed in both rat and human cerebellum. Quantitative analysis revealed that SNAP25b was the dominant isoform in all adult human brain regions examined. Conclusions: SNAP25a and SNAP25b display distinct developmental and regional expression profiles in rat and human brain. These differences might reflect distinct functions of these highly conserved isoforms in membrane fusion pathways in the brain. The antibodies generated and characterized in this study represent important tools for future analyses of these essential SNARE protein isoforms.
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differential palmitoylation regulates intracellular patterning of SNAP25
Journal of Cell Science, 2011Co-Authors: Jennifer Greaves, Luke H ChamberlainAbstract:SNAP25 regulates membrane fusion events at the plasma membrane and in the endosomal system, and a functional pool of the protein is delivered to recycling endosomes (REs) and the trans Golgi network (TGN) through an ARF6-dependent cycling pathway. SNAP25 is a peripheral membrane protein, and palmitoylation of a cluster of four cysteine residues mediates its stable association with the membrane. Here, we report that palmitoylation also determines the precise intracellular distribution of SNAP25, and that mutating single palmitoylation sites enhances the amount of SNAP25 at the RE and TGN. The farnesylated CAAX motif from Hras was ligated onto a SNAP25 mutant truncated immediately distal to the cysteine-rich domain. This construct displayed the same intracellular distribution as full-length SNAP25, and decreasing the number of cysteine residues in this construct increased its association with the RE and TGN, confirming the dominant role of the cysteine-rich domain in directing the intracellular distribution of SNAP25. Marked differences in the localisations of SNAP25-CAAX and Hras constructs, each with two palmitoylation sites, were observed, showing that subtle differences in palmitoylated sequences can have a major impact upon intracellular targeting. We propose that the cysteine-rich domain of SNAP25 is designed to facilitate the dual function of this SNARE protein at the plasma membrane and endosomes, and that dynamic palmitoylation acts as a mechanism to regulate the precise intracellular patterning of SNAP25.
Christine Salaun - One of the best experts on this subject based on the ideXlab platform.
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the linker domain of the snare protein SNAP25 acts as a flexible molecular spacer that ensures efficient s acylation
Journal of Biological Chemistry, 2020Co-Authors: Christine Salaun, Jennifer Greaves, Nicholas C O Tomkinson, Luke H ChamberlainAbstract:S-Acylation of the SNARE protein SNAP25 (synaptosome-associated protein of 25 kDa) is mediated by a subset of Golgi zinc finger DHHC-type palmitoyltransferase (zDHHC) enzymes, particularly zDHHC17. The ankyrin repeat domain of zDHHC17 interacts with a short linear motif known as the zDHHC ankyrin repeat-binding motif (zDABM) in SNAP25 (112VVASQP117), which is downstream of its S-acylated, cysteine-rich domain (85CGLCVCPC92). Here, we investigated the importance of a flexible linker region (amino acids 93-111, referred to hereafter as the "mini-linker" region) that separates the zDABM and S-acylated cysteines in SNAP25. Shortening the mini-linker did not affect the SNAP25-zDHHC17 interaction but blocked S-acylation. Insertion of additional flexible glycine-serine repeats had no effect on S-acylation, but extended and rigid alanine-proline repeats perturbed it. A SNAP25 mutant in which the mini-linker region was substituted with a flexible glycine-serine linker of the same length underwent efficient S-acylation. Furthermore, this mutant displayed the same intracellular localization as WT SNAP25, indicating that the amino acid composition of the mini-linker is not important for SNAP25 localization. Using the results of previous peptide array experiments, we generated a SNAP25 mutant predicted to have a higher-affinity zDABM. This mutant interacted with zDHHC17 more strongly but was S-acylated with reduced efficiency in HEK293T cells, implying that a lower-affinity interaction of the SNAP25 zDABM with zDHHC17 is optimal for S-acylation efficiency. These results show that amino acids 93-111 in SNAP25 act as a flexible molecular spacer that ensures efficient coupling of the SNAP25-zDHHC17 interaction and S-acylation of SNAP25.
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the linker domain of SNAP25 acts as a flexible molecular spacer to ensure efficient s acylation
bioRxiv, 2020Co-Authors: Christine Salaun, Jennifer Greaves, Nicholas C O Tomkinson, Luke H ChamberlainAbstract:ABSTRACT S-Acylation of the SNARE protein SNAP25 is mediated by a subset of Golgi zDHHC enzymes, in particular zDHHC17. The ankyrin repeat (ANK) domain of this enzyme interacts with a short linear motif known as the zDHHC ANK binding motif (zDABM) in SNAP25 (112-VVASQP-117), which is downstream of the S-acylated cysteine-rich domain (85-CGLCVCPC-92). In this study, we have investigated the importance of the flexible linker (amino acids 93-111; referred to as the “mini-linker” region) that separates the zDABM and S-acylated cysteines. Shortening the mini-linker had no effect of zDHHC17 interaction but blocked S-acylation. Insertion of additional flexible glycine-serine repeats had no effect on S-acylation, whereas extended and rigid alanine-proline repeats perturbed this process. Indeed, a SNAP25 mutant in which the mini-linker region was substituted with a flexible glycine-serine linker of the same length underwent efficient S-acylation. Furthermore, this mutant displayed the same intracellular localisation as wild-type SNAP25, showing that the sequence of the mini-linker is not important in this context. By using the results of previous peptide array experiments, we generated a SNAP25 mutant predicted to have a higher affinity zDABM, and this mutant showed enhanced interaction with zDHHC17 in cells. Interestingly, this mutant was S-acylated with reduced efficiency, implying that a lower affinity interaction of the SNAP25 zDABM with zDHHC17 is optimal for S-acylation efficiency. Overall, the results of this study show that amino acids 93-111 in SNAP25 act as a flexible molecular spacer to ensure efficient coupling of enzyme-substrate interaction and S-acylation.
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palmitoylation of the SNAP25 protein family specificity and regulation by dhhc palmitoyl transferases
Journal of Biological Chemistry, 2010Co-Authors: Jennifer Greaves, Christine Salaun, Oforiwa A Gorleku, Luke H ChamberlainAbstract:SNAP25 plays an essential role in neuronal exocytosis pathways. SNAP25a and SNAP25b are alternatively spliced isoforms differing by only nine amino acids, three of which occur within the palmitoylated cysteine-rich domain. SNAP23 is 60% identical to SNAP25 and has a distinct cysteine-rich domain to both SNAP25a and SNAP25b. Despite the conspicuous differences within the palmitoylated domains of these secretory proteins, there is no information on their comparative interactions with palmitoyl transferases. We report that membrane association of all SNAP25/23 proteins is enhanced by Golgi-localized DHHC3, DHHC7, and DHHC17. In contrast, DHHC15 promoted a statistically significant increase in membrane association of only SNAP25b. To investigate the underlying cause of this differential specificity, we examined a SNAP23 point mutant (C79F) designed to mimic the cysteine-rich domain of SNAP25b. DHHC15 promoted a marked increase in membrane binding and palmitoylation of this SNAP23 mutant, demonstrating that the distinct cysteine-rich domains of SNAP25/23 contribute to differential interactions with DHHC15. The lack of activity of DHHC15 toward wild-type SNAP23 was not overcome by replacing its DHHC domain with that from DHHC3, suggesting that substrate specificity is not determined by the DHHC domain alone. Interestingly, DHHC2, which is closely related to DHHC15, associates with the plasma membrane in PC12 cells and can palmitoylate all SNAP25 isoforms. DHHC2 is, thus, a candidate enzyme to regulate SNAP25/23 palmitoylation dynamics at the plasma membrane. Finally, we demonstrate that overexpression of specific Golgi-localized DHHC proteins active against SNAP25/23 proteins perturbs the normal secretion of human growth hormone from PC12 cells.
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the hydrophobic cysteine rich domain of SNAP25 couples with downstream residues to mediate membrane interactions and recognition by dhhc palmitoyl transferases
Molecular Biology of the Cell, 2009Co-Authors: Jennifer Greaves, Christine Salaun, Gerald R Prescott, Yuko Fukata, Masaki Fukata, Luke H ChamberlainAbstract:SNAP25 is synthesized as a soluble protein but must associate with the plasma membrane to function in exocytosis; however, this membrane-targeting pathway is poorly defined. SNAP25 contains a palmitoylated cysteine-rich domain with four cysteines, and we show that coexpression of specific DHHC palmitoyl transferases is sufficient to promote SNAP25 membrane association in HEK293 cells. siRNA-mediated knockdown of its SNARE partner, syntaxin 1A, does not affect membrane interaction of SNAP25 in PC12 cells, whereas specific cysteine-to-alanine mutations perturb membrane binding, which is restored by leucine substitutions. These results suggest a role for cysteine hydrophobicity in initial membrane interactions of SNAP25, and indeed other hydrophobic residues in the cysteine-rich domain are also important for membrane binding. In addition to the cysteine-rich domain, proline-117 is also essential for SNAP25 membrane binding, and experiments in HEK293 cells revealed that mutation of this residue inhibits membrane binding induced by coexpression with DHHC17, but not DHHC3 or DHHC7. These results suggest a model whereby SNAP25 interacts autonomously with membranes via its hydrophobic cysteine-rich domain, requiring only sufficient expression of partner DHHC proteins for stable membrane binding. The role of proline-117 in SNAP25 palmitoylation is one of the first descriptions of elements within substrate proteins that modulate DHHC specificity.
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lipid raft association of snare proteins regulates exocytosis in pc12 cells
Journal of Biological Chemistry, 2005Co-Authors: Christine Salaun, Gwyn W Gould, Luke H ChamberlainAbstract:SNAP25 and SNAP23 are plasma membrane SNARE proteins essential for regulated exocytosis in diverse cell types. Several recent studies have shown that these proteins are partly localized in lipid rafts, domains of the plasma membrane enriched in sphingolipids, and cholesterol. Here, we have employed cysteine mutants of SNAP25/SNAP23, which have modified affinities for raft domains, to examine whether raft association of these proteins is important for the regulation of exocytosis. PC12 cells were engineered that express the light chain of botulinum neurotoxin; in these cells all of the SNAP25 was cleaved to a lower molecular weight form, and regulated exocytosis was essentially absent. Exocytosis was rescued by expressing toxin-resistant SNAP25 or wild-type SNAP23, which is naturally toxin-resistant. Remarkably, a mutant SNAP25 protein with an increased affinity for rafts displayed a reduced ability to support exocytosis, whereas SNAP23 mutants with a decreased affinity for rafts displayed an enhancement of exocytosis when compared with wild-type SNAP23. The effects of the mutant proteins on exocytosis were dependent upon the integrity of the plasma membrane and lipid rafts. These results provide the first direct evidence that rafts regulate SNARE function and exocytosis and identify the central cysteine-rich region of SNAP25/23 as an important regulatory domain.
Vladimir Parpura - One of the best experts on this subject based on the ideXlab platform.
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Temporal characteristics of vesicular fusion in astrocytes: examination of synaptobrevin 2-laden vesicles at single vesicle resolution
The Journal of Physiology, 2011Co-Authors: Erik B Malarkey, Vladimir ParpuraAbstract:Non-technical summary Astrocytes have been shown to release transmitters by vesicle fusion, in a manner similar to that of neuronal exocytosis. The details of this process in astrocytes are not well understood, so we used a fluorescently labelled vesicle protein, synapto-pHluorin (spH), to track how these fusions occurred. When astrocytes were mechanically stimulated we saw a slow burst of fusions, while other stimuli caused a relatively even sustained rate of fusion. We observed two distinct types of events, transient and full fusions, the proportion of which was stimulus dependent. Similarly, stability of the vesicle fusion pore with the plasma membrane varied with the stimulus. We describe the effects on fusion events resulting from expressing variants of exocytotic proteins, synaptotagmin 1 and SNAP25B. Studying the characteristics of astrocytic exocytosis will aid in the general understanding of this process and also events at the tripartite synapse, both in health and disease. Abstract Astrocytes can release various gliotransmitters in response to stimuli that cause increases in intracellular Ca2+ levels; this secretion occurs via a regulated exocytosis pathway. Indeed, astrocytes express protein components of the vesicular secretory apparatus. However, the detailed temporal characteristics of vesicular fusions in astrocytes are not well understood. In order to start addressing this issue, we used total internal reflection fluorescence microscopy (TIRFM) to visualize vesicular fusion events in astrocytes expressing the fluorescent synaptobrevin 2 derivative, synapto-pHluorin. Although our cultured astrocytes from visual cortex express synaptosome-associated protein of 23 kDa (SNAP23), but not of 25 kDa (SNAP25), these glial cells exhibited a slow burst of exocytosis under mechanical stimulation; the expression of SNAP25B did not affect bursting behaviour. The relative amount of two distinct types of events observed, transient and full fusions, depended on the applied stimulus. Expression of exogenous synaptotagmin 1 (Syt1) in astrocytes endogenously expressing Syt4, led to a greater proportion of transient fusions when astrocytes were stimulated with bradykinin, a stimulus otherwise resulting in more full fusions. Additionally, we studied the stability of the transient fusion pore by measuring its dwell time, relation to vesicular size, flickering and decay slope; all of these characteristics were secretagogue dependent. The expression of SNAP25B or Syt1 had complex effects on transient fusion pore stability in a stimulus-specific manner. SNAP25B obliterated the appearance of flickers and reduced the dwell time when astrocytes were mechanically stimulated, while astrocytes expressing SNAP25B and stimulated with bradykinin had a reduction in decay slope. Syt1 reduced the dwell time when astrocytes were stimulated either mechanically or with bradykinin. Our detailed study of temporal characteristics of astrocytic exocytosis will not only aid the general understanding of this process, but also the interpretation of the events at the tripartite synapse, both in health and disease.
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single molecule measurements of mechanical interactions within ternary snare complexes and dynamics of their disassembly SNAP25 vs snap23
The Journal of Physiology, 2009Co-Authors: Vedrana Montana, Wei Liu, U Mohideen, Vladimir ParpuraAbstract:Regulated exocytosis is a crucial event for intercellular communication between neurons and astrocytes within the CNS. The soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor (SNARE) complex, composed of synaptobrevin 2, syntaxin and synaptosome-associated protein of 25 kDa or 23 kDa (SNAP25 or SNAP23), is essential in this process. It was reported that SNAP25 and SNAP23 have distinct roles in exocytotic release, where SNAP25, but not SNAP23, supports an exocytotic burst. It is not clear, however, whether this is due to the intrinsic properties of the ternary SNARE complex, containing either SNAP25 or SNAP23, or perhaps due to the differential association of these proteins with ancillary proteins to the complex. Here, using force spectroscopy, we show from single molecule investigations of the SNARE complex, that SNAP23A created a local interaction at the ionic layer by cuffing syntaxin 1A and synaptobrevin 2, similar to the action of SNAP25B; thus either of the ternary complexes would allow positioning of vesicles at a maximal distance of ∼13 nm from the plasma membrane. However, the stability of the ternary SNARE complex containing SNAP23A is less than half of that for the complex containing SNAP25B. Thus, differences in the stability of the two different ternary complexes could underlie some of the SNAP25/23 differential ability to control the exocytotic burst.
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single molecule mechanical probing of the snare protein interactions
Biophysical Journal, 2006Co-Authors: Wei Liu, Vedrana Montana, Jihong Bai, Edwin R Chapman, U Mohideen, Vladimir ParpuraAbstract:Exocytotic release of neurotransmitters is mediated by the ternary soluble N-ethyl maleimide-sensitive fusion protein attachment protein receptors (SNAREs) complex, comprised of syntaxin (Sx), synaptosome-associated protein of 25 kDa (SNAP25), and synaptobrevin 2 (Sb2). Since exocytosis involves the nonequilibrium process of association and dissociation of bonds between molecules of the SNARE complex, dynamic measurements at the single molecule level are necessary for a detailed understanding of these interactions. To address this issue, we used the atomic force microscope in force spectroscopy mode to show from single molecule investigations of the SNARE complex, that Sx1A and Sb2 are zippered throughout their entire SNARE domains without the involvement of SNAP25. When SNAP25B is present in the complex, it creates a local interaction at the 0 (ionic) layer by cuffing Sx1A and Sb2. Force loading rate studies indicate that the ternary complex interaction is more stable than the Sx1A-Sb2 interaction.
Jennifer Greaves - One of the best experts on this subject based on the ideXlab platform.
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the linker domain of the snare protein SNAP25 acts as a flexible molecular spacer that ensures efficient s acylation
Journal of Biological Chemistry, 2020Co-Authors: Christine Salaun, Jennifer Greaves, Nicholas C O Tomkinson, Luke H ChamberlainAbstract:S-Acylation of the SNARE protein SNAP25 (synaptosome-associated protein of 25 kDa) is mediated by a subset of Golgi zinc finger DHHC-type palmitoyltransferase (zDHHC) enzymes, particularly zDHHC17. The ankyrin repeat domain of zDHHC17 interacts with a short linear motif known as the zDHHC ankyrin repeat-binding motif (zDABM) in SNAP25 (112VVASQP117), which is downstream of its S-acylated, cysteine-rich domain (85CGLCVCPC92). Here, we investigated the importance of a flexible linker region (amino acids 93-111, referred to hereafter as the "mini-linker" region) that separates the zDABM and S-acylated cysteines in SNAP25. Shortening the mini-linker did not affect the SNAP25-zDHHC17 interaction but blocked S-acylation. Insertion of additional flexible glycine-serine repeats had no effect on S-acylation, but extended and rigid alanine-proline repeats perturbed it. A SNAP25 mutant in which the mini-linker region was substituted with a flexible glycine-serine linker of the same length underwent efficient S-acylation. Furthermore, this mutant displayed the same intracellular localization as WT SNAP25, indicating that the amino acid composition of the mini-linker is not important for SNAP25 localization. Using the results of previous peptide array experiments, we generated a SNAP25 mutant predicted to have a higher-affinity zDABM. This mutant interacted with zDHHC17 more strongly but was S-acylated with reduced efficiency in HEK293T cells, implying that a lower-affinity interaction of the SNAP25 zDABM with zDHHC17 is optimal for S-acylation efficiency. These results show that amino acids 93-111 in SNAP25 act as a flexible molecular spacer that ensures efficient coupling of the SNAP25-zDHHC17 interaction and S-acylation of SNAP25.
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the linker domain of SNAP25 acts as a flexible molecular spacer to ensure efficient s acylation
bioRxiv, 2020Co-Authors: Christine Salaun, Jennifer Greaves, Nicholas C O Tomkinson, Luke H ChamberlainAbstract:ABSTRACT S-Acylation of the SNARE protein SNAP25 is mediated by a subset of Golgi zDHHC enzymes, in particular zDHHC17. The ankyrin repeat (ANK) domain of this enzyme interacts with a short linear motif known as the zDHHC ANK binding motif (zDABM) in SNAP25 (112-VVASQP-117), which is downstream of the S-acylated cysteine-rich domain (85-CGLCVCPC-92). In this study, we have investigated the importance of the flexible linker (amino acids 93-111; referred to as the “mini-linker” region) that separates the zDABM and S-acylated cysteines. Shortening the mini-linker had no effect of zDHHC17 interaction but blocked S-acylation. Insertion of additional flexible glycine-serine repeats had no effect on S-acylation, whereas extended and rigid alanine-proline repeats perturbed this process. Indeed, a SNAP25 mutant in which the mini-linker region was substituted with a flexible glycine-serine linker of the same length underwent efficient S-acylation. Furthermore, this mutant displayed the same intracellular localisation as wild-type SNAP25, showing that the sequence of the mini-linker is not important in this context. By using the results of previous peptide array experiments, we generated a SNAP25 mutant predicted to have a higher affinity zDABM, and this mutant showed enhanced interaction with zDHHC17 in cells. Interestingly, this mutant was S-acylated with reduced efficiency, implying that a lower affinity interaction of the SNAP25 zDABM with zDHHC17 is optimal for S-acylation efficiency. Overall, the results of this study show that amino acids 93-111 in SNAP25 act as a flexible molecular spacer to ensure efficient coupling of enzyme-substrate interaction and S-acylation.
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differential palmitoylation regulates intracellular patterning of SNAP25
Journal of Cell Science, 2011Co-Authors: Jennifer Greaves, Luke H ChamberlainAbstract:SNAP25 regulates membrane fusion events at the plasma membrane and in the endosomal system, and a functional pool of the protein is delivered to recycling endosomes (REs) and the trans Golgi network (TGN) through an ARF6-dependent cycling pathway. SNAP25 is a peripheral membrane protein, and palmitoylation of a cluster of four cysteine residues mediates its stable association with the membrane. Here, we report that palmitoylation also determines the precise intracellular distribution of SNAP25, and that mutating single palmitoylation sites enhances the amount of SNAP25 at the RE and TGN. The farnesylated CAAX motif from Hras was ligated onto a SNAP25 mutant truncated immediately distal to the cysteine-rich domain. This construct displayed the same intracellular distribution as full-length SNAP25, and decreasing the number of cysteine residues in this construct increased its association with the RE and TGN, confirming the dominant role of the cysteine-rich domain in directing the intracellular distribution of SNAP25. Marked differences in the localisations of SNAP25-CAAX and Hras constructs, each with two palmitoylation sites, were observed, showing that subtle differences in palmitoylated sequences can have a major impact upon intracellular targeting. We propose that the cysteine-rich domain of SNAP25 is designed to facilitate the dual function of this SNARE protein at the plasma membrane and endosomes, and that dynamic palmitoylation acts as a mechanism to regulate the precise intracellular patterning of SNAP25.
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palmitoylation of the SNAP25 protein family specificity and regulation by dhhc palmitoyl transferases
Journal of Biological Chemistry, 2010Co-Authors: Jennifer Greaves, Christine Salaun, Oforiwa A Gorleku, Luke H ChamberlainAbstract:SNAP25 plays an essential role in neuronal exocytosis pathways. SNAP25a and SNAP25b are alternatively spliced isoforms differing by only nine amino acids, three of which occur within the palmitoylated cysteine-rich domain. SNAP23 is 60% identical to SNAP25 and has a distinct cysteine-rich domain to both SNAP25a and SNAP25b. Despite the conspicuous differences within the palmitoylated domains of these secretory proteins, there is no information on their comparative interactions with palmitoyl transferases. We report that membrane association of all SNAP25/23 proteins is enhanced by Golgi-localized DHHC3, DHHC7, and DHHC17. In contrast, DHHC15 promoted a statistically significant increase in membrane association of only SNAP25b. To investigate the underlying cause of this differential specificity, we examined a SNAP23 point mutant (C79F) designed to mimic the cysteine-rich domain of SNAP25b. DHHC15 promoted a marked increase in membrane binding and palmitoylation of this SNAP23 mutant, demonstrating that the distinct cysteine-rich domains of SNAP25/23 contribute to differential interactions with DHHC15. The lack of activity of DHHC15 toward wild-type SNAP23 was not overcome by replacing its DHHC domain with that from DHHC3, suggesting that substrate specificity is not determined by the DHHC domain alone. Interestingly, DHHC2, which is closely related to DHHC15, associates with the plasma membrane in PC12 cells and can palmitoylate all SNAP25 isoforms. DHHC2 is, thus, a candidate enzyme to regulate SNAP25/23 palmitoylation dynamics at the plasma membrane. Finally, we demonstrate that overexpression of specific Golgi-localized DHHC proteins active against SNAP25/23 proteins perturbs the normal secretion of human growth hormone from PC12 cells.
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the hydrophobic cysteine rich domain of SNAP25 couples with downstream residues to mediate membrane interactions and recognition by dhhc palmitoyl transferases
Molecular Biology of the Cell, 2009Co-Authors: Jennifer Greaves, Christine Salaun, Gerald R Prescott, Yuko Fukata, Masaki Fukata, Luke H ChamberlainAbstract:SNAP25 is synthesized as a soluble protein but must associate with the plasma membrane to function in exocytosis; however, this membrane-targeting pathway is poorly defined. SNAP25 contains a palmitoylated cysteine-rich domain with four cysteines, and we show that coexpression of specific DHHC palmitoyl transferases is sufficient to promote SNAP25 membrane association in HEK293 cells. siRNA-mediated knockdown of its SNARE partner, syntaxin 1A, does not affect membrane interaction of SNAP25 in PC12 cells, whereas specific cysteine-to-alanine mutations perturb membrane binding, which is restored by leucine substitutions. These results suggest a role for cysteine hydrophobicity in initial membrane interactions of SNAP25, and indeed other hydrophobic residues in the cysteine-rich domain are also important for membrane binding. In addition to the cysteine-rich domain, proline-117 is also essential for SNAP25 membrane binding, and experiments in HEK293 cells revealed that mutation of this residue inhibits membrane binding induced by coexpression with DHHC17, but not DHHC3 or DHHC7. These results suggest a model whereby SNAP25 interacts autonomously with membranes via its hydrophobic cysteine-rich domain, requiring only sufficient expression of partner DHHC proteins for stable membrane binding. The role of proline-117 in SNAP25 palmitoylation is one of the first descriptions of elements within substrate proteins that modulate DHHC specificity.
Dongfeng Chen - One of the best experts on this subject based on the ideXlab platform.
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microrna 210 5p contributes to cognitive impairment in early vascular dementia rat model through targeting SNAP25
Frontiers in Molecular Neuroscience, 2018Co-Authors: Zhenxing Ren, Yuqing Liu, Jianhong Zhou, Rudong Deng, Dongfeng ChenAbstract:Vascular dementia (VD) is the most common form of dementia in the elderly. However, little is understood about the role of microRNAs (miRNAs) involved in cognitive impairment in early VD. Here, VD model induced by chronic cerebral ischemia and FBS-free cell model detecting synapse formation were established to investigate the function of microRNAs in early VD. Microarray analysis and real-time reverse transcription PCR showed that miR-210-5p increased significantly in the hippocampus of rat with 4 weeks ischemia. VD model rats also displayed significant cognitive deficits and synaptic loss. Overexpression of miR-210-5p decreased the synaptic number in primary hippocampal neurons. In contrast, specific suppression of miR-210-5p resulted in formation of more synapses. Additionally, intracerebroventricular injection of mir-210-5p agomir to VD rats aggravated phenotypes of cognitive impairment and synaptic loss. These VD-induced phenotypes were effectively attenuated by mir-210 antagomir. Moreover, bioinformatic prediction revealed that SNAP25 mRNA is targeted by miR-210-5p. miR-210-5p decreased the luciferase activities of 3’ UTR of SNAP25 mRNA. Mutation of predicted miR-210-5p binding sites in the 3’ UTR of SNAP25 mRNA abolished the miR-210-5p induced decrease in luciferase activity. Western blot and immunofluorescence staining confirmed that miR-210-5p targets on SNAP25. Finally, detected by RT-qPCR and immunofluorescence staining, miR-210-5p agomir downregulated SNAP25 expression in CA1 region of VD rats’ hippocampi, in contrast, miR-210-5p antagomir upregulated SNAP25 expression. Taken together, miR-210-5p contributes to cognitive impairment in chronic ischemia-induced VD model through regulation of SNAP25 expression, which potentially offer the opportunity to develop a new therapeutic strategy for VD.
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microrna 210 5p contributes to cognitive impairment in early vascular dementia rat model through targeting SNAP25
Frontiers in Molecular Neuroscience, 2018Co-Authors: Zhenxing Ren, Yuqing Liu, Jianhong Zhou, Rudong Deng, Dongfeng ChenAbstract:Vascular dementia (VD) is the most common form of dementia in elderly people. However, little is understood about the role of microRNAs (miRNAs) involved in cognitive impairment in early VD. Here, a VD model induced by chronic cerebral ischemia and fetal bovine serum (FBS)-free cell model that detects synapse formation was established to investigate the function of miRNAs in early VD. The microarray analysis and real-time reverse transcription polymerase chain reaction (RT-PCR) showed that miR-210-5p increased significantly in the hippocampus of rats with 4 weeks of ischemia. The VD model rats also displayed significant cognitive deficits and synaptic loss. The overexpression of miR-210-5p decreased the synaptic number in primary hippocampal neurons, whereas specific suppression of miR-210-5p resulted in the formation of more synapses. Additionally, intracerebroventricular (ICV) injection of miR-210-5p agomir to VD rats aggravated phenotypes of cognitive impairment and synaptic loss. These VD-induced phenotypes were effectively attenuated by miR-210-5p antagomir. Moreover, bioinformatic prediction revealed that synaptosomal-associated protein of 25 KDa (SNAP25) mRNA is targeted by miR-210-5p. The miR-210-5p decreased the luciferase activities of 3' untranslated region (3'UTR) of SNAP25 mRNA. Mutation of predicted miR-210-5p binding sites in the 3' UTR of SNAP25 mRNA abolished the miR-210-5p-induced decrease in luciferase activity. Western blot and immunofluorescence staining confirmed that miR-210-5p targets SNAP25. Finally, RT-quantitative PCR (qPCR) and immunofluorescence staining detected that miR-210-5p agomir downregulated SNAP25 expression in the cornu ammonis1 (CA1) region of hippocampi in VD rats, whereas miR-210-5p antagomir upregulated SNAP25 expression. Altogether, miR-210-5p contributes to cognitive impairment in chronic ischemia-induced VD model through the regulation of SNAP25 expression, which potentially provides an opportunity to develop a new therapeutic strategy for VD.