The Hydrophobic Cysteine-rich Domain of SNAP25 Couples with Downstream Residues to Mediate Membrane Interactions and Recognition by DHHC Palmitoyl Transferases

Centre for Integrative Physiology, School of Biomedical Sciences, University of Edinburgh, Edinburgh EH8 9XD, United Kingdom.
Molecular biology of the cell (Impact Factor: 4.47). 02/2009; 20(6):1845-54. DOI: 10.1091/mbc.E08-09-0944
Source: PubMed


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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Available from: Christine Salaun, Oct 09, 2015
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    • "Flot1 and Flot2, we also examined SNAP-25, a well-documented HIP14 substrate (Greaves et al., 2009; Huang et al., 2004, 2009). SNAP-25, we find, is palmitoylated in HIP14-expressing cells, but not in DHHC5-expressing cells or in naive yeast cells, not expressing any mammalian PATs (Figure 2B). "
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    ABSTRACT: Protein palmitoylation, a reversible lipid modification of proteins, is widely used in the nervous system, with dysregulated palmitoylation being implicated in a variety of neurological disorders. Described below is ABE/SILAM, a proteomic strategy that couples acyl-biotinyl exchange (ABE) purification of palmitoyl-proteins to whole animal stable isotope labeling (SILAM) to provide an accurate tracking of palmitoylation change within rodent disease models. As a first application, we have used ABE/SILAM to look at Huntington's disease (HD), profiling palmitoylation change in two HD-relevant mouse mutants: the transgenic HD model mouse YAC128 and the hypomorphic Hip14-gt mouse, which has sharply reduced expression for HIP14 (Zdhhc17), a palmitoyl-transferase implicated in the HD disease process. Rather than mapping to the degenerating neurons themselves, the biggest disease changes instead map to astrocytes and oligodendrocytes (i.e., the supporting glial cells).
    Chemistry & biology 11/2013; 20(11). DOI:10.1016/j.chembiol.2013.09.018 · 6.65 Impact Factor
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    • "Singaraja et al., 2002) (Table 1a). DHHC17, also known as HIP14, is a palmitoyltransferase which palmitoylates a number of neuronal proteins, including SNAP25 (Greaves et al., 2009), PSD-95, GAD65, synaptotagmin I and huntingtin (Fang et al., 2006; Gao et al., 2009; Huang et al., 2004, 2009; Stowers and Isacoff, 2007). DHHC17 contains 7–8 ankyrin repeats in the N-terminal region (Fig. 2) that may function in its substrate recognition (Gao et al., 2009). "
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    ABSTRACT: Palmitoylation is one of the most common posttranslational lipid modifications of proteins and we now know quite a lot about it. However, the state of knowledge about the enzymes that catalyze this process is clearly insufficient. This review is focused on 23 human DHHC genes and their products - protein palmitoyltransferases. Here we describe mainly the structure and function of these proteins, but also, to a lesser degree, what the substrates of the enzymes are and whether they are related to various diseases. The main aim of this review was to catalogue existing information concerning the human DHHC family of genes/proteins, making them and their functions easier to understand.
    European journal of cell biology 12/2011; 91(2):107-17. DOI:10.1016/j.ejcb.2011.09.013 · 3.83 Impact Factor
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    • "The availability in vivo of the thiols of SNAP-25 is expected to be limited by the well-established S-palmitoylation of one or more of these thiols (Veit et al. 1996; Vogel and Roche 1999). Recent findings suggest that S-palmitoylation may promote stable association of SNAP-2 with the plasma membrane following an initial lower-affinity interaction mediated by the hydrophobicity of the cysteines and adjacent amino acid residues (Greaves et al. 2009). It is important to note, however, that the exact positions and number of cysteine residues that may be palmitoylated at any given time in SNAP-25 has not been defined. "
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    ABSTRACT: Intrachain disulfide bond formation among the cysteine thiols of SNAP-25, a component of the SNARE protein complex required for neurotransmitter release, has been hypothesized to link oxidative stress and inhibition of synaptic transmission. However, neither the availability in vivo of SNAP-25 thiols, which are known targets of S-palmitoylation, nor the tendency of these thiols to form intrachain disulfide bonds is known. We have examined, in rat brain extracts, both the availability of closely spaced, or vicinal, thiol pairs in SNAP-25 and the propensity of these dithiols toward disulfide bond formation using a method improved by us recently that exploits the high chemoselectivity of phenylarsine oxide (PAO) for vicinal thiols. The results show for the first time that a substantial fraction of soluble and, to a lesser extent, particulate SNAP-25 contain non-acylated PAO-binding thiol pairs and that these thiols in soluble SNAP-25 in particular have a high propensity toward disulfide bond formation. Indeed, disulfide bonds were detected in a small fraction of soluble SNAP-25 even under conditions designed to prevent or greatly limit protein thiol oxidation during experimental procedures. These results provide direct experimental support for the availability, in a subpopulation of SNAP-25, of vicinal thiols that may confer on one or more isoforms of this family of proteins a sensitivity to oxidative stress.
    Cellular and Molecular Neurobiology 08/2011; 32(2):201-8. DOI:10.1007/s10571-011-9748-4 · 2.51 Impact Factor
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