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P A Roche

Publications and source records attributed to P A Roche.

At least 19 recordsLinked to original sources
PubMed2001

SNAP-29 is a promiscuous syntaxin-binding SNARE.

SNARE proteins are key regulators of membrane fusion and are proposed to dictate the specificity with which particular vesicles fuse with particular target organelles. On intracellular organelles that serve as targets for transport vesicles, organelle-specific syntaxins form heterodimers with either SNAP-23 or its recently described homolog SNAP-29. We have performed a variety of in vitro and in vivo binding assays in an attempt to determine whether SNAP-23 and SNAP-29 differ in their ability to form binary SNARE complexes with different intracellular syntaxins. While SNAP-23 preferentially binds to plasma membrane-localized syntaxins, SNAP-29 binds to both plasma membrane and intracellular syntaxins equally well. Furthermore, binding to SNAP-29 augments the ability of syntaxin to bind to vesicle-associated SNAREs and the presence of vesicle SNAREs dramatically increases SNAP-29 binding to syntaxin. These data suggest that SNAP-23 preferentially regulates plasma membrane-vesicle fusion events while SNAP-29 plays a role in the maintenance of various intracellular protein trafficking pathways.

Carrier Proteins↗
PubMed2001

The last exon of SNAP-23 regulates granule exocytosis from mast cells.

SNAP-25 and its ubiquitous homolog SNAP-23 are members of the SNARE family of proteins that regulate membrane fusion during exocytosis. Although SNAP-23 has been shown to participate in a variety of intracellular transport processes, the structural domains of SNAP-23 that are required for its interaction with other SNAREs have not been determined. By employing deletion mutagenesis we found that deletion of the amino-terminal 18 amino acids of SNAP-23 (encoded in the first exon) dramatically inhibited binding of SNAP-23 to both the target SNARE syntaxin and the vesicle SNARE vesicle-associated membrane protein(VAMP). By contrast, deletion of the carboxyl-terminal 23 amino acids (encoded in the last exon) of SNAP-23 does not affect SNAP-23 binding to syntaxin but profoundly inhibits its binding to VAMP. To determine the functional relevance of the modular structure of SNAP-23, we overexpressed SNAP-23 in cells possessing the capacity to undergo regulated exocytosis. Expression of human SNAP-23 in a rat mast cell line significantly enhanced exocytosis, and this effect was not observed in transfectants expressing the carboxyl-terminal VAMP-binding mutant of SNAP-23. Despite considerable amino acid identity, we found that human SNAP-23 bound to SNAREs more efficiently than did rat SNAP-23. These data demonstrate that the introduction of a "better" SNARE binder into secretory cells augments exocytosis and defines the carboxyl terminus of SNAP-23 as an essential regulator of exocytosis in mast cells.

Animals↗
PubMed2001

Protecting genetic privacy.

This article outlines the arguments for and against new rules to protect genetic privacy. We explain why genetic information is different to other sensitive medical information, why researchers and biotechnology companies have opposed new rules to protect genetic privacy (and favour anti-discrimination laws instead), and discuss what can be done to protect privacy in relation to genetic-sequence information and to DNA samples themselves.

Employment↗
PubMed2000

Identification of syntaxin 1A as a novel binding protein for presenilin-1.

Mutations in the presenilin 1 gene have been shown to result in Alzheimer's disease. Presenilin 1 is a multi-transmembrane protein with a large hydrophilic loop near the C-terminus. This region is required for known functions of presenilin 1. We have constrained this loop within the active site of the bacterial protein, thioredoxin, to mimic its native conformational state. This hybrid protein was used as bait in a yeast two hybrid screen in an attempt to identify presenilin binding proteins. By this method syntaxin 1A, a synaptic plasma membrane protein, was identified as a novel binding protein for presenilin 1. In vitro experiments confirm the two-hybrid results suggesting that PS1 binds syntaxin under physiological conditions.

Alzheimer Disease↗
PubMed2000

Structure and chromosomal localization of the mouse SNAP-23 gene.

SNAP-23 plays an important role in the regulation of vesicle trafficking in mammalian cells. In this report, we have determined the exon/intron organization of the mouse SNAP-23 gene. The SNAP-23 gene spans 31kb of the mouse genome and consists of eight exons interrupted by seven introns. The exon organization of the mouse SNAP-23 gene is identical to that of the related SNAP-25 gene in both chicken and Drosophila, suggesting that SNAP-23 arose by duplication of the SNAP-25 gene. Primer extension analysis revealed a major transcription start site approximately 112bp upstream of the translation start site. Like many ubiquitously expressed housekeeping genes, the proximal promoter region for the mouse SNAP-23 gene lacks consensus TATA and CAAT boxes. The SNAP-23 gene was localized to mouse chromosome 2 at band 2E5 using both fluorescence in-situ hybridization and radiation hybrid panel mapping studies. The identification of the structure of the mouse SNAP-23 gene reveals that the overall exon organization of SNAP-25 family members is conserved throughout evolution.

Amino Acid Sequence↗
PubMed2000

Targeting of SNAP-25 to membranes is mediated by its association with the target SNARE syntaxin.

The docking and fusion of synaptic vesicles with the presynaptic plasma membrane require the interaction of the vesicle-associated membrane protein VAMP with the plasma membrane proteins syntaxin and SNAP-25. Both of these proteins behave as integral membrane proteins, although they are unusual in that they insert into membranes post-translationally. Whereas VAMP and syntaxin possess hydrophobic transmembrane domains, SNAP-25 does not, and it is widely believed that SNAP-25 traffics to and inserts into membranes by post-translational palmitoylation. In pulse-chase biosynthesis studies, we now show that SNAP-25 and syntaxin rapidly bind to each other while still in the cytosol of neuroendocrine and transfected heterologous cells. Cell fractionation studies revealed that cytosolic SNAP-25.syntaxin complexes then traffic to and insert into membranes. Furthermore, the association of SNAP-25 with membranes is dramatically enhanced by syntaxin, and the transmembrane domain of syntaxin is essential for this effect. Surprisingly, despite the importance of the SNAP-25 palmitoylation domain for membrane anchoring at steady state, removal of this domain did not inhibit the initial association of newly synthesized SNAP-25 with membranes in the presence of syntaxin. These data demonstrate that the initial attachment of newly synthesized SNAP-25 to membranes is a consequence of its association with syntaxin and that it is only after syntaxin-mediated membrane tethering that SNAP-25 is palmitoylated.

Animals↗
PubMed2000

Concentration of MHC class II molecules in lipid rafts facilitates antigen presentation.

The plasma membranes of eukaryotic cells are not uniform and possess distinct cholesterol- and sphingolipid-rich raft microdomains that are enriched in proteins known to be essential for cellular function. Lipid raft microdomains are important for T cell receptor (TCR)-mediated activation of T cells. However, the importance of lipid rafts on antigen presenting cells (APCs) and their role in major histocompatibility (MHC) class II-restricted antigen presentation has not been examined. MHC class II molecules were found to be constitutively present in plasma membrane lipid rafts in B cells. Disruption of these microdomains dramatically inhibited antigen presentation at limiting concentrations of antigen. The inhibitory effect of raft disruption on antigen presentation could be overcome by loading the APCs with exceptionally high doses of antigen, showing that raft association concentrates MHC class II molecules into microdomains that allow efficient antigen presentation at low ligand densities.

Animals↗
PubMed2000

Intracellular redirection of plasma membrane trafficking after loss of epithelial cell polarity.

In polarized Madin-Darby canine kidney epithelial cells, components of the plasma membrane fusion machinery, the t-SNAREs syntaxin 2, 3, and 4 and SNAP-23, are differentially localized at the apical and/or basolateral plasma membrane domains. Here we identify syntaxin 11 as a novel apical and basolateral plasma membrane t-SNARE. Surprisingly, all of these t-SNAREs redistribute to intracellular locations when Madin-Darby canine kidney cells lose their cellular polarity. Apical SNAREs relocalize to the previously characterized vacuolar apical compartment, whereas basolateral SNAREs redistribute to a novel organelle that appears to be the basolateral equivalent of the vacuolar apical compartment. Both intracellular plasma membrane compartments have an associated prominent actin cytoskeleton and receive membrane traffic from cognate apical or basolateral pathways, respectively. These findings demonstrate a fundamental shift in plasma membrane traffic toward intracellular compartments while protein sorting is preserved when epithelial cells lose their cell polarity.

Animals↗
PubMed1999

Phosphorylation of the invariant chain by protein kinase C regulates MHC class II trafficking to antigen-processing compartments.

The invariant chain (Ii) plays a critical role in the transport of newly synthesized class II molecules to endosomal Ag-processing compartments. Of the two major isoforms of human Ii, only Ii-p35 is phosphorylated in vivo, and inhibiting Ii phosphorylation inhibits the trafficking of newly synthesized class II molecules to Ag-processing compartments. We now report that a member of the protein kinase C family of serine/threonine kinases is responsible for the constitutive phosphorylation of 50% of the total cellular pool of Ii-p35 in a wide variety of APCs, including B lymphocytes, PBMC, immature dendritic cells, and mature dendritic cells. Stimulation of protein kinase C activity in APCs significantly enhanced the kinetics of degradation of class II-associated Ii in Ag-processing compartments and the binding of antigenic peptides to these class II molecules. In cells expressing an Ii-phosphorylation mutant, trafficking of class II molecules to endosomes was impaired and Ii proteolysis was inhibited, demonstrating a direct effect of Ii phosphorylation on MHC class II trafficking. These results demonstrate that phosphorylation of Ii in APCs alters the kinetics of trafficking of newly synthesized class II molecules to lysosomal Ag-processing compartments.

Antigen Presentation↗
PubMed1999

SNAP-23 and SNAP-25 are palmitoylated in vivo.

The neuronal presynaptic membrane t-SNARE complex consists of the transmembrane protein syntaxin with the palmitoylated protein SNAP-25. In non-neuronal tissues, SNAP-23 replaces SNAP-25 in the t-SNARE complex, although the mechanism of membrane anchoring of SNAP-23 has not been determined. We now report that like SNAP-25, SNAP-23 is palmitoylated in vivo on one or more cysteine residues present in a central "palmitoylation domain." Interestingly, SNAP-23 is palmitoylated less well than SNAP-25, and in vivo binding studies indicate a correlation between the extent of palmitoylation and the ability of SNAP-23 or SNAP-25 to bind to syntaxin in vivo.

Amino Acid Sequence↗
PubMed1999

SNAP-23 participates in SNARE complex assembly in rat adipose cells.

SNARE proteins are required for vesicle docking and fusion in eukaryotic cells in processes as diverse as homotypic membrane fusion and synaptic vesicle exocytosis [SNARE stands for SNAP receptor, where SNAP is soluble NSF attachment protein]. The SNARE proteins syntaxin 4 and vesicle-associated membrane protein (VAMP) 2/3 also participate in the insulin-stimulated translocation of GLUT4 from intracellular vesicles to the plasma membrane in adipose cells. We now report the molecular cloning and characterization of rat SNAP-23, a ubiquitously expressed homologue of the essential neuronal SNARE protein SNAP-25 (synaptosomal-associated protein of 25 kDa). Rat SNAP-23 is 86% and 98% identical respectively to human and mouse SNAP-23. Southern blot analysis reveals that the rat, mouse and human SNAP-23 genes encode species-specific isoforms of the same protein. Co-immunoprecipitation of syntaxin 4 and SNAP-23 shows association of these two proteins in rat adipose cell plasma membranes, and insulin stimulation does not alter the SNAP-23/syntaxin 4 complex. In addition, we demonstrate for the first time the participation of SNAP-23, along with syntaxin 4 and VAMP2/3, in the formation of 20S SNARE complexes prepared using rat adipose cell membranes and recombinant alpha-SNAP and NSF proteins. The stoichiometry of the SNARE complexes formed is essentially identical using membranes from either unstimulated or insulin-stimulated adipose cells. These data demonstrate that rat SNAP-23 associates with syntaxin 4 before insulin stimulation and is present in the SNARE complexes known to mediate the translocation of GLUT4 from intracellular vesicles to the plasma membrane of rat adipose cells.

Adipocytes↗
PubMed1999

Phosphorylation of SNAP-23 by the novel kinase SNAK regulates t-SNARE complex assembly.

The docking and fusion of cargo-containing vesicles with target membranes of eukaryotic cells is mediated by the interaction of SNARE proteins present on both vesicle and target membranes. In many cases, the target membrane SNARE, or t-SNARE, exists as a complex of syntaxin with a member of the SNAP-25 family of palmitoylated proteins. We have identified a novel human kinase SNAK (SNARE kinase) that specifically phosphorylates the nonneuronal t-SNARE SNAP-23 in vivo. Interestingly, only SNAP-23 that is not assembled into t-SNARE complexes is phosphorylated by SNAK, and phosphorylated SNAP-23 resides exclusively in the cytosol. Coexpression with SNAK significantly enhances the stability of unassembled SNAP-23, and as a consequence, the assembly of newly synthesized SNAP-23 with syntaxin is augmented. These data demonstrate that phosphorylation of SNAP-23 by SNAK enhances the kinetics of t-SNARE assembly in vivo.

Carrier Proteins↗
PubMed1999

Syntaxin 11 is associated with SNAP-23 on late endosomes and the trans-Golgi network.

SNARE proteins are known to play a role in regulating intracellular protein transport between donor and target membranes. This docking and fusion process involves the interaction of specific vesicle-SNAREs (e.g. VAMP) with specific cognate target-SNAREs (e.g. syntaxin and SNAP-23). Using human SNAP-23 as the bait in a yeast two-hybrid screen of a human B-lymphocyte cDNA library, we have identified the 287-amino-acid SNARE protein syntaxin 11. Like other syntaxin family members, syntaxin 11 binds to the SNARE proteins VAMP and SNAP-23 in vitro and also exists in a complex with SNAP-23 in transfected HeLa cells and in native human B lymphocytes. Unlike other syntaxin family members, no obvious transmembrane domain is present in syntaxin 11. Nevertheless, syntaxin 11 is predominantly membrane-associated and colocalizes with the mannose 6-phosphate receptor on late endosomes and the trans-Golgi network. These data suggest that syntaxin 11 is a SNARE that acts to regulate protein transport between late endosomes and the trans-Golgi network in mammalian cells.

Alternative Splicing↗
PubMed1998

HIV-1 tat binds TAFII250 and represses TAFII250-dependent transcription of major histocompatibility class I genes.

HIV Tat, a transactivator of viral transcription, represses transcription of major histocompatibility (MHC) class I genes. Repression depends exclusively on the C-terminal domain of Tat, although the mechanism of this repression has not been known. We now show that repression results from the interaction of Tat with the TAFII250 component of the general transcription factor, TFIID. The C-terminal domain of Tat binds to a site on TAFII250 that overlaps the histone acetyl transferase domain, inhibiting TAFII250 histone acetyl transferase activity. Furthermore, promoters repressed by Tat, including the MHC class I promoter, are dependent on TAFII250 whereas those that are not repressed by Tat, such as SV40 and MuLV promoters, are independent of functional TAFII250. Thus, Tat repression of MHC class I transcription would be one mechanism by which HIV avoids immune surveillance.

Acetyltransferases↗
PubMed1998

Phosphorylation regulates the delivery of MHC class II invariant chain complexes to antigen processing compartments.

Transport of newly synthesized MHC class II glycoproteins to endosomal Ag processing compartments is mediated by their association with the invariant chain (Ii). Targeting to these compartments is dependent upon recognition of leucine-based endo. somal/lysosomal targeting motifs in the Ii cytosolic domain. Ii, like many molecules that contain leucine-based endosomal targeting motifs, is phosphorylated in vivo. In this report we demonstrate that the cytosolic domain of the p35 Ii isoform is phosphorylated in class II Ii complexes isolated from human B lymphoblastoid cell lines or freshly obtained PBMC. Mutation of serine residue 6 or 8 prevents phosphorylation of Ii-p35 expressed in HeLa cells. Treatment of B lymphoblastoid cell lines with the serine/threonine kinase inhibitor staurosporine prevented Ii phosphorylation and significantly delayed trafficking of newly synthesized class II Ii complexes to endosomal Ag processing compartments. By contrast, staurosporine had no effect on the rate of transport of class I or class II glycoproteins through the Golgi apparatus and did not inhibit the delivery of the chimeric molecule Tac-DM, to endocytic compartments, suggesting that staurosporine does not nonspecifically inhibit protein transport to the endocytic pathway. These results demonstrate that phosphorylation regulates the efficient targeting of MHC class II Ii complexes to Ag processing compartments and strongly suggest that this effect is mediated by phosphorylation of the MHC class II-associated Ii chain.

Antigen Presentation↗
PubMed1998

Targeting of SNAP-23 and SNAP-25 in polarized epithelial cells.

SNAP-23 is the ubiquitously expressed homologue of the neuronal SNAP-25, which functions in synaptic vesicle fusion. We have investigated the subcellular localization of SNAP-23 in polarized epithelial cells. In hepatocyte-derived HepG2 cells and in Madin-Darby canine kidney (MDCK) cells, the majority of SNAP-23 was present at both the basolateral and apical plasma membrane domains with little intracellular localization. This suggests that SNAP-23 does not function in intracellular fusion events but rather as a general plasma membrane t-SNARE. Canine SNAP-23 is efficiently cleaved by the botulinum neurotoxin E, suggesting that it is the toxin-sensitive factor previously found to be involved in plasma membrane fusion in MDCK cells. The localization of SNAP-25 in transfected MDCK cells was studied for comparison and was found to be identical to SNAP-23 with the exception that SNAP-25 was transported to the primary cilia protruding from the apical plasma membrane, which suggests that subtle differences in the targeting signals of both proteins exist. In contrast to its behavior in neurons, the distribution of SNAP-25 in MDCK cells remained unaltered by treatment with dibutyryl cAMP or forskolin, which, however, caused an increased growth of the primary cilia. Finally, we found that SNAP-23/25 and syntaxin 1A, when co-expressed in MDCK cells, do not stably interact with each other but are independently targeted to the plasma membrane and lysosomes, respectively.

Animals↗
PubMed1997

SNAP-23 is not cleaved by botulinum neurotoxin E and can replace SNAP-25 in the process of insulin secretion.

The synaptosomal-associated protein of 25 kDa (SNAP-25) is expressed in neurons and endocrine cells. It has been shown to play an important role in the release mechanism of neurotransmitters and peptide hormones, including insulin. Thus, when insulin-secreting cells are permeabilized and treated with botulinum neurotoxin E (BoNT/E), SNAP-25 is hydrolyzed, and insulin secretion is inhibited. Recently SNAP-23, a more generally expressed isoform of SNAP-25, has been described. The functional role of SNAP-23 has not been investigated to date. It is now shown that SNAP-23 is resistant to cleavage by BoNT/E. It was therefore possible to test whether transfection of HIT (transformed pancreatic B-) cells with SNAP-23 reconstitutes insulin release from BoNT/E treated cells, in which SNAP-25 is inactivated by the toxin. The results show that SNAP-23 is able to replace SNAP-25 when it is overexpressed. While these results demonstrate that SNAP-23 is a functional homologue of SNAP-25, able to function in regulated exocytosis, they indicate that SNAP-23 may be inefficient in this process. This suggests that both isoforms may have their own specific binding partners and discrete, albeit mechanistically similar, functional roles within the cell.

Animals↗