Biomedical subjects

Xiaocheng Chen

Publications and source records attributed to Xiaocheng Chen.

8 recordsLinked to original sources

PubMed2006

Unraveling the mechanisms of synaptotagmin and SNARE function in neurotransmitter release.

SNARE proteins and synaptotagmin are key components of the complex machinery that controls Ca(2+)-triggered neurotransmitter release but their mechanisms of action are under debate. Recent research has shed light on which biochemical and/or biophysical properties underlie SNARE and synaptotagmin function. SNARE proteins most likely have a role in membrane fusion owing to their ability to bring the synaptic vesicle and plasma membranes together and to perturb lipid bilayers through their transmembrane regions. Synaptotagmin acts as a Ca(2+) sensor and might cooperate with the SNAREs in accelerating fusion by binding simultaneously to the two membranes. However, recent research has strongly challenged the validity of models proposing that the SNAREs (with or without synaptotagmin) constitute "minimal membrane fusion machineries" and has emphasized the essential nature of other proteins for exocytosis. Understanding the functions of these proteins will be crucial to reach a faithful description of the mechanisms of membrane fusion and neurotransmitter release.

Animals↗

PubMed2006

Close membrane-membrane proximity induced by Ca(2+)-dependent multivalent binding of synaptotagmin-1 to phospholipids.

Synaptotagmin acts as a Ca(2+) sensor in neurotransmitter release through its two C(2) domains. Ca(2+)-dependent phospholipid binding is key for synaptotagmin function, but it is unclear how this activity cooperates with the SNARE complex involved in release or why Ca(2+) binding to the C(2)B domain is more crucial for release than Ca(2+) binding to the C(2)A domain. Here we show that Ca(2+) induces high-affinity simultaneous binding of synaptotagmin to two membranes, bringing them into close proximity. The synaptotagmin C(2)B domain is sufficient for this ability, which arises from the abundance of basic residues around its surface. We propose a model wherein synaptotagmin cooperates with the SNAREs in bringing the synaptic vesicle and plasma membranes together and accelerates membrane fusion through the highly positive electrostatic potential of its C(2)B domain.

Animals↗

PubMed2005

SNARE-mediated lipid mixing depends on the physical state of the vesicles.

Reconstitution experiments have suggested that N-ethylmaleimide sensitive factor attachment protein receptor (SNARE) proteins constitute a minimal membrane fusion machinery but have yielded contradictory results, and it is unclear whether the mechanism of membrane merger is related to the stalk mechanism that underlies physiological membrane fusion. Here we show that reconstitution of solubilized neuronal SNAREs into preformed 100 nm liposomes (direct method) yields proteoliposomes with more homogeneous sizes and protein densities than the standard reconstitution method involving detergent cosolubilization of proteins and lipids. Standard reconstitutions yield slow but efficient lipid mixing at high protein densities and variable amounts of lipid mixing at moderate protein densities. However, the larger, more homogenous proteoliposomes prepared by the direct method yield almost no lipid mixing at moderate protein densities. These results suggest that the lipid mixing observed for standard reconstitutions is dominated by the physical state of the membrane, perhaps due to populations of small vesicles (or micelles) with high protein densities and curvature stress created upon reconstitution. Accordingly, changing membrane spontaneous curvature by adding lysophospholipids inhibits the lipid mixing observed for standard reconstitutions. Our data indicate that the lipid mixing caused by high SNARE densities and/or curvature stress occurs by a stalk mechanism resembling the mechanism of fusion between biological membranes, but the neuronal SNAREs are largely unable to induce lipid mixing at physiological protein densities and limited curvature stress.

Humans↗

PubMed2005

Are neuronal SNARE proteins Ca2+ sensors?

The neuronal SNARE complex formed by synaptobrevin, syntaxin and SNAP-25 plays a central role in Ca2+-triggered neurotransmitter release. The SNARE complex contains several potential Ca2+-binding sites on the surface, suggesting that the SNAREs may be involved directly in Ca2+-binding during release. Indeed, overexpression of SNAP-25 bearing mutations in two putative Ca2+ ligands (E170A/Q177A) causes a decrease in the Ca2+-cooperativity of exocytosis in chromaffin cells. To test whether the SNARE complex might function in Ca2+-sensing, we analyzed its Ca2+-binding properties using transverse relaxation optimized spectroscopy (TROSY)-based NMR methods. Several Ca2+-binding sites are found on the surface of the SNARE complex, but most of them are not specific for Ca2+ and all have very low affinity. Moreover, we find that the E170A/Q177A SNAP-25 mutation does not alter interactions between the SNAREs and the Ca2+ sensor synaptotagmin 1, but severely impairs SNARE complex assembly. These results suggest that the SNAREs do not act directly as Ca2+ receptors but SNARE complex assembly is coupled tightly to Ca2+-sensing during neurotransmitter release.

Binding Sites↗

PubMed2003

A broken alpha -helix in folded alpha -Synuclein.

alpha-Synuclein is a small cytosolic protein of presynaptic nerve terminals composed of seven 11-residue repeats and a hydrophilic tail. alpha-Synuclein misfolding and dysfunction may contribute to the pathogenesis of Parkinson's disease and neurodegenerative dementias, but its normal folding and function are unknown. In solution, alpha-synuclein is natively unstructured but assumes an alpha-helical conformation upon binding to phospholipid membranes. We now show that this conformation of alpha-synuclein consists of two alpha-helical regions that are interrupted by a short break. The structural organization of the alpha-helices of alpha-synuclein was not anticipated by sequence analyses and may be important for its pathogenic role.

Amino Acid Sequence↗

PubMed2003

Evidence for SNARE zippering during Ca2+-triggered exocytosis in PC12 cells.

SNAREs (soluble NSF attachment protein receptors) are membrane proteins that catalyze membrane fusion. SNAREs are defined by a characteristic 70 residue sequence called the SNARE motif. During synaptic vesicle fusion, the single SNARE motif of the synaptic vesicle SNARE protein synaptobrevin/VAMP associates into a four-helical bundle with SNARE motifs from the plasma membrane SNARE proteins syntaxin 1 and SNAP-25. The four SNARE motifs (one each from synaptobrevin and syntaxin, and two from SNAP-25) assume a parallel orientation in the complex, suggesting that formation of the complex initiates fusion by forcing the membranes containing the SNAREs into close proximity. It has been proposed that SNARE complexes assemble in an N- to C-terminal progression, a process referred to as zippering, but little direct evidence for zippering exists. Furthermore, the SM protein Munc18-1, which binds to syntaxin 1 and is essential for synaptic fusion, is thought to prepare SNAREs for complex formation by an unknown mechanism, possibly by nucleating zippering. We now show that fragments containing the N- and C-terminal regions of the SNARE motif from syntaxin 1A bind SNAP-25 similarly. However, in permeabilized PC12 cells which are used as a biochemical model system to study synaptic fusion, only fragments containing the N-terminal region are powerful inhibitors of fusion. Furthermore, mutations in the N-terminal part of the Syntaxin SNARE motif have only a moderate effect on SNAP-25 binding but abolish the inhibitory activity of the SNARE motif. Finally, larger fragments of syntaxin 1A that strongly bind to Munc18-1 but do not readily assemble into SNARE complexes had no effect on exocytosis in permeabilized PC12 cells. Together these results suggest that Munc18-1 acts before SNARE complex assembly, and is no longer required at the stage of fusion assayed in permeabilized PC12 cells. The selective effect of the N-terminal half of the syntaxin 1A SNARE motif on PC12 cell exocytosis shows that the SNARE motif is functionally polarized, and supports the notion that SNARE complexes assemble in an N- to C-terminal zippering reaction during fusion without a stable, partially assembled intermediate.

Animals↗

PubMed2002

Three-dimensional structure of the complexin/SNARE complex.

During neurotransmitter release, the neuronal SNARE proteins synaptobrevin/VAMP, syntaxin, and SNAP-25 form a four-helix bundle, the SNARE complex, that pulls the synaptic vesicle and plasma membranes together possibly causing membrane fusion. Complexin binds tightly to the SNARE complex and is essential for efficient Ca(2+)-evoked neurotransmitter release. A combined X-ray and TROSY-based NMR study now reveals the atomic structure of the complexin/SNARE complex. Complexin binds in an antiparallel alpha-helical conformation to the groove between the synaptobrevin and syntaxin helices. This interaction stabilizes the interface between these two helices, which bears the repulsive forces between the apposed membranes. These results suggest that complexin stabilizes the fully assembled SNARE complex as a key step that enables the exquisitely high speed of Ca(2+)-evoked neurotransmitter release.

Adaptor Proteins, Vesicular Transport↗

PubMed2002

Sly1 binds to Golgi and ER syntaxins via a conserved N-terminal peptide motif.

Sec1/munc18-like proteins (SM proteins) and SNARE complexes are probably universally required for membrane fusion. However, the molecular mechanism by which they interact has only been defined for synaptic vesicle fusion where munc18 binds to syntaxin in a closed conformation that is incompatible with SNARE complex assembly. We now show that Sly1, an SM protein involved in Golgi and ER fusion, binds to a short, evolutionarily conserved N-terminal peptide of Sed5p and Ufe1p in yeast and of syntaxins 5 and 18 in vertebrates. In these syntaxins, the Sly1 binding peptide is upstream of a separate, autonomously folded N-terminal domain. These data suggest a potentially general mechanism by which SM proteins could interact with peptides in target proteins independent of core complex assembly and suggest that munc18 binding to syntaxin is an exception.

Amino Acid Sequence↗