Bazbek Davletov

Structure of the first C2 domain of synaptotagmin I: A novel Ca2+/phospholipid-binding fold

C2 domains are regulatory sequence motifs that occur widely in nature. Synaptotagmin I, a synaptic vesicle protein involved in the Ca2+ regulation of exocytosis, contains two C2 domains, the first of which acts as a Ca2+ sensor. We now describe the three-dimensional structure of this C2 domain at 1.9 A resolution in both the Ca(2+)-bound and Ca(2+)-free forms. The C2 polypeptide forms an eight-stranded beta sandwich constructed around a conserved four-stranded motif designated as a C2 key. Ca2+ binds in a cup-shaped depression between two polypeptide loops located at the N- and C-termini of the C2-key motif.

Non-invasive detection of apoptosis using magnetic resonance imaging and a targeted contrast agent

The C2 domain of synaptotagmin I, which binds to anionic phospholipids in cell membranes, was shown to bind to the plasma membrane of apoptotic cells by both flow cytometry and confocal microscopy. Conjugation of the protein to superparamagnetic iron oxide nanoparticles allowed detection of this binding using magnetic resonance imaging. Detection of apoptotic cells, using this novel contrast agent, was demonstrated both in vitro, with isolated apoptotic tumor cells, and in vivo, in a tumor treated with chemotherapeutic drugs.

Synaptotagmin I is a high affinity receptor for clathrin AP-2: implications for membrane recycling.

In nerve terminals, Ca(2+)-stimulated synaptic vesicle exocytosis is rapidly followed by endocytosis. Synaptic vesicle endocytosis requires clathrin-coated pits similar to receptor-mediated endocytosis in fibroblasts. Binding of clathrin AP-2 (adaptor complex) to an unidentified high affinity membrane receptor appears to be necessary for coated pit assembly in fibroblasts. We now show that synaptic vesicles have a high affinity AP-2 site (KD, approximately 1 x 10(-10) M) similar to the one observed in fibroblasts. Using a combination of competition and direct binding assays, we demonstrate that synaptotagmin I, an intrinsic membrane protein of synaptic vesicles, has all of the properties of the AP-2 receptor and that AP-2 binds to the second C2 domain in the molecule. Thus, synaptotagmin I may be a multifunctional protein with a function in endocytosis in addition to the previously proposed role in exocytosis.

The synaptic vesicle protein 2C mediates the uptake of botulinum neurotoxin A into phrenic nerves

Botulinum neurotoxins (BoNTs) inhibit neurotransmitter release by selectively cleaving core components of the vesicular fusion machinery. The synaptic vesicle proteins Synaptotagmin‐I and ‐II act as receptors for BoNT/B and BoNT/G. Here we show that BoNT/A also interacts with a synaptic vesicle protein, the synaptic vesicle glycoprotein 2C (SV2C), but not with the homologous proteins SV2A and SV2B. Binding of BoNT/A occurs at the membrane juxtaposed region preceding transmembrane domain 8. A peptide comprising the intravesicular domain between transmembrane domains 7 and 8 specifically reduces the neurotoxicity of BoNT/A at phrenic nerve preparations demonstrating the physiological relevance of this interaction.

Bipartite Ca2+-binding motif in C2 domains of synaptotagmin and protein kinase C.

C2 domains are found in many proteins involved in membrane traffic or signal transduction. Although C2 domains are thought to bind calcium ions, the structural basis for calcium binding is unclear. Analysis of calcium binding to C2 domains of synaptotagmin I and protein kinase C-β by nuclear magnetic resonance spectroscopy revealed a bipartite calcium-binding motif that involves the coordination of two calcium ions by five aspartate residues located on two separate loops. Sequence comparisons indicated that this may be a widely used calcium-binding motif, designated here as the C2 motif.

Omega-3 and omega-6 fatty acids stimulate cell membrane expansion by acting on syntaxin 3

Growth of neurite processes from the cell body is the critical step in neuronal development and involves a large increase in cell membrane surface area. Arachidonic-acid-releasing phospholipases are highly enriched in nerve growth cones and have previously been implicated in neurite outgrowth. Cell membrane expansion is achieved through the fusion of transport organelles with the plasma membrane; however, the identity of the molecular target of arachidonic acid has remained elusive. Here we show that syntaxin 3 (STX3), a plasma membrane protein, has an important role in the growth of neurites, and also serves as a direct target for omega-6 arachidonic acid. By using syntaxin 3 in a screening assay, we determined that the dietary omega-3 linolenic and docosahexaenoic acids can efficiently substitute for arachidonic acid in activating syntaxin 3. Our findings provide a molecular basis for the previously established action of omega-3 and omega-6 polyunsaturated fatty acids in membrane expansion at the growth cones, and represent the first identification of a single effector molecule for these essential nutrients.

α-Latrotoxin Receptor, Latrophilin, Is a Novel Member of the Secretin Family of G Protein-coupled Receptors

α-Latrotoxin (LTX) stimulates massive exocytosis of synaptic vesicles and may help to elucidate the mechanism of regulation of neurosecretion. We have recently isolated latrophilin, the synaptic Ca2+-independent LTX receptor. Now we demonstrate that latrophilin is a novel member of the secretin family of G protein-coupled receptors that are involved in secretion. Northern blot analysis shows that latrophilin message is present only in neuronal tissue. Upon expression in COS cells, the cloned protein is indistinguishable from brain latrophilin and binds LTX with high affinity. Latrophilin physically interacts with a Gαosubunit of heterotrimeric G proteins, because the two proteins co-purify in a two-step affinity chromatography. Interestingly, extracellular domain of latrophilin is homologous to olfactomedin, a soluble neuronal protein thought to participate in odorant binding. Our findings suggest that latrophilin may bind unidentified endogenous ligands and transduce signals into nerve terminals, thus implicating G proteins in the control of synaptic vesicle exocytosis.

Mutation E46K increases phospholipid binding and assembly into filaments of human α-synuclein

Missense mutations (A30P and A53T) in α‐synuclein and the overproduction of the wild‐type protein cause familial forms of Parkinsons disease and dementia with Lewy bodies. α‐Synuclein is the major component of the filamentous Lewy bodies and Lewy neurites that define these diseases at a neuropathological level. Recently, a third missense mutation (E46K) in α‐synuclein was described in an inherited form of dementia with Lewy bodies. Here, we have investigated the functional effects of this novel mutation on phospholipid binding and filament assembly of α‐synuclein. When compared to the wild‐type protein, the E46K mutation caused a significantly increased ability of α‐synuclein to bind to negatively charged liposomes, unlike the previously described mutations. The E46K mutation increased the rate of filament assembly to the same extent as the A53T mutation. Filaments formed from E46K α‐synuclein often had a twisted morphology with a cross‐over spacing of 43 nm. The observed effects on lipid binding and filament assembly may explain the pathogenic nature of the E46K mutation in α‐synuclein.

Vesicular restriction of synaptobrevin suggests a role for calcium in membrane fusion

Release of neurotransmitter occurs when synaptic vesicles fuse with the plasma membrane. This neuronal exocytosis is triggered by calcium and requires three SNARE (soluble-N-ethylmaleimide-sensitive factor attachment protein receptors) proteins: synaptobrevin (also known as VAMP) on the synaptic vesicle, and syntaxin and SNAP-25 on the plasma membrane. Neuronal SNARE proteins form a parallel four-helix bundle that is thought to drive the fusion of opposing membranes. As formation of this SNARE complex in solution does not require calcium, it is not clear what function calcium has in triggering SNARE-mediated membrane fusion. We now demonstrate that whereas syntaxin and SNAP-25 in target membranes are freely available for SNARE complex formation, availability of synaptobrevin on synaptic vesicles is very limited. Calcium at micromolar concentrations triggers SNARE complex formation and fusion between synaptic vesicles and reconstituted target membranes. Although calcium does promote interaction of SNARE proteins between opposing membranes, it does not act by releasing synaptobrevin from synaptic vesicle restriction. Rather, our data suggest a mechanism in which calcium-triggered membrane apposition enables syntaxin and SNAP-25 to engage synaptobrevin, leading to membrane fusion.

Interaction of synaptotagmin with the cytoplasmic domains of neurexins

Synaptotagmin, a major intrinsic membrane protein of synaptic vesicles that binds Ca2+, was purified from bovine brain and immobilized onto Sepharose 4B. Affinity chromatography of brain membrane proteins on immobilized synaptotagmin revealed binding of alpha- and beta-neurexins to synaptotagmin in a Ca(2+)-independent manner. Using a series of recombinant proteins in which glutathione S-transferase was fused to the cytoplasmic domains of three different neurexins or of control proteins, it was found that synaptotagmin specifically interacts with the cytoplasmic domains of neurexins but not of control proteins. This interaction is dependent on a highly conserved, 40 amino acid sequence that makes up most of the cytoplasmic tails of the neurexins. Our data suggest a direct interaction between the cytoplasmic domains of a plasma membrane protein (the neurexins) and a protein specific for a subcellular organelle (synaptotagmin). Such an interaction could have an important role in the docking and targeting of synaptic vesicles in the nerve terminal.