Study On The Structural Basis And Mechanism Of SCGN And Doc2α In The Process Of SNARE-Mediated Membrane Fusion

Cell secretion is one of the most basic activities in cell life.It is essential for maintaining the normal cellular function.Post stimulations,relevant bioactive molecules synthesized in the cell are wrapped into vesicles,which are transported to the plasma membrane and release the active molecules into the extracellullar spaces through membrane fusion process.Many cell activities and physiological processes,such as neurotransmitter release,insulin secretion,immune response,growth and development of the body,are based on cell secretion.Cell secretion and activity depends on membrane fusion.SNARE proteins(SNAREs)are the most critical factors mediating membrane fusion in eukaryotic cells,which are firstly reported in the neurons,including Syntaxin-1(stx-1)and SNAP-25 on the presynaptic membrane and VAMP/Synaptobrevin(Syb)on the vesicles.Both syb and stx-1 have one transmembrane domain.However,SNAP-25 is a soluble protein without any transmembrane domain but a Cysteine-rich region,which can be palmitoylated and therefore helps SNAP-25translocating to the presynaptic membrane.SNAREs are very conserved from yeast to humans.So far,there are more than 60 SNAREs discovered in mammals,widely distributed in different subcellular organelles of mammalian cells,such as cytoplasmic membrane,mitochondria,endoplasmic reticulum,and peroxide bodies.SNARE motifs,which is composed of approximately 70 amino acids,are shared by the SNAREs.The SNARE motifs can form a bundle of four parallelα-helixes,where syb and stx provide oneα-helix respectively,and SNAP-25 contributes twoα-helixes.Thisα-helix bundle works as a zipper,drawing the synaptic vesicle membrane and the presynaptic membrane together,promoting membrane fusion and neurotransmitter release.SNAREs can be assembled into the correct SNARE complex,but also can form some chaotic complexes in solution.For example,stx can form homodimers or tetramers by itself,which has no function in the organism;Stx and SNAP-25 can form a heterodimer in a ratio of 2:1.Therefore,the correct assembly of SNARE complex requires regulatory factors.At present,the most well studied regulatory factors include complexin(Cpx),synaptotagmin-1(syt1),Munc18 and Munc13,which all inhibit and catalyze SNARE assembly,and regulate different membrane fusion processes.SNAREs play an important role in membrane fusion.Mutation of SNAREs or dysfunction of regulatory factors result in depression,mental retardation,movement disorders,epilepsy,etc.Neurodegenerative diseases,such as Parkinson’s syndrome and Alzheimer’s disease,are closely related to SNAREs dysfunction.Therefore,the normal function of SNAREs,including the correct assembly of SNARE complex and appropriate regulation of secretion,is essential to not only the development of the nervous and immune system but also the growth and development of organisms.Although great progress has been made in the study of SNARE functions and regulatory mechanisms in the past two decades,there are still many other secretory regulatory factors that have not been fully appreciated,such as secretagogin(SCGN)and Doc2(double C2 domain protein).SCGN is a calcium ion sensor,which contains six EF-hand domains and is highly conserved from fruit flies to humans.Human SCGN highly expresses in pancreaticβcells,brain nervous system and enteroendocrine cells.Previous studies indicated that SCGN protein can interact with SNAREs(especially SNAP-25),cytoskeleton protein,and some cargo proteins.Recent studies have shown that SCGN controls a variety of hormone secretion events in different tissues.For example,glucose-stimulated insulin secretion was significantly reduced when SCGN was disturbed in pancreaticβcells.Moreover,SCGN knockout mice showed glucose intolerance at early life-stage and ultimately developed hyperglycemia.In addition,in different neurons,SCGN is essential for the secretion of corticotropin-releasing hormone and metalloproteinase-2.In addition to regulating the secretion process,SCGN is also involved in various cellular processes,such as insulin synthesis,endoplasmic reticulum stress and protein folding.SCGN deficiency has been linked to the pathogenesis of many diseases,such as neurodegeneration diseases,autism and schizophrenia.More recently,deficiency in SCGN makes mice more susceptible to inflammatory bowel disease.Although many studies suggest that SCGN are important in cellular and physiological processes,little is known about the mechanism of SCGN regulating cell secretion and developmental processes.The normal communication between neurons depends on the fine-tuned regulation of neurotransmitter release.Action potential(AP)-evoked neurotransmitter release is the primary manner of synaptic communication between neurons.Neurotransmitter release processes includes three forms:two AP-evoked neurotransmitter release forms,rapid synchronous neurotransmitter release as well as slow asynchronous neurotransmitter release,and AP independent spontaneous release that participates in the adjustment of synaptic structure and function(such as postsynaptic excitation,synthesis of postsynaptic proteins,dendritic spines and long-term protruding plasticity).As key regulators in membrane fusion,SNAREs play an important role in all three modes of neurotransmission.Syt1 is a calcium ion sensor protein that regulates the rapid,synchronous neurotransmitter release,and the Doc2 protein(double C2domain protein)regulates the slow,asynchronous neurotransmitter release.Both Doc2protein and Syt1 contain two conserved C2 domains,C2A and C2B,which bind to the membrane relying on calcium ions.The main difference between these two proteins is that syt1 contains a transmembrane domain and is anchored to the synaptic vesicle membrane,while the Doc2 protein is a soluble cytoplasmic protein.The Doc2 protein family includes Doc2α,Doc2βand Doc2γ.The sequences of Doc2αand Doc2βare highly similar,but their expression patterns are different.Doc2αis mainly expressed in brain,while Doc2βis extensively expressed.Doc2γis very different from Doc2αand Doc2β,and predominantly expresses in heart.Interestingly,both Syt1 and Doc2can regulate spontaneous neurotransmitters release.Researches show that Doc2αand Doc2βcan interact with SNARE complexes.In addition,they can also bind to the SNARE regulatory factors,Munc13 and Munc18,promoting SNARE complex assembly and regulating cell secretion.However,the mechanism of how Doc2 regulates secretion and slow neurotransmitter transmission is currently unclear.In order to explore the regulation of SCGN on SNAREs,we used biochemical methods such as pulldown assay to verify the direct interaction between SCGN and SNARE proteins in vitro.We found that SCGN interacts with SNAP-25 but not the assembled SNARE complex.In order to detect the fragment of SNAP-25 binding to SCGN,we generated a few truncated SNAP-25 peptides and found that the C-terminal SNARE motif of SNAP-25 is the key fragment binding to SCGN.Next,we determined the crystal structure of SCGN and SNAP-25-J(143-170 aa)with calcium ions.Each asymmetric unit contains two SCGN proteins,one SNAP-25 peptide(154-170 aa)and12 calcium ions.Based on the crystal structure,SNAP-25 fragment binds to the third domain in C-terminal of SCGN(including EF5 and EF6),and each SCGN binds to six calcium ions.Compared with the structure of Holo form SCGN,we found that the overall configuration of the SCGN is still in a"V"shape,even when itself interacts with calcium ions and SNAP-25 peptide,but the conformation changes greatly.Each structural domain has a significant conformation rearrangement.When domainⅢof SCGN are aligned,its domainⅠand domainⅡare rotated nearly 180°with respect to domainⅢ,and the calcium ion binding region has changed from aβ-sheet structure to a loop structure in each domain.The conformation rearrangements of SCGN expose a hydrophobic groove on its surface to accommodate theα-helical structure of SNAP-25.We also verified the key amino acid sites of the interaction between SCGN and SNAP-25 and found that SCGN interacts SNAP-25 mainly through hydrophobic interaction and hydrogen bonds.We aligned the structures of SNAP-25 in SCGN-SNAP-25-Ca2~+complex and in SNARE complex and found that SNAP-25 adopts similar conformation in these two complexes.In vitro liposome fusion experiments showed that SCGN inhibits SNARE assembly by interacting with SNAP-25.We also found that the only EF-hand protein in Drosophila,Cbp53E,is a paralog of SCGN and interacts with SNAP-25.Since SNAP-23 is highly homologous to SNAP-25,SCGN can interact with SNAP-23,indicating that SCGN and SNAP-25 interaction is important and conserved in the regulation of neuronal activity.SCGN promotes cell secretion,we speculate that SCGN must not inhibit SNARE assembly in the whole secretion event.Therefore,we tried to study how SCGN,Doc2and SNAP-25 are regulated by each other.We found that SCGN specifically binds to the linker region of Doc2α(Doc2α-C)relying on calcium ions by in vitro pulldown assays.Through sequence alignment,we found the amino acid sequences of Doc2α-C and SNAP-25-J are highly similar to each other.Doc2αand SNAP-25 bind to SCGN in a similar manner,and Doc2αcan compete with SNAP-25 when interacts with SCGN.Liposome fusion experiments showed that Doc2αcan abort SCGN’s inhibition of SNARE assembly,thus regulate membrane fusion and cell secretion together with SCGN.In summary,we comprehensively adopted study methods of biochemistry and structural biology,obtained abundant data in vitro.We have proved that with the assistance of Doc2α,SCGN can inhibit SNARE-mediated membrane fusion.SCGN may act as both an inhibitor and an activator of SNARE activity in exocytosis.We proposed a new mechanism of SNARE in membrane fusion regulation,which laid the foundation and provided a new theoretical basis for the study of neurodegenerative diseases and secretion-related diseases.