不同血清型肉毒毒素受体结合域研究进展

肉毒毒素(botulinum neurotoxin, BoNT)是人类已知毒性最强的蛋白质之一,可以引起肌肉松弛麻痹,严重时可导致死亡。肉毒毒素共分为7种血清型(BoNT/A-BoNT/G),根据氨基酸序列差异可进一步分为40多种亚型。肉毒毒素分子结构由3个基本结构域组成：重链羧基端细胞受体结合域、氨基端的易位域和轻链催化域。在运动神经元表面,受体结合域首先与聚唾液酸神经节苷脂结合,随后与突触囊泡蛋白2或突触囊泡结合蛋白结合形成双受体复合物。每种血清型的受体结合域都必须与其相应受体结合才能发挥作用。肉毒毒素的结构功能及其对宿主的作用一直都是研究热点。近年来,因受体结合域可以促进肉毒毒素与运动神经元膜特异性结合,而成为新的研究方向。本综述将概述不同血清型肉毒毒素与受体结合过程中受体结合域结构变化和结合位点差异。通过分析不同血清型及亚型的序列以及受体结合域结构特征,可以更好地了解细胞受体结合域的序列差异和功能,并为肉毒毒素的治疗策略提供新思路。     

Identification of the synaptic vesicle glycoprotein 2 receptor binding site in botulinum neurotoxin A

Botulinum neurotoxins (BoNTs) inhibit neurotransmitter release by hydrolysing SNARE proteins. The most important serotype BoNT/A employs the synaptic vesicle glycoprotein 2 (SV2) isoforms A-C as neuronal receptors. Here, we identified their binding site by blocking SV2 interaction using monoclonal antibodies with characterised epitopes within the cell binding domain (H_C). The site is located on the backside of the conserved ganglioside binding pocket at the interface of the H_CC and H_CN subdomains. The dimension of the binding pocket was characterised in detail by site directed mutagenesis allowing the development of potent inhibitors as well as modifying receptor binding properties.     

Meta-analysis supports association of a functional SNP (rs1801133) in the MTHFR gene with Parkinson's disease

The MTHFR is a candidate risk gene for Parkinson's disease (PD), and a functional SNP (rs1801133) in the coding region of this gene has been investigated for the associations with the illness extensively among worldwide populations, but overall the results were inconsistent. Here, to assess the relationship between rs1801133 and risk of PD in general populations, we conducted a systematic meta-analysis by combining all available case–control samples in European and Asian populations, with a total of 1820 PD cases and 7530 healthy controls, and the pooled odds ratios (ORs) and 95% confidence intervals (95% CIs) for rs1801133 and PD were calculated using the Mantel–Haenszel method with a fixed-effect model. Overall, rs1801133 was significantly associated with the risk of PD (allelic model, pooled OR = 1.212 for T allele, 95% CI = 1.097–1.340, p-value = 0.0002). When stratifying for ethnicity, significant association was also observed in European (allelic model, pooled OR = 1.187 for T allele, 95% CI = 1.058–1.332, p-value = 0.004) and Asian samples (allelic model, pooled OR = 1.293 for T allele, 95% CI = 1.058–1.580, p-value = 0.012) respectively. In addition, rs1801133 was also significantly associated with MTHFR mRNA expression in both CEU (European, p-value = 0.0149) and CHB (Chinese, p-value = 0.0178) HapMap populations. Collectively, our meta-analysis suggests that rs1801133 is significantly associated with susceptibility to PD in European and Asian populations, and MTHFR is likely an authentic risk gene for PD.     

Implication of synaptotagmins 4 and 7 in activity-dependent somatodendritic dopamine release in the ventral midbrain

Dopamine (DA) neurons can release DA not just from axon terminals, but also from their somatodendritic (STD) compartment through a mechanism that is still incompletely understood. Using voltammetry in mouse mesencephalic brain slices, we find that STD DA release has low capacity and shows a calcium sensitivity that is comparable to that of axonal release. We find that the molecular mechanism of STD DA release differs from axonal release with regard to the implication of synaptotagmin (Syt) calcium sensors. While individual constitutive knockout of Syt4 or Syt7 is not sufficient to reduce STD DA release, the removal of both isoforms reduces this release by approximately 50%, leaving axonal release unimpaired. Our work unveils clear differences in the mechanisms of STD and axonal DA release.     

Complexin在囊泡运输中的作用

神经递质释放对维持生物体正常的生命活动有着重要的意义,它是由囊泡运输介导完成的.神经元细胞中囊泡运输涉及许多蛋白质间的相互作用,共同调控这一复杂的过程,可溶性小分子蛋白Complexin(Cpx)在这一过程中起着重要的作用,它同时具有抑制囊泡自发发放和促进囊泡诱发发放的功能.本文综合国内外近20年的研究,着重介绍了Cpx蛋白各部分结构域的功能,及其与一些囊泡分泌相关蛋白,如SNARE复合体、Synaptotagmin(Syt),间的相互作用机制及其最新进展.     

Chronic hypoxia stress-induced differential modulation of heat-shock protein 70 and presynaptic proteins

Chronic hypoxia exposure can cause neurobehavioral dysfunction, but the underlying cellular and molecular mechanisms remain unclear. Here, we found that adult Lymnaea stagnalis snails maintained in low O(2) (approximately 5%) for 4 days developed slowed reactions to light stimuli, and reduced righting movement. Semiquantitative immunoblotting analyses showed that hypoxia exposure induced increased expression of heat-shock protein (HSP)70 in ganglion preparations, and suppressed expression of the presynaptic proteins syntaxin I, synaptic vesicle protein 2 (SV2) and synaptotagmin I. Detailed time course analyses showed that an early moderate increase developed within 6 h, preceding a substantial up-regulation of HSP70 after 4 days; an early reduction of syntaxin I in the first 24 h; a delayed reduction of synaptotagmin I after 4 days; and a biphasic change in SV2. Using a double-stranded RNA interference approach, we demonstrated that preventing the hypoxia inducible HSP70 enhanced down-regulation of syntaxin and synaptotagmin, and aggravated motor and sensory suppression. Co-immunoprecipitation analysis revealed an interaction between HSP70 and syntaxin. We have thus provided the first evidence that early induction of HSP70 by chronic hypoxia is critical for maintaining expression levels of presynaptic proteins. These findings implicate a new molecular mechanism underlying chronic hypoxia-induced neurobehavioral adaptation and impairment.     

The Calcium-Dependent and Calcium-Independent Membrane Binding of Synaptotagmin 1: Two Modes of C2B Binding

The Ca²⁺-independent membrane interactions of the soluble C2 domains from synaptotagmin 1 (syt1) were characterized using a combination of site-directed spin labeling and vesicle sedimentation. The second C2 domain of syt1, C2B, binds to membranes containing phosphatidylserine and phosphatidylcholine in a Ca²⁺-independent manner with a lipid partition coefficient of approximately 3.0 × 10² M^− 1. A soluble fragment containing the first and second C2 domains of syt1, C2A and C2B, has a similar affinity, but C2A alone has no detectable affinity to phosphatidylcholine/phosphatidylserine bilayers in the absence of Ca²⁺. Although the Ca²⁺-independent membrane affinity of C2B is modest, it indicates that this domain will never be free in solution within the cell. Site-directed spin labeling was used to obtain bilayer depth restraints, and a simulated annealing routine was used to generate a model for the membrane docking of C2B in the absence of Ca²⁺. In this model, the polybasic strand of C2B forms the membrane binding surface for the domain; however, this face of C2B does not penetrate the bilayer but is localized within the aqueous double layer when C2B is bound. This double-layer location indicates that C2B interacts in a purely electrostatic manner with the bilayer interface. In the presence of Ca²⁺, the membrane affinity of C2B is increased approximately 20-fold, and the domain rotates so that the Ca²⁺-binding loops of C2B insert into the bilayer. This Ca²⁺-triggered conformational change may act as a switch to modulate the accessibility of the polybasic face of C2B and control interactions of syt1 with other components of the fusion machinery.     

Down-regulated expression of exocytotic proteins in pancreatic islets of diabetic GK rats

Exocytosis is regulated by exocytotic proteins, which are present in insulin-secreting beta-cells and play regulatory roles in insulin secretion. Non-insulin dependent diabetes mellitus (type 2 diabetes) is a disease characterized by impaired insulin secretion and insulin resistance. Exocytotic protein immunoreactivities were studied in pancreatic islets of type 2 diabetic Goto-Kakizaki (GK) rats using immunofluorescence histochemistry. The immunoreactivities for vesicle-associated membrane protein-2 (VAMP-2), synaptotagmin III, cysteine string protein (CSP), mammalian homologue of the unc-18 gene (Munc-18), alpha-soluble N-ethylmaleimide-sensitive attachment protein (alpha-SNAP), N-ethylmaleimide-sensitive factor (NSF) and synaptosomal-associated protein of 25 kDa (SNAP-25) exhibited weaker immunofluorescence intensity in islets of GK rats as compared to control Wistar rats. Insulin immunoreactivity was also decreased in GK rat beta-cells, whereas no detectable alterations in the expression of actin immunoreactivity could be detected. The data suggest that reduced expression of exocytotic proteins and decreased insulin content may contribute to the diabetic syndrome in the GK rat.     

Beta-strand recombination in tricalbin evolution and the origin of synaptotagmin-like C2 domains

Two protein families involved in membrane traffic, tricalbins and synaptotagmins, contain several copies of C2 domains and are related based on their sequence and domain architecture. Paradoxically, tricalbin and synaptotagmin C2 domains belong to different structural types with apparent circular permutation of terminal beta-strands. To understand whether a topological switch took place, we analyzed tricalbin and synaptotagmin-like C2 domains using two-dimensional structural analysis. We found that yeast tricalbins contain five to six C2 domains. One of these C2 domains possesses many features of synaptotagmin-like C2 domains and also carries a conserved C-terminal strand that is similar to its structural equivalent in synaptotagmin-like C2 domains, suggesting a structural permutation event. Indeed, among higher eukaryotes, animal tricalbins have evolved a C2 domain with synaptotagmin-like topology indicating that the structural conversion has taken place. Investigation of plant synaptotagmins, however, proves that they are direct tricalbin orthologs. Our analysis shows that beta-strand recombination is a possible evolutionary mechanism to generate new structural topologies with altered functional properties.     

Evolutionary conservation of complexins: from choanoflagellates to mice

Complexins are synaptic SNARE complex‐binding proteins that cooperate with synaptotagmins in activating Ca²⁺‐stimulated, synaptotagmin‐dependent synaptic vesicle exocytosis and in clamping spontaneous, synaptotagmin‐independent synaptic vesicle exocytosis. Here, we show that complexin sequences are conserved in some non‐metazoan unicellular organisms and in all metazoans, suggesting that complexins are a universal feature of metazoans that predate metazoan evolution. We show that complexin from Nematostella vectensis, a cnidarian sea anemone far separated from mammals in metazoan evolution, functionally replaces mouse complexins in activating Ca²⁺‐triggered exocytosis, but is unable to clamp spontaneous exocytosis. Thus, the activating function of complexins is likely conserved throughout metazoan evolution.