Structural analysis of the catalytic domain of tetanus neurotoxin.

Clostridium neurotoxins, comprising the tetanus neurotoxin and the seven antigenically distinct botulinum neurotoxins (BoNT/A-G), are among the known most potent bacterial protein toxins to humans. Although they have similar function, sequences and three-dimensional structures, the substrate specificity and the selectivity of peptide bond cleavage are different and unique. Tetanus and botulinum type B neurotoxins enzymatically cleave the same substrate, vesicle-associated membrane protein, at the same peptide bond though the optimum length of substrate peptide required for cleavage by them is different. Here, we present the first experimentally determined three-dimensional structure of the catalytic domain of tetanus neurotoxin and analyze its active site. The structure provides insight into the active site of tetanus toxin's proteolytic activity and the importance of the nucleophilic water and the role of the zinc ion. The probable reason for different modes of binding of vesicle-associated membrane protein to botulinum neurotoxin type B and the tetanus toxin is discussed. The structure provides a basis for designing a novel recombinant vaccine or structure-based drugs for tetanus.     

Characterization of a specific interaction between ADAM23 and cellular prion protein

ADAMs are transmembrane proteins implicated in several biological functions, including cytokine and growth factor shedding, fertilization, muscle and nervous system development. Here, we show for the first time that ADAM23, which is predominantly expressed in the central nervous system, co-localizes with cellular prion protein (PrP^C) at plasma membrane of mouse hippocampal neurons and neuroblastoma cells. Co-immunoprecipitation and pull-down assay showed a physical interaction between ADAM23 and both recombinant and endogenous PrP^C. Glycosylation seems to be not relevant to the observed interaction since both ADAM23 and PrP^C recombinant proteins expressed in bacteria or extracted from eukaryotic cells treated with tunicamycin are still able to bind each other. In vitro binding assays also suggested that the disintegrin domain of ADAM23 is able to interact directly with PrP^C. Taken together, these findings point out PrP^C as a novel molecular partner for ADAM23 in the nervous systems.     

Effect of metalloprotease inhibitors on invasion of red blood cell by Plasmodium falciparum

For successful invasion, the malaria merozoite needs to attach to the red blood cell membrane, undergo reorientation, form a junction of the apical end with the host membrane, and internalize. Malaria proteases have been implicated in the invasion process, but their specific cellular functions remain unclear. To demonstrate the involvement of metalloprotease in the process of Plasmodium falciparum merozoite entry into host red blood cell, schizont-infected red blood cells and parasitophorous vacuolar membrane-enclosed merozoite structures were treated with 1,10-phenanthroline, a metal chelator, resulting in a reduction of invasion with IC50 value of 25 and 29 microM, respectively. Absence of an accumulation of schizont stages after treatment with 1,10-phenanthroline indicated that the inhibitory effect was not due to suppression of merozoite release from red blood cells, but on the invasion step. Although treatment with GM6001, a well-known inhibitor of the mammalian matrix and disintegrin metalloprotease family, was less effective, nevertheless this study points to the importance of metal-requiring protease in the process of invasion of host red blood cell by the malaria parasite.     

BEHAB/brevican requires ADAMTS-mediated proteolytic cleavage to promote glioma invasion

Malignant gliomas are the most common and deadly primary brain tumors, due to their infiltrative invasion of the normal neural tissue that makes them virtually impossible to completely eliminate. We have previously identified and characterized the proteoglycan BEHAB/brevican in gliomas and have demonstrated that upregulation and cleavage of this CNS-specific molecule promote glioma invasion. Here, we have further investigated if the proteolytic processing of BEHAB/brevican by metalloproteases of the ADAMTS family is a necessary step in mediating its pro-invasive effect in glioma. By generating a site-specific ((396)SRG(398) --> NVY) mutant form resistant to ADAMTS cleavage, we have shown that the predominant proteolytic processing of BEHAB/brevican by glioma cells occurs only at this site. More importantly, "uncleavable" BEHAB/brevican is unable to enhance glioma cell invasion in vitro and tumor progression in vivo. In addition, our results suggest that the full-length protein and its cleavage products may act independently because the mutant form does not exert a dominant negative effect on normal BEHAB/brevican expression or cleavage. These results illustrate how the regulated processing of major components of the neural extracellular matrix has important functional implications in glioma progression. In addition, our findings underscore the relevance of the ADAMTS family of metalloproteases as attractive targets for novel pharmacological approaches in glioma therapy.     

乳腺癌钼靶X射线征象与血清相关肿瘤标志物、MMP-9相关关系研究

目的：探究乳腺癌钼靶 X 射线征象与血清相关肿瘤标志物 CEA、CA15-3、CA19-9和金属蛋白酶家族MMP-9之间关系。方法：分析2013年1月～2014年12月在我院接受治疗诊断为乳腺癌患者的临床资料，列为观察组，选取同时期在我院进行治疗的乳腺良性病变的患者作为对照组。结果：两组研究对象在是否检出毛刺征或星芒状边缘、边缘分叶、透亮环、非对称密度增高伴结构紊乱、钙化水平上，差异均具有统计学意义；钼靶 X 射线线诊断乳腺癌的敏感性为85.96％，特异性为90.38％；观察组研究对象血清中 CEA、CA15-3、CA19-9、MMP-9水平显著高于对照组，差异具有统计学意义；观察组研究对象血清中 MMP-9与 CEA、CA15-3、CA19-9均呈现正相关。结论：钼靶 X 射线线诊断乳腺癌的敏感性为85.96％，特异性为90.38％，乳腺癌患者血清中 MMP-9与 CEA、CA15-3、CA19-9呈现正相关。     

瑞舒伐他汀治疗不稳定型心绞痛疗效及对血脂、血浆金属蛋白酶的影响

目的：探讨瑞舒伐他汀对不稳定型心绞痛患者血脂和血浆金属蛋白酶（MMPs）的影响。方法：68例不稳定型心绞痛患者随机分为观察组和对照组各34例。对照组予抗凝、扩血管、降脂和改善心肌微循环等综合性对症支持治疗;观察组在此基础上加用瑞舒伐他汀片10 mg,po qd,连用6个月。观察两组血脂（TC、TG、LDL-C、HDL-C）和血浆MMP-2和MMP-8水平变化。比较两组临床总有效率。结果：观察组患者TC、TG和LDL-C水平均较前明显下降,HDL-C水平较前明显上升（P〈0.05）,对照组血脂各指标治疗前后无明显变化（P〉0.05）;治疗后两组血浆MMP-2和MMP-8水平较治疗前明显下降（P〈0.05或0.01）,且观察组下降幅度更显著（P〈0.05）。观察组临床总有效率明显优于对照组（P〈0.05）,治疗期间两组均未出现明显不良反应。结论：阿瑞舒伐他汀治疗不稳定型心绞痛具有较好的疗效,安全性较好,能明显降低血脂和血浆MMP-2和MMP-8水平,有利于动脉粥样硬化斑块的稳定,具有良好的心血管保护作用。     

Caveolin-1 negatively regulates a metalloprotease-dependent epidermal growth factor receptor transactivation by angiotensin II

A metalloprotease, ADAM17, mediates the generation of mature ligands for the epidermal growth factor receptor (EGFR). This is the key signaling step by which angiotensin II (AngII) induces EGFR transactivation leading to hypertrophy and migration of vascular smooth muscle cells (VSMCs). However, the regulatory mechanism of ADAM17 activity remains largely unclear. Here we hypothesized that caveolin-1 (Cav1), the major structural protein of a caveolae, a membrane microdomain, is involved in the regulation of ADAM17. In cultured VSMCs, infection of adenovirus encoding Cav1 markedly inhibited AngII-induced EGFR ligand shedding, EGFR transactivation, ERK activation, hypertrophy and migration, but not intracellular Ca²⁺ elevation. Methyl-β-cyclodextrin and filipin, reagents that disrupt raft structure, both stimulated an EGFR ligand shedding and EGFR transactivation in VSMCs. In addition, non-detergent sucrose gradient membrane fractionations revealed that ADAM17 cofractionated with Cav1 in lipid rafts. These results suggest that lipid rafts and perhaps caveolae provide a negative regulatory environment for EGFR transactivation linked to vascular remodeling induced by AngII. These novel findings may provide important information to target cardiovascular diseases under the enhanced renin angiotensin system.     

Effect of HUVEC apoptosis inducing proteinase from Vipera lebetina venom (VLAIP) on viability of cancer cells and on platelet aggregation

Three cancer cell lines, the human androgen independent prostate cancer PC-3, androgen dependent LNCaP prostate cancer and human chronic myeloid leukaemia cell line K-562, were treated with Sephadex G-100 sf fractions of Vipera lebetina venom and with HUVEC apoptosis inducing heterodimeric metalloproteinase (VLAIP) from the same venom. The venom was separated into nine fractions using size-exclusion chromatography on Sephadex G-100 sf. The effect of V. lebetina venom gel filtration fractions on the viability of studied cancer cells was different: high molecular mass fractions were the most effective on PC-3 cells. The viability of LNCaP cells was inhibited most strongly by the third fraction. The first and the second fractions contain different metalloproteinases including VLAIP that also most effectively reduced the viability of PC-3 cells. VLAIP decreased PC-3 cell viability in a concentration- and time-dependent manner but did not induce apoptosis as shown by DNA fragmentation assay. VLAIP induced changes in cell shape, rounding up and detachment. VLAIP inhibited the PC-3 cell adhesion to extracellular matrix proteins collagen I, fibronectin and vitronectin but not to fibrinogen. VLAIP had no significant effect on the viability of LNCaP and K-562 cells.VLAIP was also capable to inhibit ADP- and collagen-induced platelet aggregation dose-dependently. IC₅₀ was determined to be 1.89 μM and 0.94 μM, respectively.     

日本血吸虫金属蛋白酶基因的克隆和表达及其对小鼠的免疫保护性研究

目的克隆和表达日本血吸虫金属蛋白酶(metalloprotease)编码基因并纯化表达产物,观察其在成虫体内定位及在小鼠的免疫试验。方法根据表达序列标签(EST)测序的结果设计引物,从含有金属蛋白酶基因的日本血吸虫cDNA克隆(SJM2CFB04,简称SjB04)中扩增得到该编码基因片段,亚克隆至原核表达载体pET28a中表达,应用组氨酸标签亲和层析法纯化表达产物,蛋白质印迹(Western blotting)分析其免疫原性。应用间接免疫荧光法,观察金属蛋白酶在血吸虫成虫体内的分布。将18只C57BC/6小鼠随机分成两组,实验组用SjB04重组蛋白(25μg/只)免疫小鼠,共3次,每次间隔2周;对照组用佐剂免疫(50μl/只)。末次免疫后2周,每鼠腹部感染40±2条日本血吸虫尾蚴,攻击后第37天起连续收集6 d小鼠粪便,计算每克粪虫卵数和减卵率;第42天剖杀小鼠,门静脉灌注收集成虫并计算减虫率。结果获得了SjB04基因(ORF 528 bp)的编码序列,构建了原核表达载体,并在大肠埃希菌中表达,经组氨酸标签亲和层析法获得纯化重组蛋白SjB04。免疫荧光法检测结果显示,SjB04主要定位于日本血吸虫成虫的肠管表...