动脉瘤性蛛网膜下腔出血患者vWF、GMP-140及ADAMTS13的表达水平及临床意义

目的:探讨动脉瘤性蛛网膜下腔出血(aSAH)患者中血管性假血友病因子(v WF)、血小板膜糖蛋白-140(GMP-140)、血管性血友病因子裂解蛋白酶(ADAMTS13)的表达水平及临床意义。方法:选取2014年1月至2016年12月我院神经外科收治的83例aSAH患者,分为脑血管痉挛(CVS)组37例和无CVS组46例;迟发性脑缺血(DCI)组31例和非DCI组52例;根据不同动脉瘤直径分为5 mm组43例,5-10 mm组29例,10 mm组11例;预后良好组49例和预后不良组34例,检测aSAH患者血浆v WF、GMP-140、ADAMTS13水平,并分析各指标之间的相关性。结果:CVS组患者第4 d、10 d血浆v WF水平高于非CVS组,第1 d、4 d、10 d血浆GMP-140水平高于非CVS组,第1 d、10 d血浆ADAMTS13水平低于非CVS组,差异均有统计学意义(P0.05)。DCI组患者第1 d血浆v WF水平高于非DCI组,ADAMTS13水平低于非DCI组,第4 d血浆v WF、GMP-140水平高于非DCI组,差异均有统计学意义(P0.05)。10 mm组患者第1 d、4 d血浆v WF、GMP-140水平高于5 mm组和5-10 mm组,且5-10 mm组第4d的血浆v WF水平、第1 d的血浆,水平均高于5 mm组,差异有统计学意义(P0.05);10 mm组患者第1d的血浆ADAMTS13水平低于5 mm组和5-10 mm组,且5-10 mm组低于5 mm组,差异有统计学意义(P0.05)。预后良好组患者第4 d、10 d血浆v WF水平低于预后不良组,第1 d、4 d、10 d血浆GMP-140水平低于预后不良组,第1 d、4 d血浆ADAMTS13水平高于预后不良组,差异均有统计学意义(P0.05)。Pearson相关分析结果显示,第1 d、4 d血浆v WF与GMP-140呈正相关,与ADAMTS13呈负相关,GMP-140与ADAMTS13呈负相关(r=0.334、-0.426、-0.398、0.278、-0.311、-0.235,P0.05),第10 d血浆v WF、GMP-140、ADAMTS13之间无明显相关性(P0.05)。结论:v WF、GMP-140、ADAMTS13与CVS、DCI、动脉瘤直径以及预后密切相关,联合检测有助于综合评估aSAH患者病情,改善预后,值得临床推广。     

Inhibition of EGFR attenuates fibrosis and stellate cell activation in diet-induced model of nonalcoholic fatty liver disease

Nonalcoholic fatty liver disease (NAFLD) is the most common cause of chronic liver disease. NAFLD begins with steatosis and advances to nonalcoholic steatohepatitis (NASH) and cirrhosis. The molecular mechanisms involved in NAFLD progression are not understood. Based on recent studies showing dysregulation of epidermal growth factor receptor (EGFR) in animal models of liver injury, we sought to determine if inhibition of EGFR mitigates liver fibrosis and HSC activation in NAFLD. We utilized the high fat diet (HFD)-induced murine model of liver injury to study the role of EGFR in NAFLD. The lipid accumulation, oxidative stress, hepatic stellate cell (HSC) activation and matrix deposition were examined in the liver tissues. We also evaluated the EGFR signaling pathway, ROS activation and pro-fibrogenic phenotype in oxidized low density lipoproteins (ox-LDL) challenged cultured HSCs. We demonstrate that EGFR was phosphorylated in liver tissues of HFD murine model of NAFLD. Inhibition of EGFR prevented diet-induced lipid accumulation, oxidative stress, and HSC activation and matrix deposition. In cultured HSCs, we show that ox-LDL caused rapid activation of the EGFR signaling pathway and induce the production of reactive oxygen species. EGFR also mediated HSC activation and promoted a pro-fibrogenic phenotype. In conclusion, our data demonstrate that EGFR plays an important role in NAFLD and is an attractive target for NAFLD therapy.     

Human fertilin β: Identification,characterization, and chromosomal mapping of an ADAM gene family member

Fertilin α/β (PH30 α/β) is a heterodimeric sperm surface protein containing binding and fusion domains with potential for interaction with integrin receptors on the oocyte. We report the cDNA cloning, deduced amino acid sequence, tissue specificity, and chromosomal mapping of human fertilin β. Encoded by a 2205 nucleotide open reading frame, the deduced amino acid sequence of human fertilin β contains pro-, metalloprotease-like, disintegrin-like, cysteine-rich, epidermal growth factor-like (EGF) repeat, transmembrane, and cytoplasmic domains. Due to this domain organization, human fertilin β has been identified as a member of the ADAM family, which is composed of membrane-anchored proteins having A Disintegrin And Metalloprotease domain. The amino acid sequence of human fertilin β shares 90%, 56%, and 55% identity, respectively, to monkey, guinea pig, and mouse fertilin β homologs. A phenylalanine-glutamate-glutamate (FEE) binding tripeptide within the disintegrin-like domain of human fertilin β, homologous to other fertilin β RGD-like (arginine-glycine-aspartic acid) tripeptides, could compete for recognition by integrins and other receptors. Northern analysis from 16 human tissues revealed human fertilin β's 2.9 kb message only in testis, which raises interest in possible clinical applications of this molecule as a contraceptive vaccinogen. Human fertilin β maps to chromosome 8, band p11.2, by fluorescence in situ hybridization and mouse/human somatic cell hybrid Southern hybridization. Mol. Reprod. Dev. 46:363–369, 1997. © 1997 Wiley-Liss, Inc.     

Ectodomain shedding of E-cadherin and c-Met is induced by Helicobacter pylori infection

Helicobacter pylori, a microaerophilic gram-negative bacterium, colonizes the human stomach. About 50% of the world's population is infected, and this infection is considered as the major risk factor for the development of gastric adenocarcinomas in 1% of infected subjects. Carcinogenesis is characterized by the process of epithelial-to-mesenchymal transition (EMT), in the course of which fully differentiated epithelial cells turn into depolarized and migratory cells. Concomitant disruption of adherence junctions (AJ) is facilitated by growth factors like hepatocyte growth factor 1 (HGF-1), but has been also shown to depend on ectodomain shedding of E-cadherin. The aim of this study was to investigate the impact of infection with H. pylori of NCI-N87 gastric epithelial cells on the shedding of E-cadherin and HGF-receptor c-Met. Our results show that infection with H. pylori provokes shedding of the surface proteins c-Met and E-cadherin. Evidence is provided that ADAM10 contributes to the shedding of c-Met and E-cadherin.     

Inhibition of platelets and tumor cell adhesion by the disintegrin domain of human ADAM9 to collagen I under dynamic flow conditions

This work aimed to investigate the role of the disintegrin domain of the human ADAM9 (ADAM9D) on the adhesion of breast tumor cells and platelets to collagen I, in a dynamic flow assay to simulate in vivo shear conditions. Recombinant ADAM9D was able to support tumor cell adhesion through binding to the β1 integrin subunit and also to inhibit the invasion through matrigel in vitro. In a dynamic flow assay ADAM9D inhibited about 75% and 65% of MDA-MB-231 tumor cells and platelet adhesion to collagen I, respectively. In addition, it was demonstrated that αVβ3 integrin is new interacting partner for ADAM9D. In conclusion, these results suggest a role for the disintegrin domain of ADAM9 in the metastatic process. Also, ADAM9D may be a tool for investigating the role of ADAMs in metastasis and cancer progression and for the design of selective inhibitors against the adhesion and extravasation of cancer cells.     

Mechanism and significance of specific proteolytic cleavage of Reelin

Reelin is a secreted glycoprotein essential for normal brain development and function. In the extracellular milieu, Reelin is subject to specific cleavage at two (N-t and C-t) sites. The N-t cleavage of Reelin is implicated in psychiatric and Alzheimer’s diseases, but the molecular mechanism and physiological significance of this cleavage are not completely understood. Particularly, whether the N-t cleavage affects the signaling activity of Reelin remains controversial.Here, we show that the protease in charge of the N-t cleavage of Reelin requires the activity of certain proprotein convertase family for maturation and has strong affinity for heparin. By taking advantage of these observations, we for the first time succeeded in obtaining “Uncleaved” and “Completely Cleaved” Reelin proteins. The N-t cleavage splits Reelin into two distinct fragments and virtually abolishes its signaling activity. These findings provide an important biochemical basis for the function of Reelin proteolysis in brain development and function.     

Isoform-Selective Interaction of the Adaptor Protein Tks5/FISH with Sos1 and Dynamins

The adaptor protein Tks5/FISH (tyrosine kinase substrate 5/five SH3 domains, hereafter termed Tks5) is a crucial component of a protein network that controls the invasiveness of cancer cells and progression of Alzheimer's disease. Tks5 consists of an amino-terminal PX domain that is followed by five SH3 domains (SH3A-E), and two different splice variants are expressed. We identified son of sevenless-1 (Sos1) as a novel binding partner of Tks5 and found colocalization of Tks5 with Sos1 in human epithelial lung carcinoma (A549) cells and in podosomes of Src-transformed NIH 3T3 cells. We observe synergistic binding of SH3A and SH3B to Sos1 when peptide arrays are used, indicating that the tandem SH3A and SH3B domains of Tks5 can potentially bind in a superSH3 binding mode, as was described for the homologous protein p47phox. These results are further corroborated by pull-down assays and isothermal titration calorimetry showing that both intact SH3 domains are required for efficient binding to the entire proline-rich domain of Sos1. The presence of a basic insertion between the SH3A and SH3B domains in the long splice variant of Tks5 decreases the affinity to Sos1 isoforms about 10-fold as determined by analytical ultracentrifugation. Furthermore, it leads to an alteration in the recognition of binding motifs for the interaction with Sos1: While the insertion abrogates the interaction with the majority of peptides derived from the proline-rich domains of Sos1 and dynamin that are recognized by the short splice isoform, it enables binding to a different set of peptides including a sequence comprising the splice insertion in the long isoform of Sos1 (Sos1_2). In the absence of the basic insertion, Tks5 was found to bind a range of Sos1 and dynamin peptides including conventional proline-rich motifs and atypical recognition sequences. Hereby, the tandem SH3 domains in Tks5 employ two distinct types of binding modes: One class of peptides is recognized by single SH3 domains, whereas a second class of peptides requires the presence of both domains to bind synergistically. We conclude that the tandem SH3A and SH3B domains of Tks5 constitute a versatile module for the implementation of isoform-specific protein-protein interactions.     

Multimerisation of A disintegrin and metalloprotease protein-17 (ADAM17) is mediated by its EGF-like domain

A disintegrin and metalloprotease protein 17 (ADAM17) is a transmembrane zinc dependent metalloprotease. The catalytic activity of the enzyme results in the shedding of a broad range of membrane proteins. The release of the corresponding ectodomains induces a switch in various physiological and pathophysiological processes. So far there is not much information about the molecular mechanism of ADAM17 activation available. As for other transmembrane proteases, multimerisation may play a critical role in the activation and function of ADAM17. The present work demonstrates that ADAM17 indeed exists as a multimer in the cell membrane and that this multimerisation is mediated by its EGF-like domain.