CYST WALL ENDOPROTEASES OF ENTAMOEBA INVADENS AND ENTAMOEBA HISTOLYTICA

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。入侵内阿米巴和溶组织内阿米巴是相关的肠道病原体。前者是一种影响几个爬行动物分类的病原体，后者是一种人体病原体。雅各布蛋白是侵入性大肠杆菌囊壁中丰富的成分，其同源物存在于溶组织大肠杆菌中。雅各布蛋白具有保守的凝集素样结构域，可能对维持这些病原体的囊壁完整性很重要。假定的内源性蛋白酶位点在其序列中是保守的，因此蛋白水解事件可能对囊肿壁的正常功能很重要。溶组织芽孢杆菌基因组中的大多数蛋白酶都是半胱氨酸蛋白酶，这表明假定的雅各布蛋白酶属于这种类型。含有假定的内阿米巴蛋白酶位点的FITC标记肽已被构建用于使用包括MALDI-TOF质谱分析在内的方法检测蛋白酶活性的策略。本研究试图用已知的蛋白酶抑制剂鉴定溶组织芽孢杆菌细胞提取物的特异性蛋白酶活性。结果表明，特异性抑制剂将用于蛋白酶的亲和富集，蛋白质组学方法将用于鉴定活性蛋白酶。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Entamoeba invadens and Entamoeba histolytica are related enteric pathogens. The former is a pathogen affecting several reptile taxons and the later is a pathogen in man. Jacob proteins are abundant components of the cyst wall in E. invadens and homologues of it exist in E. histolytica. The Jacob proteins have conserved lectin-like domains and are likely important for maintenance of cyst wall integrity in these pathogens. Putative endoprotease sites are conserved in their sequence across Entamoeba species and therefore a proteolytic event may be important for proper function of the cyst wall. That most proteases in the E. histolytica genome are cysteine proteases suggests that the putative Jacob proteases are of this type. FITC label peptides containing the putative Entamoeba protease sites have been constructed for use in strategies to detect protease activity using approaches including MALDI-TOF mass spectrometric analysis. This study attempts to identify specific protease activities in E. histolytica cell extracts in strategies using known protease inhibitors. As results indicate, specific inhibitors will be used for affinity enrichment of the proteases and proteomics approaches will be used to identify the active protease(s).