YopT: A Yersinia Virulence Factor

描述（由申请人提供）：最近的生物恐怖主义行为强调了了解细菌发病机制的分子机制的重要性。鼠疫耶尔森氏菌是鼠疫或黑死病的病原体，以前曾被用作生物武器。人们还担心基因工程已被用于产生耶尔森氏菌的抗生素抗性菌株。该提案的重点是了解耶尔森氏菌毒力因子YopT的生物化学。我们的实验室最近证明YopT是一种半胱氨酸蛋白酶。我们建议开发一种高效和灵敏的测定YopT的催化活性。我们还将生产和纯化重组YopT、YopT/SycT复合物和与底物复合的无催化活性的YopT。纯化的蛋白质将用于获得适合使用X射线衍射进行结构分析的结晶蛋白质。我们之所以选择R21作为资助对象，是因为尽管我们在确定这种毒力蛋白的功能方面取得了很好的进展，但我们还没有关于其催化特性的初步数据。此外，尽管我们已经确定了YopT家族成员AvrPphB的结构，但为了鉴定针对该蛋白酶的潜在抑制剂，必须具有YopT的结构。

DESCRIPTION (provided by applicant): Recent acts of bioterrorism have underscored the importance of understanding the molecular mechanisms of bacterial pathogenesis. Yersinia pestis, the causative agent of the plague or the Black Death, has been used previously as a biological weapon. There is also concern that genetic engineering has been used to generate antibiotic resistant strains of Yersinia. This proposal focuses on understanding the biochemistry of the Yersinia virulence factor known as YopT. Our laboratory has recently demonstrated that YopT is a cysteine protease. We propose to develop a highly efficient and sensitive assay for YopT's catalytic activity. We will also produce and purify recombinant YopT, a YopT/SycT complex, and a catalytically inactive YopT complexed with substrate. The purified protein(s) will be used to obtain crystalline protein suitable for structural analyses using X-ray diffraction. We have chosen the R21 venue for funding because, although we have made good progress on determining the function of this virulence protein, we have no preliminary data about its catalytic properties. In addition, even though we have determined the structure of a YopT family member, AvrPphB, it will be essential to have a structure of YopT in order to identify potential inhibitors directed against this protease.