Differentiation in T.cruzi:an emerging AIDS pathogen

描述（由申请方提供）：原生动物寄生虫克氏锥虫（锥虫病的病原体）已成为HIV/AIDS相关的机会性感染。目前抗T。克氏剂具有高毒性，并且对慢性恰加斯病没有有效的治疗方法。我们对负责其分化的分子信号传导的理解的改进可能导致这些过程的有效抑制剂，从而提供新的和新颖的治疗方法。T. cruzi涉及cAMP，但其作用途径仍不清楚。与其他真核生物一样，cAMP依赖性蛋白激酶（PKA）可能是控制细胞分化的主要信号转导途径。PKA催化亚基（TcPKA-C）基因和PKA调节亚基（TcPKA-R）基因在T. cruzi的基因被我们的实验室克隆，数据表明PKA催化和调节亚基是发育调节的。这些数据支持PKA活性在T. cruzi期分化我们现在计划研究TcPKA在分化中的功能。将在转染的寄生虫中研究用组成型活性TcPKAC过表达TcPKA和用PKI克隆阻断TcPKA、显性负抑制性TcPKA-R以及条件性敲除TcPKA-C对分化和增殖的影响。TcPKA-R在T.通过TcPKA-R的N末端缺失分析和通过用质谱分析TcPKA-R的翻译后修饰来检查cruzi。为了进一步描述这一途径的组成部分，我们将确定TcPKA下游相互作用的分子，使用酵母双杂交试验和蛋白质组学方法。

DESCRIPTION (provided by applicant): The protozoan parasite Trypanosoma cruzi, the causative agent of Chagas' disease, has emerged as a HIV/AIDS-related opportunistic infection. Current anti-T. cruzi agents are highly toxic, and there is no effective treatment for chronic Chagas' disease. Improvement in our understanding of the molecular signaling responsible for its differentiation may lead to effective inhibitors of these processes providing new and novel therapeutic approaches. Differentiation of T. cruzi involves cAMP, but the pathway in which it is acting remains unknown. It is likely, that as in other eukaryotes, cAMP-dependent protein kinase (PKA) is a major signal transduction pathway controlling cell differentiation. The PKA catalytic subunit (TcPKA-C) gene and the PKA regulatory subunit gene (TcPKA-R) of T. cruzi were cloned by our laboratory and data indicates that the PKA catalytic and regulatory subunits are developmentally regulated. These data support the hypothesis that PKA activity is important in T. cruzi stage differentiation. We now plan to study the functions of TcPKA in differentiation. The effects of both overexpression of TcPKA with a constitutively active TcPKAC and blockade of TcPKA with a PKI clone, a dominant negative inhibitory TcPKA-R as well as conditional knockout of TcPKA-C on differentiation and proliferation will be studied in transfected parasites. Mechanisms of intracellular targeting of TcPKA-R in T. cruzi will be examined by deletion analysis of Nterminus of TcPKA-R and by analyzing the post-translational modifications of TcPKA-R with mass spectrometry. To further delineate the components of this pathway, we will identify the TcPKA downstream interacting molecules, using both yeast two-hybrid assays and proteomic approaches.