Differentiation in T.cruzi:an emerging AIDS pathogen

描述(申请人提供)：原生动物寄生虫锥虫克氏锥虫，恰加斯病的病原体，已成为艾滋病毒/艾滋病相关的机会性感染。目前的抗T。克鲁兹制剂毒性很大，目前还没有有效的治疗慢性恰加斯病的方法。我们对导致其分化的分子信号的了解的提高可能导致有效抑制这些过程，提供新的和新的治疗方法。克鲁兹毛滴虫的分化涉及cAMP，但它的作用途径尚不清楚。与其他真核生物一样，cAMP依赖的蛋白激酶(PKA)可能是控制细胞分化的主要信号转导途径。本实验室克隆了克鲁兹毛滴虫的PKA催化亚基基因(TcPKA-C)和PKA调节亚基基因(TcPKA-R)，结果表明PKA催化亚基和调节亚基均受发育调控。这些数据支持PKA活性在克氏锥虫幼虫分化过程中起重要作用的假说。我们现在计划研究TcPKA在分化中的功能。在转基因寄生虫中，我们将研究TcPKA的过表达和用PKI克隆阻断TcPKA、显性负抑制TcPKA-R和条件性敲除TcPKA-C对分化和增殖的影响。通过对TcPKA-R末端的缺失分析和对TcPKA-R翻译后修饰的质谱分析，将研究TcPKA-R在克鲁兹毛滴虫中的细胞内靶向机制。为了进一步描述这一途径的组成部分，我们将使用酵母双杂交分析和蛋白质组学方法鉴定TcPKA下游相互作用的分子。

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.