Differentiation in T.cruzi:an emerging AIDS pathogen

描述（由申请人提供）：原生动物寄生虫克氏锥虫，恰加斯病的病原体，已经成为HIV/ aids相关的机会性感染。当前anti-T。克鲁兹病毒具有剧毒，对慢性恰加斯病没有有效的治疗方法。我们对其分化的分子信号的理解的提高可能会导致这些过程的有效抑制剂，提供新的治疗方法。克氏锥虫的分化涉及cAMP，但其作用的途径尚不清楚。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。本实验室克隆了克氏体PKA催化亚基（TcPKA-C）基因和PKA调控亚基（TcPKA-R）基因，数据表明PKA催化亚基和调控亚基在发育过程中受到调控。这些数据支持了PKA活性在克氏体分期分化中起重要作用的假设。我们现在计划研究TcPKA在分化中的作用。本研究将在转染的疟原虫中研究TcPKA与组成活性TcPKAC的过表达、TcPKA与PKI克隆的阻断、TcPKA- r的显性负抑制以及TcPKA- c的条件敲除对其分化和增殖的影响。通过对TcPKA-R的Nterminus的缺失分析和质谱分析TcPKA-R的翻译后修饰，我们将探讨TcPKA-R在T. crozi细胞内靶向的机制。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试验和蛋白质组学方法确定TcPKA下游相互作用分子。

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.