Differentiation in T.cruzi:an emerging AIDS pathogen

克氏锥虫的分化：一种新出现的艾滋病病原体

• 批准号: 7219517
• 负责人: Huan Huang
• 金额: $ 35.41万
• 依托单位: ALBERT EINSTEIN COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-04-01 至 2010-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7219517
• 关键词: AIDS-Related Opportunistic Infections; Acquired Immunodeficiency Syndrome; Adenylate Cyclase; Appendix; Binding Sites; Catalytic Domain; Cell Differentiation process; Cells; Chagas Disease; Chronic; Condition; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Data; Development; Dictyostelium; Dominant-Negative Mutation; Elements; Eukaryota; Eukaryotic Cell; Event; Extracellular Domain; Feedback; Gene Proteins; Genes; Holoenzymes; Immune response; Knock-out; Laboratories; Lead; Life; Mass Spectrum Analysis; Mediating; Methods; Modeling; Molecular; N-terminal; Organism; Parasites; Pathway interactions; Phosphotransferases; Play; Post-Translational Protein Processing; Process; Protein Kinase; Protein Kinase C; Protein Overexpression; Proteins; Proteomics; Protozoa; Regulation; Reporting; Role; Saccharomyces cerevisiae; Signal Transduction; Signal Transduction Pathway; Site; Staging; Starvation; Stress; Temperature; Tertiary Protein Structure; Therapeutic; Trypanosoma cruzi; Two-Hybrid System Techniques; Ursidae Family; Yeasts; citrate carrier; deletion analysis; environmental change; inhibitor/antagonist; metacyclogenesis; novel; novel therapeutics; pathogen; protein kinase R; protein purification; response; yeast two hybrid system

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.

描述（由申请人提供）：原生动物寄生虫克氏锥虫是南美锥虫病的病原体，已成为一种与艾滋病毒/艾滋病相关的机会性感染。当前抗T。克鲁兹毒剂具有剧毒，慢性恰加斯病尚无有效治疗方法。我们对负责其分化的分子信号传导的理解的提高可能会导致这些过程的有效抑制剂，从而提供新的治疗方法。克氏锥虫的分化涉及 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) 很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锥虫的PKA催化亚基（TcPKA-C）基因和PKA调节亚基基因（TcPKA-R），数据表明PKA催化亚基和调节亚基受到发育调控。这些数据支持这样的假设：PKA 活性在克氏锥虫阶段分化中很重要。 We now plan to study the functions of TcPKA in differentiation.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、显性失活抑制性 TcPKA-R 阻断 TcPKA 以及条件性敲除 TcPKA-C 对分化和增殖的影响。将通过 TcPKA-R N 末端的缺失分析以及通过质谱分析 TcPKA-R 的翻译后修饰来检查 T. cruzi 中 TcPKA-R 的细胞内靶向机制。为了进一步描述该途径的组成部分，我们将使用酵母双杂交测定和蛋白质组学方法来鉴定 TcPKA 下游相互作用分子。