HUMAN T CELLS

人类T细胞

• 批准号: 6170753
• 负责人: Ajit Kumar
• 金额: $ 7.9万
• 依托单位: GEORGE WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-06-01 至 2001-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6170753
• 关键词: DNA directed RNA polymerase; T lymphocyte; complementary DNA; enzyme activity; genetic library; human immunodeficiency virus 1; immunoprecipitation; phosphoprotein phosphatase; phosphorylases; protein purification; recombinant proteins; tissue /cell culture; transcription factor; virus infection mechanism; virus protein

DESCRIPTION (adapted from applicant's abstract): Human Immunodeficiency virus type 1 (HIV-1) regulatory protein Tat increases the synthesis of full length RNA transcripts from the viral LTR. Tat interacts with transcripion factors and RNA polymerase II (RNAP II) enzyme complex to increase the efficiency of the transcript elongation process. One possible mechanism for Tat function proposes the association of Tat with specific kineses which phosphorylate the c-terminal domain (CTD) of the large subunit of RNAPII. The hypophosphorylated RNAPII is associated with the transcription initiation complex while the hyperphosphorylated RNAPII is associated with the elongation complex, and the regeneration of the hypophosphorylated form will be required for the next round of transcription. We reasoned that a Tat associated phosphatase may interact with Tat and RNAPII in the transcription complex to regenerate the hypophosphorylated form of RNAPII. We have partially characterized a Tat associated phosphatase which is sensitive to okadaic acid and decreases the CTD phosphorylation by a Tat associated CTD kinase (TTK). The long term goal of this project is to isolate the cDNA encoding the Tat associated phosphatase from human T cells and characterize the mechanism of its function in Tat trans-activation. The substrate specificity of the Tat associated phosphatase will be determined by using various phosphorylated proteins such as the large subunit of RNA polymerase II, recombinant c-terminal domain of RNAP II, dk7, and phosphorylase a. The domain requirement of Tat and the specificity of the phosphatase and Tat interaction will be studied by using mutants of Tat and an unrelated viral transactivator VP16. In viva association of the phosphatase with Tat will be studied by immunoprecipitating the Tat associated proteins from the cell Iysate of a HeLa cell line expressing HA-tagged Tat protein. Afterwards, the Tat associated phosphatase will be purified by chromatography. Antibodies will be generated using oligopeptides sepecific to the purified phosphatase. The cDNA encoding the phosphatase will be isolated by screening a cDNA expression library from T cells and its properties and functions characterized. These studies will not only help in gaining a better understanding of the mechanism of Tat response, they target issues relevant to basic mechanisms of RNA transcription.

描述（改编自申请人的摘要）：人类免疫缺陷 1 型病毒 (HIV-1) 调节蛋白 Tat 增加全合成 病毒 LTR 的 RNA 转录本长度。 Tat 与转录相互作用 因子和 RNA 聚合酶 II (RNAP II) 酶复合物，以增加 转录本延伸过程的效率。 一种可能的机制 Tat 函数提出了 Tat 与特定运动的关联， 磷酸化 RNAPII 大亚基的 c 末端结构域 (CTD)。 低磷酸化的 RNAPII 与转录相关 起始复合物，而过度磷酸化的 RNAPII 则与 延伸复合物和低磷酸化形式的再生 将需要进行下一轮转录。 我们推断一个 Tat 相关磷酸酶可能与 Tat 和 RNAPII 相互作用 转录复合物以再生低磷酸化形式的RNAPII。 我们已经部分表征了 Tat 相关磷酸酶，它是 对冈田酸敏感并通过 Tat 降低 CTD 磷酸化 相关 CTD 激酶 (TTK)。 该项目的长期目标是 从人 T 细胞中分离编码 Tat 相关磷酸酶的 cDNA 并表征其在 Tat 反式激活中的功能机制。 Tat 相关磷酸酶的底物特异性为 通过使用各种磷酸化蛋白质（例如大 RNA 聚合酶 II 亚基、RNAP II 重组 C 端结构域、dk7、 和磷酸化酶a。 Tat 的领域要求和特殊性 将使用 Tat 突变体研究磷酸酶和 Tat 相互作用 和一个不相关的病毒反式激活蛋白VP16。 活体协会 将通过免疫沉淀 Tat 来研究磷酸酶与 Tat 来自表达 HeLa 细胞系的细胞裂解物的相关蛋白 HA 标记的 Tat 蛋白。 之后，Tat 相关磷酸酶将被 通过色谱法纯化。 抗体将使用生成 纯化的磷酸酶特异的寡肽。 编码 cDNA 的 通过筛选 T 的 cDNA 表达文库来分离磷酸酶 细胞及其特性和功能的表征。 这些研究将 不仅有助于更好地理解 Tat 的机制 响应，他们针对与 RNA 基本机制相关的问题 转录。