NMR AND FUNCTIONAL ANALYSIS OF ONCOGENE PRODUCTS

癌基因产物的核磁共振和功能分析

• 批准号: 2443350
• 负责人: MARKUS W GERMANN
• 金额: $ 20.85万
• 依托单位: THOMAS JEFFERSON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-01-01 至 2001-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2443350
• 关键词: Escherichia coli; T cell receptor; affinity chromatography; animal genetic material tag; apoptosis; chimeric proteins; circular dichroism; gel electrophoresis; glucocorticoids; human genetic material tag; immunoprecipitation; lymphocytic leukemia; mass spectrometry; mutant; nuclear magnetic resonance spectroscopy; oncogenes; oncoproteins; protein engineering; protein purification; protein structure function; recombinant proteins; site directed mutagenesis; yeast two hybrid system

Clonal chromosomal aberrations observed in T cell prolymphocytic leukemias (T-PLL) involve the translocation of one T cell receptor gene to either chromosome 14q32 or Xq28. The two oncogenes involved in these translocations are hTCL-1 and hMTCP-1. The products of these two human genes are proteins of 12.6 and 13.5 kDa molecular weight, respectively, that are related by 41 percent identity in amino acid sequence and are members of a new gene family. Our preliminary data indicate that hTCL-1 functions as an inhibitor of programmed cell death. Several proteins were shown to interact with hTCL-1 by co-precipitation experiments. The objective of this proposal is to analyze the solution structure and function of hTCL-1 and hMTCP-1. The HTCL-1 mediated inhibition of apoptosis will be studied in detail; this assay provides a test bed for the functional evaluation of hTCL-1 mutants. Interacting proteins will be characterized which will provide important clues into the action of hTCL-1 and hTCL-1 mutants. Human hTCL-1 and hMTCP-1 have been expressed in E. coli and purified in milligram quantities for structural and physical analyses. In addition, we will also study the mouse homologue, MTCP-1. The purified hTCL-1, the mouse homologue mTCL-1 and the hMTCP-1 proteins will be characterized by gel electrophoresis, circular dichroism, and mass spectrometry. The solution structures will be determined by NMR spectroscopy employing 15N and 13C labeled recombinant proteins. Our concerted approach of structural and functional studies on TCL-1 provides the foundation for the understanding of its structure and function at a molecular level. The characterization of interacting proteins holds promise in the identification of additional components that are important for apoptosis. The solution structure is of particular relevance for the design of agents that act as inhibitors of oncogene function. Inhibitors of hTCL-1 and hMTCP-1 have potential for treatment of low-grade T-cell lymphomas and leukemias.

在T细胞幼淋巴细胞中观察到的克隆性染色体畸变 白血病（T-PLL）涉及一个T细胞受体基因的易位 染色体14 q32或Xq 28上。 这两个致癌基因参与了这些 易位是hTCL-1和hMTCP-1。 这两个人的产品 基因分别是12.6和13.5kDa分子量的蛋白质， 它们在氨基酸序列上有41%的相同性， 一个新的基因家族。 我们的初步数据表明，hTCL-1 作为程序性细胞死亡的抑制剂发挥作用。 几种蛋白质 通过共沉淀实验显示与hTCL-1相互作用。 的 本提案的目的是分析解决方案结构， hTCL-1和hMTCP-1的功能。 HTCL-1介导的抑制 细胞凋亡将被详细研究;该测定提供了一个测试床， hTCL-1突变体的功能评价。 相互作用的蛋白质将 这将提供重要的线索，以行动的特点， hTCL-1和hTCL-1突变体。 人hTCL-1和hMTCP-1已在大肠杆菌中表达， 在大肠大肠杆菌中，并纯化毫克量的结构和 物理分析。 此外，我们还将研究小鼠同源物， MTCP-1。 纯化的hTCL-1、小鼠同源物mTCL-1和hMTCP-1 蛋白质将通过凝胶电泳、环形电泳、电泳等来表征。 二色性和质谱法。 解决方案结构将是 采用15 N和13 C标记的重组体通过NMR光谱法测定 proteins. 我们对TCL-1的结构和功能进行了研究 为理解其结构提供了基础， 在分子水平上发挥作用。 交互作用的表征 蛋白质在鉴定其他成分方面有希望 对细胞凋亡很重要。解决方案结构为 特别是作为抑制剂的药物设计的相关性， 癌基因功能hTCL-1和hMTCP-1的抑制剂有可能 治疗低度T细胞淋巴瘤和白血病。