NMR AND FUNCTIONAL ANALYSIS OF ONCOGENE PRODUCTS

在T细胞前淋巴细胞中观察到克隆性染色体异常 白血病(T-PLL)涉及一个T细胞受体基因易位 染色体14q32或Xq28。涉及这些基因的两个癌基因 易位有hTCL-1和hMTCP-1。这两个人的产物 基因是分子量分别为12.6和13.5 kDa的蛋白质， 它们在氨基酸序列中有41%的同源性，并且是 一个新基因家族的成员。我们的初步数据表明hTCL-1 起到抑制细胞程序性死亡的作用。几种蛋白质 通过共沉淀实验证明它们与hTCL-1相互作用。这个 本提案的目标是分析解决方案结构和 HTCL-1和hMTCP-1的功能。HTCL-1介导的细胞增殖抑制作用 将对细胞凋亡进行详细研究；这一试验为 HTCL-1突变体的功能评价。相互作用的蛋白质会 这将为了解……的行动提供重要线索 HTCL-1和hTCL-1突变体。人hTCL-1和hMTCP-1的表达 在大肠杆菌中，并以毫克量纯化，用于结构和 物理分析。此外，我们还将研究老鼠的同源物， MTCP-1。纯化的hTCL-1、小鼠同系物mTCL-1和hMTCP-1 蛋白质将通过凝胶电泳法、环形电泳法进行表征 二向色性和质谱学。解决方案结构将是 用~(15)N和~(13)C标记重组核磁共振谱测定 蛋白质。 TCL-1的结构和功能研究的协同方法 为理解其结构和 在分子水平上发挥作用。互动性的表征 蛋白质有望在识别其他成分方面发挥作用 它们对细胞凋亡很重要。解决方案的结构是 与作为抑制药物的药物的设计特别相关 癌基因功能。HTCL-1和hMTCP-1的抑制剂具有潜在的 低度恶性T细胞淋巴瘤和白血病的治疗。

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.