NMR AND FUNCTIONAL ANALYSIS OF ONCOGENE PRODUCTS

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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.