Resistance to anti-neoplastic drugs is a central problem in treatment of human cancers. The cysteine protease bleomycin hydrolase was first recognized due to its ability to hydrolyze the anti-cancer drug bleomycin. The recent discovery of homologs of this protease in yeast and bacteria suggest a conserved cellular function for this protein, yet, the only known natural substrate, bleomycin, is almost never seen by any of these cells. The yeast homolog, Gal6 is an intracellular, primarily nuclear, protease that binds DNA and acts as a repressor in the Gal4 regulatory system. The cellular function of this ubiquitously-expressed protein is still unknown. In recent years, there has been considerable interest in intracellular proteases, since they seem to be involved in important regulatory processes. Bleomycin hydrolase, with its many functions including its unusual DNA-binding activity adds another dimension of complexity of these proteases. The crystal structure of the yeast bleomycin hydrolase, Gal6, revealed a hexameric structure in which the protease and DNA- binding activities are structurally intertwined. The goal of this project is to understand the functional coupling of these two activities in GaI6. These studies will foster an understanding of how bleomycin hydrolase functions in tumor drug resistance and may provide insight to what are its basic cellular functions. We will study how the single-stranded DNA recognition and how the binding effects conformation of the protein and access to the active site. The determinants of recognition and hydrolysis of bleomycin by Gal6 and how they are affected by ssDNA binding will he investigated. We will determine the substrate specificity of Gal6 protease activity, the possible regulatory role of the C-terminal arm, and whether endopeptidase activity is revealed. Finally, we will study the structural basis for tumor cell drug resistance to bleomycin by the human homolog of bleomycin hydrolase.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA071746-03
Application #
2748876
Study Section
Chemical Pathology Study Section (CPA)
Program Officer
Forry, Suzanne L
Project Start
1996-08-01
Project End
2000-07-31
Budget Start
1998-08-01
Budget End
2000-07-31
Support Year
3
Fiscal Year
1998
Total Cost
Indirect Cost
Name
Cold Spring Harbor Laboratory
Department
Type
DUNS #
065968786
City
Cold Spring Harbor
State
NY
Country
United States
Zip Code
11724