PURIFICATION, MOLECULAR-CLONING, AND CHARACTERIZATION OF TRP32, A NOVEL THIOREDOXIN-RELATED MAMMALIAN PROTEIN OF 32 KDA

We purified a protein of 32 kDa from human thymoma HPB-ALL cells that was co-purified with a catalytic fragment of MST (mammalian STE-20-lik e), a kinase of the STE20 family, which is proteolytically activated b y caspase in apoptosis (Lee, K.-K., Murakawa, M., Nishida, E., Tsubuki , S., Kawashima, S., Sakamaki, K., and Yonehara, S. (1998) Oncogene 16 , in press). Molecular cloning of the gene encoding this 32-kDa protei n (TRP32) reveals that it is a novel protein of 289 amino acid residue s and contains an NH2-terminal thioredoxin domain with a conserved thi oredoxin active site. The human and mouse TRP32 proteins have 99% homo logy, and the thioredoxin domains are completely identical. The thiore doxin domain of TRP32 has thioredoxin-like reducing activity, which ca n reduce the interchain disulfide bridges of insulin in vitro. However , the thioredoxin domain of TRP32 is more sensitive to oxidation than human thioredoxin. Northern blot analysis showed that TRP32 is express ed in all human tissues. Expression of TRP32 was also confirmed in all mammalian cell lines tested by Western blot analysis using anti-TRP32 monoclonal antibody. Subcellular fractionation and immunostaining ana lysis showed TRP32 is cytoplasmic protein. These findings suggest that TRP32 is a novel cytoplasmic regulator of the redox state in higher e ukaryotes.