The catalytic triad of testes-specific protease 50 (TSP50) is essential for its function in cell proliferation.

Testes-specific protease 50 (TSP50) is a novelly identified pro-oncogene and it shares a similar enzymatic structure with many serine proteases. Our previous results suggested that TSP50 could promote tumorigenesis through degradation of IκBα protein and activating NF-κB signaling, and the threonine mutation in its catalytic triad could depress TSP50-mediated cell proliferation. However, whether the two other residues in the catalytic triad of TSP50 play a role in maintaining protease activity and tumorigenesis, and the mechanisms involved in this process remain unclear. Here, we constructed and characterized three catalytic triad mutants of TSP50 and found that all the mutants could significantly depress TSP50-induced cell proliferation and colony formation in vitro and tumor formation in vivo, and the aspartic acid at position 206 in the catalytic triad played a more crucial role than threonine and histidine in this process. Mechanistic studies revealed that the mutants in the catalytic triad abolished the enzyme activity of TSP50, but did not change the cellular localization. Furthermore, our data indicated that all the three mutants suppressed activation of NF-κB signal by preventing the interaction between TSP50 and the NF-κB:IκBα complex. Most importantly, we demonstrated that TSP50 could interact with IκBα protein and cleave it directly as a new protease in vitro.

The threonine protease activity of testes-specific protease 50 (TSP50) is essential for its function in cell proliferation.

BACKGROUND: Testes-specific protease 50 (TSP50), a newly discovered threonine enzyme, has similar amino acid sequences and enzymatic structures to those of many serine proteases. It may be an oncogene. TSP50 is up-regulated in breast cancer epithelial cells, and ectopic expression of TSP50 in TSP50-deficient Chinese hamster ovary (CHO) cells has been found to promote cell proliferation. However, the mechanisms by which TSP50 exerts its growth-promoting effects are not yet fully understood. METHODOLOGY/PRINCIPAL FINDINGS: To delineate whether the threonine protease activity of TSP50 is essential to its function in cell proliferation, we constructed and characterized a mutant TSP50, called TSP50 T310A, which was identified as a protease-dead mutant of TSP50. By a series of proliferation analyses, colony formation assays and apoptosis analyses, we showed that T310A mutation significantly depresses TSP50-induced cell proliferation in vitro. Next, the CHO stable cell line expressing either wild-type or T310A mutant TSP50 was injected subcutaneously into nude mice. We found that the T310A mutation could abolish the tumorigenicity of TSP50 in vivo. A mechanism investigation revealed that the T310A mutation prevented interaction between TSP50 and the NF-κBIκBα complex, which is necessary for TSP50 to perform its function in cell proliferation. CONCLUSION: Our data highlight the importance of threonine 310, the most critical protease catalytic site in TSP50, to TSP50-induced cell proliferation and tumor formation.