Protease inhibitor 10 inhibits tumor necrosis factor alpha-induced cell death - Evidence for the formation of intracellular high M-r protease inhibitor 10-containing complexes

Protease inhibitor 10 (PI10) is a member of the ovalbumin family of serine protease inhibitors (ov-serpin) that is expressed at elevated levels in pat ients with acute myeloid leukemia and chronic myelomonocytic leukemia. Base d upon the ability of the related serpin plasminogen activator inhibitor 2 (PAI-8) to protect cells against tumor necrosis factor alpha (TNF alpha)-in duced cell death, this study was initiated to investigate the potential cyt oprotective activity of PI10, Two different expression systems (i.e. plasmi ds encoding either PI10 alone or PI10 fused to the tag: enhanced green fluo rescent protein, EG;FP) were utilized to stably transfect an eukaryotic mod el cell system (i.e. HeLa cells) that neither expresses PAI-2 nor PI10. The level of PI10 expression in the stable transfectants was found to correlat e with their resistance to TNF alpha-induced cell death. Immunoprecipitatio n/immunoblotting experiments demonstrated that PI10 is able to form SDS-sta ble complexes (i.e, M-r >100,000) with a cytosolic protein(s). Increased le vels of the PI10-containing complexes can be detected by TNF alpha treatmen t by preventing intracellular degradative activities with the proteasome in hibitor N-carbobenzyloxy-leucine-leucine-norvalinal. PI10-containing comple xes are dissociated with conditions known to separate classical protease-se rpin complexes (i.e., 1.5 hz ammonium hydroxide in the presence of SDS). Th ese data support a role for the regulation of intracellular protease activi ties by ov-serpins.