The Protection Function of the PAI-2 from TNF-alpha is Dependent upon Its Protein Binding Domain.

The cDNA of PAI-2 mutants, PAI-2CD and PAI-2Q were inserted into eukaryotic expression vector, pcDNA3, producing pZLE-PAI2CD and pZLE-PAI2Q respectively. The PAI-2 mutant expression plasmids were transfected into HeLa cells and the transfected cells expressing mutant proteins were selected by G418, Northern blot and ELISA assay. The ELISA assay showed that the expression level of PAI-2 mutant protein was equal to that of wild type PAI-2 in transfected cells. The biochemical characteristics of PAI-2 mutant proteins were very similar to that of the wild type PAI-2. The results of crystal violet and MTT assays showed that the PAI-2 mutants did not protect cells from the cytotoxic effects of TNF-alpha. The results of this study indicate that the protection of PAI-2 from apoptosis induced by TNF-alpha is dependent upon the protein-protein interaction mediated by the CD-interhelical domain of PAI-2. The tTG-mediated interaction between PAI-2 and cytosol protein might play a leading role in protecting from apoptosis induced by TNF-alpha.