tailieunhanh - Báo cáo khoa học: Mutational analysis of plasminogen activator inhibitor-1 Interactions of a-helix F and its neighbouring structural elements regulates the activity and the rate of latency transition

The serpin plasminogen activator inhibitor-1 (PAI-1) is a fast and specific inhibitor of the plasminogen activating serine proteases tissue-type and urokinase-type plasminogen activator and, as such, an important regulator in turnover of extracellularmatrixand infibrinolysis. PAI-1 spontaneously loses its antiproteolyticactivityby inserting its reactive centre loop (RCL) as strand 4 in b-sheet A, thereby converting to the so-called latent state. | Eur. J. Biochem. 270 1680-1688 2003 FEBS 2003 doi Mutational analysis of plasminogen activator inhibitor-1 Interactions of a-helix F and its neighbouring structural elements regulates the activity and the rate of latency transition Troels Wind Jan K. Jensen Daniel M. Dupont Paulina Kulig and Peter A. Andreasen Laboratory of Cellular Protein Science Department of Molecular Biology Aarhus University Denmark The serpin plasminogen activator inhibitor-1 PAI-1 is a fast and specific inhibitor of the plasminogen activating serine proteases tissue-type and urokinase-type plasminogen activator and as such an important regulator in turnover of extracellular matrix and in fibrinolysis. PAI-1 spontaneously loses its antiproteolytic activity by inserting its reactive centre loop RCL as strand 4 in b-sheet A thereby converting to the so-called latent state. We have investigated the importance of the amino acid sequence of a-helix F hF and the connecting loop to s3A hF s3A-loop for the rate of latency transition. We grafted regions of the hF s3A-loop from antithrombin III and a1-protease inhibitor onto PAI-1 creating eight variants and found that one of these reversions towards the serpin consensus decreased the rate of latency transition. We prepared 28 PAI-1 variants with individual residues in hF and b-sheet A replaced by an alanine. We found that mutating serpin consensus residues always had functional consequences whereas mutating nonconserved residues only had so in one case. Two variants had low but stable inhibitory activity and a pronounced tendency towards substrate behaviour suggesting that insertion of the RCL is held back during latency transition as well as during complexformation with target proteases. The data presented identify new determinants of PAI-1 latency transition and provide general insight into the characteristic loop-sheet interactions in serpins. Keywords alignment conformation mutational analysis PAI-1 proteases .