PubMed · 15031483
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Hopi E Hoekstra, Trevor Price. 2004-03-19. Evolution. Parallel evolution is in the genes.. https://doi.org/10.1126/science.1096413
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INTRODUCTION: Following thrombin cleavage of the reactive centre (P1-P1'; L444-S445) of the serpin heparin cofactor II (HCII), HCII traps thrombin (IIa) in a stable inhibitory complex. To compare HCII to other serpins we substituted: the P13-P5' residues of HCII with those of alpha(1)-proteinase inhibitor (alpha(1)-PI), alpha(1)-PI (M358R), or antithrombin (AT); the P4-P1, P3-P1, and P2-P1 residues of HCII with those of AT; and made L444A/H/K/M or R point mutations. We also combined L444R with changes in the glycosaminoglycan binding domain collectively termed MutD. MATERIALS AND METHODS: Variants were made by site-directed mutagenesis, expressed in bacteria, purified and characterized electrophoretically and kinetically. RESULTS AND CONCLUSIONS: Of the P13-P5' mutants, only the alpha(1)-PI-loop variant retained anti-IIa activity, but less than the corresponding L444M. Heparin-catalyzed rate constants for IIa inhibition were reduced vs. wild-type (WT) by at most three-fold for all P1 mutants save L444A (reduced 20-fold). L444R and L444K inhibited IIa>50- and >6-fold more rapidly than WT in heparin-free reactions, but stoichiometries of inhibition were increased for all variants. HCII-IIa complexes of all P1 variants were stable in the absence of heparin, but those of the L444K and L444R variants released active IIa over time with heparin. Limited proteolysis of these two groups of HCII-IIa complexes produced different fragmentation patterns consistent with conformational differences. The combination of either substituted AT residues at P2, P3, and P4, or the MutD mutations with L444R resulted in complex instability with or without heparin. This is the first description of HCII-IIa complexes of transient stability forming in the absence of heparin, and may explain the extent to which the reactive centre loop of HCII differs from that of AT.
The molecular pathology of bladder cancer has been the subject of considerable interest, and current efforts are targeted toward elucidating the interrelationships between individual somatic gene loss and both etiologic and prognostic factors. Mutation of the TP53 gene has been associated with more invasive bladder cancer, and evidence suggests that TP53 mutation, independent of stage, may be predictive of outcome in this disease. However, there is no consensus in the literature that bladder carcinogen exposure is associated with inactivation of the TP53 gene. Work to date has been primarily hospital based and, as such, subject to possible bias associated with selection of more advanced cases for study. We examined exposure relationships with both TP53 gene mutation and TP53 protein alterations in a population-based study of 330 bladder cancer cases in New Hampshire. Tobacco smoking was not associated with TP53 alterations. We found a higher prevalence of TP53 inactivation (i.e., mutation and nuclear accumulation) among hair dye users (odd ratio [OR] = 4.1; 95% confidence interval [CI] 1.2-14.7), and the majority of these mutations were transversions. Men who had "at risk" occupations were more likely to have mutated TP53 tumors (OR = 2.9; 95% CI 1.1-7.6). There also was a relative absence of TP53 mutation (OR = 0.4; 95% CI 0.0-2.9) and TP53 protein alterations (OR = 0.6; 95% CI 0.3-1.4) in bladder cancers from individuals with higher arsenic exposure. Our data suggest that there is exposure-specific heterogeneity in inactivation of the TP53 pathway in bladder cancers and that integration of the spectrum of pathway alterations in population-based approaches (capturing the full range of exposures to bladder carcinogens) may provide important insights into bladder tumorigenesis.
Equilibrium folding of rat liver BHMT (betaine-homocysteine methyltransferase), a TIM (triosephosphate isomerase)-barrel tetrameric protein, has been studied using urea as denaturant. A combination of activity measurements, tryptophan fluorescence, CD and sedimentation-velocity studies suggested a multiphasic process including two intermediates, a tetramer (I4) and a monomer (J). Analysis of denaturation curves for single- and six-tryptophan mutants indicated that the main changes leading to the tetrameric intermediate are related to alterations in the helix alpha4 of the barrel, as well as in the dimerization arm. Further dissociation to intermediate J included changes in the loop connecting the C-terminal alpha-helix of contact between dimers, disruption of helix alpha4, and initial alterations in helix alpha7 of the barrel, as well as in the dimerization arm. Evolution of the monomeric intermediate continued through additional perturbations in helix alpha7 of the barrel and the C-terminal loop. Our data highlight the essential role of the C-terminal helix in dimer-dimer binding through its contribution to the increased stability shown by BHMT as compared with other TIM barrel proteins. The results are discussed in the light of the high sequence conservation shown by betaine-homocysteine methyltransferases and the knowledge available for other TIM-barrel proteins.