Prognostic Value of MicroRNAs in Patients after Myocardial Infarction: A Substudy of PRAGUE-18.

BACKGROUND: The evaluation of the long-term risk of major adverse cardiovascular events and cardiac death in patients after acute myocardial infarction (AMI) is an established clinical process. Laboratory markers may significantly help with the risk stratification of these patients. Our objective was to find the relation of selected microRNAs to the standard markers of AMI and determine if these microRNAs can be used to identify patients at increased risk. METHODS: Selected microRNAs (miR-1, miR-133a, and miR-499) were measured in a cohort of 122 patients from the PRAGUE-18 study (ticagrelor vs. prasugrel in AMI treated with primary percutaneous coronary intervention (pPCI)). The cohort was split into two subgroups: 116 patients who did not die (survivors) and 6 patients who died (nonsurvivors) during the 365-day period after AMI. Plasma levels of selected circulating miRNAs were then assessed in combination with high-sensitivity cardiac troponin T (hsTnT) and N-terminal probrain natriuretic peptide (NT-proBNP). RESULTS: miR-1, miR-133a, and miR-499 correlated positively with NT-proBNP and hsTnT 24 hours after admission and negatively with left ventricular ejection fraction (LVEF). Both miR-1 and miR-133a positively correlated with hsTnT at admission. Median relative levels of all selected miRNAs were higher in the subgroup of nonsurvivors (N = 6) in comparison with survivors (N = 116), but the difference did not reach statistical significance. All patients in the nonsurvivor subgroup had miR-499 and NT-proBNP levels above the cut-off values (891.5 ng/L for NT-proBNP and 0.088 for miR-499), whereas in the survivor subgroup, only 28.4% of patients were above the cut-off values (p = 0.001). CONCLUSIONS: Statistically significant correlation was found between miR-1, miR-133a, and miR-499 and hsTnT, NT-proBNP, and LVEF. In addition, this analysis suggests that plasma levels of circulating miR-499 could contribute to the identification of patients at increased risk of death during the first year after AMI, especially when combined with NT-proBNP levels.

Replacement of Trp28 in Escherichia coli thioredoxin by site-directed mutagenesis affects thermodynamic stability but not function.

Escherichia coli thioredoxin contains two tryptophan residues (Trp28 and Trp31) situated close to the active site disulfide/dithiol. In order to probe the structural and functional roles of tryptophan in the mechanism of E. coli thioredoxin (Trx), we have replaced Trp28 with alanine using site-directed mutagenesis and expressed the mutant protein W28A in E. coli. Changes in the behavior of the mutant protein compared with the wild-type protein have been monitored by a number of physical and spectroscopic techniques and enzyme assays. As expected, removal of a tryptophan residue causes profound changes in the fluorescence spectrum of thioredoxin, particularly for the reduced protein (Trx-(SH)2), and to a lesser extent for the oxidized protein (Trx-S2). These results show that the major contribution to the strongly quenched fluorescence of Trx-S2 in both wild-type and mutant proteins is from Trp31, whereas the higher fluorescence quantum yield of Trx-(SH)2 in the wild-type protein is dominated by the emission from Trp28. The fluorescence, CD, and 1H NMR spectra are all indicative that the mutant protein is fully folded at pH 7 and room temperature, and, despite the significance of the change, from a tryptophan in close proximity to the active site to an alanine, the functions of the protein appear to be largely intact. W28A Trx-S2 is a good substrate for thioredoxin reductase, and W28A Trx-(SH)2 is as efficient as wild-type protein in reduction of insulin disulfides. DNA polymerase activity exhibited by the complex of phage T7 gene 5 protein and Trx-(SH)2 is affected only marginally by the W28A substitution, consistent with the buried position of Trp28 in the protein. However, the thermodynamic stability of the molecule appears to have been greatly reduced by the mutation: guanidine hydrochloride unfolds the protein at a significantly lower concentration for the mutant than for wild type, and the thermal stability is reduced by about 10 degrees C in each case. The stability of each form of the protein appears to be reduced by the same amount, an indication that the effect of the mutation is identical in both forms of the protein. Thus, despite its close proximity to the active site, the Trp28 residue of thioredoxin is not apparently essential to the electron transfer mechanism, but rather contributes to the stability of the protein fold in the active site region.

Sub-site preferences of the aspartic proteinase from the human immunodeficiency virus, HIV-1.

A series of synthetic, chromogenic substrates for HIV-1 proteinase with the general structure Ala-Thr-His-Xaa-Yaa-Zaa*Nph-Val-Arg-Lys-Ala was synthesised with a variety of residues introduced into the Xaa, Yaa and Zaa positions. Kinetics parameters for hydrolysis of each peptide by HIV-1 proteinase at pH 4.7, 37 degrees C and u = 1.0 M were measured spectrophotometrically and/or by reverse phase FPLC. A variety of residues was found to be acceptable in the P3 position whilst hydrophobic/aromatic residues were preferable in P1. The nature of the residue occupying the P2 position had a strong influence on kcat (with little effect on Km); beta-branched residues Val or Ile in this position resulted in considerably faster peptide hydrolysis than when e.g. the Leu-containing analogue was present in P2.