Mapping of the catalytic groove preferences of factor Xa reveals an inadequate selectivity for its macromolecule substrates

Factor Xa (FXa) hydrolyzes two peptide bonds in prothrombin having (Glu/Asp)-Gly-Arg-(Thr/Ile) for P(3)-P(2)-P(1)-P(1)' residues, but the exact preferences of its catalytic groove remain largely unknown. To investigate the specificity of FXa, we synthesized full sets of fluorescence-quenched substrates carrying all natural amino acids (except Cys) in P(3), P(2), P(1)', P(2)', and P(3)' and determined the k(cat)/K(m) values of cleavage. Contrary to expectation, glycine was not the "best" P(2) residue; peptide with phenylalanine was cleaved slightly faster. In fact, FXa had surprisingly limited preferences, barely more pronounced than trypsin; in P(2), the ratio of the k(cat)/K(m) values for the most favorable side chain over the least was 289 (12 with trypsin), but in P(1)', this ratio was only 30 (versus 80 with trypsin). This unexpected selectivity undoubtedly distinguished FXa from thrombin, which exhibited ratios higher than 19,000 in P(2) and P(1)'. Thus, with respect to the catalytic groove, FXa resembles a low efficiency trypsin rather than the highly selective thrombin. The rates of cleavage of the peptidyl substrates were virtually identical whether or not FXa was in complex with factor Va, suggesting that the cofactor did not exert a direct allosteric control on the catalytic groove. We conclude that the remarkable efficacy of FXa within prothrombinase originates from exosite interaction(s) with factor Va and/or prothrombin rather than from the selectivity of its catalytic groove.     

Cell surface expression of melanocortin-1 receptor on HaCaT keratinocytes and alpha-melanocortin stimulation do not affect the formation and repair of UVB-induced DNA photoproducts.

Ultraviolet (UV) exposure induces an up-regulation of melanocortin-1 receptor (MC1R) expression in human skin and the alpha-melanocyte-stimulating hormone (alpha-MSH) may reduce UVB-induced DNA damage in normal human melanocytes. Using high-performance liquid chromatography coupled to tandem mass spectrometry, we investigated the formation and repair of DNA lesions in UVB-irradiated HaCaT cells stably transfected with the wild type MC1R gene (HaCaT-MC1R). Similar levels of 8 bipyrimidine photoproducts including cyclobutane pyrimidine dimers (CPDs) (T<>T, T<>C, C<>T), (6-4) photoproducts ((6-4)PPs) (TT-(6-4)PPs, TC-(6-4)PPs) and their Dewar valence isomers together with 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) were found to be generated in both non-transfected and HaCaT-MC1R cells after UVB exposure. Time-course studies of DNA photoproduct yields indicated that the DNA repair ability depended upon radiation doses. It was shown that (6-4)PPs were removed from the DNA of UVB-irradiated cells much more efficiently than CPDs. The repair efficiency of 8-oxodGuo, CPDs and (6-4)PPs was relatively similar in both cell lines and was not modified by stimulation with alpha-MSH before UVB-exposure. In conclusion, cell surface-enforced expression of MC1Rs on HaCaT keratinocytes and alpha-MSH stimulation do not affect the formation of UVB-induced DNA photoproducts and their subsequent repair.     

The Israeli strain IS-98-ST1 of West Nile virus as viral model for West Nile encephalitis in the Old World

West Nile virus (WNV) recently became a major public health concern in North America, the Middle East, and Europe. In contrast with the investigations of the North-American isolates, the neurovirulence properties of Middle-Eastern strains of WNV have not been extensively characterized. Israeli WNV strain IS-98-ST1 that has been isolated from a white stork in 1998, was found to be highly neuroinvasive in adult C57BL/6 mice. Strain IS-98-ST1 infects primary neuronal cells from mouse cortex, causing neuronal death. These results demonstrate that Israeli strain IS-98-ST1 provides a suitable viral model for WNV-induced disease associated with recent WNV outbreaks in the Old World.     

Genetics of venous thrombosis: insights from a new genome wide association study

Background

Venous Thrombosis (VT) is a common multifactorial disease associated with a major public health burden. Genetics factors are known to contribute to the susceptibility of the disease but how many genes are involved and their contribution to VT risk still remain obscure. We aimed to identify genetic variants associated with VT risk.

Methodology/Principal Findings

We conducted a genome-wide association study (GWAS) based on 551,141 SNPs genotyped in 1,542 cases and 1,110 controls. Twelve SNPs reached the genome-wide significance level of 2.0×10⁻⁸ and encompassed four known VT-associated loci, ABO, F5, F11 and FGG. By means of haplotype analyses, we also provided novel arguments in favor of a role of HIVEP1, PROCR and STAB2, three loci recently hypothesized to participate in the susceptibility to VT. However, no novel VT-associated loci came out of our GWAS. Using a recently proposed statistical methodology, we also showed that common variants could explain about 35% of the genetic variance underlying VT susceptibility among which 3% could be attributable to the main identified VT loci. This analysis additionally suggested that the common variants left to be identified are not uniformly distributed across the genome and that chromosome 20, itself, could contribute to ∼7% of the total genetic variance.

Conclusions/Significance

This study might also provide a valuable source of information to expand our understanding of biological mechanisms regulating quantitative biomarkers for VT.     

Importance of correlation between gene expression levels: application to the type I interferon signature in rheumatoid arthritis

Background

The analysis of gene expression data shows that many genes display similarity in their expression profiles suggesting some co-regulation. Here, we investigated the co-expression patterns in gene expression data and proposed a correlation-based research method to stratify individuals.

Methodology/Principal Findings

Using blood from rheumatoid arthritis (RA) patients, we investigated the gene expression profiles from whole blood using Affymetrix microarray technology. Co-expressed genes were analyzed by a biclustering method, followed by gene ontology analysis of the relevant biclusters. Taking the type I interferon (IFN) pathway as an example, a classification algorithm was developed from the 102 RA patients and extended to 10 systemic lupus erythematosus (SLE) patients and 100 healthy volunteers to further characterize individuals. We developed a correlation-based algorithm referred to as Classification Algorithm Based on a Biological Signature (CABS), an alternative to other approaches focused specifically on the expression levels. This algorithm applied to the expression of 35 IFN-related genes showed that the IFN signature presented a heterogeneous expression between RA, SLE and healthy controls which could reflect the level of global IFN signature activation. Moreover, the monitoring of the IFN-related genes during the anti-TNF treatment identified changes in type I IFN gene activity induced in RA patients.

Conclusions

In conclusion, we have proposed an original method to analyze genes sharing an expression pattern and a biological function showing that the activation levels of a biological signature could be characterized by its overall state of correlation.