Deletion of various carboxy-terminal domains of Lactococcus lactis SK11 Proteinase : Effects on activity, specificity, and stability of the truncated enzyme

The Lactococcus lactis SK11 cell envelope proteinase is an extracellular, multidomain protein of nearly 2,000 residues consisting of an N-terminal serine protease domain, followed by various other domains of largely unknown function. Using a strategy of deletion mutagenesis, we have analyzed the function of several C-terminal domains of the SK11 proteinase which are absent in cell envelope proteinases of other lactic acid bacteria. The various deletion mutants were functionally expressed in L. lactis and analyzed for enzyme stability, activity, (auto)processing, and specificity toward several substrates. C-terminal deletions of first the cell envelope W (wall) and AN (anchor) domains and then the H (helix) domain leads to fully active, secreted proteinases of unaltered specificity. Gradually increasing the C-terminal deletion into the so-called B domain leads to increasing instability and autoproteolysis and progressively less proteolytic activity. However, the mutant with the largest deletion (838 residues) from the C terminus and lacking the entire B domain still retains proteolytic activity. All truncated enzymes show unaltered proteolytic specificity toward various substrates. This suggests that the main role played by these domains is providing stability or protection from autoproteolysis (B domain), spacing away from the cell (H domain), and anchoring to the cell envelope (W and AN domains). In addition, this study allowed us to more precisely map the main C-terminal autoprocessing site of the SK11 proteinase and the epitope for binding of group IV monoclonal antibodie

The Influence of Age and Sex on Genetic Associations with Adult Body Size and Shape : A Large-Scale Genome-Wide Interaction Study

Genome-wide association studies (GWAS) have identified more than 100 genetic variants contributing to BMI, a measure of body size, or waist-to-hip ratio (adjusted for BMI, WHRadjBMI), a measure of body shape. Body size and shape change as people grow older and these changes differ substantially between men and women. To systematically screen for age-and/or sex-specific effects of genetic variants on BMI and WHRadjBMI, we performed meta-analyses of 114 studies (up to 320,485 individuals of European descent) with genome-wide chip and/or Metabochip data by the Genetic Investigation of Anthropometric Traits (GIANT) Consortium. Each study tested the association of up to similar to 2.8M SNPs with BMI and WHRadjBMI in four strata (men 50y, women 50y) and summary statistics were combined in stratum-specific meta-analyses. We then screened for variants that showed age-specific effects (G x AGE), sex-specific effects (G x SEX) or age-specific effects that differed between men and women (G x AGE x SEX). For BMI, we identified 15 loci (11 previously established for main effects, four novel) that showed significant (FDR= 50y). No sex-dependent effects were identified for BMI. For WHRadjBMI, we identified 44 loci (27 previously established for main effects, 17 novel) with sex-specific effects, of which 28 showed larger effects in women than in men, five showed larger effects in men than in women, and 11 showed opposite effects between sexes. No age-dependent effects were identified for WHRadjBMI. This is the first genome-wide interaction meta-analysis to report convincing evidence of age-dependent genetic effects on BMI. In addition, we confirm the sex-specificity of genetic effects on WHRadjBMI. These results may providefurther insights into the biology that underlies weight change with age or the sexually dimorphism of body shape.Peer reviewe

Purification and some properties of malate synthase from the methylotrophic yeast Hansenuta polymorpha

Malate synthase, one of the key enzymes in the glyoxylate cycle, was purified 122-fold to homogeneity from ethanol-grown Hansenula polymorpha. SDS-polyacrylamide gel electrophoresis showed that the enzyme has a subunit size of 62000 daltons. The molecular mass of native malate synthase was determined to be 250000 daltons by gel filtration, indicating that the enzyme is a tetramer. Cell fractionation studies and immunogold staining, carried out on ultrathin sections of ethanol-grown H. polymorpha, using malate synthase-specific antibodies, showed that malate synthase was localized in the matrix of peroxisomes.