Low folding propensity and high translation efficiency distinguish in vivo substrates of GroEL from other Escherichia coli proteins

MOTIVATION: Theoretical considerations have indicated that the amount of chaperonin GroEL in Escherichia coli cells is sufficient to fold only approximately 2-5% of newly synthesized proteins under normal physiological conditions, thereby suggesting that only a subset of E.coli proteins fold in vivo in a GroEL-dependent manner. Recently, members of this subset were identified in two independent studies that resulted in two partially overlapping lists of GroEL-interacting proteins. The objective of the work described here was to identify sequence-based features of GroEL-interacting proteins that distinguish them from other E.coli proteins and that may account for their dependence on the chaperonin system. RESULTS: Our analysis shows that GroEL-interacting proteins have, on average, low folding propensities and high translation efficiencies. These two properties in combination can increase the risk of aggregation of these proteins and, thus, cause their folding to be chaperonin-dependent. Strikingly, we find that these properties are absent in proteins homologous to the E.coli GroEL-interacting proteins in Ureaplasma urealyticum, an organism that lacks a chaperonin system, thereby confirming our conclusions.     

Balancing stigma and status: racial and class identities among middle-class Haitian youth

This article examines the identity formation of middle-class Haitian youth. Segmented assimilation theory predicts that the Haitian second generation will integrate into the black American underclass or maintain strong ethnic group identities. The black middle class, however, is an unexplored pathway of cultural assimilation. This paper uses the literature on the racial and class experiences of the black American middle class as a departure point for understanding the boundary work of middle-class Haitian youth. Based on qualitative interviews and a focus group, we uncover the mechanisms of identity formation for this invisible population. Racial, ethnic and class boundaries compel Haitian youths to create strategies of either empowerment or distancing. They negotiate between their middle-class status and ethnoracial exclusion in a racially segregated neighbourhood, an ethnically homogenous church and a mixed-race school setting. This study's findings extend our theoretical understandings of middle-class immigrants and their identity work.     

A non-calcemic Vitamin D analog modulates both nuclear and putative membranal estrogen receptors in cultured human vascular smooth muscle cells

In cultured human vascular smooth muscle cells (VSMC), estradiol-17beta (E2) induced a biphasic effect on DNA synthesis, i.e., stimulation at low concentrations and inhibition at high concentrations. Additionally, E2 increased the specific activity of creatine kinase (CK) in these cells. Observations that novel protein-bound membrane impermeant estrogenic complexes could elicit inhibition of DNA synthesis, suggested interaction via membranal binding sites. Nevertheless other effects, such as increasing CK activity were only seen with native E2 but not with E2-BSA, thus indicating that the classical nuclear receptor pathway was involved. In the present report, we confirm that human VSMC express both ERalpha and ERbeta. Further, pretreatment of cultured VSMC with the Vitamin D non-calcemic analog JK 1624 F2-2 (JKF) increased ERalpha mRNA (100-200%) but decreased ERbeta mRNA (30-40%) expression as measured by real time PCR. ERalpha protein expression assessed by Western blot analysis increased (25-50%) in parallel, whereas ERbeta protein expression declines (25-55%). Using ovalbumin bound to E2 (Ov-E2) linked to Eu (Eu-Ov-E2), to assess specific membrane binding sites, we observed that membranal binding was down regulated by JKF by 70-80%. In contrast, total cell binding of 3[H] E2, that nearly entirely represents intracellular E2 binding, was increased by 60-100% by the same Vitamin D analog. The results provide evidence that the effects of JKF on ERalpha/ERbeta as well as on membranal versus nuclear binding of estrogen are divergent and show differential modulation.     

In vitro effects of calcium phosphate biomaterials on fibroblastic cell behavior

The effect of synthetic granular hydroxyapatite (HAP) on cultured fibroblastic cells (L929, human bone and gingiva cells) was studied. Phagocytosis of HAP particles and resulting morphological cell changes were demonstrated by microscopic examinations. Cell counts and [3H]thymidine uptake indicated significant increases in cell proliferation and DNA synthesis. These results could account for some of the alterations of the fibroblast behavior induced by changes in intracellular levels of calcium ions released from the material.     

Structural insights into the role of the WW2 domain on tandem WW–PPxY motif interactions of oxidoreductase WWOX

Class I WW domains are present in many proteins of various functions and mediate protein interactions by binding to short linear PPxY motifs. Tandem WW domains often bind peptides with multiple PPxY motifs, but the interplay of WW–peptide interactions is not always intuitive. The WW domain–containing oxidoreductase (WWOX) harbors two WW domains: an unstable WW1 capable of PPxY binding and stable WW2 that cannot bind PPxY. The WW2 domain has been suggested to act as a WW1 domain chaperone, but the underlying mechanism of its chaperone activity remains to be revealed. Here, we combined NMR, isothermal calorimetry, and structural modeling to elucidate the roles of both WW domains in WWOX binding to its PPxY-containing substrate ErbB4. Using NMR, we identified an interaction surface between these two domains that supports a WWOX conformation compatible with peptide substrate binding. Isothermal calorimetry and NMR measurements also indicated that while binding affinity to a single PPxY motif is marginally increased in the presence of WW2, affinity to a dual-motif peptide increases 10-fold. Furthermore, we found WW2 can directly bind double-motif peptides using its canonical binding site. Finally, differential binding of peptides in mutagenesis experiments was consistent with a parallel N- to C-terminal PPxY tandem motif orientation in binding to the WW1–WW2 tandem domain, validating structural models of the interaction. Taken together, our results reveal the complex nature of tandem WW-domain organization and substrate binding, highlighting the contribution of WWOX WW2 to both protein stability and target binding.     

Genotoxic effects of a complex mixture adsorbed onto ambient air particles on human cells in vitro; the effects of Vitamins E and C

Genotoxicity of complex mixtures of organic compounds adsorbed onto ambient air particles (extractable organic matter, EOM) collected in Teplice (Czech Republic) as well as genotoxicity of the indirectly acting carcinogens benzo[a]pyrene (B[a]P) and 5,9-dimethyl-7H-dibenzo[c,g]carbazole (5,9-diMeDBC) was studied in human HepG2 and Caco-2 cells cultured in vitro. The level of DNA breaks was detected by conventional single-cell gel electrophoresis (alkaline comet assay). The level of DNA breaks+oxidative DNA lesions was assessed by modified single-cell gel electrophoresis. The indirectly acting chemical carcinogens studied were able to induce DNA breaks as well as oxidative DNA damage in both cell lines, but stronger DNA-damaging effects were observed in HepG2 cells, which contain a higher level of metabolic enzymes. Treatment of cells with the complex mixtures showed a dose-dependent increase of DNA breaks in HepG2 cells as well as in Caco-2 cells, with seasonal differences. Winter samples of EOM from Teplice (TP-W) were more effective in inducing DNA damage than summer samples (TP-S). Both mixtures caused significant oxidative DNA damage in HepG2 cells. The effect was less evident in cells treated with higher concentrations of TP-W, since the comet assay is limited by saturation at a higher level of DNA damage. Possible reduction of B[a]P-, 5,9-diMeDBC- or EOM-induced DNA damage by Vitamins E and C was evaluated in HepG2 cells only. Pre-treatment of these cells with either one of the vitamins considerably reduced the levels of both DNA breaks and oxidative DNA lesions induced by all compounds investigated.