Overexpression of Vesicle-associated Membrane Protein (VAMP) 3, but Not VAMP2, Protects Glucose Transporter (GLUT) 4 Protein Translocation in an in Vitro Model of Cardiac Insulin Resistance

Cardiac glucose utilization is regulated by reversible translocation of the glucose transporter GLUT4 from intracellular stores to the plasma membrane. During the onset of diet-induced insulin resistance, elevated lipid levels in the circulation interfere with insulin-stimulated GLUT4 translocation, leading to impaired glucose utilization. Recently, we identified vesicle-associated membrane protein (VAMP) 2 and 3 to be required for insulin- and contraction-stimulated GLUT4 translocation, respectively, in cardiomyocytes. Here, we investigated whether overexpression of VAMP2 and/or VAMP3 could protect insulin-stimulated GLUT4 translocation under conditions of insulin resistance. HL-1 atrial cardiomyocytes transiently overexpressing either VAMP2 or VAMP3 were cultured for 16 h with elevated concentrations of palmitate and insulin. Upon subsequent acute stimulation with insulin, we measured GLUT4 translocation, plasmalemmal presence of the fatty acid transporter CD36, and myocellular lipid accumulation. Overexpression of VAMP3, but not VAMP2, completely prevented lipid-induced inhibition of insulin-stimulated GLUT4 translocation. Furthermore, the plasmalemmal presence of CD36 and intracellular lipid levels remained normal in cells overexpressing VAMP3. However, insulin signaling was not retained, indicating an effect of VAMP3 overexpression downstream of PKB/Akt. Furthermore, we revealed that endogenous VAMP3 is bound by the contraction-activated protein kinase D (PKD), and contraction and VAMP3 overexpression protect insulin-stimulated GLUT4 translocation via a common mechanism. These observations indicate that PKD activates GLUT4 translocation via a VAMP3-dependent trafficking step, which pathway might be valuable to rescue constrained glucose utilization in the insulin-resistant heart.     

Importance of tetrahedral intermediate formation in the catalytic mechanism of the serine proteases chymotrypsin and subtilisin

Two new inhibitors in which the terminal α-carboxyl groups of Z-Ala-Ala-Phe-COOH and Z-Ala-Pro-Phe-COOH have been replaced with a proton to give Z-Ala-Ala-Phe-H and Z-Ala-Pro-Phe-H, respectively, have been synthesized. Using these inhibitors, we estimate that for α-chymotrypsin and subtilisin Carlsberg the terminal carboxylate group decreases the level of inhibitor binding 3-4-fold while a glyoxal group increases the level of binding by 500-2000-fold. We show that at pH 7.2 the effective molarities of the catalytic hydroxyl group of the active site serine are 41000-229000 and 101000-159000 for α-chymotrypsin and subtilisin Carlsberg, respectively. It is estimated that oxyanion stabilization and the increased effective molarity of the catalytic serine hydroxyl group can account for the catalytic efficiency of the reaction. We argue that substrate binding induces the formation of a strong hydrogen bond or low-barrier hydrogen bond between histidine-57 and aspartate-102 that increases the pK(a) of the active site histidine, allowing it to be an effective general base catalyst for the formation of the tetrahedral intermediate and increasing the effective molarity of the catalytic hydroxyl group of serine-195. A catalytic mechanism for acyl intermediate formation in the serine proteases is proposed.     

Coordinated regulation of caveolin-1 and Rab11a in apical recycling compartments of polarized epithelial cells

Recent studies have identified caveolin-1, a protein best known for its functions in caveolae, in apical endocytic recycling compartments in polarized epithelial cells. However, very little is known about the regulation of caveolin-1 in the endocytic recycling pathway. To address this question, in the current study we compared the relationship between compartments enriched in sub-apical caveolin-1 and Rab11a, a well-defined marker of apical recycling endosomes, using polarized MDCK cells as a model. We show that caveolin-1-containing vesicles define a compartment that partially overlaps with Rab11a, and that the distribution of subapical caveolin-1 and Rab11a shows a similar dependence on microtubule disruption. Mutants of the Rab11a effector, Rab11-FIP2 also altered the localization of caveolin-1. These findings indicate that caveolin-1 is coordinately regulated with Rab11a within the apical recycling system of polarized epithelial cells, suggesting that the two proteins are components of the same pathway.     

The yeast metacaspase is implicated in oxidative stress response in frataxin-deficient cells

Friedreich ataxia is the most common recessive neurodegenerative disease and is caused by reduced expression of mitochondrial frataxin. Frataxin depletion causes impairment in iron-sulfur cluster and heme biosynthesis, disruption of iron homeostasis and hypersensitivity to oxidants. Currently no pharmacological treatment blocks disease progression, although antioxidant therapies proved to benefit patients. We show that sensitivity of yeast frataxin-deficient cells to hydrogen peroxide is partially mediated by the metacaspase. Metacaspase deletion in frataxin-deficient cells results in recovery of antioxidant capacity and heme synthesis. In addition, our results suggest that metacaspase is associated with mitochondrial respiration, intracellular redox control and genomic stability.     

Analysis of clinical ostreid herpesvirus 1 (Malacoherpesviridae) specimens by sequencing amplified fragments from three virus genome areas

Although there are a number of ostreid herpesvirus 1 (OsHV-1) variants, it is expected that the true diversity of this virus will be known only after the analysis of significantly more data. To this end, we analyzed 72 OsHV-1 "specimens" collected mainly in France over an 18-year period, from 1993 to 2010. Additional samples were also collected in Ireland, the United States, China, Japan, and New Zealand. Three virus genome regions (open reading frame 4 [ORF4], ORF35, -36, -37, and -38, and ORF42 and -43) were selected for PCR analysis and sequencing. Although ORF4 appeared to be the most polymorphic genome area, distinguishing several genogroups, ORF35, -36, -37, and -38 and ORF42 and -43 also showed variations useful in grouping subpopulations of this virus.     

Peptide Interactions Stabilize and Restructure Human Papillomavirus Type 16 E6 To Interact with p53

Human papillomavirus type 16 (HPV-16) E6 (16E6) binds the E3 ubiquitin ligase E6AP and p53, thereby targeting degradation of p53 (M. Scheffner, B. A. Werness, J. M. Huibregtse, A. J. Levine, and P. M. Howley, Cell 63:1129–1136, 1990). Here we show that minimal 16E6-binding LXXLL peptides reshape 16E6 to confer p53 interaction and stabilize 16E6 in vivo but that degradation of p53 by 16E6 requires E6AP expression. These experiments establish a general mechanism for how papillomavirus E6 binding to LXXLL peptides reshapes E6 to then act as an adapter molecule.     

HIV infection abrogates the functional advantage of natural killer cells educated through KIR3DL1/HLA-Bw4 interactions to mediate anti-HIV antibody-dependent cellular cytotoxicity

Combinations of KIR3DL1 and HLA-Bw4 alleles protect against HIV infection and/or disease progression. These combinations enhance NK cell responsiveness through the ontological process of education. However, educated KIR3DL1(+) NK cells do not have enhanced degranulation upon direct recognition of autologous HIV-infected cells. Since antibody-dependent cellular cytotoxicity (ADCC) is associated with improved HIV infection outcomes and NK cells overcome inhibition through killer cell immunoglobulin-like receptors (KIR) to mediate ADCC, we hypothesized that KIR3DL1-educated NK cells mediate anti-HIV ADCC against autologous cells. A whole-blood flow cytometry assay was used to evaluate ADCC-induced activation of NK cells. This assay assessed activation (gamma interferon [IFN-γ] production and/or CD107a expression) of KIR3DL1(+) and KIR3DL1(-) NK cells, from HLA-Bw4(+) and HLA-Bw4(-) HIV-positive and HIV-negative individuals, in response to autologous HIV-specific ADCC targets. KIR3DL1(+) NK cells were more functional than KIR3DL1(-) NK cells from HLA-Bw4(+), but not HLA-Bw4(-), healthy controls. In HIV-infected individuals, no differences in NK cell functionality were observed between KIR3DL1(+) and KIR3DL1(-) NK cells in HLA-Bw4(+) individuals, consistent with dysfunction of NK cells in the setting of HIV infection. Reflecting the partial normalization of NK cell responsiveness following initiation of antiretroviral therapy, a significant correlation was observed between the peripheral CD4(+) T-lymphocyte counts in antiretroviral therapy-treated subjects and the functionality of NK cells. However, peripheral CD4(+) T-lymphocyte counts were not correlated with an anti-HIV ADCC functional advantage in educated KIR3DL1(+) NK cells. The abrogation of the functional advantage of educated NK cells may enhance HIV disease progression. Strategies to enhance the potency of NK cell-mediated ADCC may improve HIV therapies and vaccines.     

Efficient transmission of pandemic H1N1 influenza viruses with high-level oseltamivir resistance

The limited availability of approved influenza virus antivirals highlights the importance of studying the fitness and transmissibility of drug-resistant viruses. S247N is a novel, naturally occurring N1 neuraminidase mutation that reduces oseltamivir sensitivity and greatly potentiates oseltamivir resistance in the context of the H275Y mutation. Here we show that highly oseltamivir-resistant viruses containing both the S247N and H275Y mutations transmit efficiently in the guinea pig transmission model.     

Antibody-dependent cellular cytotoxicity against primary HIV-infected CD4+ T cells is directly associated with the magnitude of surface IgG binding

Antibody (Ab)-dependent cellular cytotoxicity (ADCC) is thought to potentially play a role in vaccine-induced protection from HIV-1. The characteristics of such antibodies remain incompletely understood. Furthermore, correlates between ADCC and HIV-1 immune status are not clearly defined. We screened the sera of 20 HIV-1-positive (HIV-1(+)) patients for ADCC. Normal human peripheral blood mononuclear cells were used to derive HIV-infected CD4(+) T cell targets and autologous, freshly isolated, natural killer (NK) cells in a novel assay that measures granzyme B (GrB) and HIV-1-infected CD4(+) T cell elimination (ICE) by flow cytometry. We observed that complex sera mediated greater levels of ADCC than anti-HIV-1 envelope glycoprotein (Env)-specific monoclonal antibodies and serum-mediated ADCC correlated with the amount of IgG and IgG1 bound to HIV-1-infected CD4(+) T cells. No correlation between ADCC and viral load, CD4(+) T cell count, or neutralization of HIV-1(SF162) or other primary viral isolates was detected. Sera pooled from clade B HIV-1(+) individuals exhibited breadth in killing targets infected with HIV-1 from clades A/E, B, and C. Taken together, these data suggest that the total amount of IgG bound to an HIV-1-infected cell is an important determinant of ADCC and that polyvalent antigen-specific Abs are required for a robust ADCC response. In addition, Abs elicited by a vaccine formulated with immunogens from a single clade may generate a protective ADCC response in vivo against a variety of HIV-1 species. Increased understanding of the parameters that dictate ADCC against HIV-1-infected cells will inform efforts to stimulate ADCC activity and improve its potency in vaccinees.