22 research outputs found
Phenotypic Expressions of CCR5-Δ32/Δ32 Homozygosity
Objective: As blockade of CC-chemokine receptor 5 (CCR5) has been proposed as therapy for HIV-1, we examined whether the CCR5-Δ32/Δ32 homozygous genotype has phenotypic expressions other than those related to HIV-1. Design: Study subjects were white homosexual men or men with hemophilia who were not infected with HIV-1. In this study, 15 CCR5-Δ32/Δ32 homozygotes were compared with 201 CCR5 wild-type (+/+) subjects for a wide range of clinical conditions and laboratory assay results ascertained during prospective cohort studies and routine clinical care. CCR5-Δ32 genotype was determined by polymerase chain reaction, followed by single-stranded conformational polymorphism analysis. Results: Hypertension and conditions attributable to hemophilia were the only diagnoses frequently found in clinical records of CCR5-Δ32/Δ32 study subjects. Based on blood pressure measurement and treatment history, CCR5-Δ32/Δ32 homozygotes had a 2.8-fold higher prevalence of hypertension than age-matched CCR5-+/+ study subjects (95% confidence interval [CI], 1.2-6.4; p = .01); none of the homozygotes had severe hypertension. Hematologic measures were generally similar across the genotypes, but total lymphocyte counts were ~20% higher in CCR5-Δ32/Δ32 study subjects than in CCR5-+/+ study subjects (p \u3c .05). Among patients with hemophilia who were infected with hepatitis C virus (HCV), mean alanine aminotransferase levels were 117% higher among CCR5-Δ32/Δ32 homozygotes (p \u3c .05), but serum HCV levels did not differ by CCR5-Δ32 genotype. CCR5-Δ32/Δ32 homozygous study subjects had a lower prevalence of antibodies to measles virus than those with other genotypes, but this association was not confirmed in a group of blood donors. The prevalence of antibodies to nine other common viruses, HBV, and HCV was not related to CCR5 genotype. Conclusions: CCR5-Δ32/Δ32 homozygotes are generally similar to wild-type persons. Confirmatory investigations are required to determine whether hypertension, increased lymphocyte counts, and higher hepatic enzyme levels in the presence of HCV infection represent true phenotypic expressions of this genotype. CCR5-Δ32/Δ32 homozygosity does not provide broad protection against viral infections
Membrane-Associated RING-CH Proteins Associate with Bap31 and Target CD81 and CD44 to Lysosomes
Membrane-associated RING-CH (MARCH) proteins represent a family of transmembrane ubiquitin ligases modulating intracellular trafficking and turnover of transmembrane protein targets. While homologous proteins encoded by gamma-2 herpesviruses and leporipoxviruses have been studied extensively, limited information is available regarding the physiological targets of cellular MARCH proteins. To identify host cell proteins targeted by the human MARCH-VIII ubiquitin ligase we used stable isotope labeling of amino-acids in cell culture (SILAC) to monitor MARCH-dependent changes in the membrane proteomes of human fibroblasts. Unexpectedly, we observed that MARCH-VIII reduced the surface expression of Bap31, a chaperone that predominantly resides in the endoplasmic reticulum (ER). We demonstrate that Bap31 associates with the transmembrane domains of several MARCH proteins and controls intracellular transport of MARCH proteins. In addition, we observed that MARCH-VIII reduced the surface expression of the hyaluronic acid-receptor CD44 and both MARCH-VIII and MARCH-IV sequestered the tetraspanin CD81 in endo-lysosomal vesicles. Moreover, gene knockdown of MARCH-IV increased surface levels of endogenous CD81 suggesting a constitutive involvement of this family of ubiquitin ligases in the turnover of tetraspanins. Our data thus suggest a role of MARCH-VIII and MARCH-IV in the regulated turnover of CD81 and CD44, two ubiquitously expressed, multifunctional proteins
Diabetes induced decreases in PKA signaling in cardiomyocytes: The role of insulin.
The cAMP-dependent protein kinase (PKA) signaling pathway is the primary means by which the heart regulates moment-to-moment changes in contractility and metabolism. We have previously found that PKA signaling is dysfunctional in the diabetic heart, yet the underlying mechanisms are not fully understood. The objective of this study was to determine if decreased insulin signaling contributes to a dysfunctional PKA response. To do so, we isolated adult cardiomyocytes (ACMs) from wild type and Akita type 1 diabetic mice. ACMs were cultured in the presence or absence of insulin and PKA signaling was visualized by immunofluorescence microscopy using an antibody that recognizes proteins specifically phosphorylated by PKA. We found significant decreases in proteins phosphorylated by PKA in wild type ACMs cultured in the absence of insulin. PKA substrate phosphorylation was decreased in Akita ACMs, as compared to wild type, and unresponsive to the effects of insulin. The decrease in PKA signaling was observed regardless of whether the kinase was stimulated with a beta-agonist, a cell-permeable cAMP analog, or with phosphodiesterase inhibitors. PKA content was unaffected, suggesting that the decrease in PKA signaling may be occurring by the loss of specific PKA substrates. Phospho-specific antibodies were used to discern which potential substrates may be sensitive to the loss of insulin. Contractile proteins were phosphorylated similarly in wild type and Akita ACMs regardless of insulin. However, phosphorylation of the glycolytic regulator, PFK-2, was significantly decreased in an insulin-dependent manner in wild type ACMs and in an insulin-independent manner in Akita ACMs. These results demonstrate a defect in PKA activation in the diabetic heart, mediated in part by deficient insulin signaling, that results in an abnormal activation of a primary metabolic regulator
Depletion of MARCH-IV affects surface expression of CD81.
<p>(A): MARCH expression in HFF was analyzed using real-time PCR and the PCR products were separated on a 1% agarose gel and visualized using sybr green (left panel). MARCH mRNA expression is shown as the fold change between samples containing HFF cDNA and no template control samples (right panel). (B): HFFs were treated with siRNA against MARCH-I, -IV, or –VIII as indicated. Cells were treated with siRNA four times over the course of 7 days. The success of each siRNA treatment was determined by monitoring the reduction of MARCH mRNA levels using real time quantitative PCR. (C): In parallel, cells were harvested via trypsinization and the surface levels of CD44 and CD81 were measured using flow cytometry. Graphs are displayed as percent mean florescence intensity.</p
Depletion of Bap31 reduces surface expression of MARCH-VIII.
<p>HeLa cells were depleted of Bap31 using siRNA as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015132#s2" target="_blank">materials and methods</a> section. (A): Following Bap31 depletion, HeLa cells were transfected with a plasmid expressing MARCH-VIII. 24 hours post transfection cells were biotinylated. Following separation via SDS-PAGE gels, surface expression (Top) or total expression (Bottom) of both Bap31 and MARCH-VIII were analyzed via immunoblot. Cells depleted of Bap31 show reduced surface expression of both Bap31 and MARCH-VIII. (B): Depletion of Bap31 also reduced the levels of Bap31 associated with MARCH-VIII. Following Bap31 depletion, HeLa cells were transfected with a plasmid expressing MARCH-VIII. 24 hours post transfection cells were lysed in 1% CHAPS and MARCH-VIII was immunoprecipitated using an anti-Flag antibody. Samples were separated via SDS-PAGE and the presence of Bap31 determined by immunoblot (B Top). Expression of Bap31 and MARCH-VIII were confirmed in whole cell lysates (B Bottom).</p
Proteins displaying a differential abundance following MARCH-VIII expression.
<p>13 proteins displayed a change in abundance of more than 2 fold following transduction with Ad-MARCH-VIII in two of three replicate experiments. Listed are the Uniprot designations for each protein, as well as the average ratio observed. Ratios given are Ad-Tet:Ad-MARCH-VIII. Ratios>1 correspond to proteins that are downregulated by expression of MARCH-VIII while ratios <1 correspond to proteins that are upregulated by expression of MARCH-VIII. Also given are the number of unique peptides used to identify each protein and whether that protein was changed in 2/3 or 3/3 experiments.</p
Downregulation of CD44 and CD81 by other members of the MARCH-family.
<p>HeLa cells were transfected with plasmids expressing cDNAs for each MARCH protein and the surface expression of CD81 and CD44 were assayed by flow cytometry (A) or confocal immune fluorescence analysis (B and C).</p
CD44 and CD81 are differentially expressed following MARCH-VIII expression.
<p>(A): To confirm that CD44 and CD81 were removed from the cell surface, HFF's were infected as above. 24 hours post-infection cells were harvested and processed for flow cytometry. Samples transduced with Ad-MARCH-VIII (line) display reduced surface expression of CD44 and CD81 compared to samples infected with Ad-Tet alone (fill). Expression of MARCH-VIII was confirmed by surface staining for the known MARCH-VIII substrate TfR. (B): HFFs were transduced with Ad-WT, Ad-K5, Ad-Vpu, or Ad-MARCH-VIII. 24 hours post-transduction cells were harvested and whole cell lysates were analyzed for the abundance of CD44 and CD81 using immunoblot. Samples infected with Ad-MARCH-VIII display significantly reduced levels of both CD44 and CD81 compared to either Mock infected samples or samples infected with other control adenoviruses. Equal protein loading was confirmed by immunoblotting for the ER resident chaperone Bap31 as well as ponceau red staining.</p
MARCH-VIII reduces surface expression of Bap31.
<p>HFF's were transduced with Ad-Tet and Ad-MARCH-VIII or Ad-Tet alone. 24 hours post-infection, cells were biotinylated. Prior to purification of biotinylated proteins, 100 µl of each sample was removed for analysis of protein expression in each whole cell lysate (WCL). Expression of Bap31 was analyzed in both the biotinylated fraction and the whole cell lysate using immunoblot. Expression of MARCH-VIII and the known MARCH-VIII substrate transferrin receptor (TfR) were included as controls.</p
MARCH-VIII interacts with Bap31 through the transmembrane domains.
<p>MARCH-VIII C-Flag was expressed in HeLa cells and HFFs using either transfection (A) or adenoviral transduction (B). Cells were lysed in 1% CHAPS and MARCH-VIII was immunoprecipitated using the anti-Flag antibody. Following immunoprecipitation, samples were separated on an SDS-PAGE gel and the presence of Bap31 was analyzed via immunoblot. 5% of each sample was blotted with anti-Flag antibody to confirm MARCH-VIII expression. (C): HeLa cells were transfected with C-terminally Flag tagged versions of each MARCH protein as well as the viral MARCH proteins KSHV-K3, KSHV-K5, MYXV-M153 and the HIV-1 protein Vpu. 24 hours post transfection, each sample was lysed and analyzed as above. Expression of each transfected protein was confirmed by blotting 5% of each immunoprecipitation with the anti-Flag antibody. (D): To determine which region of MARCH proteins was required for interaction with Bap31 we used a series of previously constructed MARCH -VIII truncations. HeLa cells were transfected with C-terminally flag-tagged versions of truncated MARCH-VIII. 24 hours post transfection, samples were lysed and analyzed as above. (E): Similar experiments were performed with truncated MARCH-IV (right panel). Bap31 failed to coimmunoprecipitate following the removal of the entire C-terminus of MARCH-VIII (1–220) or MARCH-IV (1–259), removal of the entire N-terminus of MARCH-IV (89–347) or disruption of the MARCH-IV RING-CH domain by either mutation (MARCH-IV C to S) or addition of EDTA.</p