107 research outputs found
Fluorescence Proteomic Technology to Analyze Peripheral Blood Mononuclear Cells in Chronic Chagas Disease
No full textThe thiol moieties of cysteinyl residues in proteins undergo a number of modifications including nitrosylation, oxidation, persulfidation, sulfenylation, and others. These protein modifications may influence gain as well as loss of function in biological and disease conditions. Herein, we describe a quantitative approach that combines accurate, sensitive fluorescence modification of cysteinyl-S-nitrosyl (SNOFlo) groups that leaves electrophoretic mobility unaffected and offers the measurement of changes in S-nitrosylation (SNO) status relative to protein abundance. This approach has been useful in evaluating the global protein abundance and SNO profile of Chagas seropositive individuals that were categorized in clinically asymptomatic (C/A) and clinically symptomatic (C/S) subgroups and compared to normal healthy (N/H) controls. Through analyzing the proteome datasets with different bioinformatics and statistics tools, potential pathologic mechanisms in disease progression are identified. We also propose a panel of protein biomarkers that have a potential to identify the infected individuals at risk of developing clinical Chagas disease.Fil: Wiktorowicz, John E.. University of Texas Medical Branch; Estados UnidosFil: Zago, María Paola. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Salta. Instituto de Patología Experimental. Universidad Nacional de Salta. Facultad de Ciencias de la Salud. Instituto de Patología Experimental; ArgentinaFil: Garg, Nisha J.. University of Texas Medical Branch; Estados Unido
Mechanism of transmissible gastroenteritis virus nsp1-mediated translation inhibition
No full textThe non-structural protein 1 (nsp1) proteins of several coronaviruses share a common property to inhibit host gene expression, but use different mechanisms to exert this function. Unlike the nsp1 protein of SARS coronavirus, a group 2b betacoronavirus, the nsp1 protein of transmissible gastroenteritis virus (TGEV), an alphacoronavirus, inhibits the expression of host genes without associating with the 40S ribosomal subunit or inducing the endonucleolytic cleavage of mRNAs. Nsp1 of TGEV strongly inhibits the translation of capped mRNAs, as well as certain internal ribosome entry site (IRES)- containing mRNAs, in HeLa S10 extract but not in rabbit reticulocyte lysate or wheat germ extract. In this dissertation the mechanism of TGEV nsp1-mediated translation inhibition is further elucidated. Immunofluorescence analysis showed that TGEV nsp1 protein localizes to both the nucleus and cytoplasm of expressed cells. TGEV nsp1 can enter the nucleus via passive diffusion because its molecular weight (~9 kDa) is below the passive diffusion exclusion limit of the nuclear pore complex. Both TGEV infection and TGEV nsp1 protein expression inhibit cellular protein synthesis. TGEV nsp1 expression strongly inhibits the expression of reporter genes from transfected reporter plasmids. However, it fails to inhibit the translation of exogenous reporter mRNAs directly introduced into the cytoplasm. These data, along with other findings discussed in this dissertation suggest that TGEV nsp1 uses one or more factors associated with mRNA-protein complexes that originate in the cell nucleus to inhibit mRNA translation
S-Nitrosylation Proteome Profile of Peripheral Blood Mononuclear Cells in Human Heart Failure
No full textNitric oxide (NO) protects the heart against ischemic injury; however, NO- and superoxide-dependent S-nitrosylation (S-NO) of cysteines can affect function of target proteins and play a role in disease outcome. We employed 2D-GE with thiol-labeling FL-maleimide dye and MALDI-TOF MS/MS to capture the quantitative changes in abundance and S-NO proteome of HF patients (versus healthy controls, n = 30/group). We identified 93 differentially abundant (59-increased/34-decreased) and 111 S-NO-modified (63-increased/48-decreased) protein spots, respectively, in HF subjects (versus controls, fold-change | ≥1.5|, p ≤ 0.05). Ingenuity pathway analysis of proteome datasets suggested that the pathways involved in phagocytes´ migration, free radical production, and cell death were activated and fatty acid metabolism was decreased in HF subjects. Multivariate adaptive regression splines modeling of datasets identified a panel of proteins that will provide >90% prediction success in classifying HF subjects. Proteomic profiling identified ATP-synthase, thrombospondin-1 (THBS1), and vinculin (VCL) as top differentially abundant and S-NO-modified proteins, and these proteins were verified by Western blotting and ELISA in different set of HF subjects. We conclude that differential abundance and S-NO modification of proteins serve as a mechanism in regulating cell viability and free radical production, and THBS1 and VCL evaluation will potentially be useful in the prediction of heart failure.Fil: Koo, Sue Jie. University of Texas Medical Branch; Estados UnidosFil: Spratt, Heidi M.. University of Texas Medical Branch; Estados UnidosFil: Soman, Kizhake V.. University of Texas Medical Branch; Estados UnidosFil: Stafford, Susan. University of Texas Medical Branch; Estados UnidosFil: Gupta, Shivali. University of Texas Medical Branch; Estados UnidosFil: Petersen, John R.. University of Texas Medical Branch; Estados UnidosFil: Zago, María Paola. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Salta. Instituto de Patología Experimental. Universidad Nacional de Salta. Facultad de Ciencias de la Salud. Instituto de Patología Experimental; ArgentinaFil: Kuyumcu Martinez, Muge N.. University of Texas Medical Branch; Estados UnidosFil: Brasier, Allan R.. University of Texas Medical Branch; Estados UnidosFil: Wiktorowicz, John E.. University of Texas Medical Branch; Estados UnidosFil: Garg, Nisha Jain. University of Texas Medical Branch; Estados Unido
Studies on Fortilin-Prohibitin Interaction
No full textAbstract: Apoptosis, programmed cell death, is a tightly regulated process that occurs in development, in tissue maintenance and turnover, and in regulating the immune system. Alterations in apoptosis regulation is known to be involved in various diseases including cancer, autoimmune diseases, and cardiovascular diseases including atherosclerosis initiation and maintenance. Fortilin is an antiapoptotic protein with a wide tissue distribution and a wide range of functions. fortilin has no sequence homology to other regulators of apoptosis, such as the Bcl-2 Proteins or the Inhibitors of Apoptosis proteins. In elucidating the mechanism of Fortilin-mediated cellular protection, fortilin protein interactions have been previously shown to modulate the cellular response to apoptotic stimuli. The goal of this dissertation is to investigate novel fortilin protein interactions in order to further shed light on the mechanism of fortilin mediated protection. In a proteomic screen fortilin was shown to interact with the anti-proliferative protein, prohibitin. I demonstrate that fortilin specifically interacts with this protein through in vitro studies. fortilin co-localizes with prohibitin in a perinuclear distribution and subcellular studies showed that fortilin and prohibitin are found in the nucleus, cytosol, and to a lesser degree in the endoplasmic reticulum. Fortilin was shown to mutually stabilize prohibitin. Finally, cells overexpressing fortilin and prohibitin attenuate the apoptosis response of cells compared to cells overexpressing either protein alone. In summary, these findings demonstrate a novel protein interaction between fortilin and prohibitin and shows a functional significance in modulating apoptosis after inducing cell stress
High Resolution Full-Range (pI = 2.5 to 10.0) Isoelectric Focusing of Proteins and Peptides in Capillary Electrophoresis
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