Proteomic and metabonomic analysis uncovering Enterovirus A71 reprogramming host cell metabolic pathway.

Enterovirus A71 (EV71) infection can cause hand, foot, and mouth disease (HFMD) and severe neurological complications in children. However, the biological processes regulated by EV71 remain poorly understood. Herein, proteomics and metabonomics studies were conducted to uncover the mechanism of EV71 infection in rhabdomyosarcoma (RD) cells and identify potential drug targets. Differential expressed proteins from enriched membrane were analyzed by isobaric tags for relative and absolute quantitation (iTRAQ)-based proteomics technology. Twenty-six differential proteins with 1.5-fold (p < 0.05) change were detected, including 14 upregulated proteins and 12 downregulated proteins. The upregulated proteins are mainly involved in metabolic process, especially in the glycolysis pathway. Alpha-enolase (ENO1) protein was found to increase with temporal dependence following EV71 infection. The targeted metabolomics analysis revealed that glucose absorption and glycolysis metabolites were increased after EV71 infection. The glycolysis pathway was inhibited by knocking down ENO1 or the use of a glycolysis inhibitor (dichloroacetic acid [DCA]); and we found that EV71 infection was inhibited by depleting ENO1 or using DCA. Our study indicates that EV71 may reprogram glucose metabolism by activating glycolysis, and EV71 infection can be inhibited by interrupting the glycolysis pathway. ENO1 may be a potential target against EV71, and DCA could act as an inhibitor of EV71.

Increased MMAB level in mitochondria as a novel biomarker of hepatotoxicity induced by Efavirenz.

BACKGROUND: Efavirenz (EFV), a non-nucleoside reverse transcriptase inhibitor (NNRTI), has been widely used in the therapy of human immunodeficiency virus (HIV) infection. Some of its toxic effects on hepatic cells have been reported to display features of mitochondrial dysfunction through bioenergetic stress and autophagy, etc. However, alteration of protein levels, especially mitochondrial protein levels, in hepatic cells during treatment of EFV has not been fully investigated. METHODS: We built a cell model of EFV-induced liver toxicity through treating Huh-7 cells with different concentrations of EFV for different time followed by the analysis of cell viability using cell counting kit -8 (CCK8) and reactive oxygen species (ROS) using 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) and MitoSox dye. Proteomic profiles in the mitochondria of Huh-7 cells stimulated by EFV were analyzed. Four differentially expressed proteins were quantified by real time RT-PCR. We also detected the expression of mitochondrial precursor Cob(I)yrinic acid a,c-diamide adenosyltransferase (MMAB) by immunohistochemistry analysis in clinical samples. The expression levels of MMAB and ROS were detected in EFV-treated Huh-7 cells with and without shRNA used to knock down MMAB, and in primary hepatocytes (PHC). The effects of other anti-HIV drugs (nevirapine (NVP) and tenofovirdisoproxil (TDF)), and hydrogen peroxide (H2O2) were also tested. Amino acid analysis and fatty aldehyde dehydrogenase (ALDH3A2) expression after MMAB expression knock-down with shRNA was used to investigate the metabolic effect of MMAB in Huh-7 cells. RESULTS: EFV treatment inhibited cell viability and increased ROS production with time- and concentration-dependence. Proteomic study was performed at 2 hours after EFV treatment. After treated Huh-7 cells with EFV (2.5mg/L or 10 mg/L) for 2 h, fifteen differentially expressed protein spots from purified mitochondrion that included four mitochondria proteins were detected in EFV-treated Huh-7 cells compared to controls. Consistent with protein expression levels, mRNA expression levels of mitochondrial protein MMAB were also increased by EFV treatment. In addition, the liver of EFV-treated HIV infected patients showed substantially higher levels of MMAB expression compared to the livers of untreated or protease inhibitor (PI)-treated HIV-infected patients. Furthermore, ROS were found to be decreased in Huh-7 cells treated with shMMAB compared with empty plasmid treated with EFV at the concentration of 2.5 or 10 mg/L. MMAB was increased in EFV-treated Huh-7 cells and primary hepatocytes. However, no change in MMAB expression was detected after treatment of Huh-7 cells and primary hepatocytes with anti-HIV drugs nevirapine (NVP) and tenofovirdisoproxil (TDF), or hydrogen peroxide (H2O2), although ROS was increased in these cells. Finally, knockdown of MMAB by shRNA induced increases in the ß-Alanine (ß-Ala) production levels and decrease in ALDH3A2 expression. CONCLUSIONS: A mitochondrial proteomic study was performed to study the proteins related to EFV-inducted liver toxicity. MMAB might be a target and potential biomarker of hepatotoxicity in EFV-induced liver toxicity.

Recent 5-year Findings and Technological Advances in the Proteomic Study of HIV-associated Disorders.

Human immunodeficiency virus-1 (HIV-1) mainly relies on host factors to complete its life cycle. Hence, it is very important to identify HIV-regulated host proteins. Proteomics is an excellent technique for this purpose because of its high throughput and sensitivity. In this review, we summarized current technological advances in proteomics, including general isobaric tags for relative and absolute quantitation (iTRAQ) and stable isotope labeling by amino acids in cell culture (SILAC), as well as subcellular proteomics and investigation of posttranslational modifications. Furthermore, we reviewed the applications of proteomics in the discovery of HIV-related diseases and HIV infection mechanisms. Proteins identified by proteomic studies might offer new avenues for the diagnosis and treatment of HIV infection and the related diseases.