Changes in the mitochondrial proteome in human hepatocytes in response to alpha-amanitin hepatotoxicity

Wang, Mei; Chen, Yu; Guo, Zhen; Yang, Changcheng; Qi, Jiaomei; Fu, Yujuan; Chen, Zuohong; Chen, Ping; Wang, Ying

doi:10.1016/j.toxicon.2018.11.002

Cited by 23 publications

(23 citation statements)

References 36 publications

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“…The sample was added to the electrophoresis channel, and the appropriate amount of mineral oil was added to cover the strip. The program was set as follows: 50 V for 14 h, passive rehydration; 500 V for 1 h, linear; 1000 V for 1 h, rapid; 5000 V for 1 h, rapid; 8000 V for 1 h, linear; and rapid ramping to 8000 V for 60,000 Vh [10]. After isoelectric focusing, strips were equilibrated in equilibration buffer (6 M urea, 20% glycerol, 2% SDS, 25 mM Tris-HCl [pH 8.8]) containing 0.2% (w/v) dithiothreitol for 15 min and then in the same buffer containing 3.0% (w/v) iodoacetamide and 0.175% (v/v) bromophenol blue for 15 min.…”

Section: Two-dimensional (2-d) Gel Electrophoresismentioning

confidence: 99%

“…Peptide mass spectra were obtained with a 5800 MALDI TOF/TOF mass spectrometer (AB SCIEX, Foster City, USA). The MS/MS data of the peptide mass ngerprint (PMF) were submitted to the online software Mascot (Matrix Science, Boston, MA, USA) for identi cation according to the NCBIProt database [10].…”

Section: In-gel Trypsin Digestion Ms and Data Searchingmentioning

confidence: 99%

“…The identi ed proteins were subjected to subcellular localization, functional classi cation, and interaction protein queries using Gene Ontology (GO) analysis, KEGG, and STRING, and related proteins or signal pathways were analyzed. The literature was used to determine the veri cation target [10].…”

Section: Bioinformatics Analysismentioning

confidence: 99%

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Differential mitochondrial proteomic analysis of A549 cells infected with avian influenza virus subtypes H5 and H9

Yang

Zhang

Yang

et al. 2020

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Background In the past 20–30 years, both the highly pathogenic avian influenza (HPAI) H5N1 and low pathogenic avian influenza (LPAI) H9N2 viruses have been reported to cross species barriers to infect humans. However, H5N1 viruses could cause severe damage and a high death rate, but H9N2 viruses could not. In this study, we use H9N2 virus infection as a control to investigate the differential expression of mitochondrial-related proteins caused by H5N1 and H9N2 virus infections in A549 cells. Methods According to the determined viral infection titer, A549 cells were infected with 1 MOI (multiplicity of infection) virus, and the mitochondria were extracted after 24 hours of incubation. The lysed mitochondrial protein was analyzed by BCA method for protein concentration, SDS-PAGE preliminary analysis, two-dimensional gel electrophoresis, and mass spectrometry. Select different protein spots, perform Western Blot to verify the proteomics results, and then perform GO and KEGG analysis. Results In the 2-D gel electrophoresis analysis, 227 protein spots were detected in the H5N1-infected group, and 169 protein spots were detected in the H9N2-infected group. After further MS identification and removal of redundancy, 32 differentially expressed proteins were identified. Compared with the H9N2 group, the H5N1-infected group had 16 upregulated mitochondrial proteins and 16 downregulated proteins. The 70 kDa heat shock protein analogs, short-chain enoyl-CoA hydratase, malate dehydrogenase, and ATP synthase were verified by Western Blot, and the results were consistent with proteomics. Conclusions Functional analysis indicated that these differentially expressed proteins were involved mainly in apoptosis, metabolism and the cytoskeleton. The differential expression of eight mitochondrial proteins in H5N1-infected cells resulted in decreased T cell activation, decreased antigen presentation and stress response, reduced ATP synthesis, and decreased induction of apoptosis, resulting in the higher pathogenicity of H5N1 virus than H9N2 virus.These finding may provide a basis for analyzing the pathogenesis of influenza viruses with different virulence levels, identifying anti-influenza host targets and developing new influenza vaccines.

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Section: Two-dimensional (2-d) Gel Electrophoresismentioning

confidence: 99%

Section: In-gel Trypsin Digestion Ms and Data Searchingmentioning

confidence: 99%

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Differential mitochondrial proteomic analysis of A549 cells infected with avian influenza virus subtypes H5 and H9

Yang

Zhang

Yang

et al. 2020

Preprint

Self Cite

View full text Add to dashboard Cite

show abstract

“…The cells were transferred to a new 50 mL roundbottom tube, centrifuged to pellet cell debris and nuclear proteins, and centrifuged again. The supernatant obtained by centrifugation in the previous step was centrifuged at 10,300 g for 10 min at 4 ℃, and the precipitate was mitochondrial protein [17]. Centrifugation was repeated once, and the precipitate was combined with that obtained in the previous step to further maximally enrich mitochondrial protein.…”

Section: Extraction Of Mitochondrial Proteins From Virus Infectionmentioning

confidence: 99%

“…Perform regular functional analysis using the tools on the Swiss-Prot database (http://uniprot.org). The literature was used to determine the veri cation target [17,20].…”

Section: Bioinformatics Analysismentioning

confidence: 99%

Differential mitochondrial proteomic analysis of A549 cells infected with avian influenza virus subtypes H5 and H9

Yang

Zhang

Yang

et al. 2020

Preprint

Self Cite

View full text Add to dashboard Cite

Background: Both the highly pathogenic avian influenza (HPAI) H5N1 and low pathogenic avian influenza (LPAI) H9N2 viruses have been reported to cross species barriers to infect humans. However, H5N1 viruses can cause severe damage and a high mortality rate, but H9N2 viruses can not. Our purpose was to use proteomics technology to study the differential expression of mitochondrial-related proteins caused by H5N1 and H9N2 virus infections.Methods: According to the determined viral infection titer, A549 cells were infected with 1 MOI (multiplicity of infection) virus, and the mitochondria were extracted after 24 hours of incubation. The lysed mitochondrial protein was analyzed by BCA method for protein concentration, SDS-PAGE preliminary analysis, two-dimensional gel electrophoresis, and mass spectrometry. Select different protein spots, perform Western Blot to verify the proteomics results. The identified proteins use GO analysis for subcellular localization, KEGG analysis for functional classification and signal pathways, STRING analysis for functional protein association networks.Results: In the 2-D gel electrophoresis analysis, 227 protein spots were detected in the H5N1-infected group, and 169 protein spots were detected in the H9N2-infected group. After further MS identification and removal of redundancy, 32 differentially expressed proteins were identified. Compared with the H9N2 group, the H5N1-infected group had 16 upregulated mitochondrial proteins and 16 downregulated proteins. The 70 kDa heat shock protein analogs, short-chain enoyl-CoA hydratase, malate dehydrogenase, and ATP synthase were verified by Western Blot, and the results were consistent with proteomics. Functional analysis indicated that these differentially expressed proteins were involved mainly in apoptosis, metabolism.Conclusions: Compared with H9N2 group, the differential expression of eight mitochondrial proteins in H5N1 group led to T cell activation, antigen presentation, stress response, ATP synthesis and cell apoptosis reduction, leading to higher pathogenicity of H5N1 than H9N2 virus.

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Characterization of complement C3 as a marker of alpha-amanitin toxicity by comparative secretome profiling

et al. 2022

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Changes in the mitochondrial proteome in human hepatocytes in response to alpha-amanitin hepatotoxicity

Cited by 23 publications

References 36 publications

Differential mitochondrial proteomic analysis of A549 cells infected with avian influenza virus subtypes H5 and H9

Differential mitochondrial proteomic analysis of A549 cells infected with avian influenza virus subtypes H5 and H9

Differential mitochondrial proteomic analysis of A549 cells infected with avian influenza virus subtypes H5 and H9

Characterization of complement C3 as a marker of alpha-amanitin toxicity by comparative secretome profiling

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