Proteomics Analysis And Quantitative Acetylome In Human Oligodendroglia Cells In Response To Borna Disease Virus

BACKGROUNDBorna disease virus (BDV) is a negative non-segmented singlestranded (NNS) RNA virus. BDV is a neurotropic, non-cytolytic virus bypersistently infecting the central nervous system, and thereby causingneurological disorders in a variety of warm-blooded animal species,possibly including humans. In last30years, the epidemiology of BDV hasbeen carried out in the world. Although a large number of epidemiologicaldata showed that BDV infection was closely associated with humanpsychiatric disorders such as depression, schizophrenia, the association ofBDV infection with human psychiatric disorders remains a hugecontroversy. The key reason of the controversy is pathogenesis of BDVinfection remains unclear and the direct evidence of association betweenBDV and human psychiatric disorders is insufficient. Virus proteomics andprotein post-translational modification such as lysine acetylation (Kac)plays an increasingly pivotal role in exploring the interaction between virusand host, the viral pathogenesis with the development of quantitative massspectrometry techniques.OBJECTIVE1. To acquire the comprehensive proteomics data in OL cells in response to BDV infection and deepen the understanding to thepathogenesis of BDV infection through two proteomics methods(two-dimensional electrophoresis followed by mass spectrometry,SILAC-based quantitative proteomics).2. To acquire comprehensive histone Kac sites and Kcr sites in OLcells through SILAC and IP-based quantitative proteomics.3. To acquire the comprehensive acetylome in OL cells in response toBDV infection and explore the pathogenesis of BDV from acetylomethrough SILAC and IP-based quantitative proteomics.METHODS1. Human OL cells and BDV stably infected OL cells wereconstructed and cultured. Differential protein expression between the twogroups was assessed through a proteomics approach, usingtwo-dimensional electrophoresis followed by matrix-assisted laserdesorption ionization-time of flight tandem mass spectrometry. Somedifferential proteins were validated by Western blotting. The impact ofBDV infection on cell proliferation and nuclear translocation of pERK1/2was detected by CCK assay and immunofluorescence.2. Human OL cells and BDV stably infected OL cells were labeledwith SILAC kits. After six generations of passaging, lysates of crudeprotein and histone were extracted, respectively. In each independentexperiments, the equivalent protein of each heavy-or light-labeled poolwas mixed. After digestion by trypsin, the trypic peptides from cytosolicsoluble fraction were further separated with reverse-phase HPLC. Thetrypic peptides in each fraction were incubated with anti-acetyllysineagarose beads to get the enrichment of lysine acetylated peptides. Theproteins or enriched acetylated peptides were detected by LC-ESI-MS/MS. Histone crotonylation were identified by the same way.3. Using bioinformatic analysis, the impact of BDV infection onproteome, histone acetylation and acetylome of OL cells was explored tofurther reveal the possible pathogenic mechanisms.RESULTS1. A total of63differential host proteins were identified in BDVinfected OL cells compared to non-infected OL cells. We found that mostchanges referred to alterations related to pentose phosphate pathway,glyoxylate and dicarboxylate metabolism, the tricarboxylic acid (TCA)cycle, and glycolysis/gluconeogenesis. By manual querying, twodifferential proteins were found to be associated with mitogen-activatedprotein kinase (MAPK) signal transduction. Five key signaling proteins ofthis pathway (i.e., p-Raf, p-MEK, p-ERK1/2, p-RSK, and p-MSK) wereselected for Western blotting validation. p-ERK1/2and p-RSK were foundto be significantly up-regulated, and p-MSK was found to be significantlydown-regulated in BDV infected OL cells compared to non-infected OLcell. Although BDV constitutively activated the ERK-RSK pathway, hostcell proliferation and nuclear translocation of activated pERK in BDVinfected OL cells were impaired.2.4,383quantifiable non-redundant proteins were indentified in OLcells in response to BDV infection. Bioinformatic analysis indicated that4,383quantifiable proteins were functionally annotated in multiplebiological processes and pathways, including metabolism pathways,immune response, DNA replication, DNA repair and transcriptionalregulation. Moreover, the expression levels of some key histoneacetyltransferases and histone deacetylases were found significantlychanged. Indeed, the subsequent quantitative proteomic study detected30histone Kac sites, of which15histone Kac marks exhibited decreased acetylation levels. Some of the proteomic data were validated by westernblots with histone site-specific anti-Kac antibodies.3.791non-redundant Kac sites were quantified. The detected Kacsites were present in473quantifiable proteins. Annotation of the473quantifiable Kac proteins indicated that metabolic processes weresignificantly enriched in multiple biological process categories, includingbutanoate metabolism, beta Alanine metabolism, tryptophan metabolism,and fatty acid metabolism, suggesting BDV infection could markedly alterthe metabolic status in host cells. Cellular compartment analysis revealedthat acetylated substrates were markedly enriched in cell membrane andmitochondria. Molecular function analysis results showed that, towardsBDV infection, Kac substrates involved in transmembrane transporteractivity were enriched. Moreover, we observed that lysine acetylationstatus of multiple histone acetyltransferases (HATs) was altered in responseto BDV infection. All together, these results imply that BDV infectionlikely impacts HATs enzymatic activities, which may in turn regulateacetylome dynamics in host cells.4. Some histone crotonylation sites were identified tentatively.CONCLUSION1. Energy metabolism was the most significantly altered set of hostbiological pathways in BDV-infected OL cells. The downstreamERK-RSK complex of the Raf/MEK/ERK signaling cascade was found tobe activated by BDV infection which did not result in upregulated cellproliferation in OL cells. Meanwhile, although BDV produces constitutiveactivation of the ERK1/2pathway, pERK’s nuclear translocation wasimpaired.2. This study is the most comprehansive to reveal the host proteomic in BDV-infected OL cells and indicated BDV infection affected metabolicpathways, immune response, DNA replication, DNA repair, andtranscription regulation. This study is the first to reveal histone Kac profiles.Exploring the link between proteomics and histone acetylation in responseto BDV infection will deepen the understanding to the mechanism of BDVinfection.3. This study is the first comprehensive proteomic analysis to Kacmodification in response to BDV infection in OL cells and revealsmetabolic processes were significantly enriched in multiple biologicalprocess categories which indicate BDV infection regulating metabolicpathways in host cell by acetylation and the importance of metabolicchanges in the pathogenic role of BDV infection.4. BDV infection maybe regulates histone crotonylation.