Dataset Information

Mass Spectrometry-Based Characterization of the Virion Proteome, Phosphoproteome, and Associated Kinase Activity of Human Cytomegalovirus.

ABSTRACT: The assembly of human cytomegalovirus (HCMV) virions is an orchestrated process that requires, as an essential prerequisite, the complex crosstalk between viral structural proteins. Currently, however, the mechanisms governing the successive steps in the constitution of virion protein complexes remain elusive. Protein phosphorylation is a key regulator determining the sequential changes in the conformation, binding, dynamics, and stability of proteins in the course of multiprotein assembly. In this review, we present a comprehensive map of the HCMV virion proteome, including a refined view on the virion phosphoproteome, based on previous publications supplemented by new results. Thus, a novel dataset of viral and cellular proteins contained in HCMV virions is generated, providing a basis for future analyses of individual phosphorylation steps and sites involved in the orchestrated assembly of HCMV virion-specific multiprotein complexes. Finally, we present the current knowledge on the activity of pUL97, the HCMV-encoded and virion-associated kinase, in phosphorylating viral and host proteins.

SUBMITTER: Coute Y

PROVIDER: S-EPMC7357008 | biostudies-literature | 2020 May

REPOSITORIES: biostudies-literature

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Mass Spectrometry-Based Characterization of the Virion Proteome, Phosphoproteome, and Associated Kinase Activity of Human Cytomegalovirus.

Couté Yohann Y Kraut Alexandra A Zimmermann Christine C Büscher Nicole N Hesse Anne-Marie AM Bruley Christophe C De Andrea Marco M Wangen Christina C Hahn Friedrich F Marschall Manfred M Plachter Bodo B

Microorganisms 20200530 6

The assembly of human cytomegalovirus (HCMV) virions is an orchestrated process that requires, as an essential prerequisite, the complex crosstalk between viral structural proteins. Currently, however, the mechanisms governing the successive steps in the constitution of virion protein complexes remain elusive. Protein phosphorylation is a key regulator determining the sequential changes in the conformation, binding, dynamics, and stability of proteins in the course of multiprotein assembly. In t ...[more]

PMID: 32486127

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Project description:Purpose:Diverse groups of proteins play integral roles in both the physiology and pathophysiology of the retina. However, thorough proteomic analyses of retinas of experimental species are currently unavailable. The purpose of the present paper is providing the field with a comprehensive proteomic characterization of the retina of a commonly used laboratory mouse using a discovery-based mass spectrometry (MS) approach. Methods:Retinas from eight male and eight female 30-week-old outbred ND4 Swiss Webster mice were harvested and immediately processed for MS analysis on a Thermo Fisher (TF) Fusion Orbitrap MS. The retinal proteome and phosphoproteome were identified and subsequently analyzed using Proteome Discoverer 2.2 and Panther-GeneGo. SEQUEST-HT scoring was used for analysis, and the reference protein FASTA database was from Mus musculus. Specifically, three technical repeats were performed for each biological sample. For characterization, only high-scoring peptides were considered, with a false discovery rate (FDR) of <1%. Downstream bioinformatic analysis used Ingenuity Pathway Analysis (IPA; Qiagen). Results:Using Proteome Discoverer 2.2, 4,767 different proteins were identified and segregated into 26 major protein classes, 9 functional molecular classes, and 12 categories of biological processes. The five largest protein classes included the following: nucleic acid binding (17%), hydrolases (13%), enzyme modulators (10%), transferases (9%), and oxidoreductases (6%). "Binding" and "catalytic" proteins contributed to 81% of the molecular function class at 37% and 42%, respectively. "Cellular processing" and "metabolic processes" contributed the most to biologic activity, at 31% and 26%, respectively. Phosphopeptide enrichment yielded the identification of 610 additional unique proteins that were not originally identified. The two datasets combined produced an adult mouse retinal proteome consisting of 5,377 unique proteins. Overall, 41% of the retinal proteome was phosphorylated. The overwhelming diversity of retinal protein functionality was reflected through further analyses revealing 2,086 unique pathway hits across 241 different pathways (TF). A core analysis summary report was performed in IPA (Qiagen) to analyze the top signaling networks, protein-protein interaction (PPI) enrichments, and canonical pathways. Conclusions:Using this high-throughput technique, we have further deciphered and updated the diverse proteome of the mouse retina, including the phosphoproteome, thereby providing the most comprehensive proteomic profile for this tissue known to date. These findings, and the bioinformatic analyses we also provided, establish a platform for future studies, facilitating the elucidation of the relevance of these proteins to the molecular and cellular pathologies that underlie retinal function and disease.

Project description:Diabetic nephropathy is the most common chronic kidney disease in the world and the main cause of end-stage renal disease (ESRD). The structural integrity of podocytes is fundamental to the normal function of the glomerulus, and the role of glycogen synthase kinase 3β (GSK-3β) in podocytes is complicated. A thorough understanding of GSK-3β is crucial to understand the mechanism of diabetic nephropathy. To analyze the roles of GSK-3β in podocytes, GSK-3β knockdown lentivirus by clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein (Cas)9 was applied to establish stable cell lines. Mass spectrometry was utilized to search for differentially expressed proteins. Consequently, we found 34 proteins with higher levels and 115 proteins with lower levels in GSk-3β knockdown cells than in control cells and identified 581 phosphosites with higher phosphorylation levels and 288 phosphosites with lower phosphorylation levels. We performed functional enrichment analysis of these proteins and phosphorylated proteins based on public databases. Enrichment analysis revealed that GSK-3β participates in the spliceosome, Hippo signaling pathway, actin binding, structural molecule activity, and other pathways. Then, we used motif analysis of phosphate sites to determine 89 conserved motifs based on 1,068 phosphoserine (pS) sites and 15 conserved motifs in view of 104 phosphothreonine (pT) sites. Additionally, protein-protein interaction network analysis was carried out using the STRING database. Cytoscape's add-on Molecular Complex Detection (MCODE) was used to analyze key and core protein groups. In quantitative differential protein analysis, four MCODEs were obtained, and 22 MCODEs were obtained in the analysis of the phosphoproteome of differentially expressed proteins. Finally, we analyzed the kinase regulatory network in podocytes after GSK-3β knockdown and identified 299 protein kinases and 3,460 significantly changed phosphorylation modification sites on 1,574 proteins. These results will be valuable for further research on GSK-3β.

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Dataset Information

Mass Spectrometry-Based Characterization of the Virion Proteome, Phosphoproteome, and Associated Kinase Activity of Human Cytomegalovirus.

Publications

Mass Spectrometry-Based Characterization of the Virion Proteome, Phosphoproteome, and Associated Kinase Activity of Human Cytomegalovirus.

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