Project description:TMT18-labeled proteome-wide profiling of post-translational modifications (phosphorylation) in mock-infected and infected human lung epithelial cells (A549). Human lung epithelial (A549) cells were infected and mock-infected with HCoV-229E. Cells were harvested at 8, 16, and 24 hours post-infection. Extracted proteins were digested with LysC + Trypsin, then TMT18-labeled and enriched for phosphorylation. Samples were split into 6 fractions and run on the Orbitrap Exploris 480. Data was searched with MS-GF+ using PNNL's DMS Processing pipeline.
Project description:TMT18-labeled proteome-wide profiling of post-translational modifications (acetylation) in mock-infected and infected human lung epithelial cells (A549). Human lung epithelial (A549) cells were infected and mock-infected with HCoV-229E. Cells were harvested at 8, 16, and 24 hours post-infection. Extracted proteins were digested with LysC + Trypsin, then TMT18-labeled and enriched for acetylation. Samples were split into 6 fractions and run on the Orbitrap Exploris 480. Data was searched with MS-GF+ using PNNL's DMS Processing pipeline.
Project description:TMT18-labeled proteome-wide profiling of post-translational modifications (redox) in mock-infected and infected human lung epithelial cells (A549). Human lung epithelial (A549) cells were infected and mock-infected with HCoV-229E. Cells were harvested at 8, 16, and 24 hours post-infection. Extracted proteins were digested with LysC + Trypsin, then TMT18-labeled and enriched for redox PTMs. Samples were split into 6 fractions and run on the Orbitrap Exploris 480. Data was searched with MS-GF+ using PNNL's DMS Processing pipeline.
Project description:TMT18-labeled proteome-wide profiling of post-translational modifications in mock-infected and infected human lung epithelial cells (A549). Human lung epithelial (A549) cells were infected and mock-infected with HCoV-229E. Cells were harvested at 8, 16, and 24 hours post-infection. Extracted proteins were digested with LysC + Trypsin, then TMT18-labeled. Samples were split into 6 fractions and run on the Orbitrap Exploris 480. Data was searched with MS-GF+ using PNNL's DMS Processing pipeline.
Project description:Human respiratory syncytial virus (HRSV) is a major cause of pediatric lower respiratory tract disease to which there is no vaccine or efficacious chemotherapeutic strategy. Although RNA synthesis and virus assembly occur in the cytoplasm, HRSV is known to induce nuclear responses in the host cell as replication alters global gene expression. Quantitative proteomics was used to take an unbiased overview of the protein changes in transformed human alveolar basal epithelial cells infected with HRSV. Underpinning this was the use of stable isotope labeling with amino acids in cell culture coupled to LC-MS/MS, which allowed the direct and simultaneous identification and quantification of both cellular and viral proteins. To reduce sample complexity and increase data return on potential protein localization, cells were fractionated into nuclear and cytoplasmic extracts. This resulted in the identification of 1,140 cellular proteins and six viral proteins. The proteomics data were analyzed using Ingenuity Pathways Analysis to identify defined canonical pathways and functional groupings. Selected data were validated using Western blot, direct and indirect immunofluorescence confocal microscopy, and functional assays. The study served to validate and expand upon known HRSV-host cell interactions, including those associated with the antiviral response and alterations in subnuclear structures such as the nucleolus and ND10 (promyelocytic leukemia bodies). In addition, novel changes were observed in mitochondrial proteins and functions, cell cycle regulatory molecules, nuclear pore complex proteins and nucleocytoplasmic trafficking proteins. These data shed light into how the cell is potentially altered to create conditions more favorable for infection. Additionally, the study highlights the application and advantage of stable isotope labeling with amino acids in cell culture coupled to LC-MS/MS for the analysis of virus-host interactions.
Project description:The protozoan parasite Trypanosoma cruzi, the causative agent of Chagas disease, causes severe morbidity and mortality in afflicted individuals. About 30% of T. cruzi-infected individuals present with cardiac, gastrointestinal tract, and/or neurological disorders. Megacolon, one of the major pathologies of Chagas disease, is accompanied by gastrointestinal motility disorders. The molecular mechanism of T. cruzi-mediated megacolon in Chagas disease is currently unknown. To decipher the molecular mechanism of T. cruzi-induced alteration in the colon during the early infection phase, we exposed primary human colonic epithelial cells (HCoEpiC) to invasive T. cruzi trypomastigotes at multiple time points to determine changes in the phosphoprotein networks in the cells following infection using proteome profiler Human phospho-kinase arrays. We found significant changes in the phosphorylation pattern that can mediate cellular deregulations in colonic epithelial cells after infection. We detected a significant increase in the levels of phosphorylated heat shock protein (p-HSP) 27 and transcription factors that regulate various cellular functions, including c-Jun and CREB. Our study confirmed significant upregulation of phospho (p-) Akt S473, p-JNK, which may directly or indirectly modulate CREB and c-Jun phosphorylation, respectively. We also observed increased levels of phosphorylated CREB and c-Jun in the nucleus. Furthermore, we found that p-c-Jun and p-CREB co-localized in the nucleus at 180 minutes post infection, with a maximum Pearson correlation coefficient of 0.76±0.02. Increased p-c-Jun and p-CREB have been linked to inflammatory and profibrotic responses. T. cruzi infection of HCoEpiC induces an increased expression of thrombospondin-1 (TSP-1), which is fibrogenic at elevated levels. We also found that T. cruzi infection modulates the expression of NF-kB and JAK2-STAT1 signaling molecules which can increase pro-inflammatory flux. Bioinformatics analysis of the phosphoprotein networks derived using the phospho-protein data serves as a blueprint for T. cruzi-mediated cellular transformation of primary human colonic cells during the early phase of T. cruzi infection.
Project description:Telomeres play important functional roles in cell proliferation, cell cycle regulation, and genetic stability, in which telomere length is critical. In this study, quantitative proteome comparisons for the human breast epithelial cells with short and long telomeres (184-hTERTL vs. 184-hTERTS and 90P-hTERTL vs. 90P-hTERTS), resulting from transfection of the human telomerase reverse transcriptase (hTERT) gene, were performed using cleavable isotope-coded affinity tags. More than 2000 proteins were quantified in each comparative experiment, with approximately 77% of the proteins identified in both analyses. In the cells with long telomeres, significant and consistent alterations were observed in metabolism (amino acid, nucleotide, and lipid metabolism), genetic information transmission (transcription and translation regulation, spliceosome and ribosome complexes), and cell signaling. Interestingly, the DNA excision repair pathway is enhanced, while integrin and its ligands are downregulated in the cells with long telomeres. These results may provide valuable information related to telomere functions.
Project description:Polyomaviruses are a family of small DNA viruses that are associated with a number of severe human diseases, particularly in immunocompromised individuals. The detailed virus-host interactions during lytic polyomavirus infection are not fully understood. Here, we report the first nuclear proteomic study with BK polyomavirus (BKPyV) in a primary renal proximal tubule epithelial cell culture system using stable isotope labeling by amino acids in cell culture (SILAC) proteomic profiling coupled with liquid chromatography-tandem mass spectrometry. We demonstrated the feasibility of SILAC labeling in these primary cells and subsequently performed reciprocal labeling-infection experiments to identify proteins that are altered by BKPyV infection. Our analyses revealed specific proteins that are significantly up- or down-regulated in the infected nuclear proteome. The genes encoding many of these proteins were not identified in a previous microarray study, suggesting that differential regulation of these proteins may be independent of transcriptional control. Western blotting experiments verified the SILAC proteomic findings. Finally, pathway and network analyses indicated that the host cell DNA damage response signaling and DNA repair pathways are among the cellular processes most affected at the protein level during polyomavirus infection. Our study provides a comprehensive view of the host nuclear proteomic changes during polyomavirus lytic infection and suggests potential novel host factors required for a productive polyomavirus infection.
Project description:Controlled differentiation of human embryonic stem cells (hESCs) can be utilized for precise analysis of cell type identities during early development. We established a highly efficient neural induction strategy and an improved analytical platform, and determined proteomic and phosphoproteomic profiles of hESCs and their specified multipotent neural stem cell derivatives (hNSCs). This quantitative dataset (nearly 13,000 proteins and 60,000 phosphorylation sites) provides unique molecular insights into pluripotency and neural lineage entry. Systems-level comparative analysis of proteins (e.g., transcription factors, epigenetic regulators, kinase families), phosphorylation sites, and numerous biological pathways allowed the identification of distinct signatures in pluripotent and multipotent cells. Furthermore, as predicted by the dataset, we functionally validated an autocrine/paracrine mechanism by demonstrating that the secreted protein midkine is a regulator of neural specification. This resource is freely available to the scientific community, including a searchable website, PluriProt.
Project description:Infectious bursal disease virus (IBDV) enters the host cells via endocytic pathway to achieve viral replication in the cytoplasm. Here, we performed LC-MS/MS coupled with isobaric tags for relative and absolute quantification labeling of differentially abundant proteins of IBDV-infected cells using a subcellular fractionation strategy. We show that the viral infection regulates the abundance and/or subcellular localization of 3211 proteins during early infection. In total, 23 cellular proteins in the cytoplasmic proteome and 34 in the nuclear proteome were significantly altered after virus infection. These differentially abundant proteins are involved in such biological processes as immune response, signal transduction, RNA processing, macromolecular biosynthesis, energy metabolism, virus binding, and cellular apoptosis. Moreover, transcriptional profiles of the 25 genes corresponding to the identified proteins were analyzed by quantitative real-time RT-PCR. Ingenuity Pathway Analysis clustered the differentially abundant proteins primarily into the mTOR pathway, PI3K/Akt pathway, and interferon-β signaling cascades. Confocal microscopy showed colocalization of the viral protein VP3 with host proteins heterogeneous nuclear ribonucleoprotein H1, nuclear factor 45, apoptosis inhibitor 5, nuclear protein localization protein 4 and DEAD-box RNA helicase 42 during the virus infection. Together, these identified subcellular constituents provide important information for understanding host-IBDV interactions and underlying mechanisms of IBDV infection and pathogenesis.