Proteome data from a host-pathogen interaction study with Staphylococcus aureus and human lung epithelial cells
Kristin SurmannMarjolaine SimonPetra HildebrandtHenrike PförtnerStephan MichalikVishnu M. DhopleBarbara M. BrökerFrank SchmidtUwe Völker
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To simultaneously obtain proteome data of host and pathogen from an internalization experiment, human alveolar epithelial A549 cells were infected with Staphylococcus aureus HG001 which carried a plasmid (pMV158GFP) encoding a continuously expressed green fluorescent protein (GFP). Samples were taken hourly between 1.5 h and 6.5 h post infection. By fluorescence activated cell sorting GFP-expressing bacteria could be enriched from host cell debris, but also infected host cells could be separated from those which did not carry bacteria after contact (exposed). Additionally, proteome data of A549 cells which were not exposed to S. aureus but underwent the same sample processing steps are provided as a control. Time-resolved changes in bacterial protein abundance were quantified in a label-free approach. Proteome adaptations of host cells were monitored by comparative analysis to a stable isotope labeled cell culture (SILAC) standard. Proteins were extracted from the cells, digested proteolytically, measured by nanoLC-MS/MS, and subsequently identified by database search and then quantified. The data presented here are related to a previously published research article describing the interplay of S. aureus HG001 and human epithelial cells (Surmann et al., 2015 [1]). They have been deposited to the ProteomeXchange platform with the identifiers PRIDE: http://www.ebi.ac.uk/pride/archive/projects/PXD002384 for the S. aureus HG001 proteome dataset and PRIDE: http://www.ebi.ac.uk/pride/archive/projects/PXD002388 for the A549 proteome dataset.Keywords:
Proteome
Cell Sorting
Proteome
Isotopic labeling
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Abstract A diet derived agent Curcumin (Diferuloylmethane), demonstrated its clinical application in inflammation, infection and cancer conditions. Nevertheless, its impact on the proteome of epithelial cells of non-small cell lung carcinoma (NSCLC) is yet to be explored. We employed a stable isotope labeling method for cell culture (SILAC) based relative quantitative proteomics and informatics analysis to comprehend global proteome change in A549 cells treated with curcumin and/or Lipopolysaccharide (LPS). Pretreated A549 cells were infected with Mycobacterium tuberculosis H37Rv strain to monitor bacterial load. With exposure to curcumin and LPS, out of the 1492 identified proteins, 305 and 346 proteins showed deregulation respectively. The expression of BID and AIFM1 mitochondrial proteins which play critical role in apoptotic pathway were deregulated in curcumin treated cells. Higher mitochondria intensity was observed in curcumin treated A549 cells than LPS treatment. Simultaneous treatment of curcumin and LPS neutralized the effect of LPS. Curcumin and/or LPS pretreated A549 cells infected with H37Rv, showed successful bacterial internalization. LPS treated A549 cells after infection showed increased bacterial load than curcumin compared to non-treated infected cells. However, simultaneous treatment of curcumin and LPS neutralized the effect of LPS. This study generated molecular evidence to deepen our understanding of the anti-inflammatory role of curcumin and may be useful to identify molecular targets for drug discovery.
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Prohibitin
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Stable isotope labeling by amino acids in cell culture( SILAC) is an in vivo metabolic labeling technique based on the abundance ratio of mass spectrometry. Combined with different methods of proteins affinity enrichment,SILAC can obtain sensitive,accurate and quantitative information of proteins. This article reviewed the history,rationale,characteristics and application of SILAC,with special focus on its role in studying protein interactions with small molecules,quantitative proteomics and post-translational modification of proteins.
Isotopic labeling
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Mitochondrial dysfunction rapidly modulates the abundance and thermal stability of cellular proteins
Cellular functionality relies on a well-balanced, but highly dynamic proteome. Dysfunction of mitochondrial protein import leads to the cytosolic accumulation of mitochondrial precursor proteins which compromise cellular proteostasis and trigger a mitoprotein-induced stress response. To dissect the effects of mitochondrial dysfunction on the cellular proteome as a whole, we developed pre-post thermal proteome profiling. This multiplexed time-resolved proteome-wide thermal stability profiling approach with isobaric peptide tags in combination with a pulsed SILAC labelling elucidated dynamic proteostasis changes in several dimensions: In addition to adaptations in protein abundance, we observed rapid modulations of the thermal stability of individual cellular proteins. Different functional groups of proteins showed characteristic response patterns and reacted with group-specific kinetics, allowing the identification of functional modules that are relevant for mitoprotein-induced stress. Thus, our new pre-post thermal proteome profiling approach uncovered a complex response network that orchestrates proteome homeostasis in eukaryotic cells by time-controlled adaptations of the abundance and the conformation of proteins.
Proteostasis
Proteome
Isobaric labeling
Chaperone (clinical)
Tandem mass tag
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Proteome
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Background. A major goal in retroviral-based gene therapy is to establish methods that allow for selection and tracking of transduced cell populations. Green fluorescent protein (GFP) may be useful for gene therapy applications because it is a naturally fluorescent protein that can be detected using conventional flow cytometers facilitating rapid analysis and purification of transduced cell populations. However, it is unknown whether GFP can be stably expressed in vivo, particularly in multiple bone marrow-derived cell lineages. Methods. A murine retrovirus carrying the gene encoding GFP was used to infect murine bone marrow cells (BMCs). These studies were conducted to (1) directly determine whether GFP could be used as a marker of BMC transduction,(2) determine whether GFP is capable of being expressed in multiple bone marrow-derived hematopoietic cell lineages, and (3) determine whether GFP could be used to follow the fate of transduced cells in vivo. Results. Infection of BMCs with retroviruses carrying the gene encoding GFP resulted in a fluorescent signal in viable transduced cells that was detectable by flow cytometry. Expression of GFP was detected in multiple bone marrow-derived cell lineages after transduction, including stem cell antigen-positive (Sca-1+), lineage marker-negative(Lin-), cells. Using GFP as a selectable marker, we were able to enrich for transduced cells by cell sorting. Mice reconstituted with enriched populations of GFP+cells showed a significant increase in the percentage of cells expressing GFP in the periphery when compared with mice reconstituted with unenriched transduced bone marrow. Conclusions. These data indicate that GFP can be used to select for transduced BMCs in vitro, expressed in multiple bone marrow-derived cell lineages, used to select transduced cells, and follow the fate of transduced cells long-term in vivo.
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Transduction (biophysics)
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Cell Sorting
Fluorescent protein
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Proteome
Multidrug tolerance
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As mass-spectrometry-based quantitative proteomics approaches become increasingly powerful, researchers are taking advantage of well established methodologies and improving instrumentation to pioneer new protein expression profiling methods. For example, pooling several proteomes labeled using the stable isotope labeling by amino acids in cell culture (SILAC) method yields a whole-proteome stable isotope-labeled internal standard that can be mixed with a tissue-derived proteome for quantification. By increasing quantitative accuracy in the analysis of tissue proteomes, such methods should improve integration of protein expression profiling data with transcriptomic data and enhance downstream bioinformatic analyses. An accurate and scalable quantitative method to analyze tumor proteomes at the depth of several thousand proteins provides a powerful tool for global protein quantification of tissue samples and promises to redefine our understanding of tumor biology.
Proteome
Label-free Quantification
Isobaric labeling
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