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Systems Analysis of Protein Fatty Acylation in Herpes Simplex Virus-Infected Cells Using Chemical Proteomics.

Serwa RA, Abaitua F, Krause E, Tate EW, O'Hare P - Chem. Biol. (2015)

Bottom Line: Acylation also modulates the function and localization of virus-encoded proteins.Furthermore, we found that a significant fraction of the viral proteome undergoes palmitoylation; we identified a number of virus membrane glycoproteins, structural proteins, and kinases.Taken together, our results provide broad oversight of protein acylation during HSV infection, a roadmap for similar analysis in other systems, and a resource with which to pursue specific analysis of systems and functions.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, Imperial College London, Exhibition Road, London SW7 2AZ, UK.

No MeSH data available.


Related in: MedlinePlus

Quantitative Proteomics Analysis of Host Protein Fatty Acylation During HSV Infection in RPE-1 Cells(A) SILAC-based quantitative proteomics workflow.(B) Virus-induced changes to protein palmitoylation (n = 4) plotted against statistical significance of the ratio measured.(C) Virus-induced changes to protein myristoylation (n = 4) plotted against statistical significance. Black, proteins with myristoylation requirement (N-terminal Gly); red, validated NMT substrates (Broncel et al., 2015; Thinon et al., 2014).In (B) and (C) each data point represents a protein or a protein group (Cox et al., 2011).
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fig3: Quantitative Proteomics Analysis of Host Protein Fatty Acylation During HSV Infection in RPE-1 Cells(A) SILAC-based quantitative proteomics workflow.(B) Virus-induced changes to protein palmitoylation (n = 4) plotted against statistical significance of the ratio measured.(C) Virus-induced changes to protein myristoylation (n = 4) plotted against statistical significance. Black, proteins with myristoylation requirement (N-terminal Gly); red, validated NMT substrates (Broncel et al., 2015; Thinon et al., 2014).In (B) and (C) each data point represents a protein or a protein group (Cox et al., 2011).

Mentions: These results demonstrate progressive alteration in the acylated protein profiles. Nevertheless, qualitative analysis by SDS-PAGE and in-gel fluorescence, while useful as a preliminary survey, is clearly limited for the ambition of a more broad-based analysis, including target protein identification. We therefore next employed MS combined with isotopic tagging methodology, stable isotope labeling by amino acid in cell culture (SILAC) (Ong et al., 2002), to identify and quantitate HSV-induced changes to lipid fatty acylation. In this approach (Figure 3A) cells to be infected are labeled with medium containing 13C615N4-arginine and 13C615N2-lysine (heavy), whereas cells to be mock infected are incubated in medium with arginine and lysine at natural isotopic distribution (light). Cells were infected or mock infected, incubated in heavy or light medium, respectively, and pulsed with the lipid probes. Mock-infected or infected cells were then harvested (19 hr post infection [hpi]) and the proteins extracted. After determining the total concentrations, proteins were mixed in a 1:1 ratio, subject to CuAAC ligation to AzTB, and affinity enriched on NeutrAvidin beads. The labeled, purified proteins were then subject to tryptic digestion and MS measurements (see Experimental Procedures). As part of this work flow, the isotope labeling was also reversed, whereby HSV-infected cells were incubated in light medium and mock-infected cells in heavy medium, thus minimizing experimental bias and aiding identification of incidental contaminants. For each probe, biological quadruplicate experiments were performed, measurement outcomes pooled, and proteins identified on a strict filtering basis requiring presence in replicate samples for inclusion in subsequent evaluation. The data were then further subjected to statistical analysis to identify proteins with significantly different lipidation status in infected and uninfected cells.


Systems Analysis of Protein Fatty Acylation in Herpes Simplex Virus-Infected Cells Using Chemical Proteomics.

Serwa RA, Abaitua F, Krause E, Tate EW, O'Hare P - Chem. Biol. (2015)

Quantitative Proteomics Analysis of Host Protein Fatty Acylation During HSV Infection in RPE-1 Cells(A) SILAC-based quantitative proteomics workflow.(B) Virus-induced changes to protein palmitoylation (n = 4) plotted against statistical significance of the ratio measured.(C) Virus-induced changes to protein myristoylation (n = 4) plotted against statistical significance. Black, proteins with myristoylation requirement (N-terminal Gly); red, validated NMT substrates (Broncel et al., 2015; Thinon et al., 2014).In (B) and (C) each data point represents a protein or a protein group (Cox et al., 2011).
© Copyright Policy - CC BY
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4543063&req=5

fig3: Quantitative Proteomics Analysis of Host Protein Fatty Acylation During HSV Infection in RPE-1 Cells(A) SILAC-based quantitative proteomics workflow.(B) Virus-induced changes to protein palmitoylation (n = 4) plotted against statistical significance of the ratio measured.(C) Virus-induced changes to protein myristoylation (n = 4) plotted against statistical significance. Black, proteins with myristoylation requirement (N-terminal Gly); red, validated NMT substrates (Broncel et al., 2015; Thinon et al., 2014).In (B) and (C) each data point represents a protein or a protein group (Cox et al., 2011).
Mentions: These results demonstrate progressive alteration in the acylated protein profiles. Nevertheless, qualitative analysis by SDS-PAGE and in-gel fluorescence, while useful as a preliminary survey, is clearly limited for the ambition of a more broad-based analysis, including target protein identification. We therefore next employed MS combined with isotopic tagging methodology, stable isotope labeling by amino acid in cell culture (SILAC) (Ong et al., 2002), to identify and quantitate HSV-induced changes to lipid fatty acylation. In this approach (Figure 3A) cells to be infected are labeled with medium containing 13C615N4-arginine and 13C615N2-lysine (heavy), whereas cells to be mock infected are incubated in medium with arginine and lysine at natural isotopic distribution (light). Cells were infected or mock infected, incubated in heavy or light medium, respectively, and pulsed with the lipid probes. Mock-infected or infected cells were then harvested (19 hr post infection [hpi]) and the proteins extracted. After determining the total concentrations, proteins were mixed in a 1:1 ratio, subject to CuAAC ligation to AzTB, and affinity enriched on NeutrAvidin beads. The labeled, purified proteins were then subject to tryptic digestion and MS measurements (see Experimental Procedures). As part of this work flow, the isotope labeling was also reversed, whereby HSV-infected cells were incubated in light medium and mock-infected cells in heavy medium, thus minimizing experimental bias and aiding identification of incidental contaminants. For each probe, biological quadruplicate experiments were performed, measurement outcomes pooled, and proteins identified on a strict filtering basis requiring presence in replicate samples for inclusion in subsequent evaluation. The data were then further subjected to statistical analysis to identify proteins with significantly different lipidation status in infected and uninfected cells.

Bottom Line: Acylation also modulates the function and localization of virus-encoded proteins.Furthermore, we found that a significant fraction of the viral proteome undergoes palmitoylation; we identified a number of virus membrane glycoproteins, structural proteins, and kinases.Taken together, our results provide broad oversight of protein acylation during HSV infection, a roadmap for similar analysis in other systems, and a resource with which to pursue specific analysis of systems and functions.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, Imperial College London, Exhibition Road, London SW7 2AZ, UK.

No MeSH data available.


Related in: MedlinePlus