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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

In-Gel Fluorescent Analysis of Protein Fatty Acylation During HSV Infection in RPE-1 CellsCells were pulsed with YnPal or YnMyr (25 μM) for 6 hr and harvested at various times as shown. Proteins were isolated, processed for coupling to capture reagent AzTB, and analyzed for palmitoylation (A) or myristoylation (B). Arrows point to bands of increased fluorescence intensities (proteins with increased acylation levels) and arrowheads point to bands with decreased fluorescence intensities (proteins with decreased acylation levels).
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fig2: In-Gel Fluorescent Analysis of Protein Fatty Acylation During HSV Infection in RPE-1 CellsCells were pulsed with YnPal or YnMyr (25 μM) for 6 hr and harvested at various times as shown. Proteins were isolated, processed for coupling to capture reagent AzTB, and analyzed for palmitoylation (A) or myristoylation (B). Arrows point to bands of increased fluorescence intensities (proteins with increased acylation levels) and arrowheads point to bands with decreased fluorescence intensities (proteins with decreased acylation levels).

Mentions: In a series of preliminary experiments, uninfected cells were incubated with a range of concentrations (1–50 μM) of the probes for protein palmitoylation (YnPal) and myristoylation (YnMyr). The results indicated a concentration of 25 μM was optimal for both probes, with no apparent toxicity observed compared with vehicle (DMSO) over the 24-hr exposure time as measured by viable cell counting (Figure S1). Cells were then mock infected or infected with HSV-1[17] at an MOI of 5, i.e. an average of five infectious particles per cell, ensuring quantitative infection of all the cells for comparison with uninfected samples, and then pulse-labeled with YnPal and YnMyr lipid probes. Metabolically tagged, acylated proteins were subsequently ligated to AzTB, extracted, and visualized firstly by in-gel fluorescence (Figure 2). Distinct species were observed for HSV-infected versus mock-infected cells. For palmitoylation, candidate host species (Figure 2, open arrowheads) progressively reduced in abundance while a series of new species were observed, increasing with time after infection (Figure 2, solid arrows, lanes 1–4). A generally similar trend was observed for the myristoylation probe (Figure 2, solid arrows, lanes 5–8), with certain species migrating similarly to those observed with the palmitoylation probe.


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)

In-Gel Fluorescent Analysis of Protein Fatty Acylation During HSV Infection in RPE-1 CellsCells were pulsed with YnPal or YnMyr (25 μM) for 6 hr and harvested at various times as shown. Proteins were isolated, processed for coupling to capture reagent AzTB, and analyzed for palmitoylation (A) or myristoylation (B). Arrows point to bands of increased fluorescence intensities (proteins with increased acylation levels) and arrowheads point to bands with decreased fluorescence intensities (proteins with decreased acylation levels).
© Copyright Policy - CC BY
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4543063&req=5

fig2: In-Gel Fluorescent Analysis of Protein Fatty Acylation During HSV Infection in RPE-1 CellsCells were pulsed with YnPal or YnMyr (25 μM) for 6 hr and harvested at various times as shown. Proteins were isolated, processed for coupling to capture reagent AzTB, and analyzed for palmitoylation (A) or myristoylation (B). Arrows point to bands of increased fluorescence intensities (proteins with increased acylation levels) and arrowheads point to bands with decreased fluorescence intensities (proteins with decreased acylation levels).
Mentions: In a series of preliminary experiments, uninfected cells were incubated with a range of concentrations (1–50 μM) of the probes for protein palmitoylation (YnPal) and myristoylation (YnMyr). The results indicated a concentration of 25 μM was optimal for both probes, with no apparent toxicity observed compared with vehicle (DMSO) over the 24-hr exposure time as measured by viable cell counting (Figure S1). Cells were then mock infected or infected with HSV-1[17] at an MOI of 5, i.e. an average of five infectious particles per cell, ensuring quantitative infection of all the cells for comparison with uninfected samples, and then pulse-labeled with YnPal and YnMyr lipid probes. Metabolically tagged, acylated proteins were subsequently ligated to AzTB, extracted, and visualized firstly by in-gel fluorescence (Figure 2). Distinct species were observed for HSV-infected versus mock-infected cells. For palmitoylation, candidate host species (Figure 2, open arrowheads) progressively reduced in abundance while a series of new species were observed, increasing with time after infection (Figure 2, solid arrows, lanes 1–4). A generally similar trend was observed for the myristoylation probe (Figure 2, solid arrows, lanes 5–8), with certain species migrating similarly to those observed with the palmitoylation probe.

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