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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

Infection-Induced Changes to Nascent Protein Abundances(A) SILAC-based proteomic workflow.(B) Experimental results from samples prior to affinity enrichment: Log2 nascent protein abundance ratio HSV/Mock (n = 3) plotted against significance of the change measured. Red, proteins with myristoylation requirement (N-terminal Gly); black, all other proteins quantified. Each data point represents a protein or a protein group (Cox et al., 2011).
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fig4: Infection-Induced Changes to Nascent Protein Abundances(A) SILAC-based proteomic workflow.(B) Experimental results from samples prior to affinity enrichment: Log2 nascent protein abundance ratio HSV/Mock (n = 3) plotted against significance of the change measured. Red, proteins with myristoylation requirement (N-terminal Gly); black, all other proteins quantified. Each data point represents a protein or a protein group (Cox et al., 2011).

Mentions: In the next experiments, we examined alterations of nascent protein abundance in infected versus uninfected cells, and their relationship to protein acylation using a SILAC/pulse-chase protocol (Figure 4A). Cells in light medium were mock infected or infected and transferred to heavy medium, and incubation continued to compare heavy isotope incorporation into nascent proteins during the labeling period in uninfected versus HSV-infected cells. Equal amounts of the mock-infected and infected lysates were spiked with a fixed amount of protein from cells labeled by SILAC with an intermediate (medium) isotopic label, enabling normalization between samples through a standard “spike-in SILAC” approach (Geiger et al., 2011). Since we anticipated that these data might return only a small set particularly for the myristoylated proteins, we included a pulse with YnMyr prior to isotope labeling to enable enrichment and detection of this set, alongside the nascent analysis for all proteins. The spiked mixtures of total proteins were then subjected to tryptic digestion and MS analysis. In parallel, the YnMyr-tagged protein populations from the same lysate mixtures were ligated to AzTB, affinity-captured, and analyzed by on-bead digest and MS. The ratio of heavy/medium signal for mock- and HSV-infected samples yields information on changes in the abundance of individual proteins (all proteins) synthesized during the time frame of the experiment, and the affinity-captured sample gives the same information specifically for the myristoylated population. The results for nascent protein abundance in HSV- versus mock-infected cells, plotted against significance of the difference for all proteins, are shown in Figure 4B. Overall the results for 510 proteins quantitated (Table S4) revealed a significant down-regulation of protein synthesis in HSV-infected cells, in keeping with previous data (Suzutani et al., 2000), consistent with the known progression of HSV infection. To selectively examine nascent abundances for myristoylated proteins, the average fold (Log2) decrease among potential substrates (with myristoylation recognition motifs) was −1.86 ± 0.53 (33 protein groups). The average fold decrease among substrates purified by affinity enrichment for YnMyr and satisfying the criteria of possession of an N-terminal glycine and previous identification as myristoylated proteins (Broncel et al., 2015; Thinon et al., 2014) was −3.17 ± 0.23 (eight protein groups) (Table S4). Therefore, comparing the decrease in levels of nascent protein abundances from this analysis with the decrease in levels of myristoylation among validated substrates (Table S2), the effects were −2.33 ± 0.69 and −3.17 ± 0.23, respectively. These data suggest, therefore, that the apparent decrease in host protein myristoylation largely results from suppression of host protein synthesis in infected cells. In support of this, we note that total abundances of NMT1 and NMT2 in HSV- and mock-infected cells were very similar (Table S3), with average ratios of 1.13 ± 0.18 (four samples) and 1.12 ± 0.25 (seven samples), respectively.


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)

Infection-Induced Changes to Nascent Protein Abundances(A) SILAC-based proteomic workflow.(B) Experimental results from samples prior to affinity enrichment: Log2 nascent protein abundance ratio HSV/Mock (n = 3) plotted against significance of the change measured. Red, proteins with myristoylation requirement (N-terminal Gly); black, all other proteins quantified. Each data point represents a protein or a protein group (Cox et al., 2011).
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fig4: Infection-Induced Changes to Nascent Protein Abundances(A) SILAC-based proteomic workflow.(B) Experimental results from samples prior to affinity enrichment: Log2 nascent protein abundance ratio HSV/Mock (n = 3) plotted against significance of the change measured. Red, proteins with myristoylation requirement (N-terminal Gly); black, all other proteins quantified. Each data point represents a protein or a protein group (Cox et al., 2011).
Mentions: In the next experiments, we examined alterations of nascent protein abundance in infected versus uninfected cells, and their relationship to protein acylation using a SILAC/pulse-chase protocol (Figure 4A). Cells in light medium were mock infected or infected and transferred to heavy medium, and incubation continued to compare heavy isotope incorporation into nascent proteins during the labeling period in uninfected versus HSV-infected cells. Equal amounts of the mock-infected and infected lysates were spiked with a fixed amount of protein from cells labeled by SILAC with an intermediate (medium) isotopic label, enabling normalization between samples through a standard “spike-in SILAC” approach (Geiger et al., 2011). Since we anticipated that these data might return only a small set particularly for the myristoylated proteins, we included a pulse with YnMyr prior to isotope labeling to enable enrichment and detection of this set, alongside the nascent analysis for all proteins. The spiked mixtures of total proteins were then subjected to tryptic digestion and MS analysis. In parallel, the YnMyr-tagged protein populations from the same lysate mixtures were ligated to AzTB, affinity-captured, and analyzed by on-bead digest and MS. The ratio of heavy/medium signal for mock- and HSV-infected samples yields information on changes in the abundance of individual proteins (all proteins) synthesized during the time frame of the experiment, and the affinity-captured sample gives the same information specifically for the myristoylated population. The results for nascent protein abundance in HSV- versus mock-infected cells, plotted against significance of the difference for all proteins, are shown in Figure 4B. Overall the results for 510 proteins quantitated (Table S4) revealed a significant down-regulation of protein synthesis in HSV-infected cells, in keeping with previous data (Suzutani et al., 2000), consistent with the known progression of HSV infection. To selectively examine nascent abundances for myristoylated proteins, the average fold (Log2) decrease among potential substrates (with myristoylation recognition motifs) was −1.86 ± 0.53 (33 protein groups). The average fold decrease among substrates purified by affinity enrichment for YnMyr and satisfying the criteria of possession of an N-terminal glycine and previous identification as myristoylated proteins (Broncel et al., 2015; Thinon et al., 2014) was −3.17 ± 0.23 (eight protein groups) (Table S4). Therefore, comparing the decrease in levels of nascent protein abundances from this analysis with the decrease in levels of myristoylation among validated substrates (Table S2), the effects were −2.33 ± 0.69 and −3.17 ± 0.23, respectively. These data suggest, therefore, that the apparent decrease in host protein myristoylation largely results from suppression of host protein synthesis in infected cells. In support of this, we note that total abundances of NMT1 and NMT2 in HSV- and mock-infected cells were very similar (Table S3), with average ratios of 1.13 ± 0.18 (four samples) and 1.12 ± 0.25 (seven samples), respectively.

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