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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

SILAC-Based Identification of Fatty Acylated HSV-Encoded Proteins(A) SILAC-based experimental workflow.(B) Heatmap for YnPal/Pal (25 μM, 5–19 hpi) enrichment (n = 3) of proteins derived from HSV-1[17], HSV-1[KOS], and HSV-2[186]. Black to red gradation represents the degree (n = 3) of enrichment of proteins labeled with YnPal over Pal, expressed as a heatmap. Gray bars represent missing values (applied when quantification was missing for more than one out of three samples).
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fig5: SILAC-Based Identification of Fatty Acylated HSV-Encoded Proteins(A) SILAC-based experimental workflow.(B) Heatmap for YnPal/Pal (25 μM, 5–19 hpi) enrichment (n = 3) of proteins derived from HSV-1[17], HSV-1[KOS], and HSV-2[186]. Black to red gradation represents the degree (n = 3) of enrichment of proteins labeled with YnPal over Pal, expressed as a heatmap. Gray bars represent missing values (applied when quantification was missing for more than one out of three samples).

Mentions: We next directed our attention to identification of putative viral proteins that were acylated by the host machinery during infection. The workflow depicted in Figure 3A was designed to measure HSV-induced changes to the host proteome and was not optimal for an unambiguous identification of fatty acylated viral proteins. Therefore to discriminate between bona fide fatty acylated proteins and abundant HSV-encoded proteins which might non-specifically bind to the capture beads, we modified our quantitative metabolic tagging workflow (Figure 5A). Thus, heavy and light cells were infected (n = 3) with HSV and incubated with either the alkynyl fatty acid probes (heavy) or the natural fatty acids (light). The natural precursor does not contain a bio-orthogonal tag (alkyne moiety) and does not undergo chemoselective ligation to AzTB, thus significant heavy/light signal enrichment would be expected for fatty acylated proteins over non-specifically binding proteins. Using parallel pairs of probes, YnPal and Pal (palmitic acid) or YnMyr and Myr (myristic acid), nine putative palmitoylated and five putative myristoylated HSV proteins were identified in two or more samples (Figure S3; Table S7). The selective and highly significant identification of glycoproteins among the HSV proteome suggests that the assignments are highly robust. The set includes gE, gI, gG, gK, US2, US3, UL56, UL51 (TEG7), and UL24, and, consistent with a lack of myristoylation consensus in these proteins except for US2, proteins identified with YnMyr may in fact incorporate this probe through the palmitoylation pathway. Results supporting this conclusion were shown from the observed lack of sensitivity of YnMyr incorporation into HSV-encoded proteins to a specific and potent myristoylation inhibitor (Frearson et al., 2010; Goncalves et al., 2012; Table S8). In this analysis we quantified changes in the YnMyr incorporation for 9 out of 11 HSV proteins possessing a myristoylation motif. UL11 (a known myristoylated and palmitoylated protein) (Loomis et al., 2001; MacLean et al., 1989) and gN were not detected in our analyses. A reasonable explanation for this is the very limited number of tryptic peptides in these small proteins that are compatible with MS and tandem MS (MS/MS) detection.


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)

SILAC-Based Identification of Fatty Acylated HSV-Encoded Proteins(A) SILAC-based experimental workflow.(B) Heatmap for YnPal/Pal (25 μM, 5–19 hpi) enrichment (n = 3) of proteins derived from HSV-1[17], HSV-1[KOS], and HSV-2[186]. Black to red gradation represents the degree (n = 3) of enrichment of proteins labeled with YnPal over Pal, expressed as a heatmap. Gray bars represent missing values (applied when quantification was missing for more than one out of three samples).
© Copyright Policy - CC BY
Related In: Results  -  Collection

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

fig5: SILAC-Based Identification of Fatty Acylated HSV-Encoded Proteins(A) SILAC-based experimental workflow.(B) Heatmap for YnPal/Pal (25 μM, 5–19 hpi) enrichment (n = 3) of proteins derived from HSV-1[17], HSV-1[KOS], and HSV-2[186]. Black to red gradation represents the degree (n = 3) of enrichment of proteins labeled with YnPal over Pal, expressed as a heatmap. Gray bars represent missing values (applied when quantification was missing for more than one out of three samples).
Mentions: We next directed our attention to identification of putative viral proteins that were acylated by the host machinery during infection. The workflow depicted in Figure 3A was designed to measure HSV-induced changes to the host proteome and was not optimal for an unambiguous identification of fatty acylated viral proteins. Therefore to discriminate between bona fide fatty acylated proteins and abundant HSV-encoded proteins which might non-specifically bind to the capture beads, we modified our quantitative metabolic tagging workflow (Figure 5A). Thus, heavy and light cells were infected (n = 3) with HSV and incubated with either the alkynyl fatty acid probes (heavy) or the natural fatty acids (light). The natural precursor does not contain a bio-orthogonal tag (alkyne moiety) and does not undergo chemoselective ligation to AzTB, thus significant heavy/light signal enrichment would be expected for fatty acylated proteins over non-specifically binding proteins. Using parallel pairs of probes, YnPal and Pal (palmitic acid) or YnMyr and Myr (myristic acid), nine putative palmitoylated and five putative myristoylated HSV proteins were identified in two or more samples (Figure S3; Table S7). The selective and highly significant identification of glycoproteins among the HSV proteome suggests that the assignments are highly robust. The set includes gE, gI, gG, gK, US2, US3, UL56, UL51 (TEG7), and UL24, and, consistent with a lack of myristoylation consensus in these proteins except for US2, proteins identified with YnMyr may in fact incorporate this probe through the palmitoylation pathway. Results supporting this conclusion were shown from the observed lack of sensitivity of YnMyr incorporation into HSV-encoded proteins to a specific and potent myristoylation inhibitor (Frearson et al., 2010; Goncalves et al., 2012; Table S8). In this analysis we quantified changes in the YnMyr incorporation for 9 out of 11 HSV proteins possessing a myristoylation motif. UL11 (a known myristoylated and palmitoylated protein) (Loomis et al., 2001; MacLean et al., 1989) and gN were not detected in our analyses. A reasonable explanation for this is the very limited number of tryptic peptides in these small proteins that are compatible with MS and tandem MS (MS/MS) detection.

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