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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

Enrichment StrategyChemoproteomic Workflow (A), Structures of Lipid Probes (B), and Multi-functional Capture Reagent AzTB (C). Inside the cell and the test tube, colored shapes represent proteins, the triple bar represents alkyne; on the capture reagent (AzTB), red N3 represents azide, the pink star represents TAMRA fluorophore, and the blue double pentagon represents biotin.
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fig1: Enrichment StrategyChemoproteomic Workflow (A), Structures of Lipid Probes (B), and Multi-functional Capture Reagent AzTB (C). Inside the cell and the test tube, colored shapes represent proteins, the triple bar represents alkyne; on the capture reagent (AzTB), red N3 represents azide, the pink star represents TAMRA fluorophore, and the blue double pentagon represents biotin.

Mentions: Studies on the role of protein acylation during infection are frequently reported on an individual case-by-case basis rather than from systematic unbiased analysis using high-throughput techniques such as mass spectrometry (MS)-based proteomic approaches. While advances in MS enable global profiling of many post-translational modifications, identification and quantification of protein fatty acylation by standard MS remains very challenging. Acylation negatively affects solubility, chromatographic properties, and ionization of lipid-anchored tryptic peptides, rendering MS detection difficult. Furthermore, lipidated proteins often exist in low abundance in cells; therefore, lipid-specific enrichment is required to reduce sample complexity and improve discovery. An enrichment strategy that utilizes introduction of a chemical reporter via metabolic tagging with lipid probes in cell culture (Figure 1A) has become a method of choice to study protein myristoylation and palmitoylation, as well as other classes of lipidation (Berry et al., 2010; Broncel et al., 2015; Ciepla et al., 2014; Konitsiotis et al., 2015; Martin and Cravatt, 2009; Tate et al., 2015; Wilson et al., 2011; Wright et al., 2015). ω-Alkynyl fatty acid analogs (Figure 1B) can be used for metabolic tagging of acylated proteins and subsequently elaborated by a highly selective ligation reaction (copper-catalyzed azide alkyne cycloaddition [CuAAC]) (Rostovtsev et al., 2002; Tornoe et al., 2002) to a multi-functional azido capture reagent (AzTB, Figure 1C) enabling visualization (via the TAMRA component), enrichment (via the biotin component), and subsequent steps of on-bead tryptic digestion, MS measurement, software-aided peptide sequencing and identification, as well as quantification of fatty acylated proteins (Storck et al., 2013). In the work reported here, we applied chemical technologies with systems proteomic approaches to make comprehensive advances in the investigation of fatty acylation of virus and host proteins in a complex but tractable system with major clinical relevance in human disease, the human herpesviruses.


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)

Enrichment StrategyChemoproteomic Workflow (A), Structures of Lipid Probes (B), and Multi-functional Capture Reagent AzTB (C). Inside the cell and the test tube, colored shapes represent proteins, the triple bar represents alkyne; on the capture reagent (AzTB), red N3 represents azide, the pink star represents TAMRA fluorophore, and the blue double pentagon represents biotin.
© Copyright Policy - CC BY
Related In: Results  -  Collection

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

fig1: Enrichment StrategyChemoproteomic Workflow (A), Structures of Lipid Probes (B), and Multi-functional Capture Reagent AzTB (C). Inside the cell and the test tube, colored shapes represent proteins, the triple bar represents alkyne; on the capture reagent (AzTB), red N3 represents azide, the pink star represents TAMRA fluorophore, and the blue double pentagon represents biotin.
Mentions: Studies on the role of protein acylation during infection are frequently reported on an individual case-by-case basis rather than from systematic unbiased analysis using high-throughput techniques such as mass spectrometry (MS)-based proteomic approaches. While advances in MS enable global profiling of many post-translational modifications, identification and quantification of protein fatty acylation by standard MS remains very challenging. Acylation negatively affects solubility, chromatographic properties, and ionization of lipid-anchored tryptic peptides, rendering MS detection difficult. Furthermore, lipidated proteins often exist in low abundance in cells; therefore, lipid-specific enrichment is required to reduce sample complexity and improve discovery. An enrichment strategy that utilizes introduction of a chemical reporter via metabolic tagging with lipid probes in cell culture (Figure 1A) has become a method of choice to study protein myristoylation and palmitoylation, as well as other classes of lipidation (Berry et al., 2010; Broncel et al., 2015; Ciepla et al., 2014; Konitsiotis et al., 2015; Martin and Cravatt, 2009; Tate et al., 2015; Wilson et al., 2011; Wright et al., 2015). ω-Alkynyl fatty acid analogs (Figure 1B) can be used for metabolic tagging of acylated proteins and subsequently elaborated by a highly selective ligation reaction (copper-catalyzed azide alkyne cycloaddition [CuAAC]) (Rostovtsev et al., 2002; Tornoe et al., 2002) to a multi-functional azido capture reagent (AzTB, Figure 1C) enabling visualization (via the TAMRA component), enrichment (via the biotin component), and subsequent steps of on-bead tryptic digestion, MS measurement, software-aided peptide sequencing and identification, as well as quantification of fatty acylated proteins (Storck et al., 2013). In the work reported here, we applied chemical technologies with systems proteomic approaches to make comprehensive advances in the investigation of fatty acylation of virus and host proteins in a complex but tractable system with major clinical relevance in human disease, the human herpesviruses.

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