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Methods for characterizing protein acetylation during viral infection.
Methods Enzymol 2019; 626:587-620ME

Abstract

Lysine acetylation is a prevalent posttranslational modification that acts as a regulator of protein function, subcellular localization, and interactions. A growing body of work has highlighted the importance of temporal alterations in protein acetylation during infection with a range of human viruses. It has become clear that both cellular and viral proteins are decorated by lysine acetylations, and that these modifications contribute to core host defense and virus replication processes. Further defining the extent and dynamics of protein acetylation events during the progression of an infection can provide an important new perspective on the intricate mechanisms underlying the biology and pathogenesis of virus infections. Here, we provide protocols for identifying, quantifying, and probing the regulation of lysine acetylations during viral infection. We describe the use of acetyl-lysine immunoaffinity purification and quantitative mass spectrometry for assessing the cellular acetylome at different stages of an infection. As an alternative to traditional antibody-mediated western blotting, we discuss the benefits of targeted mass spectrometry approaches for detecting and quantifying site-specific acetylations on proteins of interest. Specifically, we provide a protocol using parallel reaction monitoring (PRM). We further discuss experimental considerations that are specific to studying viral infections. Finally, we provide a brief overview of the types of assays that can be employed to characterize the function of an acetylation event in the context of infection. As a method to interrogate the regulation of acetylation, we describe the Fluor de Lys assay for monitoring the enzymatic activities of deacetylases.

Authors+Show Affiliations

Department of Molecular Biology, Princeton University, Lewis Thomas Laboratory, Princeton, NJ, United States.Department of Molecular Biology, Princeton University, Lewis Thomas Laboratory, Princeton, NJ, United States.Department of Molecular Biology, Princeton University, Lewis Thomas Laboratory, Princeton, NJ, United States.Department of Molecular Biology, Princeton University, Lewis Thomas Laboratory, Princeton, NJ, United States. Electronic address: icristea@princeton.edu.

Pub Type(s)

Journal Article

Language

eng

PubMed ID

31606092

Citation

Murray, Laura A., et al. "Methods for Characterizing Protein Acetylation During Viral Infection." Methods in Enzymology, vol. 626, 2019, pp. 587-620.
Murray LA, Combs AN, Rekapalli P, et al. Methods for characterizing protein acetylation during viral infection. Meth Enzymol. 2019;626:587-620.
Murray, L. A., Combs, A. N., Rekapalli, P., & Cristea, I. M. (2019). Methods for characterizing protein acetylation during viral infection. Methods in Enzymology, 626, pp. 587-620. doi:10.1016/bs.mie.2019.06.030.
Murray LA, et al. Methods for Characterizing Protein Acetylation During Viral Infection. Meth Enzymol. 2019;626:587-620. PubMed PMID: 31606092.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Methods for characterizing protein acetylation during viral infection. AU - Murray,Laura A, AU - Combs,Ashton N, AU - Rekapalli,Pranav, AU - Cristea,Ileana M, Y1 - 2019/07/18/ PY - 2019/10/14/entrez PY - 2019/10/14/pubmed PY - 2019/10/14/medline KW - Acetylome KW - HDAC KW - Mass spectrometry KW - PRM KW - Proteomics KW - SIRT KW - Sirtuin KW - Targeted mass spectrometry SP - 587 EP - 620 JF - Methods in enzymology JO - Meth. Enzymol. VL - 626 N2 - Lysine acetylation is a prevalent posttranslational modification that acts as a regulator of protein function, subcellular localization, and interactions. A growing body of work has highlighted the importance of temporal alterations in protein acetylation during infection with a range of human viruses. It has become clear that both cellular and viral proteins are decorated by lysine acetylations, and that these modifications contribute to core host defense and virus replication processes. Further defining the extent and dynamics of protein acetylation events during the progression of an infection can provide an important new perspective on the intricate mechanisms underlying the biology and pathogenesis of virus infections. Here, we provide protocols for identifying, quantifying, and probing the regulation of lysine acetylations during viral infection. We describe the use of acetyl-lysine immunoaffinity purification and quantitative mass spectrometry for assessing the cellular acetylome at different stages of an infection. As an alternative to traditional antibody-mediated western blotting, we discuss the benefits of targeted mass spectrometry approaches for detecting and quantifying site-specific acetylations on proteins of interest. Specifically, we provide a protocol using parallel reaction monitoring (PRM). We further discuss experimental considerations that are specific to studying viral infections. Finally, we provide a brief overview of the types of assays that can be employed to characterize the function of an acetylation event in the context of infection. As a method to interrogate the regulation of acetylation, we describe the Fluor de Lys assay for monitoring the enzymatic activities of deacetylases. SN - 1557-7988 UR - https://www.unboundmedicine.com/medline/citation/31606092/Methods_for_characterizing_protein_acetylation_during_viral_infection L2 - https://linkinghub.elsevier.com/retrieve/pii/S0076-6879(19)30280-0 DB - PRIME DP - Unbound Medicine ER -