Analysis of Protein Lysine Acetylation In Vitro and In Vivo

Pelletier, Nicolas; Grégoire, Serge; Yang, Xiang‐Jiao

doi:10.1002/0471140864.ps1411s54

Cited by 2 publications

(1 citation statement)

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“…Analytical tools for lysine acetylation and related acylation have thus garnered wide interest. Prior articles provide detailed protocols for investigating acetylation of histones and non‐histone proteins in vitro with [ 3 H]‐ or [ 14 C]‐labeled acetyl coenzyme A (Pelletier et al., 2008; Pelletier et al., 2017). Those protocols are easily adaptable to analyze acylation in the presence of [ 3 H]‐ or [ 14 C]‐labeled acyl coenzyme A in vitro .…”

Section: Introductionmentioning

confidence: 99%

Analysis of Lysine Acetylation and Acetylation‐like Acylation In Vitro and In Vivo

Yan,

Mousavi,

Yang

2023

Current Protocols

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Protein lysine acetylation refers to the covalent transfer of an acetyl moiety from acetyl coenzyme A to the epsilon‐amino group of a lysine residue and is critical for regulating protein functions in almost all living cells or organisms. Studies in the past decade have demonstrated the unexpected finding that acetylation‐like acylation, such as succinylation, propionylation, butyrylation, crotonylation, and lactylation, is also present in histones and many non‐histone proteins. Acetylation and acetylation‐like acylation serve as reversible on/off switches for regulating protein function while interplaying with other post‐translational modifications (such as phosphorylation and methylation) in a codified manner. Lysine acetylation and acetylation‐like acylation are important for regulating different cellular and developmental processes in normal and pathological states. Thus, the detection of such modifications is important for related basic research and molecular diagnostics. Traditionally, lysine acetylation is detected by autoradiography, but recent decades have seen great improvement in the quality of site‐specific antibodies against acetylation (or acetylation‐like acylation), thereby providing competitive alternatives to the use of radioactive acetate and acetyl‐coenzyme A for in vivo and in vitro labeling, respectively. This article describes protocols for the detection of lysine acetylation and acetylation‐like acylation with site‐specific antibodies to complement extant autoradiography‐based methods (Pelletier et al., 2017). © 2023 Wiley Periodicals LLC. Basic Protocol 1: Acylation assays in vitro Basic Protocol 2: Determination of in vivo acylation

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Section: Introductionmentioning

confidence: 99%

Analysis of Lysine Acetylation and Acetylation‐like Acylation In Vitro and In Vivo

Yan,

Mousavi,

Yang

2023

Current Protocols

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Reconstitution of Active and Stoichiometric Multisubunit Lysine Acetyltransferase Complexes in Insect Cells

Kou

Pelletier

et al. 2011

Methods in Molecular Biology

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Protein lysine acetyltransferases (KATs) catalyze acetylation of the ε-amino group on a specific lysine residue, and this posttranslational modification is important for regulating the function and activities of thousands of proteins in diverse organisms from bacteria to humans. Interestingly, many known KATs exist in multisubunit complexes and complex formation is important for their proper structure, function, and regulation. Thus, it is necessary to reconstitute enzymatically active complexes for studying the relationship between subunits and determining structures of the complexes. Due to inherent limitations of bacterial and mammalian expression systems, baculovirus-mediated protein expression in insect cells has proven useful for assembling such multisubunit complexes. Related to this, we have adopted such an approach for reconstituting active tetrameric complexes of monocytic leukemia zinc (MOZ, finger protein, recently renamed MYST3 or KAT6A) and MOZ-related factor (MORF, also known as MYST4 or KAT6B), two KATs directly linked to development of leukemia and self-renewal of stem cells. Herein, we use these complexes as examples to describe the related procedures. Similar methods have been used for reconstituting active complexes of histone deacetylases, lysine demethylases, and ubiquitin ligases, so this simple approach can be adapted for molecular dissection of various multisubunit complexes.

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Analysis of Protein Lysine Acetylation In Vitro and In Vivo

Cited by 2 publications

References 38 publications

Analysis of Lysine Acetylation and Acetylation‐like Acylation In Vitro and In Vivo

Analysis of Lysine Acetylation and Acetylation‐like Acylation In Vitro and In Vivo

Reconstitution of Active and Stoichiometric Multisubunit Lysine Acetyltransferase Complexes in Insect Cells

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