Hydrazide Mimics for Protein Lysine Acylation To Assess Nucleosome Dynamics and Deubiquitinase Action.

A range of acyl-lysine (acyl-Lys) modifications on histones and other proteins have been mapped over the past decade but for most, their functional and structural significance remains poorly characterized. One limitation in the study of acyl-Lys containing proteins is the challenge of producing them or their mimics in site-specifically modified forms. We describe a cysteine alkylation-based method to install hydrazide mimics of acyl-Lys post-translational modifications (PTMs) on proteins. We have applied this method to install mimics of acetyl-Lys, 2-hydroxyisobutyryl-Lys, and ubiquityl-Lys that could be recognized selectively by relevant acyl-Lys modification antibodies. The acyl-Lys modified histone H3 proteins were reconstituted into nucleosomes to study nucleosome dynamics and stability as a function of modification type and site. We also installed a ubiquityl-Lys mimic in histone H2B and generated a diubiquitin analog, both of which could be cleaved by deubiquitinating enzymes. Nucleosomes containing the H2B ubiquityl-Lys mimic were used to study the SAGA deubiquitinating module's molecular recognition. These results suggest that acyl-Lys mimics offer a relatively simple and promising strategy to study the role of acyl-Lys modifications in the function, structure, and regulation of proteins and protein complexes.

• Publication year

  2018

• Venue

  Journal of the American Chemical Society

• Publication date

  2018-07-10

• Fields of study

  Biology, Medicine, Chemistry

• Identifiers
  DOI 10.1021/jacs.8b03572PMID 29991262PMCID PMC6070418
• External record

  Open on Semantic Scholar

• Source metadata

  Semantic Scholar, PubMed

• Nucleosomes containing the H2B ubiquityl-Lys mimic support analysis of molecular recognition by the SAGA deubiquitinating module.

  Confidence 0.94

  박진우 (dztg5apj7m) extractionB (s683577b42) reviewq (76h6bfydm6) review
• A ubiquityl-Lys mimic installed in histone H2B and a diubiquitin analog are cleavable by deubiquitinating enzymes.

  Confidence 0.96

  박진우 (dztg5apj7m) extractionB (s683577b42) reviewq (76h6bfydm6) review
• Acyl-Lys-modified histone H3 proteins can be reconstituted into nucleosomes to examine how modification type and site affect nucleosome dynamics and stability.

  Confidence 0.95

  박진우 (dztg5apj7m) extractionB (s683577b42) reviewq (76h6bfydm6) review
• A cysteine alkylation-based strategy installs hydrazide mimics of acetyl-Lys, 2-hydroxyisobutyryl-Lys, and ubiquityl-Lys PTMs on proteins, and the installed mimics are selectively recognized by the corresponding acyl-Lys antibodies.

  Confidence 0.98

  박진우 (dztg5apj7m) extractionB (s683577b42) reviewq (76h6bfydm6) review

• acyl-lysine ptms

  post-translational modification, modification

  Lysine side-chain acyl post-translational modifications discussed in the paper, including acetylation, 2-hydroxyisobutyrylation, and ubiquitylation.

  Aliases: acyl-Lys modifications, acyl-Lys PTMs

  박진우 (dztg5apj7m) extractionB (s683577b42) reviewq (76h6bfydm6) review
• cysteine alkylation-based method

  method

  A chemical installation strategy that uses cysteine alkylation to place lysine acylation mimics at defined sites on proteins.

  Aliases: alkylation-based method

  박진우 (dztg5apj7m) extractionB (s683577b42) reviewq (76h6bfydm6) review
• deubiquitinating enzymes

  enzyme, protein family

  Enzymes that remove ubiquitin from protein substrates and are used here to test cleavage of ubiquityl-Lys mimics.

  Aliases: DUBs

  박진우 (dztg5apj7m) extractionB (s683577b42) reviewq (76h6bfydm6) review
• diubiquitin analog

  molecule, substrate

  A synthetic two-ubiquitin mimic generated in the study as a substrate for deubiquitinating enzyme assays.

  Aliases: diubiquitin mimic

  박진우 (dztg5apj7m) extractionB (s683577b42) reviewq (76h6bfydm6) review
• histone h2b

  protein

  The histone H2B protein used as the substrate for installing a ubiquityl-Lys mimic.

  Aliases: H2B

  박진우 (dztg5apj7m) extractionB (s683577b42) reviewq (76h6bfydm6) review
• histone h3

  protein

  The histone H3 protein used as a substrate for installing acyl-Lys mimics and reconstituting modified nucleosomes.

  Aliases: H3

  박진우 (dztg5apj7m) extractionB (s683577b42) reviewq (76h6bfydm6) review
• hydrazide mimics

  chemical mimic, molecule

  Hydrazide-containing analogs used here as site-specific substitutes for lysine acyl post-translational modifications.

  Aliases: hydrazide analogs

  박진우 (dztg5apj7m) extractionB (s683577b42) reviewq (76h6bfydm6) review
• nucleosomes

  complex, chromatin particle

  Histone-DNA complexes assembled in the study to probe how modified histones affect chromatin-related properties.

  박진우 (dztg5apj7m) extractionB (s683577b42) reviewq (76h6bfydm6) review
• saga deubiquitinating module

  protein complex, module

  The SAGA-associated protein module that recognizes ubiquitinated histone substrates and mediates deubiquitination.

  Aliases: SAGA DUB module

  박진우 (dztg5apj7m) extractionB (s683577b42) reviewq (76h6bfydm6) review

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