Protein-Protein Cross-Linking by a DNA Damage-Derived Histone Modification

Area of Science:

• Biochemistry
• Molecular Biology
• Epigenetics

Background:

• Nonenzymatic covalent modification (NECM) of lysine residues, particularly the 5-methylene pyrrolone NECM (KMP) formed from oxidized abasic sites (C4-AP), can have significant physiological consequences.
• KMP formation on histones within nucleosome core particles (NCPs) is linked to DNA damage and has been observed in human cells.

Purpose of the Study:

• To investigate the formation and consequences of KMP on histones within NCPs using a synthetic molecule.
• To identify proteins that form cross-links with KMP-modified histones and understand the functional implications.

Main Methods:

• Synthesis of a molecule (1a) that directly induces KMP formation on histone lysines in NCPs.
• Incubation of KMP-containing NCPs with nuclear lysates, followed by enrichment and identification of protein-protein cross-links (PPCs) using LC-MS/MS.
• Analysis of PPCs formed by KMP generated directly on DNA via C4-AP.

Main Results:

• KMP modification occurs on all four core histones, with a preference for H3.
• NCP-protein and protein-protein cross-links were identified, indicating KMP's reactivity within the NCP environment.
• Enriched proteins were functionally biased towards DNA binding, histone binding, histone modifications, and transcription regulation.
• KMP-induced PPC formation is influenced by the NCP context and the location of C4-AP.

Conclusions:

• The synthetic molecule effectively generates KMP on histones within NCPs, enabling the study of its cross-linking potential.
• KMP-mediated protein-protein cross-linking within NCPs is specific and functionally relevant.
• Further investigation into the in vivo impact of KMP-induced cross-linking on cellular processes is warranted.