Chemical Approaches for Understanding Lysine Posttranslational Modifications by Epigenetic Proteins

Research output: ThesisPh.D. thesis

Abstract

Proteins can undergo several posttranslational modifications following their biosynthesis, which define their structure and function in the cell. These posttranslational modifications are involved in the regulation of cellular processes mostly related to gene expression. The dynamics behind such PTMs are carried out by enzymes that are responsible for the installation or removal of modifications, while binding domains recognize modifications on proteins. The focus in this thesis is to gain molecular insights into lysine-modifying enzymes and lysine-binding proteins and their target modifications, including hydroxylation, acylation and methylation. 

Chapter 1 presents an overview of the addressed lysine PTMs and related proteins in this thesis and Chapter 2 presents a summary of the research chapters. Chapter 3 of the thesis explores the substrate scope of JMJD7-catalyzed hydroxylation of DRG1-Lys22 peptide and report on the inhibition of the enzyme by the incorporation of unnatural lysine analogs into the peptide. The results inform on the high selectivity of JMJD7 for Lys, identify thialysine as an excellent mimic and reveal the inhibitory potential of Cys/Sec-containing peptide analogs, which is suggested to be mediated by the cross-linking with a Cys residue at the active site of JMJD7. Chapters 4 and 5 investigate the biomolecular recognition of crotonyllysine mimics on histone peptides by the AF9 YEATS domain. Different acyl modifications introduced via on-resin reactions at the H3K9 site were evaluated for their binding to the reader domain. All analogs were found to bind with lower affinity than the crotonyl modification, except being the bulky cinnamoyl group that was comparably binding. The naturally occurring regioisomeric methacryl modification displayed a significant decrease in binding, revealing the importance of CH-π/π-π interactions for binding crotonyllysine in the aromatic pocket of AF9 YEATS domain. Chapter 6 employs stapled H3K4 peptides to study the catalytic selectivity of the SETD7 methyltransferase. Enzymatic methylation reactions reveal that Ala1-Glu6 lactamstapled peptides outcompeted the linear H3K4 substrate. The substituted H3K4 stapled peptides were competitive inhibitors of SETD7. These results reveal that the stapled conformation of H3 peptide has a major contribution in modulating SETD7 activity. Chapter 7 employs H3K9 bis-thioether stapled peptides to study KMTD1 and KAT2B, revealing that KMT1D can catalyze the methylation of most of these peptides, whereas KAT2B displays high selectivity for the flexible H3K9 peptide by not accepting any of the stapled H3K9 peptides as substrates. Chapter 8 explores the catalytic activity of selected KATs that utilize dpAcCoA analog as cosubstrate in comparison with AcCoA in acetylation reactions of histone peptides, which reveals that KAT1 and KAT8 enzymes show similar selectivity, between these cosubsrates, and KAT3B demonstrates a significant selectivity for AcCoA. 
Original languageEnglish
Awarding Institution
  • University of Southern Denmark
Supervisors/Advisors
  • Mecinovic, Jasmin, Principal supervisor
Date of defence28. Nov 2024
Publisher
DOIs
Publication statusPublished - 20. Dec 2024

Note re. dissertation

A print copy of the thesis can be accessed at the library. 

Fingerprint

Dive into the research topics of 'Chemical Approaches for Understanding Lysine Posttranslational Modifications by Epigenetic Proteins'. Together they form a unique fingerprint.

Cite this