Recognition of stapled histone H3K4me3 peptides by epigenetic reader proteins.

The flexible N-terminal histone tails are a subject of numerous posttranslational modifications, including methylation. We report development of stapled histone peptides bearing trimethyllysine as ligands for epigenetic reader proteins. Stronger or weaker binding affinities have been observed for stapled histone peptides relative to linear histones, indicating that selectivity towards reader proteins can be achieved.

Probing lysine posttranslational modifications by unnatural amino acids.

Posttranslational modifications, typically small chemical tags attached on amino acids following protein biosynthesis, have a profound effect on protein structure and function. Numerous chemically and structurally diverse posttranslational modifications, including methylation, acetylation, hydroxylation, and ubiquitination, have been identified and characterised on lysine residues in proteins. In this feature article, we focus on chemical tools that rely on the site-specific incorporation of unnatural amino acids into peptides and proteins to probe posttranslational modifications of lysine. We highlight that simple amino acid mimics enable detailed mechanistic and functional assignment of enzymes that install and remove such modifications, and proteins that specifically recognise lysine posttranslational modifications.

Importance of Ile71 in ß-actin on histidine methyltransferase SETD3 catalysis.

SETD3-catalysed N3-methylation of His73 in ß-actin plays a key role in stabilisation of actin filaments in the metazoan cells. Overexpression and/or dysregulation of SETD3 is associated with several human pathologies, including cancer. Here, we examined the role of the Ile71 residue in ß-actin on human SETD3 catalysis. Substitution of Ile71 in ß-actin peptides by its natural and unnatural mimics reveals that the 'secondary' Ile71 binding pocket modulates the substrate efficiency of ß-actin. Our enzymatic work demonstrates that human SETD3 can accommodate structurally diverse hydrophobic side chains in its Ile71 binding pocket, providing clear limits of the size and shape of Ile analogues. Water thermodynamics calculations reveal that the Ile71 pocket is occupied by high-energy water molecules, that are released upon the Ile71 binding, contributing favourably to the SETD3-ßA complex formation. The work highlights that the hydrophobic Ile71 binding site plays an essential role in SETD3 catalysis, contributing to an ongoing effort in the design and development of chemical probes targeting SETD3.

Amide-derived lysine analogues as substrates and inhibitors of histone lysine methyltransferases and acetyltransferases.

Histone lysine methyltransferases and acetyltransferases are two classes of epigenetic enzymes that play pivotal roles in human gene regulation. Although they both recognise and posttranslationally modify lysine residues in histone proteins, their difference in histone peptide-based substrates and inhibitors remains to be firmly established. Here, we have synthesised lysine mimics that posses an amide bond linker in the side chain, incorporated them into histone H3 tail peptides, and examined synthetic histone peptides as substrates and inhibitors for human lysine methyltransferases and acetyltransferases. This work demonstrates that histone lysine methyltransferases G9a and GLP do catalyse methylation of the most similar lysine mimic, whereas they typically do not tolerate more sterically demanding side chains. In contrast, histone lysine acetyltransferases GCN5 and PCAF do not catalyse acetylation of the same panel of lysine analogues. Our results also identify potent H3-based inhibitors of GLP methyltransferase, providing a basis for development of peptidomimetics for targeting KMT enzymes.

Targeting SARS-CoV-2 spike protein by stapled hACE2 peptides.

SARS-CoV-2 Spike protein RBD interacts with the hACE2 receptor to initiate cell entry and infection. We set out to develop lactam-based i,i + 4 stapled hACE2 peptides targeting SARS-CoV-2. In vitro screening demonstrates the inhibition of the Spike protein RBD-hACE2 complex formation by the hACE221-55A36K-F40E stapled peptide (IC50: 3.6 µM, Kd: 2.1 µM), suggesting that hACE2 peptidomimetics could form the basis for the development of anti-COVID-19 therapeutics.

Trimethyllysine: From Carnitine Biosynthesis to Epigenetics.

Trimethyllysine is an important post-translationally modified amino acid with functions in the carnitine biosynthesis and regulation of key epigenetic processes. Protein lysine methyltransferases and demethylases dynamically control protein lysine methylation, with each state of methylation changing the biophysical properties of lysine and the subsequent effect on protein function, in particular histone proteins and their central role in epigenetics. Epigenetic reader domain proteins can distinguish between different lysine methylation states and initiate downstream cellular processes upon recognition. Dysregulation of protein methylation is linked to various diseases, including cancer, inflammation, and genetic disorders. In this review, we cover biomolecular studies on the role of trimethyllysine in carnitine biosynthesis, different enzymatic reactions involved in the synthesis and removal of trimethyllysine, trimethyllysine recognition by reader proteins, and the role of trimethyllysine on the nucleosome assembly.