Cracking Lysine Crotonylation (Kcr): Enlightening a Promising Post-Translational Modification

Abstract

Lysine crotonylation (Kcr) is a recently discovered post-translational modification (PTM). Both histone and non-histone Kcr-proteins have been associated with numerous diseases including cancer, acute kidney injury, HIV latency, and cardiovascular disease. Histone Kcr enhances gene expression to a larger extend than the extensively studied lysine acetylation (Kac), suggesting Kcr as a novel potential therapeutic target. Although numerous scientific reports on crotonylation were published in the last years, relevant knowledge gaps concerning this PTM and its regulation still remain. To date, only few selective Kcr-interacting proteins have been identified and selective methods for the enrichment of Kcr-proteins in chemical proteomics analysis are still lacking. The development of new techniques to study this underexplored PTM could then clarify its function in health and disease and hopefully accelerate the development of new therapeutics for Kcr-related disease. Herein we briefly review what is known about the regulation mechanisms of Kcr and the current methods used to identify Kcr-proteins and their interacting partners. This report aims to highlight the significant potential of Kcr as a therapeutic target and to identify the existing scientific gaps that new research must address.

Keywords: Chemical proteomics; Crotonyl-CoA; Enzymatic PTMs regulation; Post-translational modifications (PTMs).

Figure 1

Short lysine acylations and their discovery year: acetylation, formylation,[ ¹¹ , ¹² ] propionylation, butyrylation, crotonylation, malonylation, succinylation, glutarylation, 2‐hydroxyisobutyrylation, β‐hydroxybutyrylation, benzoylation, lactylation, methacrylation.

Figure 2

The function of Kcr in disease; increased levels of Kcr are indicated with an upwards arrow and a downwards arrow indicates downregulation of Kcr in the respective disease. The involvement of Kcr in the diseases reported in the dashed squares is only hypothesised, not yet confirmed.

Figure 3

Overview of known writers, readers, and erasers for Kcr. The only selective enzymes identified for Kcr are readers containing a YEATS domain, raising the question whether there are also selective writers and erasers for this PTM.

Figure 4

A). Crystal structure of H3K9cr in the Taf14 binding pocket, with K9cr indicated as yellow stick, showing triple π‐stacking, with the crotonyl group being sandwiched between W81 and F62; B). Composition of the K14cr‐binding β2 pocket of MOZ, with K14cr shown as yellow stick with its extended two‐hydrocarbon group highlighted in green. Images adapted from corresponding publications (PDB ID: 5IOK).

Figure 5

Possible mechanism of crotonylation. Intestinal microbiome produces the short‐chain fatty acids crotonate and butyrate through dietary fermentation. Crotonic acid enters the cytoplasm through diffusion or MCT‐1 vectors, while crotonate enters through SMCT‐1 vectors. Butyrate enters through both SMCT‐1 and MCT‐1. They are both converted into crotonyl‐CoA through ACSS2. Butyryl‐CoA, glutaryl‐CoA, and (S)−3‐hydroxybutyryl‐CoA can all be converted into crotonyl‐CoA. Enzymes in blue are the ones promoting crotonyl‐CoA production, and in red are the enzymes promoting hydrolysis of crotonyl‐CoA. Sodium crotonate and disodium crotonate are analogues of crotonic acid commonly used in experiments to upregulate crotonylation. After conversion into crotonyl‐CoA, both non‐histone proteins in the cytoplasm and histone proteins in the nucleus can be crotonylated.

Figure 6

Schematic representation of the non‐selective enrichment using the anti‐Kcr antibody compared to chemical probe enrichment.

Figure 7

A). Covalent capture of crotonylated histones by the biotinylated TCEP probe, the first chemical probe for Kcr; B). Polycrotonylated (H4pKcr), monocrotonylated (H3K18cr), polyacetylated (H4pAc), and unmodified (WT) nucleosomes were incubated with the TCEP probe and analysed by western‐blot. The same nucleosomes were analysed by western blotting employing the pan‐anti Kcr and pan‐anti Kac antibody; C). Western blot analysis showing Kcr levels of either acid extracted histones (left panel) or nuclear extract (right panel) from HEK93 cells, either grown in the presence or absence of sodium crotonate. Adapted from.

Figure 8

A). Covalent capture of Kcr‐interacting proteins via proximity‐induced cross‐linking; B). Schematic representation of the reversible formation of O‐alkylamidate and its effect on cross‐linking and decrotonylation.

Figure 9

A). Photo‐cross‐linking strategy towards the detection of Kcr‐interacting targets. B). Ester‐derived crotonyl‐mimic probe for capturing of H3K27cr interacting proteins. C). and D). Mechanistic approach for the identification of decrotonylases using fluorescent Kcr‐peptide probes.