Emerging roles of non-histone protein crotonylation in biomedicine

Abstract

Crotonylation of proteins is a newly found type of post-translational modifications (PTMs) which occurs leadingly on the lysine residue, namely, lysine crotonylation (Kcr). Kcr is conserved and is regulated by a series of enzymes and co-enzymes including lysine crotonyltransferase (writer), lysine decrotonylase (eraser), certain YEATS proteins (reader), and crotonyl-coenzyme A (donor). Histone Kcr has been substantially studied since 2011, but the Kcr of non-histone proteins is just an emerging field since its finding in 2017. Recent advances in the identification and quantification of non-histone protein Kcr by mass spectrometry have increased our understanding of Kcr. In this review, we summarized the main proteomic characteristics of non-histone protein Kcr and discussed its biological functions, including gene transcription, DNA damage response, enzymes regulation, metabolic pathways, cell cycle, and localization of heterochromatin in cells. We further proposed the performance of non-histone protein Kcr in diseases and the prospect of Kcr manipulators as potential therapeutic candidates in the diseases.

Keywords: Cell biology; Disease; Lysine crotonylation; Post-translational modification; Protein.

Fig. 1

The metabolic pathways and enzymes and modulators for lysine crotonylation (Kcr). Colon microbiota is a source of short chain fatty acids (SCFAs) which may be metabolized to acetyl/crotonyl/propionyl/butyryl-CoA in host cells. These CoAs are precursors used by enzymes to promote lysine acetylation (Kac), crotonylation (Kcr), propionylation, and butyrylation of histone and non-histone proteins. Currently characterized crotonyltransferases (KCTs) (writer) include CBP/P300, MYST, and GNAT family, and histone decrotonylases (KDCRs) (eraser) include HDAC I and HDAC III. When substrates are histone proteins, Kcr readers, including bromodomains, YEATS, and DPF domain, recognize the Kcr. Chromodomain Y-like (CDYL), acting as a crotonyl-CoA hydratase which converts crotonyl-CoA to β-hydroxybutyryl-CoA, negatively regulates crotonylation. ACCS2, acyl-CoA synthetase short chain family member 2. Inhibitors of P300/CBP include C646 and SGC-CBP30. Inhibitors of HDACs include trichostatin A (TSA), nicotinamide (NAM), suberoylanilide hydroxamic acid (SAHA), and LBH589

Fig. 2

Schematic diagram of the experimental procedures for mass spectrometry-based global analysis. Proteins are extracted from cells or tissues and digested into peptides with a protease such as trypsin. The tryptic peptides are then separated and fractionated by high pH reverse-phase high performance liquid chromatography (HPLC). Proteolysis of whole-cell protein extracts generates numerous peptides, but only a small fraction is crotonylated. To enrich lysine crotonylated peptides, pan-Kcr antibodies are applied to identify the crotonylated peptides in complex peptide mixtures using immunoaffinity purification. The resulting peptides are ionized in the electrospray source before entering the mass spectrometer. MS and MS/MS spectra are then computationally processed to deduce peptide sequences, including the presence and location of crotonylation, and to quantify the abundance of crotonylated peptides and proteins

Fig. 3

The role of RPA1 Kcr in camptothecin (CPT)-induced double-strand break (DSB) of DNA. RPA1 takes an important role in DNA repair. The Kcr levels at K88, K379, and K595 sites of RPA1 are upregulated by DNA damage but downregulated by CDYL. Sites K379 and K595 locate at the DNA binding domain of RPA1 and may be more important than site K88 for the ssDNA binding ability of RPA1. Crotonylation of RPA1 promotes the binding of RPA1 to ssDNA, recruits RPA1 to the site of DNA damage, enhances the interaction of RPA1 with homologous recombination (HR) factors (including BLM, DNA2L, Mre11, NBS1, and RAD51), and promotes the formation of RAD51 lesions after CPT injury. As a result, the Kcr of RPA1 may make the cells survive and resistant to apoptosis after DNA damage (referenced to: Yu H et al. (2020) Sci Adv 6:eaay4697)