First profiling of lysine crotonylation of myofilament proteins and ribosomal proteins in zebrafish embryos

Abstract

Zebrafish embryos are translucent and develop rapidly in individual eggs ex utero; they are widely used as models for embryogenesis and organ development for human diseases and drug discovery. Lysine crotonylation (Kcr) is a type of histone post-translational modifications discovered in 2011. Kcr dynamics are involved in gene expression regulation and acute kidney injury; however, little is known about the effects of Kcr on non-histone proteins. In the present study, we conducted the first proteome-wide profiling of Kcr in zebrafish larvae and identified 557 Kcr sites on 218 proteins, representing the Kcr event in zebrafish. We identified two types of Kcr motifs containing hydrophobic (Leu, Ile, Val) and acidic (Asp and Glu) amino acids near the modified lysine residues. Our results show that both crotonylated proteins and sites of crotonylation were evolutionarily conserved between zebrafish embryos and humans. Specifically, Kcr on ribosomal proteins and myofilament proteins, including myosin, tropomyosin and troponin, were widely enriched. Interestingly, 55 lysine crotonylation sites on myosin were distributed throughout coiled coil regions. Therefore, Kcr may regulate muscle contraction and protein synthesis. Our results provide a foundation for future studies on the effects of lysine crotonylation on aging and heart failure.

Figure 1

Experimental workflow for global proteomic analysis of lysine-crotonylated proteins. (a) Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and crotonyl-lysine immunoblot. Lines represent 72 and 120 hpf, respectively. (b) Schematic representation of the sequential steps used for global profiling of lysine crotonylation in zebrafish larvae. (c) Overlap of crotonylated sites and proteins in immunoprecipitation experiments performed in triplicate.

Figure 2

Motif analysis of all identified sites. (a) Crotonylation sequence motifs. (b) Number of identified peptides containing crotonylated lysines in each motif.

Figure 3

Enrichment analysis of crotonylated protein in zebrafish larvae. (a) GO enrichment. (b) Domain enrichment. (c) KEGG pathway enrichment analysis.

Figure 4

Sequence alignment of crotonylated myosin at coiled coil regions between zebrafish and humans.