Large-scale lysine crotonylation analysis reveals the role of TRAF6-Ecsit complex in endoplasmic reticulum stress in mud crab (Scylla paramamosain).

Lysine crotonylation (Kcr) is a newly discovered type of post-translational modification. Although Kcr has been reported in several species, its role in crustaceans remains largely unknown. In this study, Kcr in hemocytes of mud crab (Scylla paramamosain) was characterized using pan anti-crotonyllysine antibody enrichment and high-resolution liquid chromatogram-mass spectrometry analysis after SpTRAF6 or SpEcsit silencing. Altogether, 1,800 Kcr sites with six conserved motifs were identified from 512 proteins. Subcellular localization analysis showed that the identified Kcr proteins were mainly localized to the cytoplasm, nucleus, and mitochondria. The cellular components analysis showed that the 'chromosomal region' was enriched in the hemocytes of SpTRAF6-or SpEcsit-silenced mud crabs. The KEGG and PPI analyses showed that the identified Kcr proteins in the hemocytes SpTRAF6-or SpEcsit-silenced mud crabs were related to the 'protein processing in endoplasmic reticulum'; of which the marker of endoplasmic reticulum stress (Bip) was identified to be crotonylated. These datasets present the first comprehensive analysis of the crotonylome in mud crab hemocytes, providing invaluable insights into the regulatory functions of SpTRAF6 and SpEcsit in Kcr. Additionally, our findings shed light on the potential role of these proteins in activating marker proteins during endoplasmic reticulum stress in invertebrates.