关键词: ERS, 肝细胞, GRP94, 互作蛋白, 猪

Abstract:

【Background】 Extensive stress reactions often occur in pigs due to poor breeding environment, thick subcutaneous fat and lack of sweat glands in pigs. Endoplasmic reticulum (ER) stress (ERS) may be the earliest stress response, which is closely related to apoptosis and metabolism. Liver is the main metabolic organ of the body. In the process of pig breeding, the artificial operations, such as weaning, feed mildew, and inhalation of harmful gases and temperature changes, will cause ERS in pig liver, which will not only cause liver damage, but also cause liver fat metabolism disorder and extensive inflammatory reaction, affecting animal production performance and reproductive performance. The regulation of ERS in production is helpful to reduce the recessive loss in the pig breeding process. 【Objective】 Immunoprecipitation combined with mass spectrometry was used to screen the cellular proteins interacting with glucose-regulated protein 94 (GRP94) in porcine hepatic stellate cells (HSC) under ERS condition, which could lay a foundation for further study on the protective mechanism of GRP94 on biological function of HSC. 【Method】 Protein complexes bound to GRP94 bait protein were eluted, collected, and verified by SDS-PAGE gel electrophoresis. The successfully verified sample eluent was detected by liquid chromatography-tandem mass spectrometry (LC-MS/MS), and the interaction proteins of GRP94 under normal condition and ERS condition were identified. The detected proteins were analyzed by GO, KEGG and interaction network. Vimentin, one of the interacting proteins, was verified by co-immunoprecipitation. 【Result】 In porcine hepatic stellate cells, 146 proteins were interacting with GRP94 under normal condition, 76 proteins under ERS condition, and 44 proteins under both conditions. The results showed that 76 proteins interacting with GRP94 under ERS were mainly involved in the negative regulation of apoptosis process, peptide cross-linking, ubiquitin-dependent ERAD (endoplasmic reticulum associated degradation) pathway and hydrogen peroxide catabolic process, among them, the specific proteins interacting with GRP94 in the negative regulation of apoptosis process were albumin, catalase, filament A, heat shock protein family A member 5, keratin 18, and prohibin 2, indicating that GRP94 might play an anti-apoptotic role with these proteins. Besides, the vimentin protein that made up the intermediate filament was involved in multiple GO enrichment terms, which might have an important interaction with GRP94, which was further confirmed by co-immunoprecipitation test, further demonstrating that there was indeed an interactive relationship between this two. Further analysis showed that some GRP94 interacting proteins (Such as peroxiredoxin, death inducer obliterator 1, catalase, glandular kallikrein, pyruvate kinase and so on) specifically expressed under ERS were closely related to anti-apoptosis. 【Conclusion】 Under ERS, GRP94 interacting proteins were mainly involved in anti-apoptosis, folding of unfolded proteins and maintenance of intracellular homeostasis-related signal pathways. This conclusion laid a foundation for further study on the mechanism of GRP94 involved in liver ERS regulation.

Key words: endoplasmic reticulum stress, liver cells, GRP94, protein interactions, pig