Proteomics Study On Lipid Rafts Of Human Hepatocytes Reveals New HPS Mechanism

HPS is a secreted protein,mainly expressed in the liver,and a small amount is expressed in the pancreas.Early laboratory studies have shown that there are HPS receptors on hepatocytes.Using proteomics technology,combined with the results of bioinformatics analysis of the identified proteins,It was found that several molecules closely related to HPS activity(such as EGFR,Src,CAV1,etc.)are located in the lipid raft region of the cell membrane.Lipid raft refers to some membrane microregions rich in glycosphingolipids and cholesterol on the cell membrane.It has unique fluidity.Under the stimulation of extracellular signals,it can gather proteins that are far away from the cell membrane,such as receptors.Tyrosine kinases,G protein-coupled receptors,phosphatases,and other important molecules involved in Cell Signal promote the interaction between them and transmit signals downstream.We speculate that HPS may regulate the life activities of cells through changes in protein interactions in lipid rafts.This study explored the proteomic changes in the lipid raft region of human normal liver cell line WRL-68 cells stimulated by HPS,and revealed new downstream pathways of HPS through in vitro experiments.We first established a simpler method of protein extraction in the lipid raft region,and extracted the protein in the lipid raft region based on this method,and studied the effect of HPS stimulation on the caveolae protein CAV1,a lipid raft marker molecule in WRL-68 cells.In order to screen the protein interacting with HPS,we used p-Tyr antibody and chemical cross-linking agent to carry out immunoprecipitation experiments,after the immune mixture was subjected to SDS-PAGE and Coomassie brilliant blue staining,the differential protein bands were selected for mass spectrometry analysis and identification Using mass spectrometrybased proteomics technology,hundreds of HPS interacting proteins were discovered,and a preliminary study was conducted on the regulation of STAT3 pathway by HPS.We used HPS(100 ng/m L)to continuously stimulate WRL-68 at different times(0,5,15,30,60 min).Western Blot and indirect immunofluorescence were used to detect STAT3 total protein expression,phosphorylation activation,and whether nuclear transfer occurred.,EGFR and Src mediate phosphorylation activation of STAT3.The results showed that under the stimulation of HPS,there was no significant change in the total protein level of STAT3,and the phosphorylation level increased in a timedependent manner.Combined with Western Blot and indirect immunofluorescence results,HPS stimulation can significantly activate the STAT3 pathway,promote STAT3 entry into the nucleus,and up-regulate the expression of its downstream target genes MYD88,PSMB8,TRIM21 and ERAP1.After inhibiting EGFR and Src,the phosphorylation level of STAT3 was significantly reduced,indicating that HPS activation of STAT3 depends on EGFR and Src.Blocking the STAT3 pathway significantly inhibited the hepatocyte proliferation function of HPS.The PPI network analysis of the proteins detected by the two mass spectrometers also provides new clues for the study of the function and mechanism of HPS,which enriches the understanding of the mechanism of HPS.