Identification Of Interacting Proteins For A Specific Peptide Binding With Liver Cells From Mice With Septic Shock

With the characteristics of high prevalence, high mortality and high cost of treatment, sepsis has got a great attention by basic medical and clinical researchers, and has become research focus at the field of life science research. Epidemiological analysis showed about 25-30% of sepsis are caused by gram-negative bacterial infections. After released into the blood circulation, LPS bind with LPS binding protein (LBP) to form a complex, which is transported to cluster of differentiation 14 (CD 14) on the surface of monocytes, macrophages, neutrophils and vascular endothelial cell. Lacking the cytoplasmic domain, CD 14 can not transfer the LPS signal into cells, so it needs the help of Toll-like receptor 4 (TLR4) to transfer the LPS signal. With the help of MD-2, TLR4 activates myeloid differentiation protein 88 (MyD88) by activating the protein tyrosine kinase. MyD88 then activates nuclear factor-KB (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways. As a result, protein-1 (AP-1) and NF-κB are activated. After translocation from cytoplasm to the nucleus, AP-1 and NF-κB starts the transcription and expression of targeting genes. In this way, cells produce many species of cytokines such as tumor tumor necrosis factor--α(TNF--a), interferon--γ(IFN--γ), interleukin-1 (IL-1), IL-6, IL-8 and so on. Those inflammatory factors activate the second inflammatory cascade and the acquired immune system. It means that monocytes, macrophages, neutrophils and vascular endothelial cells release more cytokines and inflammatory factors, causing SIRS, sepsis, severe sepsis, septic shock (septic shock) and refractory septic shock subsequently.The liver is an easily damaged organ in sepsis. Comparing with normal liver cells, Kupffer cells and hepatic vascular endothelial cells in sepsis change their function and structure. Membrane molecules increase their expression or exposure their active sites. Those differential expressed molecules are closely related to the development of sepsis, they may be targets for septic diagnosis and treatment. We screen the specific peptides binding to those differential molecules on membrane surface. They are significant for searching antagonist for inflammatory factors, for drawing a map about specific peptides binding to liver cells in endotoxic shock, for studying the mechanisms of inflammatory signal transduction mediated by LPS, and for exploring strategies about treatment of endotoxic shock.In 1985, Smith invented the phage display technology (phage display). He inserted gene coding EcoR I endonuclease into gene coding phage capsid protein pⅢ. In this way, a fusing protein was constructed containing EcoR I endonuclease and phage capsid protein pⅢ. This fusing protein can be effectively identified by antibodies specific for EcoR I endonuclease, it means the fusing protein can maintain their natural conformation and activity. The basic principle for constructing a phage display peptide library is inserting genes coding exogenous proteins into phage genes coding capsid proteins. As a result, exogenous proteins can be fused with phage capsid protein. The principle of screening phage peptide library is based on the principle of protein-protein interaction (PPI). Protein-protein interactions depend on three-dimensional structure of proteins. Protein motifs are the key structure for protein-protein interactions. A lot of biology experiment showed the smallest amino acid protein motif only suffers three amino acid residues. But it can still simulate some of the key structural of proteins. Since the phage peptides containing the key amino acid residues, they can simulate protein-protein interactions.In 1996, Pasqualini and Ruoslahti applied phage display technology in vivo for the first time. They screened peptides specific binding to brain and kidney in normal mice. Since then, they studied tumor targeted therapy by using this technology. According to their research, peptides obtained by in vivo phage display had many important biological functions. Their research also showed molecules on the surface of vascular endothelial cells in different organs are heterogeneous. So they proposed the concept of vascular proteomics. Applying in vivo phage display one can obtain a specific binding peptides library for certain organ or tissue. This technology includes attaching, elution and amplification repeating 3 to 5 rounds. As a simple, efficient, quick technology, it has been widely applied to study of cancer, chronic inflammatory disease. And researchers have got many remarkable achievements.In view of the critical position of liver cells in the development of endotoxin shock, and the efficiency of in vivo phage display, in our preliminary studies, we have screened a specific binding peptide library for the live cells of mice with endotoxic shock by using in vivo phage display. After bioinformatics analysis, we drew a map for the live cells of mice with endotoxic shock. In this study, we selected the highest abundant short peptide LTTWAPA from the library mentioned above. After bioinformatics analysis and target validating, we searched its interacting proteins. the aim of bioinformatics analysis is predict which transmembrane and secreted proteins it may simulate. The basic process of analysis:First, by using the U.S. National Center for Biotechnology Information (center for biotechnology information, NCBI) online BLAST, we searched top 100 most similarity proteins according their amino acid sequence similarity, then, by using the Danish University Centre for Biological Sequence Analysis signal peptide developed prediction software SignalP and cut point prediction software TMHMM, we predicted secreted proteins and transmembrane proteins, finally, by combining with the Swiss Institute of Bioinformatics Expert Protein Analysis System (expert protein analysis system, EXPASY), NCBI, and the literatures about those secreted proteins, we found proteins having functions in endotoxic shock. The purpose of targeting validation is to verify LTTWAPA specific binding to liver in mice with endotoxic shock. We achieved this goal by using phage transfusion count and competitive inhibition experiments. The final objective is to find and identify its interacting proteins. After discovering its interacting proteins by using pull down technology, we applied SDS-PAGE and 2D-DIGE to separated those interacting proteins, finally we identified them by mass spectrometry after separationAccording to the result of our research, we can draw the following conclusions: Firstly, the secreted proteins simulated by LTTWAPA are bactericidal permeability-increasing protein (BPI), fibronectin (FN), complement C5, granulocyte colony-stimulating factor receptor (G-CSF-R), the membrane protein simulated by LTTWAPA is protein spinster homolog 1(Spns1). They are all related to the onset of endotoxic shock; secondly, LTTWAPA specifically binds to the shock mouse liver; thirdly, Migration inhibitory factor-related protein 14 (Mrp14) may interact with LTTWAPA, so the identification of Mrp14 may play an important role in diagnosis and treatment of endotoxin shock.