Pathways Important Molecular .pi3k/akt Interaction Screening And A Preliminary Study

Liver, as the major "biochemistry factory" within the body, plays a key role in kinds of physiological processes. Elucidation of liver protein-protein interaction patterns will provide an important basic data set in the functional analysis of liver proteome, providing insights into individual protein functions, pathways, molecular machines, functional protein modules and evolution.Growth factor signaling is of central importance during liver development, injury, regeneration as well as functional regulation. HGF, EGF, insulin, TGFs activate downstream pathways, which include MAPK, PDK/Akt, SMAD, and regulate various processes such as cell proliferation, survival, migration in the liver. Growth factor signaling provides a perfect starting point for and is an important part of the research of CNHLPP protein-protein interaction network.The serine/threonine kinase Akt regulates multiple biological processes including cell survival, proliferation, growth, apoptosis and glycogen metabolism. In this project, we performed a primary research on PI3K/Akt pathway related protein network in human by modular-scale yeast two-hybrid library screening, for further explanation and annotation of mechanisms of liver metabolism and regeneration on protein-protein interaction level.Twenty eight baits were screened against human liver cDNA library, including the critical nodes, kinases and negative regulators. 488 candidate colonies were isolated initially and their AD-Y identities were determined with interaction sequence tags (ISTs) obtained by sequencing the corresponding polymerase chain reaction (PCR) products. The AD-Y reading frame was verified for each IST to avoid the recovery of out-of-frame peptides. In total, 150 ISTs were obtained, forming 101 different protein interaction pairs involved 93 proteins. Positives were subsequently retested in fresh yeast cells, 44 interaction pairs out of 58 were recovered.To estimate the coverage of the Y2H data sets, we manually searched the baits screened here for known interactors in PubMed and HPRD. This search gave rise to 8 interactions. Our overall rate of coverage for the data set is 8.6% (8/93). To evaluate the accuracy of the Y2H data sets, a representative sample of Y2H interaction pairs was randomly selected, and tested in a Co-immunoprecipitation (Co-IP) assay. Bait and prey ORFs were transiently transfected into 293T cells as FLAG-bait and Myc-prey fusions, respectively. For potential interaction pairs where both proteins were expressed at detectable levels, the Co-IP success rates were 10 out of 15 (67%).In addition to experimental screens, we also performed in silico analysis to estimate the fidelity of the Y2H data sets and presented the interactions in visible network graphs with Osprey network visualization system, where there was a clustering module related to cell stress. By integrating the experimental and various confidence evaluation information, as well as literature research, we performed a comprehensive analysis of the biological relevance of some interactions.We confirmed the interaction between a ubiquitin ligase SIAH1 and a scaffold protein TRB3 and found that SIAH1 bound to and targeted TRB3 for proteasome-dependent degradation. Cotransfection of SIAH1 could rescue down-regulation of phosphorylated Akt by TRB3 upon insulin stimulating, which suggested that prevention of SIAH1 induced degradation of TRB3 represents a potential therapeutic target to type 2 diabetes.In conclusion, this dissertation presents a primary network for human liver proteins related to PI3K/Akt pathway by Y2H library screening, which will help to understand the complicated regulation of liver functions and the physiological and pathological processes with various liver disorders.