The Establishment Of High-throughput Yeast Two-hybrid Platform And Its Applications In Protein-protein Interaction Among The Group Of School

Functional proteins are connected in a dynamic and highly organized network in living cells. And it remains a problem how to parse the complex network and find more meaningful interactions that are related with human diseases. The results are valuable for drug discovery in pharmaceutical industry. Classic methods, such as the yeast two hybrid, are laborious and time-consuming, and it is a mission impossible for researchers finally build up a reliable and integrated protein interaction network even in human cells. Those methods do not meet the needs of proteomics any more. Thus, we have developed the high through-put yeast two hybrid system aiming to build up protein interaction networks in various species including human. It can liberate scientists from repeated operations and help them in exploring the more deep mechanism inside living cells. On setting up this platform, we have built up a interaction network of human proteins and further we focused on human protein MSK1 and we screened its preys and one of them is MRKβ. MRKβis a member of MAPKKK kinases. It has an N terminal catalytic domain which is responsible for the phosphorylation of its substrates. MSK1 is also an important kinase in the MAPK pathway and its activation depends on the phosphorylation of serines and threonines by ERK1/2 or p38. Compared with its relative RSK protein families, MSK1 should also be activated by some certain kinase besides ERK1/2 or p38. However, no such kinases have been found up to now. Our results show that MRKβcould interact with and then activate MSK1 through phosphorylation. Thus, downstream transcription factor CREB can be activated by the active MSK1 at Ser 133. This results in the gene expression in the nucleus responding to the extra signal. We have also use mass spectrum method to detect the phosphorylation sites in MSK1 by MRKβ. MRKβcan possibly phosphorylate MSK1 at Ser 436, Ser522 and Thr523. More research is needed to find out the in-depth mechanism and its physiological meaning.We have also identified MRKβcan also interact with YWHAZ which is a member of 14-3-3 protein family. GST pull-down and Co-IP resuts show that they interact with each other. The co-localization of these two proteins may imply an interaction-induced trans-localization of MRKβ. More details are waiting to be discovered.We also found human CtBP2 interacts with CCNH in Hela cell nucleus.The high through-put yeast two hybrid platform has been applied into the research of interactomics successfully and at the same time we have also found problems in it. Modification and optimization are needed.Bioinformatics is now in its blossom. Computational methods are now applied in many fields of biological research. Protein informatics is one of them. Identification of novel domains and the verification of their functions are focus of the third part in this thesis. We have developed a free of charge web server KemaDom to identify domains in proteins and we hope this could be of help to experimental scientists in labs. On the basis, we have identified a C₄ zinc ribbon domain in the C terminus of the phiC31 integrase which is successfully applied in the fundamental research of human gene therapy. We have also used motif finding methods to identify the recognition motif of the phiC31 integrase and thus we calculate the distribution of the motif in the human genome which help us in a conclusion that this system is fairly safe while some hidden risks are still inside the genome. SerRD is a database we have developed to collect information about the serine integrase and we hope this free database could help researchers in the study of this protein family.Protein interactomics is a crucial component of the proteomics. By constructing different protein-protein interaction map, researchers could discover those meaningful protein interactions. High through-put yeast two hybrid system inherits from the pervious normal yeast two hybrid system and it has already been applied in the large-scale identification of protein interactions as a main technique platform. It offers a great number of reliable data for researchers to take a comprehensive analysis in the protein function and evolution from the view of system biology. At the same time, the platform also gives reasonable clues to researchers in the medical research and drug targets discovery. Opportunities for both computational scientists and lab researchers are now on the table. On the one hand, we produce high through-put data by using automated equipments and then make detailed verifications in molecular methods; on the other hand, we use computational methods on the accumulated data to find those statistical results which can not be easily be discovered by conventional lab methods. This is a new era in the life science, and we need to be more ready to think computationally and do corporately.