Structure Based Engineering Of A Novel Luciferase NanoLuc And The Functions Of Tyrosine Phosphatase SHP2 In HCT-116 Colon Cancer Cell Line

This thesis is divided into two parts: First,the structure studies and applications of the novel luciferase NanoLuc was investigated.Bioluminescence refers to the generation and emission of light through living organisms.It is a special form of chemiluminescence and is widely found in nature.In recent years,due to its sensitive light detection,high conversion efficiency,small toxic and side effects,and ease of use,it has attracted the attention of researchers.More and more research is devoted to the development of new bioluminescence systems and their application in genetic engineering and biomedical research.The main applications of bioluminescence include imagingin vivo,cell viability assays,and reporter gene assays.The bioluminescence plays very important roles in both medical and laboratory research.Currently,research on bioluminescent systems mainly focused on the development of highly stable luciferases.NanoLuc luciferase(NLuc)is a luciferase,which is extracted from deep sea shrimp Oplophorus gracilirostris and subjected to three mutagenesis.NanoLuc’s molecular weight is only 19.1 kDa and it is the smallest protein involved in light formation.And it does not rely on ATP,it produces fluorescence through oxidizing coelenterazine analog--furimazin,and the flurescence intensity is about 150 times more than firefly luciferase and Renilla luciferase.It is the latest luciferase that has been used in biological research.In this paper,we overexpressed NanoLuc protein tagged with C terminal His in E.coli expression system,and purified protein through Nickel column and gel filtration,then the purified protein was processed crystal growth,and collected data using Argonne Photon Synchrotron(APS,Chicago)LS-CAT.We obtained the crystal structure of NanoLuc full-length protein with 2.36? resolution.The structure of NanoLuc consists of 11 antiparallel β-sheets forming a β-barrel structure with a catalytic activity centered on the central cavity of the β-barrel structure,where Nanoluc binds to the substrate and catalyzes its oxidation reaction.We explored the structure of NanoLuc and substrate co-crystallization,and analyze the structure to provides theoretical basis and further applications of NanoLuc.In addition,due to the mutation stability of NanoLuc,we mutated three glycines to alanine to get the stable NanoLuc mutation protein which as a lower background and has potential to be a detection tool for alanine to glycine mistranslation caused by AlaRS.The second part of the article is to understand the relationship between tyrosine phosphatase SHP2 activity and colon cancer HCT-116 cell proliferation.Protein tyrosine phosphorylation is controlled by the coordinated actions of protein tyrosine kinases(PTKs)and phosphatases(PTPs).Deregulation of tyrosine phosphorylation is often implicated in cancer development.Consequently,many anti-cancer drugs targeting oncogenic PTKs have been found to be very effective in treating various cancers.Recently,PTPs have also become attractive targets for anti-cancer drug development.SHP2 is a member of the PTP super-family and is encoded by the proto-oncogene PTPN11.SHP2 is ubiquitously expressed.And acting downstream of receptors for growth factors,cytokines,and cell adhesion molecules,SHP2 plays crucial roles in several cell signaling pathways,most notably the Ras-Raf-MEK-ERK pathway,where it regulates a multitude of cellular processes such as growth,differentiation,and proliferation.Progressive studies have successfully profiled it as an oncoprotein with a leading role in tumorigenesis when actively mutated.Gain-of-function mutation of SHP2 causes cancer,which makes SHP2 a potential target for anti-cancer drug development.Recently,several selective and potent SHP2 inhibitors have been discovered.One inhibitor,SHP099,has been shown to be particularly effective at mitigating aberrant SHP2 activity.It was shown to be effective for receptor tyrosine kinase-driven cancers.More recent studies demonstrated a central and indispensable role for SHP2 in oncogenic KRAS-driven tumors.In this study,we employed a colorectal cancer cell line HCT-116 that carries a heterozygous mutation of KRAS-G13 D.Interestingly,SHP2 inhibitors failed to inhibit HCT-116 growth and ERK1/2 activation but on the contrary had the opposite effect.We further generated SHP2 knockout cells by using the CRISPR technique and provided further evidence that loss of SHP2 activity promotes growth of HCT-116 cells in vitro and in vivo.Our study suggests that targeting SHP2 may have adverse effects on certain cancers,which may have major implications for further development of anti-cancer drugs targeting SHP2.