Protein-Protein Interaction Between CGB And MT2

In this research, yeast two hybrid screening system was adopted to find interaction proteins with CGB (Cytoglobin). We identified MT2 (Metallothioneinâ…¡) as a novel CGB binding protein in a human fetal brain cDNA library. Then, the interaction was confirmed by yeast two and collocalization in COS7 cells. Moreover, Reverse transcription polymerase chain reaction technique (RT-PCR) was used to test CGB mRNA expression levels change and fluorescence microscope was used to detect the colocalizition change of the interaction between CGB and MT2 in oxidation damage models. Furthermore, we also expressed CGB in E.coli successfully. This research contains the following studies.1. Identification of MT2 as CGB-interacting protein by yeast two-hybrid screening system. CGB yeast plasmid was constructed, and was expressed successfully in AH109 while no self-activation detected. The human fetal brain cDNA library was then screened through the yeast two-hybrid technique. One of the selected 56 clones encoding MT2 was obtained.2. The interaction between CGB and MT2 was confirmed in yeast two and co-localization studies in vivo. The result showed that MT2 without self-transcriptional activity alone, which confirmed CGB could interact with MT2 in yeast two hybrid screening system; Confocal laser microscopy demonstrated that overexpression of CGB was mainly localized in cytoplasm and partially in the nucleus alone, while MT2 was mainly localized in cytoplasm. However, the two proteins were colocalized both in cytoplasm and nucleus in a punctuate pattern, which indicated that the expression of CGB could affect the main localization of MT2; CGB seems to cluster the MT2 translocation into nucleus from cytoplasm.3. The changes of CGB mRNA expression levels and colocalization of the interaction between CGB and MT2. In our study, CGB mRNA expression levels were increased at a dose- and time-dependent manner in ZnSO4-induced oxidation damage of the COS7 cells. Interestingly, the colocalization of CGB and MT2 were totally in nucleus in ZnSO4-induced COS7 cells. Which indicated the interaction between CGB and MT2 was related with the oxidation damage to the DNA damage response.