Dissertation > Biological Sciences > Molecular Biology > Genetic engineering (genetic engineering)

Screening of CUEDC2-interacting Proteins by Yeast Two-hybrid Assasy and Functional Analysis of the Protein Complex

Author LiuHui
Tutor ZhangXueMin
School PLA Military Academy of Medical Sciences
Course Cell Biology
Keywords CUEDC2 GADD34 p53 Yeast two-hybrid Interaction Transcriptional activity
Type Master's thesis
Year 2007
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CUEDC2 (CUE domain containing 2), is a the CUE domain protein, its biological function is unclear. CUE domain of about 40 amino acids, with the ubiquitin-conjugating, widely exist in a variety of eukaryotic proteins. The CUE domain with a single ubiquitin, polyubiquitin there are interaction, and play a different function. Ubiquitination is an important form of regulation of protein function, is an important regulatory mechanism of cellular signal transduction. Order to reveal the biological function of the CUEDC2, we applied the yeast two-hybrid screening technology CUEDC2 interacting proteins were screened and conducted a preliminary study its function. As bait, we to CUEDC2 gene is cDNA library interacting proteins from human breast. We screened 185 candidate clones seven positive clones CUEDC2 there is interaction. The sequencing results showed that, seven clones were four genes Source: Homo sapiens the THAP domain containing, apoptosis associated protein. 3 (THAP3); Homo sapiens suppressor of cytokine signaling 3 (SOCS3); apoptosis associated protein of Homo sapiens, growth arrest and of DNA damage-inducible gene 34 (GADD34); Homo sapiens IkB kinase-beta (IKKβ). CDNA sequences of the four proteins from human breast library fish, ready for the next step of the structural and functional studies. Interaction between CUEDC2 four proteins then by the GST pull-down experiments in vitro and in vivo immune coprecipitation experiments verified. To further clarify the biological function of the CUEDC2 we the yeast filter results based on research CUEDC2 and GADD34 interacting biological significance. According to the literature, GADD34 is able to promote the transcriptional activation of p53 activity, we first use the dual fluorescent reporter system verified GADD34 role of p53 transcriptional activity in eukaryotic cells in MCF-7. We will CUEDC2 separately transferred MCF-7 cells, the detection of p53 transcriptional activity, found, CUEDC2 no effect on p53 activity, however, can significantly enhance the co-transfected CUEDC2 and GADD34 GADD34 promoting effect on p53 activity. We found that the CUEDC2 increase in mammalian cells capable of specifically GADD34 protein expression levels. Prompt CUEDC2 may GADD34 interaction, promote GADD34 protein expression, thus affecting GADD34 promote the ability of p53 transcriptional activity. In conclusion, our experiments show that, have CUEDC2 by binding GADD34, and affect the expression level of the GADD34 protein, thereby affecting the activity of p53. Found CUEDC2 GADD34 interaction complex for us to study the biological functions of the CUEDC2 revelation, further research may find CUEDC2 involved in the regulation of signal transduction mechanisms.

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