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Cloning and functional study of the human genome hUBC16 and GPx7

Author: YinGang
Tutor: MaoYuMin
School: Fudan University
Course: Genetics
Keywords: Ubiquitin-conjugating enzyme GPx biquitination enzyme active site subcellular localization cell cycyle Yeast Two-hybrid Screen
CLC: Q78
Type: PhD thesis
Year: 2006
Downloads: 111
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Abstract


The large-scale cDNA library cloning and sequencing analysis in our lab lead to the accumulation of plenty of full-length human genes. Using various kinds of bioinformatics analysis, we are trying to screen from the library and study the function of potential genes closely related to important physiological pathways and human diseases. On this basis, the two genes we have screened out are hUBC16 and GPx7.The hUBC16 gene is 2252 base pairs in length, encoding a putative 162 amino acids protein. Protein sequence analysis reveals that hUBC16 has an important functional domain: the ubiquitin-conjugating enzyme domain (UBC domain). This cDNA sequence has been submitted in GeneBank, and the accession number is AY948289. Bioinformatics analysis also predicts that hUBC16 shares high homology to Arabidopsis thaliana ubiquitin-conjugating enzyme 16, suggesting its role as a new member of human ubiquitin-conjugating enzyme protein family.The ubiquitin-conjugating enzyme (E2) is an essential part of the ubiquitination machinery. As a bridging element, E2s receive activated ubiquitin from ubiquitin-activating enzymes E1s, form a thiol ester with the C terminus of the ubiquitin and transfer it to down stream ubiquitin-ligases E3s or the immediate substrate proteins. When the target protein is modified by ubiquitin, it will undergo degradation or protein activity modification. Bioinformatics prediction found out that hUBC16 may be localized in the nucleus. Blast n and blast p analysis by NCBI database showed two nuclear localization signals (NLSs) in the C terminus of hUBC16 protein sequence and a putative enzymatic site at the 102 amino acid cysteine, which is further proved through in vitro and in vivo experiments. The result in subcellular localization accords with the bioinformatics prediction that hUBC16 is mainly localized in the nucleus. We experimentally proved that the active site and NLSs are both important components for the normal localization of hUBC16. Yeast two-hybrid analysis screened out several potential interacting proteins with hUBC16, which are ERAL1, UBE1v2, RNF167, KIAA1972 and RNF8.Cell cycle test found out that up regulation of hUBC16 expression leads to an increase in the S phase of the cell cycle, and down regulation causesdecrease in the S phase cells. All of the cell cycle test results show that hUBC16 can promotes cell cycle progression, while the molecular mechanism of which is still unclear and needs further study.Glutathione peroxidases (GPxs) are believed to protect cells against constitutive oxidative damages induced by reactive oxygen species (ROS) generated from aerobic metabolism, and play a role in the regulation of cell cycle and signaling pathways. Here we cloned a human cDNA sequence, encoding a 187 amino acids protein containing a GSHPx domain and a BtuE motif using bioinformatics analysis. Through gene analysis and analysis of the structure of the encoded protein, this sequence turns out to be an NPGPx gene. Using EST and tissue expression pattern analysis, this gene is proved to be widely expressed in normal tissues while specifically expressed in tumor tissues. We cloned the full-length GPx7 cDNA. Subcellular localization prediction and experiment proved the cytoplasmic localization of GPx7.Two of the yeast two-hybrid analysis screen-outs are RAN and EIF3S12. Sequence analysis shows that RAN is a member of the Ras oncogene super family and promotes cell proliferation. EIF3S12 is reported as a subunit of human eukaryotic translation initiation factor 3 and may have a potential function in the transcription of eukaryotic genes. Cell cycle experiments proved that up regulation of GPx7 leads to the shrink in S phase and increase in G1 phase in cell cycle, and the S phase cells are less when RAN and GPx7 are both expressed than RAN alone, suggesting a role of GPx7 in cell cycle inhibition.In a word, we cloned a novel human ubiquitin-conjugating enzyme gene hUBC16 and a reported glutathione peroxidase gene GPx7. we conducted functional studies on them and found out both of them play a role in cell cycle. While hUBC16 might promote cell cycle progression, the up regulation of GPx7 might inhibit cell proliferation, while the molecular mechanisms of both cases need further study.

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