Research of Screening and Function of P53 Regulators in KZNF (AxZ Type、SABZ Type and ACxZ Type) Family
|School||Anhui Medical University,|
|Keywords||KZNF family Apak selective regulation of p53 KRAB domain|
P53 tumor suppressor protein is known as one of the most closely related with human carcinogenesis, and has become the key protein in the protein-protein network controlling cell cycle progression and cell apoptosis. The activity of P53 as a transcriptional factor, is rapidly increased when it’s involving in various types of stresses including DNA damage,abnormal proliferation and oncogene activation, and selectively regulats the expression of its target genes,thus leading to cell cycle arrest and or cell apoptosis. In recent years, more investigations are focusing on the selective regulation of P53. Several members from Hzf, hCAS/CSE1L, TP53INP1 and the ASPP family have been identified,revealing their regulations on P53.We believe the increased members which change P53 activity will provide more clues to understanding the mechanism of normal cell growth and carcinogenesis.The zinc finger proteins containing the KRAB (Kruppel-associated box) domain (KZNF) are the single largest class of transcription factors in the human genome. They repress transcription in conjunction with the co-repressor, KAP-1 and other transcriptional factors. Apak (ATM and P53 associated KZNF protein) belongs to KZNF family, this family. previous study revealed that Apak is a negative regulator of P53, which specifically downregulates pro-apoptotic genes,including Puma,p53AIP,Noxa and Fas, and remarkably suppresses P53-mediated apoptosis by attenuating acetylation of P53. But Apak had no significant effects on P53-mediated cell cycle arrest., cell metabolism and other physiological functions.Earlier results revealed that Apak has important functions in the selective regulation of P53. There are more 400 members of the KZNF superfamily, which is only found in four-legged vertebrate. The members of KZNFs family contain a conserved KRAB domain in their N terminal contain, and the variable numbers of C2H2-type zinc finger ranging from the central to C terminal,KRAB domain with strong transcriptional repression function mediates protein - protein interactions . .Based on different types of KRAB, the screened members from KZNFs family are categorized as seven groups, namely, KRAB-A, KRAB-AB, KRAB-AC, KRAB-SAB, KRAB-ABL,KRAB-SA, KRAB-S, among of them, KRAB-A, KRAB-AB, KRAB-SAB have more members. It is of interest for us to how many members of KZNF family could negatively regulate p53 alike Apak and what are the connections between these members.We found 48 members containing potential ATM phosphorylation site, SQ/TQ motif from 423 members of KZNF family except the member of ABxZ type.. Then we successfully constructed the expression plasmids of 18 members and confirmed their expression in mammalian cells. We detected that 7 members (ZNF205,ZNF475,ZNF420, ZNF589, ZNF498,ZNF417,ZNF202) could strongly downregulate the transcriptional activity of p53. Interesting, ZNF136 can strongly upregulate the transcriptional activity of P53.We defined thses molecules’effects on P53 and it’s downstream targeting genes by monitoring protein expression levels changes with westernblot..Among the four candidates we showeed.ZNF205, ZNF597 can selectively regulate P53-mediated apoptosis, whereas ZNF475,ZNF498 can selectively regulate p53-mediated cell metabolism.In addition, appling the technique of Co-IP, the results show that four of the five family members which significantly inhibited the transcriptional activity of P53 can interact with P53.Taken together, this paper investigated the possible roles of members of the KZNF family in P53 regulation. We totally obtained 48 potential ATM substrates among KZNF family and constructed the expression plasmids of 16 members. Several members were shown to repress P53 activity significantly, their function seems to be related to the association with P53 . These findings might extend our understandings of the physiological roles of KZNF family and highlight the spatio-temporal complexity of P53 regulation.