| The Pax6genes first to be cloned are from human, mouse, zebrafish and quail. It regulates eye, brain and pancreas development. Homozygous Pax6mutations are fatal at birth, leading to deficiency of brain and absence of eyes and nose. Heterozygous mutations result in small eye (Sey) in mouse and aniridia in human, probably cataract or glaucoma in th future. Peter’s anomaly is also caused by Pax6mutation which results in a central corneal opacity and physical connection of the lens and cornea. All these Pax6mutation phenotypes are involved in eyes and nose defects. In homozygous phenotypes, the absence of eyes and nose indicates that Pax6is an essential factor in early embryo development and that its loss can not be compensated by other genes which function similarly.Pax6is a transcription factor. It has two DNA binding domain, which are paired domain (PD) in the N-terminal consisting of128amino acids and homeodomain (HD) in the C-terminal consisting of60amino acids. The paired domain of all Pax6proteins displays a high degree of sequence conservation. The vertebrate Pax6proteins share an identical paired domain, with the exception of zebrafish Pax-6, in which a single amino acid is changed. The similarity of paired domain is almost over90%when comparing the invertebrate Pax6proteins with mouse Pax6. The homeodomain of all Pax6proteins is also highly conserved. The vertebrate Pax6homeodomains are identical, while invertebrate share more than90%homology to the mouse sequence. The high conservation of Pax6sequence implies that it plays a crucial role in development. Pax6has four isoforms which are named p48, p46, p43, p32according to their molecular masses. It is reported that p46and p48are from alternative splicing while p32is transcribed from a second, internal promoter and hence lacks the paired domain. We’ve already confirmed that p43was formed by combination of p32and a small protein named SUMO1in human and mouse eye and that sumoylation was important in maintaining the transcription activity of p32.SUMO, small ubiquitin-related modifier, is an important class of ubiquitin-like proteins that has three family members which are SUMO1, SUMO2and SUMO3. They share a common structural fold, core conjugation but different sequence and function. SUMO is structurally related to ubiquitin and can also ligate to lysine residues of its target proteins. Three enzymes are involved in sumoylation, which are SUMO El activating enzyme, SUMO E2conjugation enzyme and SUMO E3ligase.We’ve confirmed that in vitro, sumoylation of p32can activate its regulatory activity but the physiological function is not quite clear. Therefore, We utilized constructed stable pancreatic cancer cell lines overexpressing wild type p32and mutated p32and carried out cell proliferation experiment, nude mice experiment, tumor slice and cell cycle related proteins analysis to find out the regulatory effect of p32toward tumorigenesity after sumoylation. It turned out that cells expressing wild type p32proliferated the quickest and obviously much quicker than those expressing mutated p32. Tumors from the nude mice which were injected with cells expressing wild type p32were larger than those from mice which were injected with cells expressing mutated p32and had more vessels in the outer surface. Some tumors had holes inside which were filled with blood. Tumor slide revealed that cells from wild type p32Pax6tumor, most of which were at mitosis, were much more than those from sumoylation site mutated p32Pax6tumor. Blood vessels were obviously formed at the external surface of wild type p32Pax6tumor, provided nutrition to it and promoted its growth. The analysis of cell cycle related proteins displayed that the expression of p27and chk2in sumoylation site mutated p32Pax6cells was higher than that in wild type p32Pax6cells. It was supposed that the mutation at sumoylation site could release the inhibition of p27and chk2, then slower cell cycle and promote apoptosis. In a word, sumoylation of p32Pax6is very important in tumorigenesity. |
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