Study On The Function Of A Novel Decarboxylase PDD87 With Golgi Localization Which Is Up Regulated In Pten Conditional Knockout Mice And Proteomic Analysis Of A Strain Of MEF Cells With Pten Deletion

Pten is a tumor suppressor gene with phosphatase activity. The mouse with pten conditionally deleted in brain has a larger brain, and negative regulation of neural stem/progenitor cell proliferation by the Pten tumor suppressor gene in vivo was found using this mouse model. To further pursue the mechanisms behind this phenomenon, the gene expression profiles of the cerebrum of the normal mouse embryo at day 14 and that of the pten conditional knockout mouse embryo were compared using Affymetrix gene chips, and a novel cDNA (Accession Number BC005541) was identified to be up-regulated in the cerebrum of the pten conditional knockout mouse embryo. Northern blot confirmed the upregulation and further work revealed that this gene is also up-regulated in two pten-/- cell lines and the breast cancer tissues of pten conditional knockout mice. The transcript of this gene exists in a veriety of mouse tissues.BC005541 is 3749bp long encoding a 787 amino acids protein, bioinformatic analysis revealed that its molecular weight is about 87.2 Kd, and it bears a conserved pyridoxal-dependent decarboxylase domain. So we named it as pdd87 temporarily, which stands for pjridoxal-dependent decarboxylase with MW 87.2. Pdd87 gene spans over 67Kb on chromosome 16 and contains 23 exons and 22 introns. Bioinformatic analysis also revealed that this gene is more or less similar to the glutamate decarboxylases, which takes part in the production of gamma-Aminobutyric acid (GABA), a important neurotransmitter in the vertebrate brain.To determine the subcellular localization of the encoded protein of pdd87, fusion protein expression vector pEGEP-C1-pdd87 was constructed and transfected into NIH3T3 cells and cells were stained with BODIPY TR C5-Ceramide to display Golgi body. Microscope observation showed that PDD87-GFP fusion protein was located in Golgi body, while GFP protein was expressed in nucleus and cytoplasm and had permeating distribution, indicating that the protein encoded by the gene pdd87 was located in the Golgi body.Using HPLC assay, we detected the level of GABA in a mouse embryonic fibroblast (MEF) cell line MEF1/pten-/- and its normal counterpart (MEF1 cell line).The result showed that MEF1/pten-/- cell had more GABA than MEF1 cell, We postulated that may be associated with the pdd87 gene.We analysesed the decarboxylase activity of PDD87 expressed in E.coli. The result showed that more GABA were produced when PDD87 is induced in E.coli. But purified PDD87 is needed to confirm its decarboxylase activity, until then we can say pdd87 has the decarboxylase activity.Three expression plasmids: pET-28a-pdd87, pET-28a-pdd87-404 and pMXB10-pdd87, were constructed. pET-28a-pdd87 and pMXB10-pdd87 contain the entire murine pdd87 gene ORP. pET-28a-pdd87-404 contains the C-terminal 404 amino acids of the PDD87 protein. Induced by IPTG, the three plasmids were all expressed. PDD87 and PDD87-404 with His6 tag were purified by Ni-NTA affinity chromatography. PDD87 protein without His6 tag was purified by chitin affinity chromatography. PDD87-404 was used as antigen to immunize rabbits to get antibody, but unfortunately high titer antibody with good specificity hasn't been produced. PDD87 protein without His6 tag can be used for characterizing the decarboxylase activity of PDD87 in future experiments.Using FACS analysis, we found MEF2/Pten-/- (DK05 cell line) andMEF1/Pten-/-(27 cell line) had more S phase cells than their normal counterparts. DK05 cell line had more S phase cells than MEF1/Pten-/-(27 cell line). The western blot results showed that the levels of AKT protein did not changed but its phosphate levels were all raised in both cell lines. The levels of phosphated downstream molecules are not same between the two Pten-/- cell lines, which may be the reason of their cell cycle difference.The development of 2-DE and MS allows us to analyze the protein profiles of MEF1 (Pten+/+) and MEF1/Pten-/- cell lines. We identified that the CuZn-SOD, peroxisomal membrane protein 20 and peroxiredoxin 6 were down-regulated in 27(Pten...