Abstract
Ornithine decarboxylase (ODC), a key enzyme in the biosynthesis of polyamines, is a labile protein that is regulated by interacting with antizymes (AZs), a family of polyamine-induced proteins. Recently, a novel human gene highly homologous to ODC, termed ODC-like or ODC-paralogue (ODCp), was cloned, but the studies aimed to determine its function rendered contradictory results. We have cloned the mouse orthologue of human ODCp and studied its expression and possible function. mRNA of mouse Odcp was found in the brain and testes, showing a conserved expression pattern with regard to the human gene. Transfection of mouse Odcp in HEK 293T cells elicited an increase in ODC activity, but no signs of arginine decarboxylase activity were evident. On the other hand, whereas the ODCp protein was mainly localized in the mitochondrial/membrane fraction, ODC activity was found in the cytosolic fraction and was markedly decreased by small interfering RNA against human ODC. Co-transfection experiments with combinations of Odc, Az1, Az2, Az3, antizyme inhibitor (Azi), and Odcp genes showed that ODCp mimics the action of AZI, rescuing ODC from the effects of AZs and prevented ODC degradation by the proteasome. A direct interaction between ODCp and AZs was detected by immunoprecipitation experiments. We conclude that mouse ODCp has no intrinsic decarboxylase activity, but it acts as a novel antizyme inhibitory protein (AZI2).
Highlights
Ated by ornithine decarboxylase (ODC)2 (EC 4.1.1.17) through the decarboxylation of L-ornithine
Our results indicate that the murine orthologue of human ODCp displays an expression pattern similar to its human orthologue and, more importantly, that the murine ODCp protein acts as an AZ inhibitory protein, at least in HEK 293 cells
This conclusion is based on our data showing the following. (a) The expression of ODCp in co-transfected HEK 293T cells may abolish the inhibitory effect produced by the co-expression of any member of the AZ family on ODC activity. (b) The action of ODCp on ODC affected ODC activity and ODC stability, because the expression of ODCp appears to prevent the degradation of ODC protein mediated by AZ. (c) ODCp can directly interact with the three AZs
Summary
L-[1-14C]Ornithine was purchased from Moravek Biochemicals Inc. (Brea, CA). L-[U-14C]arginine (specific activity from different lots ranged from 240 –320 mCi/mmol) was supplied by American Radiolabeled Chemicals Inc. Thirty PCR rounds (denaturation for 1 min at 95 °C, annealing for 2 min at 64 °C, and extension for 2 min at 72 °C, followed by a final 10-min extension at 72 °C) were performed using brain cDNA as template, 5 M of each primer, 200 M of each dNTP, and 1.5 units of the proofreading Pfu polymerase. The amplification product was purified, digested, and inserted in the expression vector pcDNA3 (Invitrogen) by the added restriction sites (underlined) and used to transform competent DH5␣ E. coli cells following standard procedures [53]. Mouse ODC, AZ1, AZ2, AZ3, and AZI coding sequences were cloned by a similar procedure using the following primers: ODC The pcDNA3 plasmids containing ODC, ODCp, or AZI coding sequences inserted between the restriction sites EcoRI and XbaI of the polylinker were opened by digestion with BamHI and EcoRI enzymes. The sequences of the constructs were verified as described above
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
