Prolidase releases both proline and hydroxyproline from imidodipeptides. A body of robust evidence supports the role of proline in programmed cell death and bioenergetics (Phang J. <I>et al., J. Nutr. </I>138:2008S, 2008), but the other imino acid released by prolidase, hydroxyproline, was also investigated. Hydroxyproline oxidase, an enzyme distinct from proline oxidase, and encoded by a distinct gene, was shown to be a p53-induced gene which generates ROS and activates apoptosis (Cooper, S.K. <I>et al., J. Biol. Chem.</I>283:10485, 2008). These findings further emphasize the importance of prolidase in catalyzing the release of both imino acids from the degradation of extracellular matrix. Previous studies showed that nitric oxide (NO) increases prolidase activity by stimulating its serine/threonine phosphorylation. The effect of NO can be produced either by treatment with an exogenous donor (DETA/NO) or by transfection of cells with iNOS to increase endogenous production of NO. (Surazynski <I>et al., Int. J. Cancer </I> 122:1435, 2008). The effect is mediated through the cGMP-Protein Kinase G pathway rather than the MAP kinase pathway. Since defective wound healing is a prominent finding in patients with inherited prolidase deficiency, we focused on prolidase-dependent mechanisms and showed that prolidase-dependent regulation occurs by least two pathways. First, we showed that the release of proline from collagen degradation increases the rate of collagen synthesis (Surazynski A. <I>et al., Amino Acids </I>35:731, 2008). Presumably this occurs because substrate proline may be rate limiting for collagen synthesis during wound healing. Another effect of prolidase is mediated through HIF-1alpha. We obtained RKO colorectal cancer cells stably transfected with expression of prolidase 15-20 fold that of vector controls. The prolidase expressing cells (PL) have increased levels of vascular endothelial growth factor (VEGF) in whole cell extracts by western blots and in conditioned media as measured by ELISA. Glucose transporter-1 (Glut-1) is also increased in PL cells. HIF-1 was much more active in PL cells, and the degradation of the oxygen-dependent domain (ODD) was markedly decreased That these mechanisms are due to the catalytic activity of prolidase was shown by the finding that medium proline and hydroxyproline could augment the effect. Furthermore, inhibition of prolidase activity by N-benzyloxycarbonyl-L-proline (Cbz-Pro) markedly decreased the angiogenic signaling with increased prolidase. In conclusion, accompanying the activation of MMP and increased ECM degradation as a source of stress substrates (proline/hydroxyproline), signals are generated to activate angiogenesis to augment the nutrient supply (Surazynski A. <I>et al., Int. J. Cancer </I>122:1435, 2008). The defective wound healing in patients with prolidase deficiency is of considerable interest since cancer has been considered """"""""a wound that never heals."""""""" Thus, we initiated a project to develop prolidase knockout mice. Embryonic stem cells with the prolidase gene knocked out were purchased from BayGenomics. These ES cells were targeted with a gene-trap vector containing a splice acceptor sequence upstream of a reporter gene, beta-geo, a fusion of beta-galactosidase and neomycin phosphotransferase II. The construct, randomly inserted into the murine genome was then inserted into intron 11 of the prolidase gene on chromosome 7 in the mouse genome. Following karyotyping, the targeted ES cells were microinjected into C57Bl/6 blastocysts to obtain chimeric mice. Although we have obtained heterozygotes, we obtained no completely knocked-out mice. Prolidase enzyme activity was only 90% knocked out and we did not obtain a convincing metabolic phenotype. We are undertaking an alternative strategy provided by the Knockout Mouse Project (KOMP) which uses an alternative strategy and have ES with Pepd (prolidase) knocked out and are in the process of expanding the ES cells. The We expect to obtain ES cells with their genotype verified for injection in October, 2009.
| Phang, James Ming; Liu, Wei; Hancock, Chad et al. (2012) The proline regulatory axis and cancer. Front Oncol 2:60 |
| Phang, James M; Liu, Wei (2012) Proline metabolism and cancer. Front Biosci 17:1835-45 |
| Jung, Seungwoo; Silvius, Derek; Nolan, Katherine A et al. (2011) Developmental cardiac hypertrophy in a mouse model of prolidase deficiency. Birth Defects Res A Clin Mol Teratol 91:204-17 |
| Phang, James M; Liu, Wei; Zabirnyk, Olga (2010) Proline metabolism and microenvironmental stress. Annu Rev Nutr 30:441-63 |
| Tokar, Erik J; Qu, Wei; Liu, Jie et al. (2010) Arsenic-specific stem cell selection during malignant transformation. J Natl Cancer Inst 102:638-49 |
| Hong, Sam Y; Borchert, Gregory L; Maciag, Anna E et al. (2010) The Nitric Oxide Prodrug V-PROLI/NO Inhibits Cellular Uptake of Proline. ACS Med Chem Lett 1:386-389 |
| Surazynski, A; Miltyk, W; Palka, J et al. (2008) Prolidase-dependent regulation of collagen biosynthesis. Amino Acids 35:731-8 |
