Extracellular Matrix and Stress Substrates: the Role of Prolidase

脯氨酸酶从酰二肽中释放脯氨酸和羟脯氨酸。大量有力证据支持脯氨酸在程序性细胞死亡和生物能量学中的作用（Phang J. <I>et al.， J. Nutr.）。</I>138:2008S, 2008)，但对脯氨酸酶释放的另一种亚胺酸羟基脯氨酸也进行了研究。羟脯氨酸氧化酶是一种不同于脯氨酸氧化酶的酶，由不同的基因编码，被证明是p53诱导的基因，产生ROS并激活细胞凋亡（Cooper， S.K. <I>et al.， J. Biol.）。化学。;/ I&gt 283:10485, 2008)。这些发现进一步强调了增殖酶在细胞外基质降解中催化两种亚胺酸释放的重要性。先前的研究表明，一氧化氮（NO）通过刺激其丝氨酸/苏氨酸磷酸化来增加脯氨酸酶的活性。NO的作用可以通过外源性供体（DETA/NO）处理或用iNOS转染细胞以增加内源性NO的产生而产生。(苏拉津斯基&lt；I>et al.；[j] .癌症&lt；/I> 122:1435, 2008]。这种作用是通过cgmp -蛋白激酶G途径而不是MAP激酶途径介导的。由于伤口愈合缺陷是遗传性脯氨酸酶缺乏症患者的重要发现，我们重点研究了脯氨酸酶依赖机制，并表明脯氨酸酶依赖调节至少通过两种途径发生。首先，我们发现胶原降解释放的脯氨酸增加了胶原合成的速率（Surazynski A. <I>et al., Amino Acids </I>35:731, 2008）。据推测，这是因为底物脯氨酸可能是伤口愈合过程中胶原合成的速率限制。增殖酶的另一个作用是通过hif -1介导的。我们获得了稳定转染的RKO结直肠癌细胞，其增殖酶的表达是载体对照的15-20倍。通过western blots和ELISA检测，增殖酶表达细胞（PL）在全细胞提取物和条件培养基中血管内皮生长因子（VEGF）水平升高。葡萄糖转运蛋白-1 （Glut-1）也在PL细胞中升高。HIF-1在PL细胞中更活跃，氧依赖结构域（ODD）的降解明显减少，这些机制是由于脯氨酸酶的催化活性，通过发现中脯氨酸和羟脯氨酸可以增强这种作用。此外，n -苄氧羰基- l-脯氨酸（Cbz-Pro）对脯氨酸酶活性的抑制显著降低了血管生成信号，增加了脯氨酸酶。总之，作为应激底物（脯氨酸/羟脯氨酸）的来源，伴随着MMP的激活和ECM降解的增加，会产生激活血管生成的信号，以增加营养供应(Surazynski a . <I>et al.；[j] .癌症&lt；/I> 122:1435, 2008]。自癌症被认为是“永不愈合的伤口”以来，增殖酶缺乏症患者的伤口愈合缺陷引起了相当大的兴趣。因此，我们启动了一个项目，开发增殖酶敲除小鼠。敲除脯氨酸酶基因的胚胎干细胞是从BayGenomics购买的。这些胚胎干细胞被一种基因诱捕载体靶向，该载体含有报告基因β -geo上游的剪接受体序列，这是β -半乳糖苷酶和新霉素磷酸转移酶II的融合。该构建体随机插入小鼠基因组，然后将其插入小鼠基因组7号染色体上的脯氨酸酶基因11内含子中。核型分析后，将目标胚胎干细胞微注射到C57Bl/6囊胚中获得嵌合小鼠。虽然我们获得了杂合子，但我们没有获得完全被敲除的小鼠。脯氨酸酶活性只有90%被敲除，我们没有获得令人信服的代谢表型。我们正在采用敲除小鼠项目（KOMP）提供的替代策略，该项目使用替代策略，敲除带有Pepd（增殖酶）的胚胎干细胞，并正在扩增胚胎干细胞。我们期望在2009年10月获得基因型验证的胚胎干细胞用于注射。

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.