Extracellular Matrix and Stress Substrates: the Role of Prolidase

我们首先发现，一氧化氮（NO），一种多功能的炎症信号，增加脯氨酰二肽酶的活性，通过增加其丝氨酸/苏氨酸残基的磷酸化。在NIH 3 T3小鼠成纤维细胞刺激NO供体（DETA/NO）或在细胞转染iNOS激活内源性NO的生产，氨脯氨酸二肽酶活性增加2倍以上，而不增加氨脯氨酸二肽酶蛋白。由于磷酸化是一种可能性，我们免疫沉淀氨酰基脯氨酸二肽酶，发现增加免疫检测磷酸化丝氨酸/苏氨酸。由于cGMP-PKG途径是NO作用的主要介质，我们用cGMP激动剂8-溴-cGMP处理细胞，发现它刺激脯氨酰二肽酶的磷酸化。cGMP激酶抑制剂Rp-8-pCPT-cGMP可阻断DETA/NO的作用，而ERK 1/2信号通路抑制剂UO 126则不能阻断DETA/NO的作用。因此，NO通过cGMP-PKG信号传导介导的丝氨酸/苏氨酸残基的磷酸化而不是通过MAP激酶激活氨酰基脯氨酸二肽酶活性。这些发现为氨酰基脯氨酸二肽酶对炎症信号的反应提供了分子基础。在死后的组织学检查中，脯氨酰二肽酶缺乏症患者的伤口愈合缺陷被描述为血管病性。这在不愈合的溃疡和远处器官部位都有发现。由于脯氨酰二肽酶的产物（脯氨酸、羟脯氨酸）参与了许多调节机制，我们询问脯氨酰二肽酶是否可能在血管生成中发挥作用。为了检验这一假设，我们通过用氨酰基脯氨酸二肽酶cDNA表达质粒转染RKO结直肠癌细胞并分离稳定的转染子获得了功能获得性实验模型，所述稳定的转染子表达载体对照的15-20倍，如通过酶活性和蛋白质印迹所确定的。通过蛋白质印迹法和ELISA法测量，氨酰基脯氨酸二肽酶表达细胞（PL）在全细胞提取物中具有增加的血管内皮生长因子（VEGF）水平。PL细胞中葡萄糖转运蛋白-1（Glut-1）也增加。由于VEGF和Glut-1都是HIF-1的靶基因，我们使用缺氧反应元件（HRE）荧光素酶构建体测量HIF-1的转录活性，发现HIF-1在PL细胞中更活跃。正如预期的那样，PL细胞核提取物中的HIF-1 α水平较高。由于HIF-1 α主要由氧依赖性降解结构域（ODD）的脯氨酰羟基化后的降解调节，因此我们使用ODD-荧光素酶构建体来研究PL对脯氨酰羟基化酶依赖性蛋白酶体降解的影响。我们发现，ODD降解显着减少PL细胞。脯氨酸和羟脯氨酸可增强脯氨酰二肽酶的催化活性，这表明脯氨酰二肽酶的催化活性是上述作用机制的基础。此外，通过N-苄氧羰基-L-脯氨酸（Cbz-Pro）抑制脯氨酰二肽酶活性显著降低了血管生成信号，增加了脯氨酰二肽酶。总之，伴随MMP的活化和作为应激底物（脯氨酸/羟脯氨酸）的来源的ECM降解增加，产生信号以活化血管生成以增加营养供应。我们认为，HIF-1 α降解减少伴随脯氨酰二肽酶过表达的机制是由于脯氨酰羟化酶的抑制。我们的工作假设是脯氨酸二肽酶的产物，即脯氨酸和羟脯氨酸，分别被脯氨酸和羟脯氨酸氧化酶降解，产生吡咯啉-5-羧酸和羟基-吡咯啉-5-羧酸。这些代谢中间体分别与谷氨酸-γ-半醛和羟基-谷氨酸-γ-半醛处于互变异构平衡。这两种5-碳化合物都类似于α-酮戊二酸，但它们各自的γ碳是亲核羰基。我们建议，这些化合物是脯氨酰羟化酶的抑制剂在α酮戊二酸的结合位点。使用下拉分析的羟基化的氧依赖性降解结构域，我们有初步的证据表明，脯氨酰羟化酶的活性受到抑制，特别是由羟基-吡咯啉-5-羧酸

We first showed that nitric oxide (NO), a versatile inflammatory signal, increases prolidase activity by increasing its phosphorylation on serine/threonine residues. In NIH 3T3 mouse fibroblasts stimulated with NO donors (DETA/NO) or in cells transfected with iNOS to activate endogenous NO production, prolidase activity was increased more than 2-fold, without an increase in prolidase protein. Since phosphorylation was a possibility, we immunoprecipitated prolidase and found increased immunodetectible phosphorylated serine/threonine. Since cGMP-PKG pathway is the main mediator of NO effects, we treated cells with 8-bromo-cGMP, a cGMP agonist, and found that it stimulated the phosphorylation of prolidase. Furthermore, the effects of DETA/NO could be blocked by Rp-8-pCPT-cGMP, an inhibitor of cGMP kinase, but not by UO126, an inhibitor of ERK1/2 signaling. Thus, NO activates prolidase activity through phosphorylation of serine/threonine residues mediated by cGMP-PKG signaling and not through MAP kinases. These findings provide a molecular basis for a prolidase response to inflammatory signals. The wound-healing defect in patients with prolidase deficiency has been described as angiopathic in histologic examinations postmortem. This was found both in nonhealing ulcers and at distant organ sites. Since the products of prolidase (proline, hydroxy-proline) are involved in a number of regulatory mechanisms, we asked whether prolidase might play a role in angiogenesis. To test this hypothesis, we obtained a gain-of-function experimental model by transfecting RKO colorectal cancer cells with a prolidase cDNA expression plasmid and isolating stable transfectants which express 15-20 fold that of vector controls as determined by enzyme activity and western blots. 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. Since both VEGF and Glut-1 are target genes of HIF-1, we measured HIF-1 transcriptional activity using a hypoxia response element (HRE) luciferase construct and found that HIF-1 was much more active in PL cells. As expected, HIF-1alpha levels were higher in nuclear extracts from PL cells. Since HIF-1alpha is regulated primarily by degradation after prolyl hydroxylation of the oxygen dependent degradation domain (ODD), we used an ODD-luciferase construct to examine the effect of PL on prolyl hydroxylase-dependent proteasomal degradation. We found that ODD degradation was markedly decreased in PL cells. That these mechanisms are due to the catalytic activity of prolidase was shown by the finding that proline and hydroxyproline could augment the effects. 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. We propose that the mechanism for decreased degradation of HIF-1alpha accompanying overexpression of prolidase is due to inhibition of prolyl hydroxylase. Our working hypothesis is that products of prolidase i.e. proline and hydroxyproline, are degraded by proline and hydroxyproline oxidase, respectively to produce pyrroline-5-carboxylate and hydroxy-pyrroline-5-carboxylate. These metabolic intermediates are in tautomeric equilibrium with glutamic-gamma semialdehyde and hydroxy-glutamic-gamma-semialdehyde, respectively. Both of these 5-carbon compunds are similar to alpha-ketoglutarate but their respective gamma carbons are nucleophilic carbonyl groups. We propose that these compounds are inhibitors of prolyl hydroxylase at the binding site for alpha ketoglutarate. Using a pull-down assay for the hydroxylated oxygen-dependent degradation domain, we have preliminary evidence that prolyl hydroxylase activity is inhibited, especially by hydroxy-pyrroline-5-carboxylate