Extracellular Matrix and Stress Substrates: the Role of Prolidase

我们首次发现，一氧化氮(NO)是一种多功能的炎症信号，它通过增加丝氨酸/苏氨酸残基上的磷酸化来增加脯氨酸酶的活性。在NO供体(DETA/NO)刺激的NIH3T3小鼠成纤维细胞中，或在iNOS转染的细胞中激活内源性NO产生的细胞中，Pro酶活性增加2倍以上，而Prolidase蛋白没有增加。由于磷酸化是一种可能性，我们免疫沉淀了Prolidase，并发现免疫可检测到的磷酸化丝氨酸/苏氨酸增加。由于cGMP-PKG途径是NO作用的主要介导者，我们用cGMP激动剂8-bromo-cGMP处理细胞，发现它能刺激酪氨酸氨酸酶的磷酸化。此外，cGMP激酶抑制剂RP-8-PCPT-cGMP可阻断DETA/NO的作用，而ERK1/2信号通路抑制剂UO126则不能阻断这一作用。因此，NO通过cGMP-PKG信号转导的丝氨酸/苏氨酸残基的磷酸化而不是通过MAP激酶来激活Pro酶活性。这些发现为脯氨酸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，脯氨酸酶缺乏患者的伤口愈合缺陷被描述为血管病变。在无法愈合的溃疡和遥远的器官部位都发现了这一点。由于脯氨酸酶的产物(脯氨酸、羟基脯氨酸)参与了多种调节机制，因此我们想知道它是否可能在血管生成中发挥作用。为了验证这一假说，我们通过用Prolidase cDNA表达载体转染RKO结直肠癌细胞，并分离到稳定的转染体，通过酶活性和免疫印迹检测，获得了一个功能增强的实验模型，其表达是载体对照组的15-20倍。经免疫印迹法检测，表达脯氨酸酶的细胞(PL)在全细胞提取液和条件培养上清液中的血管内皮生长因子(VEGF)水平均升高。在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，我们使用低氧反应元件(HRE)荧光素酶构建来检测HIF-1的转录活性，发现HIF-1在PL细胞中的活性要高得多。正如预期的那样，PL细胞的核提取液中的HIF-1α水平更高。由于HIF-1α主要受氧依赖降解结构域(ODD)羟基化后的降解调节，我们使用ODD-荧光素酶结构来检测PL对依赖于Pro羟基酶的蛋白酶体降解的影响。我们发现PL细胞的ODD降解率明显降低。这些机制是由于Pro酶的催化活性所致，这一发现表明，Pro和羟脯氨酸可以增强这种作用。此外，N-苄氧甲酰基-L-脯氨酸(CBZ-Pro)抑制酪氨酸氨基转移酶的活性，使血管生成信号显著减少，同时增加了酪氨酸苷酶的活性。总之，伴随着基质金属蛋白酶的激活和作为应激底物(脯氨酸/羟脯氨酸)来源的ECM降解的增加，信号被产生以激活血管生成以增加营养供应。我们认为HIF-1α降解减少的机制是由于抑制了Pro羟基酶的表达。我们的工作假设是，脯氨酸酶的产物，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，生成吡咯烷-5-羧酸盐和羟基-吡咯烷-5-羧酸盐。这些代谢中间体分别与谷氨酸-伽马半醛和羟基-谷氨酸-伽马-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，但它们各自的伽马碳都是亲核的羰基。我们认为这些化合物是α-酮戊二酸结合部位的脯氨酸羟基酶的抑制剂。使用羟化氧依赖降解结构域的下拉试验，我们有初步证据表明，Pro羟基酶活性受到抑制，尤其是羟基吡咯烷-5-羧酸盐

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