Imidodipeptides and Amino Acid Metabolites in Cell Regulation and Carcinogenesis

P53依赖的细胞凋亡诱导过程中伴随着Pro氧化酶的诱导，提示Pro代谢途径在细胞程序性死亡和癌变中起着重要作用。脯氨酸的一个重要来源是金属蛋白酶对细胞外基质的降解。我们正在研究这些代谢途径的水平：a)它的诱导及其在细胞凋亡中的作用；b)细胞信号中的吡咯酸5-羧酸(P5C)和P5C还原酶；以及c)Pro：一种连接细胞外基质降解、炎症和细胞凋亡的机制。 A)脯氨酸氧化酶--其诱导作用及其在细胞凋亡中的作用。在经历细胞凋亡的过程中，脯氨酸氧化酶被诱导，而添加Pro可刺激活性氧(ROS)的形成。重要的是，在过表达脯氨酸氧化酶的p53阴性细胞中，添加脯氨酸就足以诱导细胞凋亡。为了确定特定的活性氧物种，我们在表达POX的细胞中共表达了Ad-MnSOD、Ad-CuZnSOD或Ad-CAT。这些实验表明，POX会产生超氧自由基，而超氧阴离子自由基是细胞凋亡的中介。另一方面，过氧化氢似乎具有抗凋亡作用，可能是因为它参与了增殖信号的传递。POX的表达不仅激活了线粒体内部的凋亡途径，也激活了外源性的死亡受体途径。最近的研究表明，在某些肿瘤中，面对p53、p21和Bax的正常表达，Pro氧化酶的表达是沉默的，这表明Pro氧化酶可能是一种癌症抑制因子。我们正在鉴定脯氨酸氧化酶启动子，以确定其在分子水平上的调控。PPARGamma及其配体在上调POX表达方面非常活跃。这些配体不仅激活了荧光素酶分析中的POX启动子，还增加了内源性POX的表达，这一发现将POX与生物能量学和肥胖症联系在一起。此外，我们正在使用实时聚合酶链式反应和癌细胞和肿瘤面板来监测参与这一代谢模式的基因的表达。PRODH和PRODH2这两种酶的表达存在差异，PRODH编码的是脯氨酸氧化酶，而PRODH2编码的是羟脯氨酸氧化酶。PRODH和PRODH2在肾组织中高水平表达。相比之下，几种肿瘤细胞株这两种氧化酶基因的表达水平都很低。 B)作为信号和调节分子的吡咯烷5-羧酸盐(P5C)。P5C是脯氨酸氧化酶、鸟氨酸转氨酶和谷氨酸合成酶的产物，具有调节活性。最近的研究表明，P5C还原酶(P5CR)可能在受体介导的调节中发挥作用。P5CR的两种已知同工酶可能具有不同的功能。P5CR1是一种依赖于NADH的酶，被调节来产生脯氨酸，而P5CR2是一种依赖于NADPH的酶，与氧化还原调节相耦合。我们正在使用酵母双杂交系统(Myriad)定义P5CR2与调节蛋白的相互作用。以P5CR2为诱饵，已鉴定出几种有趣的蛋白质，目前正在研究中。 C)Prolidase：连接细胞外基质降解、炎症和细胞凋亡的机制基质金属蛋白酶(MMPs)对细胞外基质(ECM)的降解与炎症、癌变和肿瘤侵袭有关。基质蛋白，尤其是胶原蛋白，在营养胁迫条件下可作为生物能量学底物的脯氨酸和羟脯氨酸的来源。基质蛋白成分的释放，特别是脯氨酸和羟脯氨酸的代谢后果，几乎没有引起人们的注意。脯氨酸酶催化基质降解的最后一步，因为它独特地水解亚氨基二肽，在羧基末端含有脯氨酸或羟脯氨酸。

The induction of proline oxidase accompanying p53-dependent induction of apoptosis suggests that the proline metabolic pathway plays a role in programmed cell death and carcinogenesis. An important source of proline is from degradation of extracellular matrix (ECM) by metalloproteinases. We are studying these metabolic pathways at the level of : a) proline oxidase - its induction and its role in apoptosis; b) pyrroline 5-carboxylate (P5C) and P5C reductase in cell signaling; and c) prolidase: a mechanism linking extracellular matrix degradation, inflammation and apoptosis a) Proline oxidase - its induction and its role in apoptosis. In cells undergoing apoptosis, proline oxidase is induced and added proline stimulates the formation of reactive oxygen species (ROS). Importantly, in p53 negative cells transfected to overexpress proline oxidase, the addition of proline is sufficient to induce apoptosis. To define the specific reactive oxygen species, we co-expressed Ad-MnSOD, Ad-CuZnSOD or Ad-catalase in cells expressing POX. These experiments showed that POX generates superoxide radicals which is the mediator of apoptosis. Hydrogen peroxide, on the other hand, appears to be antiapoptotic perhaps due to its involvement in proliferative signaling. POX expression not only activates the intrinsic mitochondrial apoptosis pathway, but also the extrinsic, death receptor pathway. Recently, it has been shown that the expression of proline oxidase is silenced in certain tumors in the face of normal expression of p53, p21 and BAX suggesting that proline oxidase may act as a cancer suppressor. We are characterizing the proline oxidase promoter to determine its regulation on a molecular level. PPARgamma and its ligands are very active in upregulating POX expression. These ligands not only activate the POX promoter in luciferase assays, but also increase endogenous POX expression, findings which link POX with bioenergetics and obesity. In addition, we are monitoring the expression of genes participating in this metabolic paradigm using real-time PCR and panels of cancer cells and tumors. There is differential expression of the two oxidase enzymes, PRODH which codes for proline oxidase and PRODH2 which codes for hydroxyproline oxidase. PRODH and PRODH2 are expressed at high levels in renal tissue. In contrast, several tumor cell lines have low expression of both oxidase genes. b) Pyrroline 5-carboxylate (P5C) as a signaling and regulatory molecule. P5C, the product of proline oxidase, ornithine aminotransferase and glutamate synthase, has been shown to have regulatory activities. Recent work suggests that P5C reductase (P5CR) may play a role in receptor-mediated regulation. The two known isozymes of P5CR may serve distinct functions. P5CR1 is a NADH-dependent enzyme regulated to produce proline, whereas P5CR2 is a NADPH-dependent enzyme coupled to redox regulation. We are defining the interaction of P5CR2 with regulatory proteins using the yeast two-hybrid system (Myriad). Using P5CR2 as bait, several interesting proteins have been identified and are currently being characterized. c) Prolidase: a mechanism linking extracellular matrix degradation, inflammation and apoptosis The degradation of extracellular matrix (ECM) by matrix metalloproteinases (MMPs) is associated with inflammation, carcinogenesis and tumor invasion. Matrix proteins, especially collagen, can be a source of proline and hydroxyproline as substrate for bioenergetics under conditions of nutritional stress. The metabolic consequences of the release of the constituents of matrix proteins, especially proline and hydroxyproline, have received little attention. Prolidase catalyzes the final step in matrix degradation because it uniquely hydrolyzes imidodipeptides containing proline or hydroxyproline at the carboxyl terminus.