Imidodipeptides and Amino Acid Metabolites in Cell Regulation and Carcinogenesis

酰亚胺二肽和氨基酸代谢物在细胞调节和癌变中的作用

• 批准号: 7283948
• 负责人: JAMES M PHANG
• 金额: --
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7283948
• 关键词: amine oxidoreductase; aminoacid; apoptosis; biological signal transduction; carcinogenesis; cell line; enzyme activity; extracellular matrix; free radical oxygen; gene expression; genetic promoter element; metalloendopeptidases; nitrogen compounds; polymerase chain reaction; proline; protein protein interaction; superoxide dismutase; yeast two hybrid system

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.

脯氨酸氧化酶的诱导伴随p53依赖性诱导细胞凋亡表明脯氨酸代谢途径在程序性细胞死亡和癌变中起作用。脯氨酸的一个重要来源是通过金属蛋白酶降解细胞外基质（ECM）。我们正在研究这些代谢途径的水平：a）脯氨酸氧化酶-它的诱导和它在细胞凋亡中的作用; B）吡咯啉5-羧酸（P5 C）和P5 C还原酶在细胞信号传导中的作用;和c）脯氨酰二肽酶：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-其诱导及其在细胞凋亡中的作用。在经历凋亡的细胞中，脯氨酸氧化酶被诱导，并且加入的脯氨酸刺激活性氧簇（ROS）的形成。重要的是，在p53阴性细胞转染过表达脯氨酸氧化酶，加入脯氨酸足以诱导凋亡。为了定义特定的活性氧，我们在表达POX的细胞中共表达Ad-MnSOD、Ad-CuZnSOD或Ad-过氧化氢酶。这些实验表明，POX产生超氧自由基，这是细胞凋亡的介质。另一方面，过氧化氢似乎是抗凋亡的，可能是由于其参与增殖信号传导。POX的表达不仅激活了内在的线粒体凋亡途径，也激活了外在的死亡受体途径。最近，已经表明，在某些肿瘤中，在p53、p21和BAX正常表达的情况下，脯氨酸氧化酶的表达被沉默，这表明脯氨酸氧化酶可能充当癌症抑制剂。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表达中非常活跃。这些配体不仅在荧光素酶测定中激活POX启动子，而且还增加内源性POX表达，这些发现将POX与生物能量学和肥胖联系起来。此外，我们正在使用实时PCR和癌细胞和肿瘤组监测参与这种代谢模式的基因的表达。存在两种氧化酶的差异表达，即编码脯氨酸氧化酶的PRODH和编码羟脯氨酸氧化酶的PRODH 2。PRODH和PRODH 2在肾组织中以高水平表达。相反，几种肿瘤细胞系具有两种氧化酶基因的低表达。 B）吡咯啉5-羧酸酯（P5 C）作为信号传导和调节分子。P5 C是脯氨酸氧化酶、鸟氨酸氨基转移酶和谷氨酸合酶的产物，已被证明具有调节活性。最近的研究表明，P5 C还原酶（P5 CR）可能在受体介导的调节中发挥作用。P5 CR的两种已知同工酶可能具有不同的功能。P5 CR 1是一种受调节产生脯氨酸的NADPH依赖性酶，而P5 CR2是一种与氧化还原调节偶联的NADPH依赖性酶。我们正在使用酵母双杂交系统（Myriad）定义P5 CR2与调节蛋白的相互作用。使用P5 CR2作为诱饵，已经鉴定了几种有趣的蛋白质，目前正在进行表征。 c）脯氨酰二肽酶：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、致癌和肿瘤侵袭有关。基质蛋白，特别是胶原蛋白，可以是脯氨酸和羟脯氨酸的来源，作为营养应激条件下生物能量学的底物。基质蛋白的成分，特别是脯氨酸和羟脯氨酸的释放的代谢后果，很少受到关注。氨酰基脯氨酸二肽酶催化基质降解的最后一步，因为它独特地水解羧基末端含有脯氨酸或羟脯氨酸的亚氨基二肽。