Imidodipeptides/Amino Acid Metabolite in Cell Regulation

细胞调节中的酰亚胺二肽/氨基酸代谢物

• 批准号: 6557519
• 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/6557519
• 关键词: Xenopus; amine oxidoreductase; apoptosis; biological signal transduction; carboxylate; carcinogenesis; cell growth regulation; colorectal neoplasms; dipeptides; embryogenesis; enzyme activity; enzyme induction /repression; genetically modified animals; laboratory mouse; nutrition aspect of cancer; nutrition related tag; ornithine; oxidative stress; oxidoreductase; proline; tissue /cell culture; transaminases

The induction of proline oxidase accompanying p53-dependent induction of apoptosis suggests that the proline metabolic pathway plays a role in programmed cell death. We are studying this metabolic pathway 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) ornithine aminotransferase in embryonic development. a) Proline oxidase - its induction and its role in apoptosis. We have focused on proline oxidase, the enzyme which catalyzes the conversion of proline to pyrroline 5-carboxylate. The enzyme is bound to mitochondrial inner membranes and donates electrons to complex II. Recently others have shown that proline oxidase (POX) is one of only 14 genes (from 7202 genes monitored by SAGE) highly induced accompanying p53-dependent induction of apoptosis in colorectal tumor cells. To investigate the role of POX, we first used RT-PCR to examine several cell types and the stimuli which will result in POX induction. In human colorectal cancer (LoVo) cells, mouse colonic epithelial cells transformed by temperature sensitive SV40 (YAMC), as well as a variety of carcinoma cell lines, we found that serum starvation as well as cytotoxic drugs induced proline oxidase in a p53-dependent fashion. We proposed that proline oxidase may contribute to the metabolic events accompanying apoptosis by generating reactive oxygen species (ROS). This possibility was examined in LoVo and YAMC cells by using a fluorescent probe and laser cytometry to monitor ROS. Importantly, we included DLD1-POX, a p53-negative colon tumor cell line stably transfected with POX. Interestingly, the addition of proline markedly increased ROS generation in a concentration-dependent manner but only when POX was expressed. Glutamate, on the other hand, was without effect. Importantly, the proline-POX effect is sufficient to induce mitochondria-mediated apoptosis. b) Pyrroline 5-carboxylate (P5C) as a signaling and regulatory molecule. Pyrroline 5-carboxylate (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 with redox-related regulation. In lymphocytes activated by lectins, it appears that P5CR2 is induced. We are defining the interaction of P5CR2 with membrane receptors. c) Ornithine aminotransferase in embryonic development. Ornithine aminotransferase (OAT) converts ornithine to P5C and inherited deficiencies of this enzyme results in degeneration of the neuroretina. Using Xenopus embryos as a model, we have shown that OAT is expressed during a critical stage in neural development. In situ hybridization with Xenopus OAT cDNA shows that expression of OAT correlates with critical stages of neural development. Since the pathway for neurodevelopment has been characterized, the interaction of OAT or its products in modulating this pathway has been investigated.

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中起作用。我们正在研究这一代谢途径的水平：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，和c）鸟氨酸氨基转移酶在胚胎发育。 a）脯氨酸氧化酶-其诱导及其在细胞凋亡中的作用。我们已经集中在脯氨酸氧化酶，催化脯氨酸转化为吡咯啉5-羧酸的酶。该酶与线粒体内膜结合，并向复合物II提供电子。最近，其他人已经表明，脯氨酸氧化酶（POX）是只有14个基因（从7202基因监测SAGE）高度诱导伴随p53依赖性诱导大肠癌细胞凋亡。为了研究POX的作用，我们首先使用RT-PCR检测几种细胞类型和导致POX诱导的刺激。在人结肠癌（LoVo）细胞、温度敏感性SV 40转化的小鼠结肠上皮细胞（YAMC）以及多种癌细胞系中，我们发现血清饥饿以及细胞毒性药物以p53依赖的方式诱导脯氨酸氧化酶。我们提出脯氨酸氧化酶可能通过产生活性氧（ROS）参与伴随细胞凋亡的代谢事件。在LoVo和YAMC细胞中通过使用荧光探针和激光细胞术监测ROS来检查这种可能性。重要的是，我们包括DLD 1-POX，一种用POX稳定转染的p53阴性结肠肿瘤细胞系。有趣的是，除了脯氨酸显着增加ROS的产生浓度依赖性的方式，但只有当POX表达。另一方面，谷氨酸盐没有效果。重要的是，脯氨酸-POX效应足以诱导细胞凋亡。 B）吡咯啉5-羧酸酯（P5 C）作为信号传导和调节分子。吡咯啉5-羧酸（P5 C），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NADH依赖性酶，而P5 CR2是一种与氧化还原相关调节偶联的NADPH依赖性酶。在由凝集素激活的淋巴细胞中，似乎P5 CR2被诱导。我们正在定义P5 CR2与膜受体的相互作用。 c）胚胎发育中的鸟氨酸转氨酶。鸟氨酸氨基转移酶（OAT）将鸟氨酸转化为P5 C，这种酶的遗传缺陷导致神经视网膜变性。使用非洲爪蟾胚胎作为模型，我们已经表明，OAT在神经发育的关键阶段表达。与非洲爪蟾OAT cDNA的原位杂交表明，OAT的表达与神经发育的关键阶段相关。由于神经发育的途径已经被表征，因此已经研究了OAT或其产物在调节该途径中的相互作用。