Imidodipeptides/Amino Acid Metabolite in Cell Regulation

脯氨酸氧化酶的诱导伴随p53依赖性的细胞凋亡，提示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-其诱导及其在细胞凋亡中的作用，b) 5-羧酸吡啶（P5C）和P5C还原酶在细胞信号传导中的作用，c)鸟氨酸转氨酶在胚胎发育中的作用。a)脯氨酸氧化酶的诱导及其在细胞凋亡中的作用。脯氨酸氧化酶是一种催化脯氨酸转化为5-羧酸吡咯啉的酶。该酶与线粒体内膜结合，并向复合体II提供电子。最近其他研究表明，脯氨酸氧化酶（POX）是仅有的14个基因（SAGE监测的7202个基因中）高度诱导结直肠肿瘤细胞伴随p53依赖性诱导凋亡的基因之一。为了研究POX的作用，我们首先使用RT-PCR检测了几种细胞类型和导致POX诱导的刺激。在人类结直肠癌（LoVo）细胞、温度敏感SV40 （YAMC）转化的小鼠结肠上皮细胞以及多种癌细胞系中，我们发现血清饥饿和细胞毒性药物以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，通过荧光探针和激光细胞术监测ROS来检验这种可能性。重要的是，我们纳入了DLD1-POX，这是一种p53阴性的结肠肿瘤细胞系，稳定地转染了POX。有趣的是，脯氨酸的添加以浓度依赖的方式显著增加ROS的产生，但仅在POX表达时。另一方面，谷氨酸没有效果。重要的是，脯氨酸-痘效应足以诱导线粒体介导的细胞凋亡。b) 5-羧酸吡咯啉（P5C）作为信号和调控分子。脯氨酸氧化酶、鸟氨酸转氨酶和谷氨酸合成酶的产物吡咯啉5-羧酸酯（P5C）已被证明具有调控活性。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。两种已知的P5CR同工酶可能具有不同的功能。P5CR1是nadh依赖的酶，被调节产生脯氨酸，而P5CR2是nadph依赖的酶，与氧化还原相关的调节相结合。在被凝集素激活的淋巴细胞中，P5CR2似乎是被诱导的。我们正在定义P5CR2与膜受体的相互作用。

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.