Imidodipeptides/Amino Acid Metabolite in Cell Regulation

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中起着重要作用。我们正在研究这一代谢途径的水平：a)它的诱导及其在细胞凋亡中的作用，b)吡咯烷5-羧酸(P5C)和P5C还原酶在细胞信号传递中的作用，以及c)鸟氨酸转氨酶在胚胎发育中的作用。A)脯氨酸氧化酶--其诱导作用及其在细胞凋亡中的作用。我们重点研究了脯氨酸氧化酶，它是一种催化脯氨酸转化为吡咯酸的酶。该酶结合在线粒体内膜上，并向复合体II提供电子。最近，其他研究表明，从SAGE监测的7202个基因中，仅有14个基因高度诱导伴随着p53依赖的P53诱导结直肠肿瘤细胞的凋亡。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将导致POX诱导的刺激。在人类结直肠癌(LoVo)细胞、温敏性SV40(YAMC)转化的小鼠结肠上皮细胞以及多种癌细胞系中，我们发现血清饥饿以及细胞毒药物以一种P53依赖的方式诱导Pro氧化酶。我们认为，脯氨酸氧化酶可能通过产生活性氧(ROS)而参与伴随细胞凋亡的代谢事件。通过使用荧光探针和激光细胞术监测ROS，在LoVo和YAMC细胞中检测了这种可能性。重要的是，我们纳入了DLD1-POX，一种稳定转染POX的P53阴性结肠肿瘤细胞株。有趣的是，添加Pro以浓度依赖的方式显著增加ROS的产生，但仅当POX表达时。另一方面，谷氨酸则没有效果。重要的是，Pro-POX效应足以诱导线粒体介导的细胞凋亡。B)作为信号和调节分子的吡咯烷5-羧酸盐(P5C)。吡咯烷5-羧酸(P5C)是由脯氨酸氧化酶、鸟氨酸氨基转移酶和谷氨酸合成酶共同作用的产物，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NADH依赖的酶，被调节来产生脯氨酸，而P5CR2是一种NADPH依赖的酶，与氧化还原相关的调节相耦合。在由凝集素激活的淋巴细胞中，似乎诱导了P5CR2。我们正在定义P5CR2与膜受体的相互作用。 C)胚胎发育中的鸟氨酸转氨酶。鸟氨酸转氨酶(OAT)将鸟氨酸转化为P5C，该酶的遗传缺陷导致神经视网膜退化。以非洲爪哇胚胎为模型，我们发现OAT在神经发育的关键阶段表达。与非洲爪哇OAT基因的原位杂交表明，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确定，燕麦或其产物在调节这一途径中的相互作用已被研究。

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.