Imidodipeptides and Amino Acid Metabolites in Cell Regul

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。我们正在从以下水平研究这一代谢途径：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还原酶在细胞信号转导中的作用；c)Pro酶在细胞生理中的作用。 A)脯氨酸氧化酶--其诱导作用及其在细胞凋亡中的作用。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细胞凋亡的作用。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。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。最后，我们正在研究这一代谢途径在低氧反应中的作用，已知的低氧反应是由HIF-1α的Pro-羟化调节的。P5C是HIF-1α-Pro羟化酶的抑制剂，POX的表达激活了HIF-1α与其反应元件的结合。综上所述，这些研究表明，脯氨酸和吡咯烷-5-羧酸可能在发育、增殖和凋亡的代谢信号中起重要作用。 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)镍对脯氨酸酶的抑制作用。脯氨酸酯酶是一种金属酶，它能与羧基末端的脯氨酸一起水解亚胺二肽。镍可导致人类的肺纤维化和癌症，是一种有效的脯氨酸酶活性抑制剂。动力学研究表明，镍是与锰相互作用的竞争性抑制剂，锰是最有效的催化活性活化剂。这种抑制作用不仅可以在无细胞系统中表现出来，也可以在完整的CHO-K1细胞中表现出来，CHO-K1细胞是脯氨酸营养缺陷体，而且可以在GLY-PRO上生长。镍对这些细胞在GLY-PRO上的生长有明显的抑制作用，但对PRO上的生长无明显影响。我们正在利用这些细胞来干扰亚胺二肽和其他含有脯氨酸的多肽的处理，以研究它们在细胞信号转导中的潜在作用。

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) the function of prolidase in cell physiology. 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. 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. 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. Finally, we are investigating the role for this metabolic pathway in the hypoxic response which is known to be regulated by the prolyl hydroxylation of HIF-1 alpha. P5C is an inhibitor of HIF-1 alpha prolyl hydroxylase and expression of POX activates the binding of HIF-1 alpha to its response element. In summary, these studies show that proline and pyrroline-5-carboxylate may be important in metabolic signaling in development, proliferation, and apoptosis. 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 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) Inhibition of prolidase by nickel. Prolidase hydrolyzes imidodipeptides with carboxyl terminus proline and is a metalloenzyme. Nickel which can cause pulmonary fibrosis and cancer in humans, is a potent inhibitor of prolidase activity. Kinetic studies show that nickel is a competitive inhibitor of the interaction with manganese, the most potent activator of catalytic activity. The inhibitory effect can be demonstrated not only in cell-free systems, but also in intact CHO-K1 cells which are proline auxotrophs but which also can grow on GLY-PRO. Nickel markedly inhibits growth of these cells on GLY-PRO but not on PRO. We are using these cells to perturb the processing of imidodipeptides and other proline containing peptides to investigate their potential role in cell signaling.