Regulation of Proline Oxidase for Bioenergetics during Nutrient Stress

我们的特点是调节脯氨酸氧化酶的POX启动子荧光素酶报告构建测试各种转录因子的功能作用。在测试的转录因子中，PPARgamma是最有效的。此外，PPARgamma的药理学配体，即噻唑烷二酮类，通常用于2型糖尿病的药物，进一步增加了POX的诱导。我们发现，PPARgamma结合其响应元件在POX启动子使用电泳迁移率变动分析和染色质免疫沉淀试验。曲格列酮是一种强效噻唑烷二酮，通过PPARgamma依赖性和非依赖性机制诱导POX。后者通过p53间接介导。POX与PPARgamma的偶联强烈表明POX参与生物能量学的调节和对营养胁迫的响应，这一发现使我们考虑mTOR-AMPK信号通路。该途径整合来自生长因子、营养素、能量水平和细胞应激的信号，以调节蛋白质翻译和细胞生长。该途径中的突变与许多肿瘤表型相关。我们测试了雷帕霉素（一种mTOR抑制剂）、LY 294002（一种PI 3-K/Akt抑制剂）和5-氨基-4-甲酰胺呋喃核糖苷（AICAR）（一种激活AMP导向蛋白激酶（AMPK）的嘌呤类似物）的作用。我们发现，这些在3个不同位点阻断mTOR信号传导的药物均显著激活POX活性。此外，雷帕霉素通过阻断mTOR抑制蛋白质翻译和细胞生长，并伴随增加细胞ATP水平，推测是为了维持植物生存状态。有趣的是，通过POX siRNA阻断POX表达或用脱氢脯氨酸抑制POX催化活性显著抑制雷帕霉素诱导的细胞ATP增加。这些研究表明，脯氨酸可以作为一种应激底物的PPARgamma和mTOR/AMPK信号通路的调节下。虽然我们表明，POX表达产生ATP的营养胁迫条件下，ATP的生化来源需要阐明。葡萄糖是培养细胞中ATP的主要来源，因此我们测试了POX过表达是否会增加糖酵解。令人惊讶的是，通过（5）-3H-葡萄糖转化为3 H2O测量的糖酵解不随POX过表达而改变。与此相反，戊糖磷酸分流（PPS）增加超过5倍时，POX诱导。此外，限制葡萄糖（0.05 mM），ATP水平逐渐下降。然而，当POX被诱导时，在存在或不存在添加的脯氨酸的情况下维持ATP水平。推测内源脯氨酸的循环可能介导了这种效应。这些发现表明，当葡萄糖受限时，POX通过PPS促进葡萄糖的代谢，并且脯氨酸的循环将分流产生的NADPH穿梭到ATP产生的还原电位的来源。在营养胁迫条件下，脯氨酸（和羟脯氨酸）的来源是细胞外基质中的胶原蛋白。胶原蛋白是人体中最丰富的蛋白质，胶原蛋白中25%的氨基酸残基是脯氨酸或羟脯氨酸。因此，胶原蛋白的降解将提供脯氨酸和羟脯氨酸作为应激底物。在已发表的使用皮肤肿瘤发生模型的研究中，真皮胶原蛋白迅速减少。在组织培养研究中，我们已经表明引起POX上调的条件与基质金属蛋白酶（MMP 2，MMP-9）表达的增加和细胞内脯氨酸的增加相关。尽管POX水平增加，但脯氨酸的积累表明脯氨酸碳没有被完全氧化。更有可能的是，脯氨酸在磷酸戊糖途径中与葡萄糖代谢连锁循环，使得磷酸戊糖途径产生的还原电位（NADPH）可以作为脯氨酸穿梭进入线粒体以产生ATP

We characterized the regulation of proline oxidase by making a POX promoter-luciferase reporter construct to test the functional role of various transcriptional factors. Among the transcriptional factors tested, PPARgamma was the most potent. Additionally, pharmacologic ligands of PPARgamma, i.e. the thiazolidinediones, commonly used drugs for type 2 diabetes, further increased the induction of POX. We showed that PPARgamma bound to its response element in the POX promoter using electrophoretic mobility shift analysis and chromatin immuno-precipitation assays. Troglitazone, a potent thiazolidinedione, induced POX by PPARgamma-dependent and -independent mechanisms. The latter is mediated indirectly through p53. The coupling of POX to PPARgamma strongly suggests that POX is involved in regulation of bioenergetics and responses to nutrient stress, a finding which led us to consider the mTOR-AMPK signaling pathway. This pathway integrates signals from growth factors, nutrients, energy levels and cellular stress to regulate protein translation and cell growth. Mutations in this pathway have been associated with a number of neoplastic phenotypes. We tested the effects of Rapamycin, an inhibitor of mTOR, LY 294002, an inhibitor of PI3-K/Akt, and 5-amino-4-carboxamide ribofuranoside (AICAR), a purine analog which activates AMP-directed protein kinase (AMPK). We found that these agents which block mTOR signaling at 3 different sites, all markedly activated POX activity. Additionally, Rapamycin by blocking mTOR inhibited protein translation and cell growth and concomitantly increased cellular ATP levels presumably to sustain a vegetative survival state. Interestingly, blockade of POX expression by POX siRNA or inhibiting POX catalytic activity with dehydroproline markedly inhibited the Rapamycin induced increase in cellular ATP. These studies suggest that proline can function as a stress substrate under the regulation of PPARgamma and the mTOR/AMPK signaling pathways. Although we showed that POX expression generated ATP under conditions of nutrient stress, the biochemical source for the ATP required elucidation. Glucose is the main source for ATP in cultured cells; therefore we tested whether glycolysis was increased by POX overexpression. Surprisingly, glycolysis measured by the conversion of (5)-3H-glucose to 3H2O was not changed with POX overexpression. In contrast, the pentose phosphate shunt (PPS) was increased more than 5-fold when POX was induced. Furthermore, with limiting glucose (.05 mM), ATP levels progressively fell. However, when POX was induced, ATP levels were maintained in the presence or absence of added proline. Presumably, the cycling of endogenous proline could mediate the effect. These findings suggest that when glucose is limiting, POX promotes the metabolism of glucose through the PPS and the cycling of proline shuttles the NADPH generated from the shunt into a source of reducing potential for ATP generation. The source of the proline (and hydroxyproline) under conditions of nutrient stress is collagen in extracellular matrix. Collagen is the most abundant (by mass) protein in the body and 25% of the amino acid residues in collagen is either proline or hydroxyproline. Thus, degradation of collagen would provide proline and hydroxyproline as stress substrates. In published studies using the skin tumorigenesis model, dermal collagen is rapidly decreased. In tissue culture studies we have shown that the conditions which cause upregulation of POX are correlated with an increase in expression of matrix metalloproteinases (MMP2, MMP-9) and an increase in intracellular proline. The accumulation of proline in spite of an increased level of POX suggests that proline carbons are not totally oxidized. More likely, proline is cycling in a metabolic interlock with glucose metabolism in the pentose phosphate pathway so that the reducing potential (NADPH) generated by the pentose phosphate pathway can be shuttled into mitochondria as proline to generate ATP