Regulation of Proline Oxidase for Bioenergetics during Nutrient Stress

我们通过构建痘启动子-荧光素酶报告基因结构来表征脯氨酸氧化酶的调控，以测试各种转录因子的功能作用。在所检测的转录因子中，PPARgamma是最有效的。此外，PPARgamma的药理学配体，即2型糖尿病常用的噻唑烷二酮类药物，进一步增加了POX的诱导。我们通过电泳迁移率转移分析和染色质免疫沉淀分析发现，PPARgamma与痘启动子中的应答元件结合。曲格列酮是一种有效的噻唑烷二酮类药物，通过依赖和不依赖的机制诱导痘。后者通过p53间接介导。POX与pppargamma的偶联强烈提示POX参与了生物能量学的调节和对营养胁迫的反应，这一发现促使我们考虑mTOR-AMPK信号通路。该通路整合来自生长因子、营养物质、能量水平和细胞应激的信号，调节蛋白质翻译和细胞生长。这一途径的突变与许多肿瘤表型有关。我们测试了Rapamycin （mTOR抑制剂）、LY 294002 （PI3-K/Akt抑制剂）和5-氨基-4-carboxamide核糖呋喃苷（AICAR）（激活AMP-directed protein kinase （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信号的药物都显著激活了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，同时增加细胞ATP水平，可能维持植物生存状态。有趣的是，用痘siRNA阻断痘表达或用脱氢脯氨酸抑制痘的催化活性可显著抑制雷帕霉素诱导的细胞ATP的增加。这些研究表明脯氨酸可以在PPARgamma和mTOR/AMPK信号通路的调控下作为胁迫底物发挥作用。虽然我们发现在营养胁迫条件下，痘表达产生ATP，但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，我们检测了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3H2O测量的糖酵解并没有随着POX过表达而改变。与此相反，经痘诱导后，戊糖磷酸分流（PPS）增加了5倍以上。此外，限制葡萄糖(。0.05 mM)， ATP水平逐渐下降。然而，当诱导痘时，ATP水平在添加脯氨酸或不添加脯氨酸的情况下保持不变。据推测，内源性脯氨酸的循环可能介导了这种效应。这些发现表明，当葡萄糖限制时，POX通过PPS促进葡萄糖代谢，而脯氨酸的循环将从分流产生的NADPH转移到减少ATP生成的潜在来源。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。胶原蛋白是人体中含量最多的蛋白质，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，导致痘上调的条件与基质金属蛋白酶（MMP2, MMP-9）的表达增加和细胞内脯氨酸的增加有关。尽管POX水平升高，但脯氨酸的积累表明脯氨酸碳并未完全氧化。更有可能的是，在戊糖磷酸途径中，脯氨酸与葡萄糖代谢在代谢联锁中循环，因此戊糖磷酸途径产生的还原电位（NADPH）可以作为脯氨酸穿梭到线粒体中产生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