Tumor suppressing pathways in renal cancer

肾透明细胞癌(Ccrcc)是军事人员，特别是男性以外的严重健康问题。 40岁，包括退伍军人。根据1995-2004年国防医学流行病学数据库 针对40岁以上军人的肾癌发病率急剧增加到8.5%。 相比之下，每10万人年的总发病率为1.5例。该提案直接调查 肾癌背景下选择性自噬和葡萄糖代谢之间的机制联系 细胞。自噬是一个严格控制的自我消化过程。自噬小体的形成需要 微管相关蛋白1轻链A、B和C的脂基化和插入(MAP1LC3A、B、C，参考文献 LC3A、LC3B和LC3C3)进入自噬体膜。我们确定了LC3C自噬是 VHL下游的肿瘤抑制和功能，肿瘤抑制因子在肾透明细胞癌中缺失。 Lc3c是一种进化晚期基因，只存在于高等灵长类动物和人类中，它包含一种独特的和 保守的C-末端20个氨基酸，在启动LC3C自噬过程中被切割。C端子 LC3c的多肽有一个类似于低氧诱导因子αS的典型基序的Pro羟化基序，其中Pro是 羟化由2-羟基戊二酸(2OG)依赖的EGLN脯氨酸羟基酶。我们的初步数据显示 LC3C肽中的P133在自噬过程中被EGLN3 Pro羟基酶羟化。 依赖的态度。最近我们发现，LC3C自噬需要葡萄糖代谢流。反过来，损失 增加糖酵解和磷酸戊糖代谢产物的稳态水平，代表 致癌代谢形式的特征(Warburg效应)与ccRCC特别相关。我们决定 自溶体降解苹果酸/2-羟基戊二酸(2OG)的免疫共沉淀物和靶标 天冬氨酸/谷氨酸逆向转运蛋白SCL25A11和SLC25A13分别是线粒体的一部分 苹果酸-天冬氨酸穿梭机(MAS)。MAS在线粒体和细胞质之间转移还原等价物， 产生线粒体NADH用于ATP合成，同时产生胞质NAD以维持糖酵解。另外， 航天飞机交换谷氨酸和天冬氨酸，这有助于生物合成潜力。我们提出了一部小说， 肿瘤抑制LC3C活性的代谢偶联机制：LC3C自噬靶向MAS蛋白 溶酶体在有丝分裂过程中的降解。这是糖酵解的检查点，通过调节 胞浆NAD/NADH比率，以及SLC25A13介导的天冬氨酸从线粒体输出。那 提示Lc3c在代谢上与VHL抑制糖酵解的转录效应相结合。此外，我们 假设在线粒体携带者附近选择性激活LC3C自噬是由 2OG通过TCA循环从葡萄糖中提取并通过SLC25A11转运，SLC25A11激活 EGLN3，导致P133羟化。在这里，我们将机械地研究代谢输入和功能 在肾癌的背景下，关于MAS的LC3C自噬的结果。目标1将决定LC3C的角色 SLC25A11/13自噬降解中的结构元素及其对MAS的功能影响 活动。我们假设LC3C端多肽和P133对此是必要的，并且可能是足够的 活动。目的2鉴定LC3C抑瘤活性的代谢效应。我们假设LC3C将 (I)抑制NAD/NADH再生和糖酵解活性；(Ii)抑制线粒体天冬氨酸的输出 以及它对生物合成途径的可用性。目标3将确定葡萄糖的分子机制 监管LC3C的活动。我们认为，LC3C自噬是通过检测线粒体20G水平来激活的 通过依赖于EGLN3的C端肽中P133的羟基化。EGLN3-LC3C使用的2OG是 来源于葡萄糖，并通过SLC25A11退出线粒体，表明葡萄糖氧化是 LC3C自噬活动。

Clear cell renal cell carcinoma (ccRCC) is a serious health concern for military personnel, particularly males beyond 40 years of age, including military veterans. According to The Defense Medical Epidemiology Database for 1995-2004 the incidence of RCC specifically for military members after the 4th decade of life is dramatically increased to 8.5 as compared to 1.5 cases per 100,000 person-years of the overall incidence. This proposal investigates direct mechanistic connection between selective autophagy and glucose metabolism in the context of renal cancer cells. Autophagy is a tightly regulated process of self-digestion. Formation of an autophagosome requires lipidation and insertion of microtubule associated protein 1 light chains A, B and C (MAP1LC3A, B, C, referred to as LC3A, LC3B, and LC3C) into the autophagosomal membrane. We established that LC3C autophagy is tumor suppressing and functions downstream form VHL, tumor suppressor lost in clear cell renal cell carcinoma. LC3C is an evolutionary late gene, present only in higher primates and humans, that contains a unique and conserved C-terminal 20 amino acid peptide that is cleaved during initiation of LC3C autophagy. The C-terminal peptide of LC3C has a proline hydroxylation motif similar to the canonical motifs in HIFαs, where prolines are hydroxylated by 2-oxyglutarate (2OG)-dependent EGLN proline hydroxylases. Our preliminary data indicate that P133 within the LC3C peptide undergoes hydroxylation by EGLN3 proline hydroxylase in an autophagy- dependent manner. Recently we discovered that LC3C autophagy requires glucose metabolic flux. In turn, loss of LC3C increases the steady-state levels of glycolytic and pentose phosphate metabolites, representing hallmarks of oncogenic form of metabolism (Warburg effect) particularly relevant in ccRCC. We determined that LC3C co-immunoprecipitates and targets for autolysosomal degradation malate/2-oxyglutarate(2OG) and aspartate /glutamate antiporters, SCL25A11 and SLC25A13, respectively that are part of the mitochondrial malate-aspartate shuttle (MAS). MAS transfers reducing equivalents between mitochondria and cytoplasm, yielding mitochondrial NADH for ATP synthesis while generating cytosolic NAD to sustain glycolysis. Additionally, the shuttle exchanges glutamate and aspartate that contributes to biosynthetic potential. We propose a novel, metabolism-coupled mechanism of tumor suppressing LC3C activity: LC3C autophagy targets MAS proteins for lysosomal degradation in the process of mitophagy. This acts as a checkpoint for glycolysis by regulating cytosolic NAD/NADH ratio, as well as for SLC25A13-mediated export of aspartate from mitochondria. That indicates that LC3C metabolically partners with transcriptional effects of VHL inhibiting glycolysis. Moreover, we hypothesize that selective activation of LC3C autophagy in the proximity to mitochondrial carriers is caused by 2OG derived from glucose through the TCA cycle and transported through the SLC25A11 which activates EGLN3, leading to P133 hydroxylation. Here we will mechanistically investigate metabolic inputs and functional output of LC3C autophagy in regard to MAS in the context of renal cancer. Aim 1 will determine role of LC3C structural elements in the autophagic degradation of SLC25A11/13 and in functional consequences for MAS activity. We hypothesize that LC3C C-terminal peptide and P133 are necessary and potentially sufficient for this activity. Aim 2 will identify metabolic effects of LC3C tumor suppressing activity. We hypothesize that LC3C will (i) inhibit NAD/NADH regeneration and glycolytic activity and (ii) suppress export of aspartate from mitochondria and its availability for biosynthetic pathways. Aim 3 will determine molecular mechanism by which glucose regulates LC3C activity. We propose that LC3C autophagy is activated by sensing mitochondrial 2OG levels through EGLN3-dependent hydroxylation of P133 in the C-terminal peptide. 2OG utilized by EGLN3-LC3C is derived from glucose and exits mitochondria through SLC25A11, indicating that glucose oxidation is critical for LC3C autophagic activity.