Mechanisms of selective autophagy

在这项建议中，我们研究了选择性自噬的机制，在一个新的，非正则途径 肾癌的肿瘤抑制活性。自噬是一种严格控制的自我消化过程，在 癌症可以同时具有肿瘤抑制和致癌活性。自噬小体的形成需要 微管相关蛋白1轻链A、B和C(MAP1LC3A、B、C，简称LC3A、B和C)。LC3S 通过受体上的LIR基序与货物受体结合。透明细胞肾 摘要细胞癌(Ccrcc)是最常见的以von hippel-linau基因缺失为特征的肾癌。 (VHL)。VHL功能丧失导致低氧诱导转录因子(HIF)激活和血管内皮细胞 血管生成和代谢。我们的实验室发现VHL调节自噬。VHL抑制LC3B 自噬，这在肾细胞癌中是致癌的。相比之下，VHL诱导肿瘤抑制，LC3C自噬 涉及HIF去除转录抑制的机制。Lc3c是一个进化的晚期基因，目前 仅在高等灵长类动物和人类中进化为多方面的自噬调节因子，更复杂的是 与LC3B/A相比，它保持了与规范LIR的结合位点，与其他LC3类似，但它获得了一个新的 Lc3c特异的LIR，CLIR和一个高度保守的C末端20个氨基酸的结合位点，裂解在 甘氨酸脂化的过程。我们的初步结果表明，LC3c自噬需要新的非规范 预引发复合体，包括ULK3、BECN1、UVRAG和PIK3C2A。我们确认了两个直接 通过LC3C自噬选择性降解的靶点：(I)分割后的中体环(PDMB)，中体残留物 在胞质分裂过程中产生的结构。PDMB在癌细胞中的积聚促进干细胞样状态和癌症 进步。(Ii)小窝蛋白1(CAV1)，一种脂筏胆固醇结合蛋白。我们发现，依赖于LC3C的 货物的降解需要LC3C上的C-末端多肽和包括接头的蛋白质复合体 蛋白质、TSG101和CHMP2B。令人惊讶的是，VHL与LC3C中的核心自噬装置相互作用- 这是一种依赖的方式，表明它直接参与了LC3C自噬小体的形成。在目标1中，我们 将决定LC3C预引发、引发和接头复合体的活性。我们假设LC3c 与LC3B相比，自噬是由不同的和选择性的介体调控的 复合体和TSG101将LC3C锚定在选择性货物上。在目标2中，我们将确定VHL的直接作用 在对LC3c自噬的调控中。我们建议VHL通过以下方式锚定在LC3C监管综合体 C-端肽中的羟基脯氨酸，是与核心自噬蛋白结合所必需的 蛋白质。在目标3中，我们将确定Lc3c的独特结构在货物选择性降解中的作用 通过非典型途径和在肿瘤抑制活性中的LC3C。我们假设这个过程 需要进化中的最新C-端肽。

In this proposal we investigate mechanism of selective autophagy in a novel, noncanonical pathway which has tumor suppressing activity in renal cancer. Autophagy is a tightly regulated process of self-digestion, which in cancer can have both tumor suppressing and oncogenic activities. Formation of an autophagosome requires microtubule associated protein 1 light chains A, B and C (MAP1LC3A, B, C referred to as LC3A, B and C). LC3s bind with cargo receptors through the LC3C-interacting regions (LIR) motifs on the receptors. Clear cell renal cell carcinoma (ccRCC) is the most frequent renal cancer characterized by the loss the von Hippel-Lindau gene (VHL). Loss of VHL function leads to activation of Hypoxia Inducible Transcription Factors (HIF) and changes in angiogenesis and metabolism. Our laboratory discovered that VHL regulates autophagy. VHL inhibits LC3B autophagy, which is oncogenic in ccRCC. In contrast, VHL induces tumor suppressing, LC3C autophagy in a mechanism that involves removal of transcription repression by HIF. LC3C is an evolutionary late gene, present only in higher primates and humans that evolved into multifaceted autophagic regulator, more complicated as compared to LC3B/A. It maintains binding site for the canonical LIRs, similar to other LC3s, but it gained a new binding site for LC3C-specific LIR, CLIR, and a highly conserved C-terminal, 20 amino acid peptide, cleaved in the process of glycine lipidation. Our preliminary results show that LC3C autophagy requires novel non-canonical pre- and -initiation complexes, which include ULK3, BECN1, UVRAG and PIK3C2A. We identified two direct targets selectively degraded by LC3C autophagy: (i) Postdivision Midbody Rings (PDMBs), remnants of midbody structures created during cytokinesis. PDMB accumulation in cancer cells promotes stem-like state and cancer progression. (ii) Caveolin 1(CAV1), a lipid raft cholesterol-binding protein. We found that LC3C-dependent degradation of the cargo requires C-terminal peptide on LC3C and a protein complex that includes adapter proteins, TSG101 and CHMP2B. Surprisingly, VHL interacts with the core autophagy apparatus in an LC3C- dependent manner, an indication that it directly serves in the formation of LC3C autophagosomes. In Aim 1, we will determine activity of the LC3C preinitiation, initiation, and adapter complexes. We hypothesize that LC3C autophagy is regulated by distinctive and selective mediators, as compared to LC3B, of the pre- and initiation complexes and TSG101 anchors LC3C to the selective cargo. In Aim 2 we will determine the direct role of VHL in regulation of LC3C autophagy. We propose that VHL anchors at the LC3C regulatory complex through the hydroxylated proline in the C-terminal peptide and is necessary for the association with the core autophagic proteins. In Aim 3 we will determine the role of unique structures of LC3C in the selective degradation of cargo by the non-canonical pathway and in tumor suppressing activity of LC3C. We hypothesize that this process requires the evolutionary recent C-terminal peptide.