Mechanisms of selective autophagy

选择性自噬机制

• 批准号: 10240490
• 负责人: Maria F Czyzyk-Krzeska
• 金额: $ 32.1万
• 依托单位: UNIVERSITY OF CINCINNATI
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-12 至 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10240490
• 关键词: Adaptor Signaling Protein; Amino Acids; Autophagocytosis; Autophagosome; Binding; Binding Sites; C-terminal; Cells; Chromosomes; Clear cell renal cell carcinoma; Cleaved cell; Complex; Cytokinesis; Data; Digestion; Event; Excision; Genes; Glycine; Human; Hydroxylation; Hypoxia; Kidney; Laboratories; Light; Lipids; MAP1 Microtubule-Associated Protein; Malignant Neoplasms; Mediating; Mediator of activation protein; Membrane; Membrane Microdomains; Metabolism; Modeling; Molecular; Oncogenic; Pathology; Pathway interactions; Peptides; Primates; Process; Proline; Proteins; Regulation; Renal carcinoma; Role; Sorting - Cell Movement; Structure; TSG101 gene; Tumor Suppression; Tumor Suppressor Genes; Tumor Suppressor Proteins; VHL gene; Work; angiogenesis; cancer cell; cancer therapy; caveolin 1; cholesterol-binding protein; gene function; gene repression; insight; member; novel; programs; protein complex; receptor; recruit; stem; theories; transcription factor; tumor; tumor progression

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.

在此提案中，我们研究了一种具有选择性自噬的机制 肿瘤抑制肾癌活性。自噬是一个严格调节的自我消化过程，在 癌症既可以抑制肿瘤和致癌活性。自噬体的形成需要 微管相关蛋白1光链A，B和C（MAP1LC3A，B，C称为LC3A，B和C）。 LC3S 通过受体上的LC3C相互作用区域（LIR）与货物受体结合。透明细胞肾脏 细胞癌（CCRCC）是最常见的肾癌，其特征是损失von hippel-lindau基因 （VHL）。 VHL功能的丧失导致缺氧诱导转录因子（HIF）激活和变化 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，在CCRCC中具有致癌性。相反，VHL诱导抑制肿瘤，LC3C自噬在A 涉及HIF去除转录抑制的机制。 LC3C是一个进化的晚期基因，存在 只有在多方面的自噬调节剂中演变成多方面的高级灵长类动物和人类中，更复杂 与LC3B/A相比。它维护着规范LIR的绑定位点，类似于其他LC3，但获得了新的。 LC3C特异性LIR，CLIR和高度保守的C末端，20氨基酸肽的结合位点，裂解 甘氨酸脂质的过程。我们的初步结果表明，LC3C自噬需要新颖的非典型 前和 - 启动复合物，包括ULK3，BECN1，UVRAG和PIK3C2A。我们确定了两个直接 LC3C自噬选择性降解的靶标：（i）分区后中体戒指（PDMBS），中体残留物 在细胞因子过程中产生的结构。 PDMB在癌细胞中的积累促进了干状状态和癌症 进展。 （ii）小窝蛋白1（CAV1），一种脂质筏胆固醇结合蛋白。我们发现LC3C依赖性 货物的降解需要在LC3C上进行C末端肽和包括适配器的蛋白质复合物 蛋白质，TSG101和CHMP2B。令人惊讶的是，VHL与LC3C-中的核心自噬设备相互作用 依赖方式，这表明它直接用于LC3C自噬体的形成。在AIM 1中，我们 将确定LC3C预处理，起始和适配器复合物的活性。我们假设LC3C 与LC3B相比，自噬受到独特和选择性介体的调节 复合物和TSG101将LC3C固定在选择性货物上。在AIM 2中，我们将确定VHL的直接作用 在LC3C自噬的调节中。我们建议VHL通过 C末端肽中的羟基化脯氨酸是与核心自噬的关联所必需的 蛋白质。在AIM 3中，我们将确定LC3C独特结构在货物的选择性降解中的作用 通过非经典途径和LC3C抑制肿瘤活性。我们假设这个过程 需要进化最近的C末端肽。