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.

在这个提议中，我们研究了一种新的非经典途径中的选择性自噬机制， 肾癌的肿瘤抑制活性。自噬是一种严格调控的自我消化过程， 癌症可以具有肿瘤抑制和致癌活性。自噬体的形成需要 微管相关蛋白1轻链A、B和C（MAP 1 LC 3 A、B、C称为LC 3 A、B和C）。LC 3s 通过受体上的LC 3C相互作用区（LIR）基序与货物受体结合。透明细胞肾 细胞癌（ccRCC）是最常见的肾癌，其特征在于von Hippel-Lindau基因的丢失 （VHL）. VHL功能的丧失导致缺氧诱导转录因子（HIF）的激活和VHL基因表达的改变。 血管生成和代谢。我们的实验室发现VHL调节自噬。VHL抑制LC 3B 自噬，其在ccRCC中是致癌的。相比之下，VHL诱导肿瘤抑制，LC 3C自噬在一个细胞中， 这一机制涉及通过HIF消除转录抑制。LC 3C是一个进化晚期基因，存在于 只有在高等灵长类动物和人类进化成多方面的自噬调节器，更复杂的是， 与LC 3B/A相比。与其他LC 3类似，它保留了典型LIR的结合位点，但它获得了一个新的 LC 3C特异性LIR、CLIR的结合位点和高度保守的C末端20个氨基酸肽，在 甘氨酸脂化的过程。我们的初步结果表明，LC 3C自噬需要新的非经典的 前-和-起始复合物，其包括ULK 3、BECN 1、UVRAG和PIK 3C 2A。我们发现了两个 通过LC 3C自噬选择性降解的靶标：（i）分裂后中间体环（PDMB），中间体的残余物 细胞质分裂过程中产生的结构。癌细胞中PDMB的积累促进干细胞样状态和癌症 进展(ii)Caveolin 1（CAV 1）是一种脂筏胆固醇结合蛋白。我们发现LC 3C依赖性 货物的降解需要LC 3C上的C-末端肽和包括衔接子的蛋白质复合物 蛋白质，TSG 101和CHMP 2B。令人惊讶的是，VHL与LC 3C-3中的核心自噬装置相互作用。 依赖性的方式，这表明它直接在LC 3C自噬体的形成。目标1： 将决定LC 3C预起始、起始和衔接子复合物的活性。我们假设LC 3C 与LC 3B相比，自噬由独特的和选择性的介体调节， TSG 101将LC 3C锚定至选择性货物。在目标2中，我们将确定VHL的直接作用 LC 3C自噬的调控。我们认为，VHL锚定在LC 3C调控复合物，通过 羟化脯氨酸的C-末端肽，是必要的协会与核心自噬 proteins.在目标3中，我们将确定LC 3C的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LC 3C的肿瘤抑制活性。我们假设这个过程 需要进化最近的C末端肽。