Mechanisms of selective autophagy

选择性自噬机制

基本信息

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
项目类别：
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

项目摘要

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.

在本提案中，我们研究了一种新颖的非典型途径中的选择性自噬机制，该途径具有肾癌的肿瘤抑制活性。自噬是一个严格调控的自我消化过程，癌症可以同时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（MAP1LC3A、B、C简称LC3A、B和C）。 LC3 通过受体上的 LC3C 相互作用区 (LIR) 基序与货物受体结合。肾透明细胞细胞癌 (ccRCC) 是最常见的肾癌，其特征是 von Hippel-Lindau 基因缺失（VHL）。 VHL 功能的丧失会导致缺氧诱导转录因子 (HIF) 的激活以及血管生成和代谢。我们实验室发现VHL调节自噬。 VHL 抑制 LC3B 自噬，在 ccRCC 中具有致癌作用。相比之下，VHL 在肿瘤抑制中诱导 LC3C 自噬涉及消除 HIF 转录抑制的机制。 LC3C是一个进化晚期基因，存在仅在高等灵长类动物和人类中进化成多方面的自噬调节因子，更复杂的是与 LC3B/A 相比。与其他 LC3 类似，它保留了规范 LIR 的结合位点，但它获得了新的 LC3C 特异性 LIR、CLIR 和高度保守的 C 端 20 个氨基酸肽的结合位点，在甘氨酸脂化过程。我们的初步结果表明 LC3C 自噬需要新颖的非规范预起始复合物和起始复合物，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) 分裂后中体环 (PDMB)、中体残余物胞质分裂过程中产生的结构。 PDMB 在癌细胞中的积累促进干细胞样状态和癌症进展。 (ii) Caveolin 1 (CAV1)，一种脂筏胆固醇结合蛋白。我们发现 LC3C 依赖货物的降解需要 LC3C 上的 C 端肽和包含接头的蛋白质复合物蛋白质、TSG101 和 CHMP2B。令人惊讶的是，VHL 与 LC3C- 中的核心自噬装置相互作用依赖性方式，表明它直接参与 LC3C 自噬体的形成。在目标 1 中，我们将确定 LC3C 预引发、引发和接头复合物的活性。我们假设 LC3C 与 LC3B 相比，自噬受到独特且选择性的介质的调节。复合物和 TSG101 将 LC3C 锚定到选择性货物上。在目标 2 中，我们将确定 VHL 的直接作用 LC3C 自噬的调节。我们建议 VHL 通过以下方式锚定于 LC3C 监管复合体： C 端肽中的羟基化脯氨酸对于与核心自噬的关联是必需的蛋白质。在目标 3 中，我们将确定 LC3C 独特结构在货物选择性降解中的作用通过非经典途径和 LC3C 的肿瘤抑制活性。我们假设这个过程需要进化最近的C端肽。