Uncovering kinase cascade mechanisms that target organelles for destruction by selective autophagy

揭示通过选择性自噬破坏靶向细胞器的激酶级联机制

• 批准号: 9215087
• 负责人: Vladimir Denic
• 金额: $ 32.87万
• 依托单位: HARVARD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-04-01 至 2021-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9215087
• 关键词: ATP phosphohydrolase; Address; Animal Model; Autophagocytosis; Autophagosome; Binding; Biochemical; Biogenesis; Biological; Biological Assay; CRISPR interference; Candidate Disease Gene; Case Study; Cell Extracts; Cells; Complex; Cytoplasm; Decision Making; Defect; Drug Targeting; Encapsulated; Engineering; Eukaryotic Cell; Fluorescence; Fluorescence Microscopy; Gene Expression; Genes; Genetic; Genetic Screening; Genetic study; Goals; Grant; Guide RNA; Health; Human; Hypoxia; Integral Membrane Protein; K562 Cells; Lysosomes; Mammalian Cell; Measures; Medical; Membrane; Modeling; Nerve Degeneration; Organelles; Pathway interactions; Patients; Phosphorylation; Phosphotransferases; Process; Protein Kinase; Proteins; Publishing; RNA library; Regulation; Reporter; Role; Saccharomyces cerevisiae; Saccharomycetales; Scaffolding Protein; Signal Transduction; Starvation; Structure; Substrate Specificity; Testing; Time; Vesicle; Vesicle Transport Pathway; Viral; Work; Yeasts; casein kinase I; chemical genetics; comparative; fluorescence imaging; follow-up; genetic analysis; genetic approach; high throughput screening; human disease; improved; in vitro Assay; insight; mutant; novel; peroxisome; peroxisome membrane; protein aggregate; public health relevance; receptor; receptor binding; receptor function; scaffold

PROJECT SUMMARY/ABSTRACT Macroautophagy (hereafter autophagy) is an umbrella term for vesicle transport pathways that target cytoplasmic components to the lysosome for destruction. There are two classes of autophagy: nonselective and selective. During nonselective autophagy, bulk cytoplasm gets packaged into specialized vesicles, called autophagosomes, which fuse with the lysosome to degrade their encapsulated contents. By contrast, during selective autophagy, specific toxic structures – such as ubiquitinated protein aggregates – are recognized by autophagy receptors, which also interact with the nascent autophagosome membrane to enable vesicle engulfment of the receptor-target complex. In a recently published work, we discovered that autophagy receptors also have an unanticipated regulatory role as activators of the dedicated kinase that controls initiation of autophagosome formation. This work fits nicely with other studies showing that receptor activity is not constitutive but rather directly controlled by general kinases. To gain further insights into receptor function, we have hypothesized that receptors enable transduction of selective autophagy signals down kinase cascades – from general kinases to the dedicated autophagy kinase – to make decisions about the destruction of selective autophagy targets – which specific target should be destroyed and when? This proposal uses selective autophagy of peroxisomes (pexophagy) to build a case study for the kinase signal cascade hypothesis. The bulk of the proposed work will be performed using budding yeast because this model organism has facile genetics, is tractable biochemically, and accessible cell biologically to imaging by fluorescence microscopy. The remainder of the work comprises pioneering genetic screens in mammalian cells for pexophagy mutants. Our strong track record in doing follow- up mechanistic work on the GET pathway components, which were identified originally by yeast high- throughput screens, will help guide us from screen hits towards new mammalian pexophagy components and mechanistic insights that are missing from the current picture. This latter work also has direct medical relevance because in certain Zellweger Spectrum patients, peroxisomes can form normally, but are degraded by autophagy.

项目总结/摘要 巨自噬（以下简称自噬）是一个总括性术语，囊泡转运途径，靶向 将细胞质成分转移到溶酶体进行破坏。自噬有两种类型：非选择性自噬 而且有选择性。在非选择性自噬过程中，大量的细胞质被包装成专门的小泡，称为 自噬体，其与溶酶体融合以降解其包封的内容物。相比之下， 在选择性自噬中，特异性毒性结构-如泛素化蛋白质聚集体-被 自噬受体，也与新生的自噬体膜相互作用，使囊泡 吞噬受体-靶复合物。 在最近发表的一项研究中，我们发现自噬受体也有一种意想不到的调节作用， 作为控制自噬体形成起始的专用激酶的激活剂的作用。这项工作符合 与其他研究表明受体活性不是组成性的，而是直接受 一般激酶为了进一步了解受体功能，我们假设受体能够 选择性自噬信号的转导激酶级联-从一般激酶到专用激酶 自噬激酶-决定选择性自噬靶点的破坏-哪些特定的 目标应该在何时被摧毁该提案使用过氧化物酶体的选择性自噬（pexophagy）， 为激酶信号级联假说建立一个案例研究。大部分拟议的工作将在 使用芽殖酵母，因为这种模式生物具有简单的遗传学，在生物化学上易于处理， 生物学上可接近的细胞通过荧光显微镜成像。其余工作包括 在哺乳动物细胞中进行食粪突变体的遗传筛选的先驱。我们在跟踪方面的良好记录- 在GET途径组分上进行了机械工作，这些组分最初由酵母高- 通量筛选，将有助于指导我们从筛选命中到新的哺乳动物pexophagy组件， 机械的洞察力，这是从目前的图片失踪。这后一项工作也有直接的医疗 相关性，因为在某些Zellweger Spectrum患者中，过氧化物酶体可以正常形成，但被降解 通过自噬。