Functional assessment of Chaperone Mediated Autophagy during aging in Drosophila

果蝇衰老过程中伴侣介导的自噬的功能评估

• 批准号: 8769895
• 负责人: ANDREAS JENNY
• 金额: $ 20.88万
• 依托单位: ALBERT EINSTEIN COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-09-01 至 2016-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8769895
• 关键词: Address; Age; Aging; Animal Model; Animals; Autophagocytosis; Autophagosome; Bioinformatics; Biosensor; Birds; Caenorhabditis elegans; Cell physiology; Cells; Cellular Stress; Cytoplasm; Data; Disease; Drosophila genus; Drosophila melanogaster; Eukaryota; Fat Body; Genes; Genetic; Genetic Screening; Genome; Health; Homologous Gene; Human; Invertebrates; Lead; Life; Longevity; Lysosomes; Mammalian Cell; Mammals; Mediating; Membrane; Modeling; Molecular Chaperones; Mus; Normal Cell; Nutrient; Organ; Organelles; Organism; Orthologous Gene; Pathway interactions; Phosphoric Monoester Hydrolases; Phosphotransferases; Process; Proteins; RNA Interference; Rattus; Regulation; Reporter; Resistance; Role; Starvation; Stress; System; Testing; Transgenic Organisms; Vesicle; Yeasts; Zebrafish; age related; energy balance; fly; improved; in vivo; knock-down; liver function; multicatalytic endopeptidase complex; mutant; novel; overexpression; prevent; public health relevance; receptor; success

DESCRIPTION (provided by applicant): Proper turnover of organelles and proteins is essential for normal cell function during life. Damaged or altered cytosolic proteins are cleared by the proteasome and autophagy. Importantly, autophagy has the additional role of providing nutrients to cells under stress conditions such as starvation, and is thus essential for energy balance. Not surprisingly, autophagy declines with age, associated with the accumulation of altered, defective components in all model organisms including mice and Drosophila melanogaster. During macroautophagy (MA), membrane structures in the cytoplasm engulf bulk-regions of cytoplasm including organelles in a double membrane vesicle (autophagosome). Autophagosomes fuse to lysosomes leading to the degradation of the engulfed cytoplasm. Most autophagy related genes were originally identified in cellular stress-related genetic screens in yeast and are conserved through the animal kingdom including in Drosophila and humans. Recent genetic screens in C. elegans and Drosophila discovered novel macroautophagy genes not present in yeast but conserved in higher eukaryotes, underscoring the importance of using multicellular organisms to study autophagy. In contrast to MA, less is known about Chaperone Mediated Autophagy (CMA). CMA specifically degrades single proteins that contain a CMA targeting motif (KFERQ related sequences). Substrates containing a KFERQ targeting motif are recognized by the cytoplasmic Hsc70 (and co-chaperones), which then interacts with the lysosomal receptor LAMP-2A, the limiting component of CMA in mammalian cells. To date, LAMP-2A has been identified only in birds and mammalian species including rat, mouse and humans. Due to the inability to identify LAMP-2A by bioinformatics, studies to address whether or not CMA or a CMA-like process exists in other species including zebrafish and invertebrates have not been performed yet. On the other hand, Hsc70, the obligate cytoplasmic chaperone recognizing the KFERQ targeting motif, as well as KFERQ targeting motifs of known CMA substrates are conserved between Drosophila and humans, thus supporting the possibility a CMA-like pathway exists in invertebrates. Consistent with the natural decrease of MA and CMA with age, experimental reduction of autophagy in animal models such as mice or flies leads to reduced organ function and shortened life span. Conversely, overexpressing the central component of CMA, LAMP-2A, prevents natural, age related reduction of CMA and concomitantly rescues the decrease in liver function normally observed in old mice, demonstrating the significance of CMA mediated autophagic clearance of damaged material at old age. The aims of this proposal thus are to functionally test the existence of CMA or a CMA-like process in Drosophila in vivo and to assess its change over the lifespan of flies. To this end we generated transgenic flies expressing an artificial CMA target (KFERQ-PA-GFP). Furthermore, we will systematically knock-down kinases and phosphatases in the Drosophila fat body to identify novel components regulating a potential CMA-like process in vivo, an approach feasible only in a genetically tractable organism with a short life span.

描述（由申请人提供）：细胞器和蛋白质的适当周转对于生命期间的正常细胞功能至关重要。受损或改变的胞浆蛋白由蛋白酶体和自噬清除。重要的是，自噬还具有在饥饿等应激条件下为细胞提供营养的额外作用，因此对于能量平衡至关重要。毫不奇怪，自噬随着年龄的增长而下降，这与包括小鼠和果蝇在内的所有模型生物体中改变的、有缺陷的成分的积累有关。在巨自噬 (MA) 过程中，细胞质中的膜结构吞噬细胞质的大部分区域，包括双膜囊泡（自噬体）中的细胞器。自噬体与溶酶体融合，导致被吞噬的细胞质降解。大多数自噬相关基因最初是在酵母中与细胞应激相关的遗传筛选中发现的，并且在包括果蝇和人类在内的动物界中是保守的。最近对线虫和果蝇的遗传筛选发现了酵母中不存在但在高等真核生物中保守的新型巨自噬基因，强调了使用多细胞生物研究自噬的重要性。与 MA 相比，人们对伴侣介导的自噬 (CMA) 知之甚少。 CMA 特异性降解含有 CMA 靶向基序（KFERQ 相关序列）的单一蛋白质。含有 KFERQ 靶向基序的底物被细胞质 Hsc70（和共伴侣）识别，然后与溶酶体受体 LAMP-2A（哺乳动物细胞中 CMA 的限制成分）相互作用。迄今为止，仅在鸟类和哺乳动物（包括大鼠、小鼠和人类）中发现了 LAMP-2A。由于无法通过生物信息学识别 LAMP-2A，因此尚未进行关于斑马鱼和无脊椎动物等其他物种中是否存在 CMA 或类似 CMA 过程的研究。另一方面，识别 KFERQ 靶向基序的专性细胞质伴侣 Hsc70 以及已知 CMA 底物的 KFERQ 靶向基序在果蝇和人类之间是保守的，因此支持无脊椎动物中存在 CMA 样途径的可能性。与 MA 和 CMA 随着年龄的增长而自然减少一致，在小鼠或果蝇等动物模型中实验性减少自噬会导致器官功能下降和寿命缩短。相反，过度表达 CMA 的核心成分 LAMP-2A，可以防止 CMA 自然的、与年龄相关的减少，同时挽救老年小鼠中通常观察到的肝功能下降，这证明了 CMA 介导的老年受损物质自噬清除的重要性。因此，该提案的目的是在果蝇体内功能测试 CMA 或类似 CMA 的过程的存在，并评估其在果蝇生命周期中的变化。为此，我们生成了表达人工 CMA 靶标 (KFERQ-PA-GFP) 的转基因果蝇。此外，我们将系统地敲低果蝇脂肪体中的激酶和磷酸酶，以确定体内调节潜在的 CMA 样过程的新成分，这种方法仅在寿命较短的遗传易驯化生物体中可行。