Functional assessment of Chaperone Mediated Autophagy during aging in Drosophila

描述（由申请人提供）：在生命中，细胞器和蛋白质的适当周转是正常细胞功能所必需的。受损或改变的细胞质蛋白被蛋白酶体和自噬清除。重要的是，自噬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，这与包括小鼠和黑腹果蝇在内的所有模式生物中改变的、有缺陷的成分的积累有关。在巨噬过程中，细胞质中的膜结构吞噬细胞质的大块区域，包括双膜囊泡中的细胞器（自噬体）。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的细胞应激相关基因筛选中发现的，并在包括果蝇和人类在内的动物王国中保存下来。最近在秀丽隐杆线虫和果蝇的遗传筛选中发现了新的巨噬基因，这些基因在酵母中不存在，但在高等真核生物中保守，强调了利用多细胞生物研究自噬的重要性。与MA相反，对伴侣介导的自噬（CMA）知之甚少。CMA特异性地降解含有CMA靶向基序的单个蛋白质（KFERQ相关序列）。含有KFERQ靶向基元的底物被细胞质Hsc70（和共伴侣）识别，然后与溶酶体受体LAMP-2A相互作用，后者是哺乳动物细胞中CMA的限制性成分。到目前为止，LAMP-2A仅在鸟类和哺乳动物物种中被发现，包括大鼠、小鼠和人类。由于无法通过生物信息学鉴定LAMP-2A，目前还没有研究表明CMA或CMA样过程是否存在于包括斑马鱼和无脊椎动物在内的其他物种中。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，从而支持了无脊椎动物中存在CMA样途径的可能性。与MA和CMA随年龄的自然下降一致，小鼠或苍蝇等动物模型中自噬的实验性减少导致器官功能下降和寿命缩短。相反，过表达CMA的核心成分LAMP-2A，可以阻止CMA自然的、与年龄相关的减少，并同时挽救通常在老年小鼠中观察到的肝功能下降，证明CMA介导的老年损伤物质自噬清除的意义。因此，本提案的目的是在果蝇体内功能性测试CMA或CMA样过程的存在，并评估其在果蝇寿命中的变化。为此，我们培育了表达人工CMA靶点（KFERQ-PA-GFP）的转基因果蝇。此外，我们将系统地敲除果蝇脂肪体中的激酶和磷酸酶，以确定体内调节潜在cma样过程的新成分，这种方法仅在寿命短的遗传易感生物中可行。

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.