Functional assessment of Chaperone Mediated Autophagy during aging in Drosophila

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。受损或改变的胞质蛋白通过蛋白酶体和自噬被清除。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，这与所有模型生物（包括小鼠和黑腹果蝇）中改变的缺陷成分的积累有关。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的细胞应激相关遗传筛选中鉴定的，并且在包括果蝇和人类在内的动物界中是保守的。最近的遗传筛选C。线虫和果蝇发现了酵母中不存在但在高等真核生物中保守的新的大自噬基因，强调了使用多细胞生物研究自噬的重要性。与MA相比，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Hsc 70（和共分子伴侣）识别，然后与溶酶体受体LAMP-2A（哺乳动物细胞中CMA的限制性组分）相互作用。到目前为止，LAMP-2A仅在鸟类和哺乳动物物种（包括大鼠、小鼠和人类）中被鉴定。由于无法通过生物信息学鉴定LAMP-2A，尚未进行关于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的研究。另一方面，Hsc 70，识别KFERQ靶向基序的专性细胞质伴侣，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，从而支持在无脊椎动物中存在CMA样途径的可能性。与MA和CMA随年龄的自然减少一致，在动物模型如小鼠或苍蝇中自噬的实验减少导致器官功能降低和寿命缩短。相反，过表达CMA的中心组分LAMP-2A防止CMA的自然的、年龄相关的减少，并且伴随地挽救了通常在老年小鼠中观察到的肝功能的降低，证明了CMA介导的自噬清除老年时受损物质的重要性。因此，本提案的目的是功能测试CMA或CMA样过程在果蝇体内的存在，并评估其在果蝇寿命的变化。为此，我们产生了表达人工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CMA样过程的新组分，这种方法仅适用于寿命短的遗传易处理生物体。

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.