Regulatory mechanisms of lysosomal degradation in neurodegenerative disease

项目摘要 细胞通过激活自噬来应对营养缺乏，自噬是一种受调节的过程， 功能失调的细胞成分，允许蛋白质，糖和蛋白质的有序降解和再循环， 脂质。虽然众所周知，饥饿诱导巨自噬（通常简称为巨自噬）， 自噬），一个涉及形成称为自噬体的双膜结构的过程，其他 自噬途径，例如，内体微自噬，也发生饥饿的组成部分， 这种反应有助于细胞科普营养缺乏引起的压力。作为自噬的关键步骤， 溶酶体中的蛋白质降解对于合成所需的氨基酸的再生至关重要。 支持缺乏营养的细胞存活的必需核心蛋白质。然而，溶酶体降解 对营养缺乏的反应是不清楚的。我们发现了一种新的调控途径 以谷氨酰胺酶水解为中心的溶酶体降解和 铵的在进食条件下，谷氨酰胺的丰度支持铵的产生， 逆转溶酶体腔的碱化，通过保持溶酶体 在次优条件下水解酶。在氨基酸或谷氨酰胺撤出后， 产生后立即引起溶酶体酸化和蛋白质降解加速。我们 进一步发现，饥饿后溶酶体降解的增加是由加速的 通过激活混合谱系激酶结构域样假激酶（MLKL）， 以前主要因其在死亡受体下游的坏死性细胞死亡中的作用而已知的蛋白质， 受体相互作用丝氨酸/苏氨酸蛋白激酶1和3（RIPK 1和RIPK 3）。我们发现饥饿 通过Ca 2 +-钙调蛋白依赖性激酶II（CaMKII）独立于RIPK 3激活MLKL， 寡聚化MLKL通路靶向自噬体，而不是质膜，在那里它 支持吞噬细胞闭合，这是自噬体融合前成熟所需的关键步骤 与溶酶体形成自噬体，在自噬体中发生自噬物质的分解。我们的目标是 定义这种新途径在神经元中的功能意义， 降解，强烈影响神经元细胞的存活和死亡（目的I），并进一步阐明如何多 调节机制协调细胞对氨基酸缺乏的早期反应科普 饥饿的压力（目标二）。由于自噬和溶酶体的重要参与， 在许多类型的神经退行性疾病的功能障碍，这项探索性和基础性的研究， 促进自噬和溶酶体的新的调节机制的早期和概念阶段 监管，将可能导致神经科学的重要领域的突破。

PROJECT SUMMARY Cells respond to nutrient shortage by activating autophagy, regulated processes of removing unnecessary or dysfunctional cellular components that allow orderly degradation and recycling of proteins, sugars, and lipids. While it is well known that starvation induces macroautophagy (often simply referred to as just autophagy), a process involving the formation of double-membraned structure called autophagosomes, other autophagic pathways, e.g., endosomal microautophagy, also occur as integral parts of the starvation response to help the cell cope with the stress caused by the nutrient deficiency. As a key step of autophagy, protein degradation in the lysosomes is crucial for regeneration of amino acids needed for the synthesis of essential core proteins that support survival of nutrient-deprived cells. However, how lysosomal degradation is regulated in response to nutrient deprivation is not clear. We have uncovered a novel regulatory pathway of lysosomal degradation centered around glutamine hydrolysis by glutaminases and the production of ammonium. Under fed conditions, the abundance of glutamine supports the ammonium production and in turn alkalization of the lysosome lumen, which slows down protein degradation by keeping lysosomal hydrolases in suboptimal conditions. Upon amino acid or glutamine withdrawal, the loss of ammonium production immediately causes acidification of the lysosomes and acceleration of protein degradation. We further found that this increase in lysosomal degradation following starvation is facilitated by accelerated autolysosome formation through activation of Mixed Lineage Kinase Domain Like Pseudokinase (MLKL), a protein previously mainly known for its role in necroptotic cell death downstream of death receptors and receptor-interacting serine/threonine-protein kinases 1 and 3 (RIPK1 and RIPK3). We show that starvation activates MLKL through Ca2+-calmodulin-dependent kinase II (CaMKII) independently of RIPK3 and this pathway targets the oligomerized MLKL to autophagosomes, instead of plasma membrane, where it supports phagophore closure, a key step required for the maturation of autophagosomes before they fuse with lysosomes to form autolysosomes where the breakdown of autophagosome cargoes occurs. We aim to define the functional significance of this new pathway in neurons where autophagy, including lysosomal degradation, strongly impacts neuronal cell survival and death (Aim I) and further elucidate how multiple regulatory mechanisms orchestrate the early response of the cells to amino acid shortage in order to cope with the stress of starvation (Aim II). Because of the critical involvement of autophagy and lysosomal dysfunction in many types of neurodegenerative diseases, this exploratory and foundational research, fostering the early and conceptual stages of a novel regulatory mechanism of autophagy and lysosomal regulation, will likely lead to breakthroughs in important areas of neuroscience.