Cellular Mechanisms of Neuronal Metal Transport and Toxicity

描述（由申请人提供）：锌在细胞中起着基础和多种作用，但过量的游离锌与金属细胞毒性有关。为了平衡这些相反的作用，细胞进化出了控制胞质金属浓度的普遍机制。细胞通过将锌挤压到细胞外空间、与细胞质伴侣螯合或在细胞内隔离来实现这一目标。最后一种机制是较少探索的过程，它构成了我们建议的主要焦点。锌在突触生理学以及急性和慢性病理条件中发挥着重要作用，从兴奋毒性到神经退行性疾病特有的淀粉样蛋白聚集体的形成。尽管锌的这些基本作用，我们对细胞内隔室在金属隔离和平衡中的贡献的理解是有限的。在神经元中，细胞器锌通过突触囊泡特异性锌转运体ZnT3的活性储存在突触囊泡（SVs）中。我们分离了一个富含ZnT3的囊泡群体，并对其进行了蛋白质组学分析。在这些囊泡中，我们已经确定了约140个分子靶点，其中一些或上调或下调囊泡内体锌的储存。这些分子提供了一套独特的工具来评估细胞内细胞器，特别是核内体和SV在正常和病理金属稳态中的作用。我们的研究表明，ZnT3转运蛋白的转运功能受其所在的胞室性质的调控。与这一观点一致，我们已经确定了三种控制ZnT3亚细胞定位的靶向机制，这些机制有可能调节ZnT3锌的转运功能。在本提案中，我们将专门探讨这些新的调节范式，以验证内核体特异性锌转运蛋白相互作用调节锌转运蛋白活性和对金属诱导的细胞毒性的抗性的假设。

DESCRIPTION (provided by applicant): Zinc plays fundamental and diverse roles in cells yet excess free zinc is associated with metal cytotoxicity. To balance these opposing effects, cells have evolved universal mechanisms controlling cytoplasmic metal concentration. Cells accomplish this goal by zinc extrusion into the extracellular space, chelation by cytosolic chaperones, or sequestration within intracellular compartments. This last mechanism is the less explored process and it constitutes the main focus of our proposal. Zinc plays fundamental roles in synaptic physiology as well as in acute and chronic pathological conditions, ranging from excitotoxicity to the formation of amyloid aggregates characteristic of neurodegenerative diseases. Despite these fundamental roles of zinc, our understanding of the contribution of intracellular compartment in metal sequestration and homoeostasis is limited. In neurons, organellar zinc is stored in synaptic vesicles (SVs) by the activity of a synaptic vesicle specific zinc transporter, ZnT3. We have isolated and characterized by proteomics a ZnT3- enriched vesicle population. In these vesicles, we have identified ~ 140 molecular targets, several of which either up- or down-regulate vesicular endosomal zinc stores. These molecules provide a unique set of tools to assess the role of intracellular organelles, en particular endosomes and SV, in normal and pathological metal homeostasis. Our studies suggest that ZnT3 transport function are regulated by the nature of the compartment in which the ZnT3 transporter resides. Consistent with this notion, we have identified three targeting mechanisms that control ZnT3 subcellular localization that have the potential to regulate ZnT3 zinc transport function. In this proposal, we will specifically explore these novel regulatory paradigms testing the hypothesis that endosome-specific zinc transporter interactions regulate zinc transporter activity and resistance to metal-induced cytotoxicity.