Cellular Mechanisms of Neuronal Metal Transport and Toxicity

说明(申请人提供)：锌在细胞中起着基本的和不同的作用，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影响，细胞进化出控制细胞质金属浓度的普遍机制。细胞通过锌排入细胞外空间、胞液伴侣蛋白的螯合或细胞内隔膜来实现这一目标。最后这一机制是较少被探讨的进程，它构成了我们提案的主要重点。锌在突触生理学以及急性和慢性病理条件下发挥着重要作用，从兴奋性毒性到神经退行性疾病所特有的淀粉样聚集体的形成。尽管锌有这些基本的作用，但我们对细胞内隔室在金属隔离和稳态中的作用的了解是有限的。在神经元中，细胞器锌通过突触小泡特异性锌转运体--锌转运蛋白3的活性储存在突触小泡(SVS)中。我们分离了富含锌的囊泡，并用蛋白质组学对其进行了鉴定。在这些囊泡中，我们已经确定了大约140个分子靶点，其中几个分子靶点上调或下调囊泡内体锌储存。这些分子提供了一套独特的工具来评估细胞内细胞器，尤其是内小体和SV在正常和病理性金属动态平衡中的作用。我们的研究表明，锌转运蛋白的转运功能受锌转运蛋白所在隔室的性质调节。与这一概念一致，我们已经确定了三种靶向机制，它们控制着ZnT3亚细胞的定位，有可能调节ZnT3的锌转运功能。在这个提案中，我们将专门探索这些新的调控范例，以检验内体特异性锌转运体相互作用调节锌转运体活性和对金属诱导的细胞毒性的抵抗力的假设。

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.