Intracellular Pathways of manganese Trafficking

描述(由申请人提供)：锰是一种基本的营养物质，也可能是有毒的，接触环境中的锰会导致神经毒性。尽管它对公众健康很重要，但人们对锰的动态平衡知之甚少。该计划的长期目标是利用面包师酵母作为真核生物的模型，以了解锰的代谢和毒性。目前的提案涉及锰生物学的三个不同但相关的方面：(目标1)在锰胁迫期间对锰的感知；(目标2)作为锰毒性的关键决定因素的磷酸盐；(目标3)线粒体铁对与抗氧化酶超氧化物歧化酶2(SOD2)结合的锰的影响。在目标1中，“锰胁迫”被定义为锰饥饿或锰中毒的极端条件。初步研究表明，对这两种压力的锰感应发生在不同的细胞隔间，但在这两种情况下，细胞通过翻译后控制锰转运体Smf1p进行反应。拟议的研究将检验Smf1p本身是锰的传感器的假设，并将研究Smf1p的定位和表达受锰胁迫控制的机制。对于目标2，高的胞内磷酸盐被发现对酵母造成严重的锰毒性，这种毒性通过染色质重塑因子被逆转。可能的表观遗传效应将通过酵母遗传筛选和转录图谱研究来探索。此外，在专家合作者的帮助下，将通过31P-核磁共振和Endor光谱分析获得伴随锰毒性的细胞内锰-磷相互作用的概况。最后，目标3探索了一个专门的线粒体铁池是如何与锰竞争结合到SOD2上的。分子遗传学实验将验证这一假说，即这种SOD2活性铁来自铁-S生物发生途径，并通过抑制锰与SOD2的结合，在铁超载疾病中促进线粒体损伤。SOD2活性铁的性质将通过线粒体亚铁的比色分析和金属配位环境的XANES分析(由我们的合作者J.Penner-Hahn)进行评估。对SOD2活性铁的这种研究将为了解SOD2通常如何选择锰提供重要线索。总体而言，这种理解锰离子生物学的多学科方法应该为有毒营养物质的动态平衡提供新的见解。

DESCRIPTION (provided by applicant): Manganese is an essential nutrient that can also be toxic, and exposure to environmental manganese can cause neurotoxicity. In spite of its public health importance, little is known regarding manganese homeostasis. The long-term goals of this program are to employ bakers' yeast as a model eukaryote to understand manganese metabolism and toxicity. The current proposal addresses three distinct but related aspects of manganese biology: (Aim 1) the sensing of manganese during manganese stress; (Aim 2) phosphate as a key determinant of manganese toxicity; and (Aim 3) the impact of mitochondrial iron on manganese binding to the anti-oxidant enzyme superoxide dismutase 2 (SOD2). In Aim 1, "manganese stress" is defined as extreme conditions of manganese starvation or manganese toxicity. Preliminary studies indicate that manganese sensing for these two stresses occurs in separate cell compartments, but in both cases, cells respond through post-translational control of a manganese transporter, Smf1p. Proposed studies will test the hypothesis that Smf1p itself is a sensor for manganese and will examine the mechanism by which Smf1p localization and expression are controlled by manganese stress. Regarding Aim 2, high intracellular phosphate was found to cause profound manganese toxicity in yeast, and this toxicity was reversed through chromatin remodeling factors. The possible epigenetic effects will be probed through yeast genetic screens and transcription profiling studies. Additionally, the profile of manganese-phosphate interactions inside the cell that accompany manganese toxicity will be obtained through 31P-NMR and ENDOR spectroscopy with help from expert collaborators. Finally, Aim 3 explores how a specialized pool of mitochondrial iron competes with manganese for binding to SOD2. Molecular genetics experiments will test the hypothesis that this SOD2-reactive iron is derived from the Fe-S biogenesis pathway, and by inhibiting manganese binding to SOD2, it contributes to mitochondrial damage in a disease of iron overload. The nature of SOD2-reactive iron will be evaluated through a colorimetric assay for mitochondrial ferrous iron and through XANES analyses (by our collaborator J. Penner-Hahn) of the metal coordination environment. Such investigations into SOD2-reactive iron will provide important clues as to how SOD2 normally selects manganese. Overall, this multi-disciplinary approach to understanding manganese ion biology should provide novel insight into the homeostasis of a toxic nutrient.