Principles of selectivity and translocation in transition metal transporter

摘要 铁、铜、锌等过渡金属是生命必需的微量元素， 催化、结构和信号功能。调节载体金属摄取的跨膜转运蛋白 和挤压穿过细胞膜在控制金属体内平衡中起着看门人的作用。他们的活动 确保金属水平得到严格控制，以满足不可或缺的细胞需求， 毒性水平。 母公司MIRA项目的目标是主要的活性过渡金属泵和溶质载体（SLC）： 研究了第一、第二和第三行过渡金属的金属选择性原理，以及（ii）它们的金属 配位化学;（iii）确定金属易位途径;（iv）解决 分子水平的能量转换过程。我们调查已知的和新的转运蛋白家族 参与金属稳态和疾病进展的酶包括：1）P1 B型ATP酶，主要活性 转运蛋白控制人类细胞内铜水平，并调节铜的浓度， 病原菌中的其他过渡金属; 2）TMEM 205，一种新型的与铜有关的人体转运蛋白 挤压和抗癌铂络合物的运输和阻力; 3）IroT转运蛋白，推定的铁调节 溶质载体负责铁（II）的收购和致病原核生物的毒力。 在本提案中，我们建议收购Biacore T200表面等离子体共振仪器，以识别 并表征转运蛋白和负责金属递送的分子伴侣之间的相互作用 和被研究的转运蛋白的摄取。该仪器将用于定量测定 生物分子与金属相互作用的动力学、亲和性、特异性、选择性和热力学参数， 分子伴侣供体/受体，并筛选小分子文库以鉴定新的转运蛋白调节剂。的 该单元在分析物解离阶段具有样品回收模式，并将集成到蛋白质组学中。 和金属组学工作流程，以允许从细胞中鉴定、表征和金属形态形成新的结合剂， 提取物和分级裂解物。该仪器在我们的实验工作流程中的实施是预期的 在负责金属输送和激活的关键途径中提供前所未有的分子见解， 原核生物和真核生物中的跨膜金属转运蛋白。

Abstract Transition metals, such as iron, copper, and zinc, are essential trace elements for life, playing fundamental catalytic, structural and signaling functions. Transmembrane transporters that regulate the vectorial metal uptake and extrusion across cellular membranes play a gatekeeper role in controlling metal homeostasis. Their activity guarantees that metal levels are tightly regulated to meet indispensable cellular requirements without reaching toxic levels. The parent MIRA project targets primary active transition metal pumps and solute carriers (SLC) towards: (i) investigating the principles of metal selectivity for first, second- and third- row transition metals and (ii) their metal coordination chemistry; (iii) determining the metal translocation pathway; (iv) addressing the mechanisms of energy transduction processes at a molecular level. We investigate known and novel transporter families involved in metal homeostasis and disease progression including: 1) P1B-type ATPases, primary active transporters controlling intracellular copper levels in humans, and modulating the concentrations of copper and other transition metals in pathogenic bacteria; 2) TMEM205, a novel human transporter involved in copper extrusion and anti-cancer Pt-complexes transport and resistance; 3) IroT transporters, putative iron-regulated solute carriers responsible for iron(II) acquisition and virulence in pathogenic prokaryotes. In this proposal we propose the acquisition of a Biacore T200 Surface Plasmon Resonance instrument to identify and characterize the interactions between transporters and molecular partners responsible for metal delivery and uptake to/from the investigated transporters. This instrumentation will be utilized to quantitatively determine kinetic, affinity, specificity, selectivity, and thermodynamic parameters of biomolecular interactions with metal- chaperone donors/acceptors, and screen small molecule libraries to identify novel transporter modulators. The unit features a sample recovery modality in the analyte dissociation phase and will be integrated in a proteomic and metallomic workflow to allow identification, characterization and metal speciation of novel binders from cell extracts and fractionated lysates. The implementation of this instrument in our experimental workflow is expected to provide unprecedented molecular insights in key pathways responsible for metal delivery and activation of transmembrane metal transporters in prokaryotes and eukaryotes.