Principles of selectivity and translocation in transition metal transporter

摘要 过渡金属，如铁、铜和锌，是生命所必需的微量元素，起着基础作用。 催化、结构和信号功能。调节载体金属摄取的跨膜转运蛋白 而跨细胞膜的挤出在控制金属动态平衡方面起着把关作用。他们的活动 保证金属水平受到严格控制，以满足必要的蜂窝要求，而不会达到 毒性水平。 母公司MIRA项目的目标是主要活跃的过渡金属泵和溶质载体(SLC)：(I) 研究第一、第二和第三排过渡金属的金属选择性原理和(Ii)它们的金属 配位化学；(Iii)确定金属移位途径；(Iv)探讨 分子水平上的能量传递过程。我们调查了已知和新的转运蛋白家族 参与金属动态平衡和疾病进展包括：1)P1B型ATPase，初级活跃 转运体控制人类细胞内铜水平，并调节铜和铜的浓度 病原菌中的其他过渡金属；2)TMEM205，一种与铜有关的新的人类转运蛋白 排出与抗癌铂复合体的转运和抗性；3)IroT转运体，可能是铁调节的 致病原核生物中负责铁(II)获取和毒力的溶质载体。 在这项提案中，我们提议收购Biacore T200表面等离子体共振仪，以确定 并描述了转运体和负责金属输送的分子伙伴之间的相互作用 以及向/从被调查的转运体摄取。该仪器将被用于定量地确定 生物分子与金属相互作用的动力学、亲和力、特异性、选择性和热力学参数- 伴侣供体/受体，并筛选小分子文库以确定新的转运蛋白调节子。这个 单元以分析物分离阶段的样品回收为特征，并将被整合到蛋白质组中 和金属工作流程，以实现电池中新型粘结剂的鉴定、表征和金属形态形成 萃取物和分级裂解物。该仪器在我们的实验工作流程中的实现是可望的 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洞察力 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.