Metal Transfer Reactions between Stable Isotopically Doped Zn Proteins

描述（由申请人提供）：该项目的广泛目标是开发新技术，以经验确定细胞选择性分化和掺入金属的机制，以利用这些金属赋予受体分子的额外空间，催化和氧化还原特性。最近实现的Zn在生命过程中的重要性，强调了需要更好地了解控制胞质游离Zn 2+离子活性的调节过程，从而使细胞过程同时受到保护，免受这种金属的缺乏和有毒过量。金属硫蛋白（MT）是一类与金属稳态有关的蛋白质。这个古老的金属结合蛋白家族在所有哺乳动物中普遍表达，除了Zn和Cu稳态外，还参与保护细胞免受有毒金属（Cd，Hg），电离辐射，活性氧，亲电子抗癌药物和诱变剂的影响。MT的四种主要亚型存在于人类中，它们在空间和发育上对各种刺激都有差异表达，尽管它们的具体和相互作用尚不清楚。现有技术允许仅从一个供体监测细胞Zn转移反应。因此，不知道当一致表达时，与每个特定同种型相关的金属是否在动力学上和/或代谢上彼此分离。这些信息对于确定它们在金属稳态和解毒中的个体或集体作用是重要的。我们提出了直接耦合的HPLC-ICP-MS的实用程序的初步数据，经验研究五种不同的供体蛋白质之间的锌转移反应。该项目的具体目的是使用这种新技术，用66/67/68/70 Zn对各种MT亚型进行差异化标记，用于多重转移反应，旨在阐明它们在细胞内Zn分布中的个体和相互作用。这些实验将：（i）确定特定同种型和潜在受体脱辅基酶之间的Zn交换;（ii）确定这些转移是否需要配体-配体相互作用;（iii）确定同种型间交换是否需要游离Zn和通过硫醇桥连的异二聚化;（iii）利用双同位素Zn/Cd MT同种型来研究每种同种型在不同氧化还原条件下从Zn中区别处理潜在毒性Cd的能力。 生命已经进化到利用金属，很难想象任何已知的细胞途径，无论是发育，防御，认知，调节，信号传导，基因激活/表达，代谢，运输，该研究旨在测试一种新的技术，该技术可以定量监测多种金属在不同细胞之间的同时运动。分子。该技术的未来发展及其在蛋白质组学中的应用将允许在相对复杂的样品中研究与细胞质环境相当的金属-蛋白质相互作用，这是了解金属在体内蛋白质功能和细胞代谢中的调节作用的重要一步。

DESCRIPTION (provided by applicant): The broad term goals of this project are to develop new techniques to empirically determine the mechanisms by which cells selectively differentiate and incorporate metals to take advantage of the additional steric, catalytic and redox properties these metals confer to acceptor molecules. The recent realization of the importance of Zn in life processes has underscored the need for a better understanding of the regulatory processes controlling the cytosolic free Zn2+ ion activity such that cellular processes are simultaneous protected from deficiencies and toxic excesses of this metal. One class of protein that have been implicated in metal homeostasis is metallothionein (MT). This ancient family of metal-binding proteins is ubiquitously expressed in all mammals and, in addition to Zn and Cu homeostasis, is though be involved in protecting cells against toxic metals (Cd, Hg), ionizing radiation, reactive oxygen species, electrophilic anticancer drugs and mutagens. Four major isoforms of MT exist in humans that are differentially expressed both spatially and developmentally in response to various stimuli, although their specific and interactive roles are unknown. Existing technologies allow cellular Zn transfer reactions to be monitored from only one donor. Consequently it is not known whether the metals associated with each specific isoform are kinetically and/or thermodynamically isolated from each other when concordantly expressed. This information is important in determining their individual or collective roles in metal homeostasis and detoxification. We present preliminary data on the utility of directly coupled HPLC- ICP-MS for empirically studying Zn transfer reactions between five different donor proteins. The specific aim of this project is to use this new technology to differentially label the various MT isoforms with 66/67/68/70 Zn for multiplexing transfer reactions designed to elucidate their individual and interactive roles in Zn distribution within the cell. These experiments will: (i) define Zn exchange between the specific isoforms and potential recipient apo-enzymes; (ii) determine if these transfers require ligand- ligand interaction; (iii) establish if inter-isoform exchange requires free Zn and heterodimerization via thiol bridging; (iii) utilize dual-isotopically Zn/Cd MT isoforms to study the ability of each isoform to differentially process potentially toxic Cd from Zn under varying redox conditions. Life has evolved to utilize metals and it is hard to envisage of any known cellular pathway, be it developmental, defense, cognitive, regulatory, signaling, gene activation/expression, metabolic, transport, growth etc. that does not have metals implicitly and intricately tied into it. The proposed research aims to test a novel technique that can quantitatively monitor the simultaneous movement of numerous metals between different cellular molecules. Future development of the technique and its application to proteomics will allow the study of metal-protein interactions in relatively complex samples comparable to the cytoplasmic milieu, which is an important step to understanding the regulatory role of metals in protein functioning and in cellular metabolism in vivo.