Metal Transfer Reactions between Stable Isotopically Doped Zn Proteins

描述（由申请人提供）：该项目的长期目标是开发新技术，以经验确定细胞选择性分化和结合金属的机制，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。最近认识到锌在生命过程中的重要性，强调需要更好地理解控制胞质游离Zn2+离子活性的调节过程，以便同时保护细胞过程免受这种金属的缺乏和毒性过量。一类与金属稳态有关的蛋白质是金属硫蛋白（MT）。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，除了锌和铜的稳态外，还参与保护细胞免受有毒金属（Cd, Hg），电离辐射，活性氧，亲电抗癌药物和诱变剂的侵害。MT的四种主要同工异构体存在于人类中，它们在空间和发育上对各种刺激的反应存在差异，尽管它们的具体作用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锌转移反应。因此，不知道与每个特定异构体相关的金属在一致表达时是否在动力学和/或热力学上彼此分离。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。我们提出了直接偶联HPLC- ICP-MS对五种不同供体蛋白之间锌转移反应的实证研究的初步数据。该项目的具体目的是利用这项新技术，用66/67/68/70 Zn区分标记各种MT异构体，用于多路转移反应，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)确定特定亚型和潜在受体载脂蛋白酶之间的锌交换；（ii）确定这些转移是否需要配体-配体相互作用；（iii）确定异构体间交换是否需要通过巯基桥接进行自由锌和异源二聚化；（iii）利用双同位素Zn/Cd MT异构体研究在不同氧化还原条件下，每种异构体对Zn中潜在毒性Cd的不同处理能力。

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.