Metal Transfer Reactions between Stable Isotopically Doped Zn Proteins

稳定同位素掺杂锌蛋白之间的金属转移反应

• 批准号: 7304911
• 负责人: ANDREW Z MASON
• 金额: $ 21.53万
• 依托单位: CALIFORNIA STATE UNIVERSITY LONG BEACH
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-08-02 至 2012-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7304911
• 关键词: Antineoplastic Agents; Binding; Binding Proteins; Cd-Zn-metallothionein; Cell physiology; Cells; Class; Cognitive; Complex; Condition; Coupled; Data; Development; Dialysis procedure; Dimerization; Drug Metabolic Detoxication; Enzymes; Family; Future; Gene Activation; Glutathione Disulfide; Goals; Growth; Heterodimerization; High Pressure Liquid Chromatography; Homeostasis; Human; Incubated; Individual; Ionizing radiation; Ions; Kinetics; Label; Life; Ligands; Mammals; Mass Spectrum Analysis; Metabolic; Metabolism; Metallothionein; Metals; Molecular Sieve Chromatography; Monitor; Mono-S; Movement; Mutagens; Numbers; Oxidation-Reduction; Oxidative Stress; Pathway interactions; Physical Dialysis; Plasma; Process; Property; Protein Isoforms; Proteins; Proteomics; Reaction; Reactive Oxygen Species; Relative (related person); Research; Role; Sampling; Signal Transduction; Solutions; Specificity; Stimulus; Stress; Sulfhydryl Compounds; Techniques; Technology; Testing; Thermodynamics; Variant; design; human MT3 protein; in vivo; metalloenzyme; new technology; novel; prevent; protein function; research study; response; segregation; stable isotope; stoichiometry; technique development; toxic metal

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.

描述（由申请人提供）：该项目的广义目标是开发新技术，以经验确定细胞选择性区分并掺入金属以利用其他空间，催化和氧化还原特性的机制，这些金属赋予受体分子。 。 Zn在生命过程中的重要性的最新意识强调了需要更好地理解控制胞质游离Zn2+离子活性的调节过程，以便细胞过程同时保护这种金属的缺陷和毒性过剩。与金属稳态有关的一类蛋白质是金属硫硫蛋白（MT）。这种古老的金属结合蛋白家族在所有哺乳动物中均普遍表达，除了Zn和Cu稳态外，还参与了保护细胞免受有毒金属（CD，HG），电离辐射，反应性氧的保护，电力性氧气，电动抗癌药物，电力抗癌药物和诱变剂。在人类中，MT的四个主要同工型存在于各种刺激上在空间和发育上差异表达，尽管它们的特定和互动作用尚不清楚。现有技术允许仅从一个供体中监视细胞Zn转移反应。因此，尚不清楚与每种特定同工型相关的金属是否在一致表达时在动力学和/或热力学上彼此分离。这些信息对于确定其在金属稳态和排毒中的个人或集体角色很重要。我们介绍了有关直接耦合HPLC-ICP-MS实用性的初步数据，以研究五种不同的供体蛋白之间的Zn转移反应。该项目的具体目的是使用这项新技术来差异标记具有66/67/68/70 Zn的各种MT同工型，用于旨在阐明其在单元内Zn分布中的个体和互动角色的多路复用转移反应。这些实验将：（i）定义特定的同工型和潜在受体中酶之间的Zn交换； （ii）确定这些转移是否需要配体配体相互作用； （iii）确定是否需要通过硫醇桥梁进行游离Zn和异二聚化； （iii）利用双重Zn/CD MT同工型研究每种同工型在不同的氧化还原条件下从Zn中差异化的差异毒性CD的能力。 生命已经发展为利用金属，很难设想任何已知的细胞途径，无论是发育，防御，认知，调节性，信号传导，基因激活/表达，代谢，运输，生长等，都没有隐含金属的金属。绑在其中。拟议的研究旨在测试一种可以定量监测不同细胞分子之间大量金属同时运动的新技术。该技术的未来开发及其在蛋白质组学中的应用将允许研究与细胞质环境相当的相对复杂样品中的金属 - 蛋白质相互作用，这是了解金属在蛋白质功能和体内细胞代谢中的调节作用的重要步骤。