Effective Methods to Identify the Toxic Metal Proteome

识别有毒金属蛋白质组的有效方法

• 批准号: 8769739
• 负责人: DAVID Harold PETERING
• 金额: $ 21.85万
• 依托单位: UNIVERSITY OF WISCONSIN MILWAUKEE
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-07-01 至 2016-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8769739
• 关键词: Ablation; Acute; Address; Affinity; Amines; Animals; Area; Binding; Binding Sites; Biochemistry; Carcinogens; Cd-Zn-metallothionein; Cell Extracts; Cells; Chemicals; Chronic; Coupled; Development; Diabetes Mellitus; Disease Outbreaks; Exposure to; Face; Family suidae; Fanconi Syndrome; Gel; Gene Expression; Genomic Instability; Glycine decarboxylase; Human; Imidazole; In Vitro; Ions; Kidney; Kidney Failure; Knowledge; LLC-PK1 Cells; Lasers; Learning; Ligand Binding; Maintenance; Mass Spectrum Analysis; Measures; Messenger RNA; Metabolic; Metalloproteins; Metallothionein; Metals; Methodology; Methods; Molecular; Movement; Nephrotoxic; Nucleic Acids; Oxidative Stress; Paper; Pathway interactions; Phase; Plasma; Polyacrylamide Gel Electrophoresis; Population; Procedures; Protein Binding; Protein Biosynthesis; Proteins; Proteome; Proteomics; Publishing; Reaction; Reporting; Research; Resolution; Signal Transduction; Site; Societies; Sodium Dodecyl Sulfate; Solid; Sulfhydryl Compounds; Technology; Testing; Time; Toxic effect; Tubular formation; Water; Xenobiotics; base; bone; cadmium-binding protein; cell injury; cofactor; consumer demand; glucose uptake; in vivo; phosphodiester; pollutant; protein complex; protein function; public health relevance; research study; success; toxic metal; trafficking; transcription factor; zinc thionein

DESCRIPTION (provided by applicant): The cellular metallome includes thousands of Zn-proteins and hundreds of Fe- and Cu-proteins. When toxic metals interact with cells, it is hypothesized that they bind to sensitive sites and, thereby, elicit their deleterious effects by altering protein function. Binding may occur through exchange with metals present in pre-existent metalloproteins or to other sites that also display substantial affinity for metals. Much remains to be learned about the native metalloproteome and little is known about the binding distribution of toxic metals within the proteome. Without such information, it is not possible to understand at a chemical level how xenobiotic agents exert their cellular impact. Research in these areas has been severely hindered by the lack of methods that provide high resolution separation of the proteome with the retention of bound metals to native proteins. This proposal will test the applicability of a new method of sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) called native (N)SDS-PAGE that utilizes milder conditions to achieve both the excellent separation of SDS-PAGE and the retention of metals bound to proteins in their native state. Once dispersed on the gel, metalloproteins will be detected using laser ablation inductively coupled plasma mass spectrometry and then identified by mass spectrometry. For this test, the hypothesis will be examined that Cd2+ binds to proteome through metal exchange reactions with Zn-proteins, resulting in the formation of Cd- proteins with displacement of Zn2+. Further, the contraction of the proteomic distribution of Cd-proteins in the presence of the major Cd-binding protein, metallothionein (MT), will be investigated. The secondary hypothesis will be explored that Cd-proteins and Zn-MT undergo metal exchange to produce Cd-MT and Zn- proteins. In this context, the overall objective of the project is to determine whether NSDS-PAGE in conjunction with LA-ICP-MS can be used as the central methodology to support mechanistic studies that focus on the reaction of exogenous metals with cells and their distribution among cell biomolecules. The following specific aims address this objective: [1] Test and finalize the procedures for conducting NSDS-PAGE followed by LA-ICP-MS. [2] Characterize the Cd-proteome formed during in vitro reaction of Cd2+ with the isolated proteome of pig kidney LLC-PK1 cells. [3] Determine how the in vitro Cd-proteome reacts with apo- and Zn-metallothionein. [4] Compare in vitro and in vivo reactivity of the proteome with Cd2+ in the absence and presence of metallothionein. Success will open the nascent field of metalloproteomics to a wide range of experimentation into the mechanism of action of toxic metals.

描述(申请人提供)：细胞金属组包括数千个锌蛋白和数百个铁和铜蛋白。当有毒金属与细胞相互作用时，假设它们与敏感部位结合，从而通过改变蛋白质功能来引起它们的有害影响。结合可以通过与预先存在的金属蛋白中的金属交换发生，或者通过与其他也对金属有很大亲和力的位置进行交换来进行。对天然金属蛋白质组的研究还很多，对有毒金属在蛋白质组中的结合分布知之甚少。如果没有这样的信息，就不可能在化学水平上理解异生物质如何发挥其细胞影响。这些领域的研究由于缺乏提供高分辨率分离蛋白质组并将结合金属保留在天然蛋白质上的方法而受到严重阻碍。这项提议将测试一种名为Native(N)SDS-PAGE的新的十二烷基硫酸钠-聚丙烯酰胺凝胶电泳法(SDS-PAGE)的适用性，该方法利用较温和的条件实现SDS-PAGE的出色分离和与蛋白质结合的金属在其天然状态下的保留。金属蛋白一旦分散在凝胶上，将用激光烧蚀电感耦合等离子体质谱检测，然后用质谱仪进行鉴定。在这个测试中，我们将检验这一假设，即Cd~(2+)通过与锌蛋白的金属交换反应与蛋白质组结合，形成置换了锌~(2+)的Cd~(2+)蛋白。此外，还将研究主要的镉结合蛋白金属硫蛋白(MT)存在时，镉蛋白蛋白质组分布的收缩情况。第二个假设是Cd-蛋白质和锌-MT发生金属交换，产生Cd-MT和锌-蛋白质。在这方面，该项目的总体目标是确定NSDS-PAGE和LA-ICP-MS是否可以用作中心方法学，以支持侧重于外源金属与细胞的反应及其在细胞生物分子中的分布的机制研究。以下具体目标针对这一目标：[1]测试并最终确定进行NSDS-PAGE和LA-ICP-MS的程序。[2]鉴定Cd~(2+)与猪肾LLC-PK1细胞分离蛋白质组在体外反应中形成的CD-蛋白质组。[3]确定体外CD-蛋白质组如何与脱脂蛋白和锌-金属硫蛋白反应。[4]比较该蛋白质组在无金属硫蛋白和有金属硫蛋白存在的情况下与Cd~(2+)的体外和体内反应性。这项研究的成功将开启金属蛋白质组学的新生领域，为有毒金属的作用机制进行广泛的实验。