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（由申请人提供）：细胞金属组包括数千种锌蛋白和数百种铁蛋白和铜蛋白。当有毒金属与细胞相互作用时，假设它们与敏感位点结合，从而通过改变蛋白质功能引起其有害作用。结合可以通过与存在于预先存在的金属蛋白中的金属交换或与也显示出对金属的实质性亲和力的其他位点交换而发生。关于天然金属蛋白质组还有很多有待了解，而关于蛋白质组内有毒金属的结合分布知之甚少。如果没有这些信息，就不可能在化学水平上了解外源性物质如何发挥其细胞影响。这些领域的研究受到了严重的阻碍，缺乏的方法，提供高分辨率的蛋白质组分离与保留结合的金属天然蛋白质。该提案将测试一种称为非变性（N）SDS-PAGE的十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE）新方法的适用性，该方法利用更温和的条件实现SDS-PAGE的出色分离和保留与蛋白质结合的金属。一旦分散在凝胶上，金属蛋白将使用激光烧蚀电感耦合等离子体质谱法检测，然后通过质谱法鉴定。对于本试验，将检查以下假设：Cd 2+通过与Zn-蛋白质的金属交换反应与蛋白质组结合，导致Zn 2+置换形成Cd-蛋白质。此外，收缩的蛋白质组分布的镉蛋白的主要镉结合蛋白，金属硫蛋白（MT）的存在下，将进行研究。第二个假设将探讨镉蛋白和锌MT进行金属交换，产生镉MT和锌蛋白。在这种情况下，该项目的总体目标是确定NSDS-PAGE与LA-ICP-MS结合是否可以用作支持机制研究的中心方法，重点是外源金属与细胞的反应及其在细胞生物分子中的分布。以下具体目标实现了这一目标：[1]测试并最终确定进行NSDS-PAGE和LA-ICP-MS的程序。[2]表征Cd 2+与猪肾LLC-PK 1细胞分离蛋白质组体外反应期间形成的Cd蛋白质组。[3]确定体外镉蛋白质组如何与载脂蛋白和锌金属硫蛋白反应。[4]比较在体外和体内的蛋白质组与Cd 2+的金属硫蛋白的存在和不存在的反应。这一成功将开启金属蛋白质组学这一新生领域，为有毒金属的作用机制进行广泛的实验研究。