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Zn-蛋白质以及数百种Fe-和Cu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。结合可能通过与先前存在的金属蛋白中存在的金属或其他对金属具有实质性亲和力的位点交换。关于天然金属蛋白质组的尚待了解，关于蛋白质组中有毒金属的结合分布知之甚少。没有这样的信息，就无法在化学水平上理解异种生物剂如何发挥其细胞影响。由于缺乏将蛋白质组高分辨率分离而与本机蛋白保留的方法，这些方法在这些领域的研究受到了严重阻碍。该提案将测试一种新方法的新方法十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE），称为天然（N）SDS-PAGE，该方法利用温和的条件来实现SDS-PAGE的良好分离和与本机状态蛋白质结合的金属的出色分离。一旦分散在凝胶上，将使用激光消融电感耦合等离子体质谱法检测金属蛋白，然后通过质谱法鉴定。对于此测试，将检查假设CD2+通过与Zn-蛋白质的金属交换反应与蛋白质组结合，从而形成了CD-蛋白，并取代了Zn2+的位移。此外，将研究CD蛋白在主要CD结合蛋白Metallothionein（MT）的情况下的蛋白质组学分布的收缩。将探讨次要假设，即CD蛋白和Zn-MT经历了金属交换以产生CD-MT和Zn-蛋白。在这种情况下，该项目的总体目标是确定是否可以将NSDS-PAGE与LA-ICP-MS结合起来作为支持机械研究的中心方法，以侧重于外源金属与细胞的反应及其在细胞生物分子之间的分布。以下具体目的解决了此目标：[1]测试并最终确定进行NSDS-PAGE的程序，然后测试LA-ICP-MS。 [2]表征在CD2+与猪肾脏LLC-PK1细胞分离的蛋白质组体外反应过程中形成的CD-蛋白酶。 [3]确定体外CD-蛋白酶与apo-和Zn-Metallothionein的反应。 [4]在缺乏和存在金属硫蛋白的情况下，将蛋白质组与CD2+的体外和体内反应性进行比较。成功将使金属蛋白质组学的新生场对有毒金属的作用机理进行广泛的实验。