Protein Microcharacterization

蛋白质微观表征

• 批准号: 8734189
• 负责人: Jason Williams
• 金额: $ 92.87万
• 依托单位: NATIONAL INSTITUTE OF ENVIRONMENTAL HEALTH SCIENCES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8734189
• 关键词: Adenosine Diphosphate; Adenosine Diphosphate Ribose; Advanced Glycosylation End Products; Affinity; Alleles; Allergens; Amino Acids; Antibodies; Base Excision Repairs; Binding; Biochemical; Candidate Disease Gene; Cell Extracts; Cell Line; Cell physiology; Cells; Centrifugation; Cleaved cell; Complement; Complex; Core Facility; DNA; DNA Damage; DNA Repair; DNA glycosylase; DNA ligase III; Data; Databases; Defect; Enzymes; Family; Family member; Fibroblasts; Flagella; Gene Expression; Genes; Glutamates; Glycolysis; Hand; Histocompatibility Testing; Human; Hydrolysis; Hypersensitivity; In Vitro; Infertility; Inositol; Intramural Research; Isoenzymes; Laboratories; Lesion; Link; Lipids; Location; London; Mammalian Cell; Manuscripts; Mediating; Methyl Methanesulfonate; Minor; Modeling; Modification; Molecular; Molecular Biology; Molecular Weight; Mono-S; Mus; Mutation; N-terminal; National Institute of Environmental Health Sciences; Nerve Degeneration; Nucleotides; One-Step dentin bonding system; Oxidation-Reduction; Oxidative Stress; PARP inhibition; Pathway interactions; Patients; Peanuts - dietary; Peptides; Phosphorylation; Phosphotransferases; Polymerase; Polynucleotide 5' -Hydroxyl-Kinase; Post-Translational Modification Site; Post-Translational Protein Processing; Procedures; Process; Protein Family; Proteins; Proteomics; Publishing; RTH-1 Nuclease; Reaction; Recycling; Regulation; Reporting; Research Personnel; Resources; Roentgen Rays; Role; Sampling; Scientist; Services; Sperm Count Procedure; Sperm Motility; Sperm Tail; Spermatogenic Cell; Structure; Sucrose; Testis; Type I DNA Topoisomerases; Wild Type Mouse; Work; XRCC1 gene; cell motility; cytotoxicity; disulfide bond; embryonic stem cell; endonuclease; enolase; gag Gene Products; human XRCC1 protein; in vivo; male; mutant; novel; pol genes; postnatal; protein complex; protein expression; receptor; repaired; response; sperm cell; tyrosyl-DNA phosphodiesterase

A variety of service and collaborative projects in protein characterization have been or are being carried out with the Protein Microcharacterization Core Facility (PMCF) with approximately 6000 samples analyzed from 60 scientists representing 27 principle investigators from 8 laboratory branches. One large effort is in support of the Protein Expression Core Facility (PECF) and Dr. Bob Petrovich. The Role of the PMCF is to confirm gene expression at the protein level prior to the PECF handing materials over to their users. Other unpublished projects that are still ongoing include: Identification of binding partners and sites of post-translational modifications (PTMs) on lipid and inositol kinases - Steve Shears Identification of proteins in the BAF complexes from a variety of tissue types and/or conditions - Trevor Archer Glis family members modifications and binding partners Anton Jetten Other recently published projects or projects in press include: Enolase: Sperm utilize glycolysis to generate ATP required for motility, and several spermatogenic cell-specific glycolytic isozymes are associated with the fibrous sheath (FS) in the principal piece of the sperm flagellum. We used proteomics and molecular biology approaches to confirm earlier reports that a novel enolase is present in mouse sperm. We then found that a pan-enolase antibody, but not antibodies to ENO2 and ENO3, recognized a protein in the principal piece of the mouse sperm flagellum. Database analyses identified two previously uncharacterized enolase family-like candidate genes, 64306537H0Rik and Gm5506. Northern analysis indicated that 64306537H0Rik (renamed Eno4) was transcribed in testes of mice by Postnatal Day 12. To determine the role of ENO4, we generated mice using embryonic stem cells in which an Eno4 allele was disrupted and male mice homozygous for the disrupted allele were infertile. Epididymal sperm numbers were 2-fold lower and sperm motility was reduced substantially compared to wild-type mice. Sperm from these mice had a coiled flagellum and a disorganized FS. The Gm5506 gene encodes a protein identical to ENO1 and also is transcribed at a low level in testis. We conclude that ENO4 is required for normal assembly of the FS and provides most of the enolase activity in sperm and that Eno1 and/or Gm5506 may encode a minor portion of the enolase activity in sperm. -Mitch Eddy TARG1: Adenosine diphosphate (ADP)-ribosylation is a post-translational protein modification implicated in the regulation of a range of cellular processes. A family of proteins that catalyse ADP-ribosylation reactions are PARPs. PARPs covalently attach an ADP-ribose nucleotide to target proteins and some PARP family members can subsequently add additional ADP-ribose units to generate a PAR chain. The hydrolysis of PAR chains is catalysed by PARG. PARG is unable to cleave the mono(ADP-ribose) unit directly linked to the protein and although the enzymatic activity that catalyses this reaction has been detected in mammalian cell extracts, the protein(s) responsible remain unknown. Here, we report the homozygous mutation of the c6orf130 gene in patients with severe neurodegeneration, and identify C6orf130 as a PARP-interacting protein that removes mono(ADP-ribosyl)ation on glutamate amino acid residues in PARP-modified proteins. X-ray structures and biochemical analysis of C6orf130 suggest a mechanism of catalytic reversal involving a transient C6orf130 lysyl-(ADP-ribose) intermediate. Furthermore, depletion of C6orf130 protein in cells leads to proliferation and DNA repair defects. Collectively, our data suggest that C6orf130 enzymatic activity has a role in the turnover and recycling of protein ADP-ribosylation, and we have implicated the importance of this protein in supporting normal cellular function in humans. R. Scott Williams XRCC1: PARP-1 binds intermediates of base excision repair (BER) and becomes activated for PAR synthesis. PAR mediates recruitment and functions of the key BER factors XRCC1 and DNA pol that in turn regulate PAR. Yet, the molecular mechanism and implications of coordination between XRCC1 and pol in regulating the level of PAR are poorly understood. A complex of PARP-1, XRCC1 and pol is found in vivo, and it is known that pol and XRCC1 interact through a redox-sensitive binding interface in the N-terminal domain of XRCC1. We confirmed here that both oxidized and reduced forms of XRCC1 are present in mouse fibroblasts. To further understand the importance of the C12-C20 oxidized form of XRCC1 and the interaction with pol , we characterized cell lines representing stable transfectants in Xrcc1(-/-) mouse fibroblasts of wild-type XRCC1 and two mutants of XRCC1, a novel reduced form with the C12-C20 disulfide bond blocked (C12A) and a reference mutant that is unable to bind pol (V88R). XRCC1-deficient mouse fibroblasts are extremely hypersensitive to methyl methanesulfonate (MMS), and transfected wild-type and C12A mutant XRCC1 proteins similarly reversed MMS hypersensitivity. However, after MMS exposure the cellular PAR level was found to increase to a much greater extent in cells expressing the C12A mutant than in cells expressing wild-type XRCC1. PARP inhibition resulted in very strong MMS sensitization in cells expressing wild-type XRCC1, but this sensitization was much less in cells expressing the C12A mutant. The results suggest a role for the oxidized form of XRCC1 in the interaction with pol in (1) controlling the PAR level after MMS exposure and (2) enabling the extreme cytotoxicity of PARP inhibition during the MMS DNA damage response. Sam Wilson Pol beta: During mammalian base excision repair (BER) of lesion-containing DNA, it is proposed that toxic strand-break intermediates generated throughout the pathway are sequestered and passed from one step to the next until repair is complete. This stepwise process is termed substrate channeling. A working model evaluated here is that a complex of BER factors may facilitate the BER process. FLAG-tagged pol was expressed in mouse fibroblasts carrying a deletion in the endogenous pol gene, and the cell extract was subjected to an 'affinity-capture' procedure using anti-FLAG antibody. The pol affinity-capture fraction (ACF) was found to contain several BER factors including polymerase-1, X-ray cross-complementing factor1-DNA ligase III and enzymes involved in processing 3'-blocked ends of BER intermediates, e.g. polynucleotide kinase and tyrosyl-DNA phosphodiesterase 1. In contrast, DNA glycosylases, apurinic/aprymidinic endonuclease 1 and flap endonuclease 1 and several other factors involved in BER were not present. Some of the BER factors in the pol ACF were in a multi-protein complex as observed by sucrose gradient centrifugation. The pol ACF was capable of substrate channeling for steps in vitro BER and was proficient in in vitro repair of substrates mimicking a 3'-blocked topoisomerase I covalent intermediate or an oxidative stress-induced 3'-blocked intermediate. Sam Wilson Two additional manuscripts are in press. One on the role of advanced glycation end products on peanut allergen proteins and their interactions with the receptor for advanced glycation end products (Mueller and London) and the other on the location and role of phosphorylation of the drosophilla gag protein (J. Mason). Additional projects that have required more than negligible resources include efforts performed with the Adelman, Armstrong, Blackshear, Cidlowski, Hall, Hu, and R.S. Williams laboratories.

蛋白质表征的各种服务和协作项目已经或正在使用蛋白质微含量核心设施（PMCF）进行，其中大约有6000个样本，分析了来自60位科学家的6000个样本，代表来自8个实验室分支机构的27名主要研究人员。 一项巨大的努力是支持蛋白质表达核心设施（PECF）和Bob Petrovich博士。 PMCF的作用是在将PECF将材料移交给使用者之前确认蛋白质水平的基因表达。 其他仍在进行的未发表的项目包括： 在脂质和肌醇激酶上鉴定结合伴侣和翻译后修饰（PTMS）的位点-Steve Shears 来自多种组织类型和/或条件的BAF复合物中蛋白质的鉴定-Trevor Archer Glis家族成员修改和约束伙伴Anton Jetten 媒体上最近发布的其他项目或项目包括： 烯醇酶：精子利用糖酵解来产生运动性所需的ATP，并且几种精子特异性糖溶解同工酶与精子鞭毛的主要部分中的纤维鞘（FS）相关。我们使用蛋白质组学和分子生物学方法来确认较早的报道，即小鼠精子中存在一种新颖的烯醇酶。然后，我们发现一种泛烯酚酶抗体，而不是与ENO2和ENO3的抗体，在小鼠精子鞭毛的主要片段中识别出一种蛋白质。数据库分析确定了两个先前未表征的烯醇酶家庭样候选基因，分别为64306537H0RIK和GM5506。北部分析表明，通过产后第12天将64306537H0RIK（更名为ENO4）转录在小鼠的睾丸中。为了确定ENO4的作用，我们使用Eno4等位基因中断的胚胎干细胞产生了小鼠，其中破坏了雌性小鼠，而雄性小鼠则因弱化的等位基因不遗传。附睾精子数低2倍，与野生型小鼠相比，精子运动大大降低。这些小鼠的精子有一个盘绕的鞭毛和一个混乱的FS。 GM5506基因编码与ENO1相同的蛋白质，并且在睾丸中也以低水平转录。我们得出的结论是，FS的正常组装需要ENO4，并提供精子中的大部分烯醇酶活性，ENO1和/或GM5506可能会编码精子中烯醇酶活性的一小部分。 -Mitch Eddy TARG1：二磷酸腺苷（ADP） - 介酰化是一种翻译后的蛋白质修饰，涉及一系列细胞过程的调节。催化ADP-核糖基化反应的蛋白质家族是PARP。 PARP共价将ADP-核糖核苷酸连接到靶蛋白上，一些PARP家族成员随后可以添加其他ADP-核糖单元以生成PAR链。 PAR链的水解由PARG催化。 PARG无法裂解与蛋白质直接相关的单声道（ADP-核糖）单元，尽管在哺乳动物细胞提取物中检测到了催化这种反应的酶促活性，但负责的蛋白质仍然未知。在这里，我们报告了严重神经退行性的患者中C6ORF130基因的纯合突变，并将C6ORF130鉴定为PARP相互作用的蛋白质，可在PARP-Modyified蛋白中去除谷氨酸氨基酸残基上的单声道（ADP-核糖基）。 C6ORF130的X射线结构和生化分析提出了一种涉及瞬时C6ORF130赖氨酸（ADP-核糖）中间体的催化反转机制。此外，细胞中C6ORF130蛋白的耗竭会导致增殖和DNA修复缺陷。总的来说，我们的数据表明C6ORF130酶活性在蛋白质ADP-核糖基化的周转和回收中起作用，并且我们暗示了该蛋白质在支持人类正常细胞功能方面的重要性。 R. Scott Williams XRCC1：PARP-1结合碱基切除修复（BER）的中间体，并激活以进行PAR合成。 PAR介导关键因素XRCC1和DNA POL的募集和功能，而DNA POR反过来调节PAR。然而，对调节PAR水平的XRCC1和POL之间的协调的分子机制和含义尚不清楚。在体内发现了PARP-1，XRCC1和POL的复合物，众所周知，POL和XRCC1通过XRCC1的N末端结构域中的氧化还原敏感的结合界面相互作用。我们在这里证实，XRCC1的氧化和还原形式都存在于小鼠成纤维细胞中。为了进一步了解XRCC1的C12-C20氧化形式和与POL的相互作用的重要性，我们表征了野生型XRCC1的XRCC1（ - / - ）小鼠成纤维细胞中稳定转染物的细胞系，XRCC1的成纤维细胞和XRCC1的两个突变体的XRCC1突变体，一种与C12-C20二胰岛键合（C12-C20二胰岛素）的新型XRCC1的突变体（ （v88r）。缺乏XRCC1的小鼠成纤维细胞对甲基甲磺酸甲酯（MMS）极为高敏，转染的野生型和C12A突变体XRCC1蛋白类似地反转MMS超敏。但是，在表达C12A突变体的细胞中，MMS暴露后，细胞PAR水平比表达野生型XRCC1的细胞更大。 PARP抑制作用导致表达野生型XRCC1的细胞中非常强大的MMS敏化，但是在表达C12A突变体的细胞中，这种敏化的效果要少得多。结果表明，XRCC1氧化形式在与POL的相互作用中的作用（1）控制MMS暴露后的PAR水平，（2）在MMS DNA损伤响应期间实现PARP抑制的极端细胞毒性。山姆·威尔逊 POL BETA：在含有病变的DNA的哺乳动物碱基切除修复（BER）期间，提议在整个途径中产生的有毒链破裂中间体被隔离，并从一个步骤到下一步，直到修复完成为止。此逐步过程称为基材通道。此处评估的工作模型是，一系列的ber因子可能有助于BER过程。在内源性POL基因中携带缺失的小鼠成纤维细胞中表达了FLAG标记的POL，并使用抗FLAG抗体对细胞提取物进行了“亲和力捕获”程序。发现POL亲和捕获量馏分（ACF）包含几个BER因子，包括聚合酶1，X射线交叉汇编因子1-DNA连接酶III和参与处理BER中间体3'Blocked端的酶，例如。多核苷酸激酶和酪酶-DNA磷酸二酯酶1。相比之下，DNA糖基酶，肾上腺素/丙烯蛋白核酸内核酸酶1和瓣核酸内核酸酶1和BER中涉及的其他几个因素。 POL ACF中的某些BER因子在多蛋白质复合物中，如蔗糖梯度离心观察到。 POL ACF能够底物在体外的台阶通道，并精通模仿3'Block topoisomerase i共价中间体或氧化应激诱导的3'Block的中间体的底物的体外修复。山姆·威尔逊 印刷中还有另外两个手稿。一种是关于高级糖化终产物在花生过敏原蛋白上的作用及其与晚期糖基化终产物的相互作用（Mueller和London）的作用，而另一个在果蝇GAG GAG蛋白（J. Mason）的位置和作用上的作用（Mueller和London）。 除了可忽略不计的资源所需的其他项目还包括与阿德曼，阿姆斯特朗，黑皮，西德洛夫斯基，霍尔，胡和R.S.所做的努力。威廉姆斯实验室。