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.

蛋白质表征方面的各种服务和合作项目已经或正在利用蛋白质微表征核心设施 (PMCF) 进行，分析了来自 8 个实验室分支机构的 27 位主要研究人员的 60 名科学家的约 6000 个样品。 其中一项重大工作是支持蛋白质表达核心设施 (PECF) 和 Bob Petrovich 博士。 PMCF 的作用是在 PECF 将材料交给用户之前在蛋白质水平上确认基因表达。 其他仍在进行中的未发布项目包括： 脂质和肌醇激酶上结合伴侣和翻译后修饰 (PTM) 位点的鉴定 - Steve Shears 从各种组织类型和/或条件中鉴定 BAF 复合物中的蛋白质 - Trevor Archer Glis 家族成员修改并绑定合作伙伴 Anton Jetten 其他最近发表的项目或正在出版的项目包括： 烯醇化酶：精子利用糖酵解产生运动所需的 ATP，并且几种生精细胞特异性糖酵解同工酶与精子鞭毛主要部分的纤维鞘 (FS) 相关。我们使用蛋白质组学和分子生物学方法来证实早期的报道，即小鼠精子中存在一种新型烯醇化酶。然后我们发现，泛烯醇酶抗体（而非 ENO2 和 ENO3 抗体）识别小鼠精子鞭毛主要部分中的蛋白质。数据库分析确定了两个先前未表征的烯醇酶家族样候选基因：64306537H0Rik 和 Gm5506。 Northern 分析表明，64306537H0Rik（重命名为 Eno4）在出生后第 12 天在小鼠睾丸中转录。为了确定 ENO4 的作用，我们使用 Eno4 等位基因被破坏的胚胎干细胞产生了小鼠，并与被破坏的等位基因纯合的雄性小鼠不育。与野生型小鼠相比，附睾精子数量减少了 2 倍，精子活力也显着降低。这些小鼠的精子具有卷曲的鞭毛和杂乱的 FS。 Gm5506 基因编码与 ENO1 相同的蛋白质，并且也在睾丸中以低水平转录。我们得出结论，ENO4 是 FS 正常组装所必需的，并提供精子中的大部分烯醇酶活性，而 Eno1 和/或 Gm5506 可能编码精子中的一小部分烯醇酶活性。 ——米奇·艾迪 TARG1：二磷酸腺苷 (ADP)-核糖基化是一种翻译后蛋白质修饰，涉及一系列细胞过程的调节。 PARP 是催化 ADP-核糖基化反应的蛋白质家族。 PARP 将 ADP-核糖核苷酸共价连接到靶蛋白上，一些 PARP 家族成员随后可以添加额外的 ADP-核糖单位以生成 PAR 链。 PAR 链的水解由 PARG 催化。 PARG 无法裂解与蛋白质直接连接的单（ADP-核糖）单元，尽管已在哺乳动物细胞提取物中检测到催化该反应的酶活性，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疾病患者中 c6orf130 基因的纯合突变，并将 C6orf130 鉴定为 PARP 相互作用蛋白，可消除 PARP 修饰蛋白中谷氨酸氨基酸残基的单（ADP-核糖基）化。 C6orf130 的 X 射线结构和生化分析表明，催化逆转机制涉及瞬态 C6orf130 赖氨酰-(ADP-核糖) 中间体。此外，细胞中 C6orf130 蛋白的消耗会导致增殖和 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-核糖基化的周转和循环中发挥作用，并且我们暗示了该蛋白质在支持人类正常细胞功能中的重要性。 R·斯科特·威廉姆斯 XRCC1：PARP-1 结合碱基切除修复 (BER) 的中间体并被激活以进行 PAR 合成。 PAR 介导关键 BER 因子 XRCC1 和 DNA pol 的募集和功能，进而调节 PAR。然而，XRCC1 和 pol 协调调节 PAR 水平的分子机制和意义尚不清楚。在体内发现了 PARP-1、XRCC1 和 pol 的复合物，并且已知 pol 和 XRCC1 通过 XRCC1 N 端结构域中的氧化还原敏感结合界面相互作用。我们在此证实，XRCC1 的氧化形式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 C12-C20 氧化形式的重要性以及与 pol 的相互作用，我们表征了野生型 XRCC1 和 XRCC1 的两个突变体的 Xrcc1(-/-) 小鼠成纤维细胞中代表稳定转染子的细胞系。与 C12-C20 二硫键封闭 (C12A) 和无法结合 pol (V88R) 的参考突变体形成。 XRCC1 缺陷的小鼠成纤维细胞对甲磺酸甲酯 (MMS) 极其敏感，转染的野生型和 C12A 突变型 XRCC1 蛋白同样可以逆转 MMS 过敏。然而，在暴露于 MMS 后，发现表达 C12A 突变体的细胞中的细胞 PAR 水平比表达野生型 XRCC1 的细胞中增加得更大。 PARP 抑制导致表达野生型 XRCC1 的细胞中产生非常强的 MMS 敏化，但这种敏化在表达 C12A 突变体的细胞中要弱得多。结果表明，XRCC1 的氧化形式在与 pol 相互作用中发挥着以下作用：(1) 在 MMS 暴露后控制 PAR 水平，以及 (2) 在 MMS DNA 损伤反应期间实现 PARP 抑制的极端细胞毒性。萨姆·威尔逊 Pol beta：在哺乳动物对含有病变的 DNA 进行碱基切除修复 (BER) 过程中，有人提出，整个途径中产生的有毒链断裂中间体会被隔离，并从一个步骤传递到下一个步骤，直到修复完成。这个逐步过程被称为基底沟道。这里评估的工作模型是复杂的 BER 因素可以促进 BER 过程。 FLAG 标记的 pol 在内源性 pol 基因缺失的小鼠成纤维细胞中表达，并使用抗 FLAG 抗体对细胞提取物进行“亲和捕获”程序。发现 pol 亲和捕获级分 (ACF) 含有多种 BER 因子，包括聚合酶 1、X 射线交叉互补因子 1-DNA 连接酶 III 和参与处理 BER 中间体 3' 封闭末端的酶，例如多核苷酸激酶和酪氨酰-DNA 磷酸二酯酶 1。相比之下，DNA 糖基化酶、脱嘌呤/脱氨嘧啶核酸内切酶 1 和瓣状核酸内切酶 1 以及其他几个与 BER 相关的因子不存在。通过蔗糖梯度离心观察到，pol ACF 中的一些 BER 因子位于多蛋白复合物中。 pol ACF 能够在体外 BER 中进行底物通道，并且擅长模拟 3' 封闭的拓扑异构酶 I 共价中间体或氧化应激诱导的 3' 封闭中间体的底物体外修复。萨姆·威尔逊 另外两份手稿正在印刷中。一篇关于高级糖基化终产物对花生过敏原蛋白的作用及其与高级糖基化终产物受体的相互作用（Mueller 和 London），另一篇关于果蝇 gag 蛋白磷酸化的位置和作用（J. Mason）。 其他需要大量资源的项目包括与 Adelman、Armstrong、Blackshear、Cidlowski、Hall、Hu 和 R.S. 合作进行的项目。威廉姆斯实验室。