Protein Microcharacterization

蛋白质微观表征

• 批准号: 8929838
• 负责人: Jason Williams
• 金额: $ 103.23万
• 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/8929838
• 关键词: 26S proteasome; Alanine; Alleles; Allergens; Antibodies; Aspartic Acid; Binding; Biochemical; Biological Assay; Camptothecin; Cell Nucleus; Cells; Characteristics; Co-Immunoprecipitations; Complex; Core Facility; Cysteine; DNA; Dendritic Cells; Detergents; Development; Diabetes Mellitus; Disease; Down-Regulation; Drosophila genus; Elements; Family member; Gagging; Gene Expression; Genetic Transcription; Glucose; Hand; Hippocampus (Brain); Histocompatibility Testing; Hormones; Hybrids; IgE; In Vitro; Inflammatory; Insulin; Interferon Activation; Intramural Research; Laboratories; Mass Spectrum Analysis; Mediating; Metabolism; Modification; Molecular Weight; Mus; Mutation; Mutation Analysis; N-terminal; National Institute of Environmental Health Sciences; Nitric Oxide; Nuclear; Nuclear Hormone Receptors; Pathway interactions; Peanuts - dietary; Peptides; Phosphorylation; Phosphorylation Site; Phosphotyrosine; Point Mutation; Polyubiquitination; Post-Translational Modification Site; Production; Protein Tyrosine Kinase; Proteins; Publications; Publishing; Raja; Regulation; Reporting; Research Personnel; Resistance; Resources; Role; Sampling; Scientist; Serine; Services; Signal Transduction; Site-Directed Mutagenesis; Sodium Chloride; Spectrophotometry; Suggestion; Synapses; THRB gene; Thyroid Hormone Receptor; Time; Transactivation; Ubiquitin; Western Blotting; Xylose; Yeasts; Zinc Fingers; gag Gene Products; hippocampal pyramidal neuron; novel; nuclease; overexpression; polyarginine; postnatal; promoter; protein degradation; protein expression; research study; src-Family Kinases; therapeutic target; transcription factor; ubiquitin-protein ligase

A variety of service and collaborative projects in protein characterization have been or are being carried out with the Protein Micro-characterization Core Facility (PMCF) with approximately 6500 samples analyzed from approximately 50 scientists representing 26 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 Other unpublished projects that are still ongoing include: Identification of binding partners and sites of post-translational modifications (PTMs) on transcription factors Raja Jothi Identification of binding partners and sites of post-translational modifications (PTMs) on transcription complexes Karen Adelman Identification of proteins in the BAF complexes from a variety of tissue types and/or conditions - Trevor Archer Puf family members binding partners - Traci Hall Other projects that have been recently published, or have been submitted and/or accepted for publication include: A report that TOPO1 is polynitrosylated by nitric oxide derived species, most likely at cysteine residues C300, C504, C505, and C630, resulting in significant down regulation of the protein via ubiquitin/26S proteasome pathway. Importantly, this down-regulation of TOPO1 resulted in a significant resistance to camptothecin. This resistance to camptothecin following nitrosylation did not result in the loss of the activity as there were no significant differences in TOPO1-induced DNA cleavage in vitro or in cells. B. Sinha and R. Mason A project showing that, in the absence of hormone, the nuclear receptor TRβ forms a cytoplasmic complex with the p85 subunit of PI3K and the Src family tyrosine kinase, Lyn, which depends on two canonical phosphotyrosine motifs in the second zinc finger of TRβ that are not conserved in TRα. We found that when hormone is added, TRβ dissociates and moves to the nucleus, and PIP3 production goes up rapidly. When the rapid signaling mechanism was blocked chronically throughout development in mice by a targeted point mutation in both alleles of THRB and the mutation significantly impaired maturation and plasticity of the Schaffer collateral synapses on CA1 pyramidal neurons in the postnatal hippocampus. Thus, phosphotyrosine-dependent association of TRβ with PI3K provides a potential mechanism for integrating regulation of development and metabolism by thyroid hormone and receptor tyrosine kinases. - D. Armstrong We identified by mass-spectrometry and yeast 2-hybrid analyses several proteins that interact with the N-terminal region of Glis3. These include the WW-domain-containing HECT E3 ubiquitin ligases, Itch, Smurf2, and NEDD4. The interaction between Glis3 and the HECT E3 ubiquitin ligases was verified by co-immunoprecipitation assays and mutation analysis. All three proteins interact through their WW-domains with a PPxY motif located in the Glis3 N-terminus and promote Glis3 polyubiquitination. However, only Itch significantly reduced Glis3 stability by enhancing its proteasomal degradation. Transcription analyses demonstrated that Itch dramatically inhibited Glis3-mediated transactivation and endogenous Ins2 expression by increasing Glis3 protein turnover. Taken together, our study identifies Itch as a critical negative regulator of Glis3-mediated transcriptional activity. This regulation provides a novel mechanism to modulate insulin gene expression and could provide a potential therapeutic target for Glis3-associated diseases, such as diabetes. A. Jetten We show PPIP5K2 has a conserved, polyarginine nuclear localization sequence (NLS), the function of which is supervised by Ser1006 phosphorylation. Mutation of either the NLS or Ser1006 alters the size of the nuclear pool of PPIP5K2 and impairs its ability to promote a pro-inflammatory activation of interferon-β promoter activity. This demonstration of a mechanism to regulate PPIP5K2 function offers new directions for understanding nuclear PP-InsP activities. S. Shears Peanut extract was characterized by mass spectrometry (MS)and specific AGE modifications were found in raw and roasted peanuts and on rAra h 1 that was artificially gylcated by incubation with glucose or xylose. The binding of the RAGE-V1C1 domain to peanut allergens was assessed by PAGE and Western blotting with anti-Ara h 1, 2, and 3 antibodies. IgE binding to rAra h1 was assessed by PAGE and Western Blotting and RAGE was demonstrated to selectively interact with AGE modified rAra h 1. If the suggestion that sensitization to peanut allergens occurs in dendritic cells via RAGE is correct, these cells are likely interacting with modified Ara h 1, and Ara h 3, and not Ara h 2. G. Mueller and R. London We characterized some biochemical characteristics of the HeT-A Gag protein encoded by the HeT-A element of Drosophila. The HeT-A Gag protein when overexpressed in S2 cells was localized to the nucleus but was resistant to high salt, detergents and nuclease extraction treatments. Analysis of the HeT-A Gag protein by tandem mass spectrophotometry revealed that serines 216 and 221 are phosphorylated. Substituting these serines with alanine or aspartic acid by site-directed mutagenesis did not result in any changes in HeT-A Gag translocation across the nucleus, suggesting that phosphorylation of these sites is not associated with HeT-A Gag translocation, but time course experiments showed that these phosphorylation sites are important for Gag-protein stability. Additional projects that have required more than negligible resources include efforts performed with the Hu, Wilson, and R.S. Williams laboratories.

蛋白质微观表征核心设施 (PMCF) 已经或正在开展各种蛋白质表征服务和合作项目，分析了来自 8 个实验室分支机构的 26 位主要研究人员的约 50 名科学家的约 6500 个样本。 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： 转录因子 Raja Jothi 上的结合伴侣和翻译后修饰 (PTM) 位点的鉴定 识别转录复合物上的结合伴侣和翻译后修饰 (PTM) 位点 Karen Adelman 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 Puf 家族成员绑定合作伙伴 - Traci Hall 最近发表或已提交和/或接受发表的其他项目包括： 一份报告指出，TOPO1 被一氧化氮衍生物质多亚硝基化，最有可能在半胱氨酸残基 C300、C504、C505 和 C630 处，导致通过泛素/26S 蛋白酶体途径显着下调该蛋白质。 重要的是，TOPO1 的下调导致了对喜树碱的显着耐药性。 亚硝基化后对喜树碱的这种抗性不会导致活性丧失，因为在体外或细胞内 TOPO1 诱导的 DNA 裂解没有显着差异。 B.辛哈和R.梅森 该项目表明，在没有激素的情况下，核受体 TRβ 与 PI3K 的 p85 亚基和 Src 家族酪氨酸激酶 Lyn 形成细胞质复合物，该复合物依赖于 TRβ 第二锌指中的两个典型磷酸酪氨酸基序，而这些基序在 TRα 中不保守。我们发现，当添加激素时，TRβ解离并移动到细胞核，PIP3的产量迅速增加。当快速信号传导机制在小鼠的整个发育过程中被 THRB 两个等位基因的靶向点突变长期阻断时，该突变显着损害了出生后海马 CA1 锥体神经元上 Schaffer 侧支突触的成熟和可塑性。因此，TRβ 与 PI3K 的磷酸酪氨酸依赖性关联为甲状腺激素和受体酪氨酸激酶整合发育和代谢调节提供了潜在的机制。 ——D·阿姆斯特朗 我们通过质谱法和酵母 2-hybrid 分析鉴定了几种与 Glis3 N 末端区域相互作用的蛋白质。其中包括含有 WW 结构域的 HECT E3 泛素连接酶、Itch、Smurf2 和 NEDD4。 Glis3 和 HECT E3 泛素连接酶之间的相互作用通过免疫共沉淀测定和突变分析得到验证。 所有三种蛋白均通过其 WW 结构域与位于 Glis3 N 末端的 PPxY 基序相互作用，并促进 Glis3 多泛素化。然而，只有 Itch 通过增强蛋白酶体降解而显着降低了 Glis3 的稳定性。 转录分析表明，Itch 通过增加 Glis3 蛋白周转来显着抑制 Glis3 介导的反式激活和内源性 Ins2 表达。 综上所述，我们的研究确定 Itch 是 Glis3 介导的转录活性的关键负调节因子。这种调节提供了一种调节胰岛素基因表达的新机制，并且可以为 Glis3 相关疾病（例如糖尿病）提供潜在的治疗靶点。 A·杰顿 我们发现 PPIP5K2 具有保守的多聚精氨酸核定位序列 (NLS)，其功能受到 Ser1006 磷酸化的监督。 NLS 或 Ser1006 的突变会改变 PPIP5K2 核库的大小，并损害其促进干扰素-β 启动子活性的促炎激活的能力。这种调节 PPIP5K2 功能的机制的演示为理解核 PP-InsP 活动提供了新的方向。 S·希尔斯 通过质谱 (MS) 对花生提取物进行了表征，在生花生和烤花生以及通过与葡萄糖或木糖孵育人工糖化的 rAra h 1 上发现了特定的 AGE 修饰。 RAGE-V1C1 结构域与花生过敏原的结合通过 PAGE 和使用抗 Ara h 1、2 和 3 抗体的蛋白质印迹进行评估。通过 PAGE 和蛋白质印迹评估 IgE 与 rAra h1 的结合，并且 RAGE 被证明选择性地与 AGE 修饰的 rAra h 1 相互作用。如果通过 RAGE 在树突状细胞中发生对花生过敏原的敏感性的建议是正确的，那么这些细胞可能与修饰的 Ara h 1 和 Ara h 3 相互作用，而不是与 Ara h 2 相互作用。G. Mueller 和 R. London 我们表征了果蝇 HeT-A 元件编码的 HeT-A Gag 蛋白的一些生化特征。 HeT-A Gag 蛋白在 S2 细胞中过度表达时定位于细胞核，但对高盐、去污剂和核酸酶提取处理具有抗性。通过串联质谱法对 HeT-A Gag 蛋白的分析表明，丝氨酸 216 和 221 被磷酸化。通过定点诱变用丙氨酸或天冬氨酸取代这些丝氨酸不会导致 HeT-A Gag 跨核易位发生任何变化，表明这些位点的磷酸化与 HeT-A Gag 易位无关，但时程实验表明这些磷酸化位点对于 Gag 蛋白的稳定性很重要。 其他需要大量资源的项目包括与 Hu、Wilson 和 R.S. 合作进行的项目。威廉姆斯实验室。