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 Other unpublished projects that are still ongoing include: Identification of binding partners and sites of post-translational modifications (PTMs) on transcription factors Raja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.

蛋白质微表征核心设施（PMCF）已经或正在进行蛋白质表征方面的各种服务和合作项目，分析了来自8个实验室分支的约50名科学家代表26名主要研究者的约6500份样品。 其中一个很大的努力是支持蛋白质表达核心设施（PECF）和鲍勃彼得罗维奇博士。 PMCF的作用是在PECF将材料移交给其用户之前确认蛋白质水平的基因表达。 其他尚未公布的项目仍在进行中，包括： Raja Jothi转录因子上的结合配偶体和翻译后修饰（PTM）位点的鉴定 识别转录复合物上的结合配偶体和翻译后修饰（PTM）位点 凯伦·阿德尔曼 从各种组织类型和/或条件中鉴定BAF复合物中的蛋白质-特雷弗阿彻 Puf家庭成员绑定合作伙伴- Traci Hall 最近出版或已提交和/或接受出版的其他项目包括： 一份报告称，TOPO 1被一氧化氮衍生物质多亚硝基化，最可能在半胱氨酸残基C300、C504、C505和C630处，导致蛋白质通过泛素/26 S蛋白酶体途径显著下调。 重要的是，TOPO 1的这种下调导致对喜树碱的显著抗性。 这种对喜树碱的耐药性在亚硝基化后并没有导致活性的丧失，因为在体外或细胞中TOPO 1诱导的DNA切割没有显著差异。 B。Sinha和R.梅森 一个项目显示，在没有激素的情况下，核受体TR与PI 3 K的p85亚基和Src家族酪氨酸激酶林恩形成胞质复合物，该复合物依赖于TR第二锌指中的两个典型磷酸酪氨酸基序，该基序在TR中不保守。我们发现，当添加激素时，TR解离并移动到细胞核，PIP 3的产生迅速增加。当快速信号传导机制在小鼠的整个发育过程中被THRB的两个等位基因中的靶点突变长期阻断时，该突变显着损害了出生后海马CA 1锥体神经元上Schaffer侧支突触的成熟和可塑性。因此，TR与PI 3 K的磷酸酪氨酸依赖性关联提供了甲状腺激素和受体酪氨酸激酶整合发育和代谢调节的潜在机制。- D.阿姆斯特朗 我们通过质谱和酵母双杂交分析确定了几种与Glis 3的N-末端区域相互作用的蛋白质。这些包括含有WWW结构域的HECT E3泛素连接酶、Itch、Smurf 2和NEDD 4。Glis 3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来验证。 所有三种蛋白质通过其WW-domain与位于Glis 3 N-末端的PPxY基序相互作用，并促进Glis 3聚泛素化。然而，只有瘙痒显着降低Glis 3的稳定性，通过增强其蛋白酶体降解。 转录分析表明，痒显着抑制Glis 3介导的反式激活和内源性Ins 2的表达，增加Glis 3蛋白周转。 总而言之，我们的研究确定瘙痒是Glis 3介导的转录活性的关键负调节因子。这种调节提供了一种新的机制来调节胰岛素基因的表达，并可能提供一个潜在的治疗靶点Glis 3相关疾病，如糖尿病。 A. Jetten 我们发现PPIP 5 K2具有保守的多聚精氨酸核定位序列（NLS），其功能受Ser 1006磷酸化的监督。NLS或Ser 1006的突变改变了PPIP 5 K2的核库的大小，并损害了其促进干扰素启动子活性的促炎活化的能力。这种调节PPIP 5 K2功能的机制的展示为理解核PP-InsP活动提供了新的方向。 S.剪 花生提取物，其特征在于通过质谱（MS）和特定的AGE修饰被发现在生的和烤花生和rAra h 1，这是人工gylcated与葡萄糖或木糖孵育。 RAGE-V1 C1结构域与花生过敏原的结合通过PAGE和用抗Ara h 1、2和3抗体的Western印迹来评估。通过PAGE和Western Blotting评估IgE与rAra h 1的结合，并证明IgE选择性地与AGE修饰的rAra h 1相互作用。 如果花生过敏原的致敏发生在树突状细胞中的建议是正确的，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Ara h 2。 G. Mueller和R.伦敦 我们表征了果蝇HeT-A元件编码的HeT-A Gag蛋白的一些生化特性。HeT-A Gag蛋白在S2细胞中过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。通过串联质谱法分析HeT-A Gag蛋白揭示丝氨酸216和221被磷酸化。通过定点突变用丙氨酸或天冬氨酸取代这些丝氨酸并没有导致HeT-A Gag跨核易位的任何变化，这表明这些位点的磷酸化与HeT-A Gag易位无关，但是时间过程实验表明这些磷酸化位点对于Gag蛋白的稳定性是重要的。 其他项目所需的资源可忽略不计，包括与Hu、Wilson和R.S.合作开展的工作。威廉姆斯实验室。