Protein Microcharacterization

蛋白质微观表征

• 批准号: 9143532
• 负责人: Jason Williams
• 金额: $ 104.83万
• 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/9143532
• 关键词: 26S proteasome; Advanced Glycosylation End Products; Affect; Algorithms; Allergy to peanuts; Binding; Biological Assay; Camptothecin; Cells; Chemicals; Co-Immunoprecipitations; Complex; Core Facility; Cysteine; DNA; Data Set; Diabetes Mellitus; Disease; Down-Regulation; Entropy; Family member; Gene Expression; Genetic Transcription; Glucose; Hand; Histocompatibility Testing; Hybrids; Hypersensitivity; Immune response; In Vitro; Inflammatory; Insulin; Interferon Activation; Intramural Research; Ions; Label; Laboratories; Lead; Mass Spectrum Analysis; Mediating; Modification; Molecular Weight; Mutation; Mutation Analysis; N-terminal; National Institute of Environmental Health Sciences; Nitric Oxide; Nuclear; Pathway interactions; Peptides; Phosphorylation; Polyubiquitination; Population; Post-Translational Modification Site; Principal Investigator; Proteins; Publications; Publishing; RNA; Raja; Recombinants; Regulation; Reporting; Research Personnel; Resistance; Resources; Role; Sampling; Scientist; Services; Structure; Testing; Transactivation; Ubiquitin; Water; Xylose; Yeasts; base; novel; polyarginine; promoter; protein degradation; protein expression; research study; 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 principal investigators from 7 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 Other published and unpublished projects that are still ongoing include: Identification of binding partners and sites of post-translational modifications (PTMs) on transcription factors Raja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 and RNA structure specific 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 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 allergy affects 1-2% of the population of the U.S. It is hypothesized that roasting of peanuts influences the allergenicity of the peanut proteins due to formation of advanced glycation end products (AGE). In efforts to evaluate the AGE composition of peanut proteins we performed nanoLC-ESI-MS and MS/MS analyses of peanut extracts as well as recombinant peanut proteins. Initial experiments showed that in both raw and roasted peanut extracts one of the common modifications was the Amadori product (mass gain of 162 Da). The ions matched in mass to expected peptides +162 Da and yielded fragments in the MS/MS that were included neutral loss of 2, 3, and 4 waters and a loss of 3 waters and HCHO. Due to the absence of b- and y-ions from the CID MS/MS of these glycated peptides, standard search algorithms do not perform well in the identification of these peptides. Hence, we developed an algorithm that applies filters based upon information content (Shannon entropy) and the loss of water and HCHO. Results with a test data set show that the algorithm finds the correct spectra with high precision. The flagged spectra contained all of the Amadori product spectra with a high (66%) false positive rate. Isotopic labeling with 13C xylose and 13C glucose confirmed that these spectra contained Amadori products. If dry roasting enhances allergenicity, identification of the chemical differences between raw and roasted peanuts will lead to a better understanding of the chemical pathways that skew immune responses toward allergy. G. Mueller and R. London 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7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。 其他已公布和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家族成员和RNA结构特异性结合伴侣- 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 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。 总之，我们的研究确定痒作为一个关键的负调节Glis 3介导的转录活性。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 花生过敏影响1-2%的美国人口。据推测，由于形成晚期糖基化终产物（AGE），花生的烘烤影响花生蛋白的过敏性。 为了评估花生蛋白的AGE组成，我们对花生提取物以及重组花生蛋白进行了nanoLC-ESI-MS和MS/MS分析。 最初的实验表明，在生的和烤花生提取物的共同修改之一是Amadori产品（162 Da的质量增益）。 离子在质量上与预期肽+162 Da匹配，并在MS/MS中产生片段，包括2、3和4沃茨的中性损失以及3沃茨和HCHO的损失。 由于这些糖化肽的CID MS/MS中不存在B-和y-离子，标准搜索算法在鉴定这些肽时表现不佳。 因此，我们开发了一种算法，该算法应用基于信息内容（香农熵）和水和HCHO的损失的过滤器。 实验结果表明，该算法能以较高的精度找到正确的光谱。 标记的光谱包含具有高（66%）假阳性率的所有Amadori产物光谱。 同位素标记13 C木糖和13 C葡萄糖证实，这些光谱包含Amadori产品。 如果干烤增强了过敏性，那么识别生花生和烤花生之间的化学差异将有助于更好地了解使免疫反应偏向过敏的化学途径。 G.穆勒和R.伦敦 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。