Protein Microcharacterization

蛋白质微观表征

基本信息

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
项目类别：
财政年份：
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

项目摘要

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 Other published and unpublished projects that are still ongoing include: Identification of binding partners and sites of post-translational modifications (PTMs) on transcription factors Raja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

蛋白质表征方面的各种服务和协作项目已与蛋白质微型特征核心设施（PMCF）一起进行，其中大约有6500个样本，分析了大约50名科学家，代表来自7个实验室分支机构的26位主要研究人员。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。仍在进行的其他已发表和未发表的项目包括：在转录因子Raja Jothi上的结合伴侣和位点的鉴定（PTMS）转录复合物上翻译后修饰（PTM）的结合伙伴和位点的鉴定Karen Adelman 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 PUF家庭成员和RNA结构特定的约束伙伴-Traci Hall 最近已发布或已提交和/或接受出版的其他项目包括：一份报告说，托托1通过一氧化氮衍生的物种进行聚硝基化，最有可能在半胱氨酸残基C300，C504，C505和C630中，从而通过泛素/26S蛋白酶体途径对蛋白质进行了显着降低调节。重要的是，TOPO1的下调导致对凸轮皮蛋白具有显着的抗性。亚硝基化后对喀普霉素的这种耐药性并没有导致活性的丧失，因为在体外或细胞中TOPO1诱导的DNA裂解没有显着差异。 B. Sinha和R. Mason 我们通过质谱法和酵母2杂交分析鉴定了几种与GLIS3的N末端区域相互作用的蛋白质。其中包括含WW域的Hect E3泛素连接酶，瘙痒，蓝精灵和NEDD4。通过共免疫沉淀分析和突变分析来验证GLIS3与HECT E3泛素连接酶之间的相互作用。所有三种蛋白质通过其WW-domain与位于Glis3 N末端的PPXY基序相互作用，并促进Glis3多泛素化。但是，只有瘙痒通过增强其蛋白酶体降解可显着降低GLIS3的稳定性。转录分析表明，瘙痒通过增加GLIS3蛋白更新，极大地抑制GLIS3介导的反式激活和内源性INS2表达。综上所述，我们的研究将瘙痒确定为GLIS3介导的转录活性的关键负调节剂。该调节提供了一种调节胰岛素基因表达的新型机制，并可以为GLIS3相关疾病（例如糖尿病）提供潜在的治疗靶标。 A. Jetten 我们显示PPIP5K2具有保守的多拉宁核定位序列（NLS），其功能由Ser1006磷酸化监督。 NLS或SER1006的突变会改变PPIP5K2的核库的大小，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功能的机制的证明为理解核PP-INSP活动提供了新的方向。 S.剪花生过敏会影响美国人口的1-2％，假设花生的烘烤会影响由于形成晚期糖基化终产物（年龄）而影响花生蛋白的过敏性。为了评估花生蛋白的年龄组成，我们对花生提取物和重组花生蛋白进行了纳米糖 - ESI-MS和MS/MS分析。最初的实验表明，在原始花生提取物和烤花生提取物中，常见的修改之一是Amadori产品（质量增益为162 DA）。与预期肽+162 DA相匹配的离子在MS/MS中产生的片段包括2、3和4水的中性损失，以及3个水和HCHO的损失。由于这些糖化肽的CID MS/MS缺乏B和Y-ION，因此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，该算法根据信息含量（香农熵）以及水和HCHO的损失应用过滤器。测试数据集的结果表明，该算法以高精度找到正确的光谱。被标记的光谱包含所有Amadori产品光谱，其假阳性率高（66％）。用13C木糖和13C葡萄糖的同位素标记证实了这些光谱含有Amadori产物。如果干烤增强了过敏性，则鉴定生花生和烤花生之间的化学差异将使人们更好地了解对过敏的免疫反应偏离免疫反应的化学途径。 G. Mueller和R. London 除了可忽略的资源所需的其他项目还包括与HU，Wilson和R.S.所做的努力。威廉姆斯实验室。