Protein Microcharacterization

蛋白质微观表征核心设施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7个实验室分支机构26名首席研究人员的约50名科学家的约6500个样本。 一项巨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。 其他仍在进行中的已发布和未发布项目包括： Raja Jothi转录因子的翻译后修饰(PTM)结合伙伴和结合位点的鉴定 鉴定转录复合体Karen Adelman上的结合伙伴和翻译后修饰(PTM)的位置 鉴定来自各种组织类型和/或条件的BAF复合体中的蛋白质--Trevor Archer PUF家族成员和RNA结构特异性结合伙伴--Traci Hall 最近出版或已提交和/或接受出版的其他项目包括： 有报道称，TOPO1是由一氧化氮衍生的物种多亚硝化的，最可能发生在半胱氨酸残基C300、C504、C505和C630上，导致该蛋白通过泛素/26S蛋白酶体途径显著下调。重要的是，TOPO1的这种下调导致了对喜树碱的显著耐药。这种对亚硝化后喜树碱的抗性不会导致活性的丧失，因为TOPO1在体外或细胞内诱导的DNA裂解没有显着差异。B.辛哈和R.梅森 我们通过质谱学和酵母双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。这些包括含有WW结构域的Hect E3泛素连接酶、Itch、SMurf2和NEDD4。免疫共沉淀实验和突变分析证实了GLIS3与Hect E3泛素连接酶之间的相互作用。这三种蛋白质都通过它们的WW结构域与位于GLIS3 N端的PPxY基序相互作用，促进GLIS3的多泛素化。然而，只有Itch通过促进其蛋白酶体的降解而显著降低了GLIS3的稳定性。转录分析表明，Itch显著抑制了GLIS3介导的反式激活和内源性Ins2的表达，这是通过增加GLIS3蛋白的周转来实现的。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控因子。这种调节提供了一种新的机制来调节胰岛素基因的表达，并可能为糖尿病等GLIS3相关疾病提供潜在的治疗靶点。A.Jetten 我们发现PPIP5K2具有一个保守的多聚精氨酸核定位序列(NLS)，其功能受Ser1006磷酸化的调控。NLS或Ser1006的突变改变了PPIP5K2核池的大小，并削弱了其促进干扰素启动子活性的促炎激活的能力。这一调控PPIP5K2功能的机制的论证为理解核PP-INSP活性提供了新的方向。S.Shears 花生过敏影响美国1-2%的人口。据推测，花生的烘焙通过形成晚期糖基化终产物(AGE)来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LC-ESI-MS和MS/MS分析。初步实验表明，在生花生和烤花生提取物中，常见的修饰之一是Amadori产品(质量增加162Da)。离子的质量与预期的+162Da相匹配，并在MS/MS中产生片段，包括2、3和4个水的中性损失和3个水的损失和HCHO。由于这些糖化多肽的CID-MS/MS中没有b-和y-离子，标准的搜索算法在这些多肽的鉴定中不能很好地执行。因此，我们开发了一种算法，根据信息含量(香农熵)以及水和甲醛的损失来应用过滤器。测试数据集的实验结果表明，该算法具有较高的识别精度。标记的光谱包含所有Amadori产物光谱，假阳性率高(66%)。~(13)C木糖和~(13)C葡萄糖的同位素标记证实了这些光谱中含有Amadori产物。如果干烤可以增强过敏性，识别生花生和烤花生之间的化学差异将有助于更好地理解导致免疫反应偏向过敏的化学途径。G.穆勒和R.伦敦 其他需要超过微不足道的资源的项目包括与胡、威尔逊和R.S.威廉姆斯实验室进行的工作。

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 Other published and unpublished projects that are still ongoing include: Identification of binding partners and sites of post-translational modifications (PTMs) on transcription factors Raja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