Protein Microcharacterization

National Institute Of Environmental Health Sciences

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Abstract

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 Other published and unpublished projects that are still ongoing include: Identification of binding partners and sites of post-translational modifications (PTMs) on transcription factors Raja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