Protein Microcharacterization

National Institute Of Environmental Health Sciences

Investigators

Linked publications, trials & patents

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 4000 samples analyzed from more than 40 scientists representing 22 principal investigators or core heads from 6 laboratory branches and the DNTP. 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 Other published and unpublished projects that are still ongoing include: Characterization of dust and proteins from allergens in collaboration - Don Cook, Geoffrey Mueller, Robert London 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 shows that mammalian Tyrosyl-DNA phosphodiesterase 2 (Tdp2) reverses Topoisomerase 2 (Top2) DNA-protein crosslinks that are triggered by Top2 engagement of DNA damage or poisoning by anticancer drugs. Tdp2 deficiencies are linked to neurological disease and cellular sensitivity to Top2 poisons. We demonstrated that a dynamic Tdp2 active site lid and deep substrate binding trench are well-suited for engaging the diverse DNA damage triggers of abortive Top2 reactions. Modeling of a proposed Tdp2 reaction coordinate, combined with mutagenesis and biochemical studies support a single Mg(2+)-ion mechanism assisted by a phosphotyrosyl-arginine cation- interface. We further identify a Tdp2 active site SNP that ablates Tdp2 Mg(2+) binding and catalytic activity, impairs Tdp2 mediated NHEJ of tyrosine blocked termini, and renders cells sensitive to the anticancer agent etoposide. Collectively, our results provide a structural mechanism for Tdp2 engagement of heterogeneous DNA damage that causes Top2 poisoning, and indicate that evaluation of Tdp2 status may be an important personalized medicine biomarker informing on individual sensitivities to chemotherapeutic Top2 poisons. The PMCF contributed to this work by identifying sites of Tdp2 that are susceptible to proteolysis in the presence and absence of DNA and in the context of the active site SNP. These mass spectrometry data helped to identify and characterize the active site lid. R.S. Williams 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 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 Blackshear, Hu, Stanley, Negishi, Wilson, and R.S. Williams laboratories.