A variety of service and collaborative projects in protein characterization have been or are being carried out with the Protein Microcharacterization Core Facility (PMCF). ? ? 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 Other unpublished projects that are still ongoing include:? ? Identifications of sites of phosphorylation on the glucocorticoid receptor.? ? Identification of binding partners and sites of post-translational modifications (PTMs) on lipid and inositol kinases.? ? Identification of polymerase beta associated proteins and those involved in base excision repair.? ? Identification of binding partners and PTMs of metal responsive transcription factor 1.? ? Identification of binding partners and PTMs of tristetraprolin and related proteins.? ? Identification of binding partners and PTMs of sirtuin proteins.? ? Identification of binding partners and PTMs of CD34.? ? Identification of binding partners and PTMs of KCNH2, TRbeta, and slowpoke.? ? Identification of binding partners of TBX1.? ? The core also is performing value added research in separations (off-gel electrophoresis) and affinity techniques (IMAC and MOAC)to aid in protein and PTM identifications.? ? Other published projects include:? Deleted in Split hand/Split foot 1 was previously identified as a (TPA)-inducible gene with possible involvement in skin carcinogenesis. The mechanisms by which human DSS1 (HsDSS1) exerts its biological effects via regulation of the ubiquitin-proteasome system are currently unknown. We demonstrated that HsDSS1 regulates the human proteasome by associating with it in the cytosol and nucleus via the RPN3/S3 subunit of the 19S regulatory particle (RP). Molecular anatomy of HsDSS1 revealed an RPN3/S3-interacting motif (R3IM), located at amino acid residues 15 to 21. Negative charges of the R3IM motif are required for proteasome interaction and binding to poly-ubiquitinated substrates. The R3IM motif of HsDSS1 protein is sufficient to pull down proteasome complexes and protein substrates with ubiquitin conjugates. The PMCF first showed that DSS1 interacts with the proteasome and resulted in authorship of the manuscript: Wei, S.J., Williams, J.G., Dang, H., Darden, T.A., Betz, B.L., Humble, M.M., Trempus, C.S., Johnson, K.L., Cannon, R.E., and Tennant, R.W. in press J. Mol. Bio. Identification of a Specific Motif of the DSS1 Protein Required for Proteasome Interaction and p53 Protein Degradation? ? Polymorphisms in the human hERG1 gene are associated with cardiac arrhythmias. The Kv11.1 channels encoded by hERG1 are also essential for rhythmic excitability of the pituitary, where they are regulated by thyroid hormone through a signal transduction cascade involving the phosphatidylinositol 3-kinase and the Ser/Thr-directed protein phosphatase, PP5. We showed that the hERG1 polymorphism at codon 897, which is read as a Thr instead of a Lys, creates a new phosphorylation site for the Akt protein kinase on the Kv11.1 channel. Consequently hormonal signaling through the PI3K signaling cascade, which normally stimulates K897 channels through PP5-mediated dephosphorylation, inhibits T897 channels through Akt-mediated phosphorylation. Thus, hormonal regulation of Kv11.1 in humans with the T897 polymorphism is predicted to prolong the QT interval of cardiac myocytes. The PMCF first identified phosphorylation as T897 which led to authorship of the paper: Gentile, S., Martin, N., Scappini, E., Smutko, P., Williams, J., Erxleben, C., Armstrong, D.L. in press P.N.A.S. Human Gene Polymorphisms Reprogram Hormonal Signaling by Altering Kinase Recognition Sites? ? Nuclear receptor coactivator 6 (NCOA6) anchors a complex of cofactors and functions as a transcriptional coactivator for certain nuclear receptors. We identified NCOA6 as a hepatic nuclear factor 4alpha (HNF4alpha)-interacting protein. CYP2C9 is an important enzyme that metabolizes both commonly used therapeutic drugs and important endogenous compounds. We have shown previously that constitutive androstane receptor (CAR) up-regulates the CYP2C9 promoter through binding to a distal site, whereas HNF4alpha transcriptionally up-regulates CYP2C9 via proximal sites. We demonstrated ligand-enhanced synergistic cross-talk between CAR and HNF4alpha. We identified NCOA6 as crucial to the underlying mechanism of this cross-talk. NCOA6 was identified as an HNF4alpha-interacting protein using a yeast two-hybrid screen and GST pull-down assays. Furthermore, we identified NCOA6, CAR, and other coactivators as part of a mega complex of cofactors associated with HNF4alpha in HepG2 cells. Chromatin immunoprecipitation analysis revealed that NCOA6 can pull down both the proximal HNF4alpha and distal CAR binding sites of the CYP2C9 promoter and provides the basis for the recruitment of other cofactors. We conclude that the coactivator NCOA6 mediates the mechanism of the synergistic activation of the CYP2C9 gene by CAR and HNF4alpha. The PMCF was involved in protein identifications from the pull-down experiments and the core was acknowledged in the subsequent manuscript: Surapureddi S, Rana R, Reddy JK, Goldstein JA. (2008) Mol Pharmacol 74(3):913-23 Nuclear receptor coactivator 6 mediates the synergistic activation of human cytochrome P-450 2C9 by the constitutive androstane receptor and hepatic nuclear factor-4alpha? ? Homeobox genes encode transcription factors whose expression organizes programs of development. A number of homeobox genes expressed in reproductive tissues have been identified recently, including a colinear cluster on the X chromosome in mice. The Eddy group identified and characterized a novel homeobox gene of the paired-like class on the X chromosome distal to the reproductive homeobox (Rhox) cluster in mice. The Rhox13 gene encodes a 25.3 kDa protein expressed in the adult testis in germ cells at the basal aspect of the seminiferous epithelium. The PMCF confirmed western blot identity and hence was acknowledged in the paper: Geyer C.B., Eddy E.M. in press Gene. Identification and characterization of Rhox13, a novel X-linked mouse homeobox gene testis, ovary, spermatogenesis, transcription, X chromosome? ? Constitutive active/androstane receptor (CAR) activates transcription of numerous hepatic genes upon exposure to therapeutic drugs and environmental pollutants. Sequestered in the cytoplasm, this receptor signals xenobiotic exposure, such as phenobarbital (PB), by translocating into the nucleus. Unlike other hormone receptors, translocation can be triggered indirectly without binding to xenobiotics. The Negishi lab and PMCF identified a membrane-associated subunit of protein phosphatase 1 (PPP1R16A, or abbreviated as R16A) as a novel CAR-binding protein. When CAR and R16A are coexpressed in mouse liver, CAR translocates into the nucleus. R16A at the membrane may mediate the PB signal to initiate CAR nuclear translocation, through a mechanism including its dimerization and inhibition of PP1beta activity, providing a novel model for the translocation of nuclear receptors in which direct interaction of ligands and the receptors may not be crucial. The PMCF identified binding partners of CAR which led to acknowledgement in the paper: Sueyoshi T., Moore R., Sugatani J., Matsumura Y., Negishi M. (2008) Mol Pharmacol. 73(4):1113-21 PPP1R16A, the membrane subunit of protein phosphatase 1beta, signals nuclear translocation of the nuclear receptor constitutive active/androstane receptor.? ? Additional projects that have required more than negligible resources include efforts performed with the Adelman, Archer, Birnbaumer, Garantziotis, Hall, London, J. Mason, Pederson, Pritchard, Van Houten, Wade, and Yakel laboratories.
| Kaminski, Andrea M; Tumbale, Percy P; Schellenberg, Matthew J et al. (2018) Structures of DNA-bound human ligase IV catalytic core reveal insights into substrate binding and catalysis. Nat Commun 9:2642 |
| Schellenberg, Matthew J; Lieberman, Jenna Ariel; Herrero-Ruiz, Andrés et al. (2017) ZATT (ZNF451)-mediated resolution of topoisomerase 2 DNA-protein cross-links. Science 357:1412-1416 |
| Johnson, Katina L; Williams, Jason G; Maleki, Soheila J et al. (2016) Enhanced Approaches for Identifying Amadori Products: Application to Peanut Allergens. J Agric Food Chem 64:1406-13 |
| Mueller, G A; Maleki, S J; Johnson, K et al. (2013) Identification of Maillard reaction products on peanut allergens that influence binding to the receptor for advanced glycation end products. Allergy 68:1546-54 |
| Dhungana, Suraj; Williams, Jason G; Fessler, Michael B et al. (2009) Epitope mapping by proteolysis of antigen-antibody complexes. Methods Mol Biol 524:87-101 |
| Cuthbertson, Brandon J; Deterding, Leesa J; Williams, Jason G et al. (2008) Diversity in penaeidin antimicrobial peptide form and function. Dev Comp Immunol 32:167-81 |
