Browsing by Subject "Aryl Hydrocarbon Receptor Nuclear Translocator"
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Item A Fragile Native State Structure: An Aryl Hydrocarbon Receptor Nuclear Translocator (ARNT) Variant Exhibits Slow Interconversion Kinetics Between two Different Folds(2009-09-04) Evans, Matthew Ryan; Gardner, Kevin H.The aryl hydrocarbon receptor nuclear translocator (ARNT) is a promiscuous basic helix-loop-helix Period/ARNT/Single-minded (bHLH-PAS) protein that controls various biological pathways by forming heterodimeric transcriptional regulator complexes with several other bHLH-PAS proteins via the beta-sheet surfaces of its two PAS domains. The beta-sheets of PAS domains are involved in many intermolecular interactions with other proteins and natural cofactors in order to detect environmental changes in sensor PAS proteins. As part of a study of the HIF-2 alpha and ARNT PAS-B heterodimer, site-directed mutagenesis was performed on the ARNT PAS-B domain. Interestingly, one point mutation on the ARNT beta-sheet surface (Y456T) resulted in a new conformation of the domain that existed in equimolar concentrations with the wild-type conformation. Subsequent studies demonstrated that the two conformations are in equilibrium and that relative populations of the two conformations can be perturbed by additional mutations. Using solution NMR spectroscopy, we solved the high resolution solution structure of a mutant ARNT PAS-B domain in the new conformation, demonstrating that it contains a three-residue slip in register and accompanying inversion of the central beta-strand. In addition, this new conformer has a greater than hundred-fold reduction in affinity for HIF-2 alpha PAS-B, disrupting the hypoxia response pathway. Solution NMR measurements of the interconversion kinetics have let us establish that these two conformations interconvert slowly (40 min at RT) with a linear Arrhenius temperature-dependence of the interconversion rate. Stopped-flow unfolding experiments using GdmHCl on Y456T, revealed a similarly slow unfolding rate (25 min at RT) and an energy barrier to unfold of approximately 13 kcal/mol, which is nearly identical to that for the interconversion process. These data indicate that the protein must undergo a global unfolding process in order to interconvert between conformations. Lastly, these relative populations of Y456T can be affected by compound preferentially binding into the core of one of these conformations. This discovery highlights the malleability of PAS beta-sheets and suggests ARNT may act as a regulatory switch to control different biological pathways. Furthermore, this system presents a great opportunity to further understand the structural and kinetic impact of beta-strand slips observed in nature.Item Of Apoptosomes and Oncogenes: Repurposing a Death Machine & Deconstructing the Action of P53 Mutations(2014-04-14) D'Brot, Alejandro; Scaglioni, Pier Paolo; Shay, Jerry W.; Yu, HongtaoIt is now well appreciated that the apoptosome, which governs caspase-dependent cell death, also drives nonapoptotic caspase activation to remodel cells. However, determinants that specify whether the apoptosome acts to kill or remodel have yet to be identified. I show here that Tango7 genetically interacts with the apoptotic machinery but instead of regulating cell death, collaborates with the apoptosome to drive caspase-dependent cellular remodeling. Specifically, Tango7 is required for non-apoptotic caspase activity during spermatid remodeling and localizes to the active apoptosome compartment in these cells via its C terminus. Furthermore, Tango7 directly stimulates activity of reconstituted apoptosomes in vitro. These and other data presented here suggest that Tango7 physically recruits the apoptosome to specify this complex for nonapoptotic cellular remodeling. In vivo genetic model systems are powerful tools to deconstruct activity of genes driving human disease. The tumor suppressor p53 is mutated more than any other gene in human cancer, but unlike other tumor suppressors, it acquires missense mutations which encode oncogenic variants. These gain-of-function mutants promote more aggressive and metastatic cancers in vivo but their oncogenic activity is not well understood. To address this problem, I have exploited Drosophila as a platform to study and stratify human p53 (hp53) mutants. I replaced fly p53 with either wild-type hp53 or 5 of the most prevalent hp53 mutations in cancer. In this system, hp53 is under control of endogenous Dp53 regulatory elements and can regulate in vivo transcriptional activation and apoptosis like its fly counterpart. Furthermore, wild-type hp53 forms foci in the germline that localize to the same subnuclear compartment as Dp53 foci. This property is compromised in all of the gain-of-function mutants and can thus be used to distinguish oncogenic variants from wild-type hp53. Future studies aim at finding whether this and other properties shared among the 5 mutants can help stratify oncogenic p53 mutations found in human cancer.Item PAS Domain-Mediated Dimerization of the Aryl Hydrocarbon Receptor Nuclear Translocator (ARNT) in the Hypoxia Response Pathway(2005-12-19) Card, Paul B.; Gardner, Kevin H.PAS (Per-ARNT-SIM) domains are versatile protein-protein interaction domains that are often used as regulatory modules in a variety of important biological pathways. Although their importance in several of these pathways has been well established, only sparse structural data exists that could help elucidate a general mode of PAS-PAS interaction. As such, more examples of these domains must be studied using a variety of techniques to understand how these domains carry out viable functions within the cell.The Aryl Hydrocarbon Receptor Nuclear Translocator (ARNT) is a constituent of heterodimeric transcriptional activation complexes used in several important biochemical pathways. One such complex is the hypoxia inducible transcription factor (HIF), which allows mammalian cells to respond to changes in oxygen availability. In HIF, ARNT dimerizes with HIFa to upregulate genes involved in the hypoxia response. The dimerization of ARNT and HIFa involves interactions between PAS domains in both proteins, but details regarding how these domains interact in this and other heterodimeric complexes have not previously been well characterized. To investigate these interactions within the context of the HIF/ARNT heterodimer, we expressed the C-terminal PAS domains from both proteins and characterized the complex using NMR-based methods. Solution structures of each domain are presented, as well as a model of the ARNT/HIF heterodimeric PAS complex. This model was used to identify of key interfacial residues, and the roles of these were tested in a variety of ways by sitedirected mutagenesis. In addition, extended constructs from ARNT that include other components of the full-length protein were investigated to establish the validity of a reductionist approach in the study of individual PAS domains from this system. In many biological systems, PAS domains bind small molecules to regulate proteinprotein interactions. With this in mind, we also subjected PAS domains from HIFa and ARNT to an NMR-based screen against an in-house library of 800 compounds to determine the potential of these domains to bind small molecules. With have used this approach to identify compounds that can disrupt PAS-PAS interactions in the hypoxia pathway in order to help elucidate some of the molecular details of PAS domain-mediated signaling.Item Structural Characterization and Chemical Inhibition of the ARNT/TACC3 Complex(2015-04-13) Guo, Yirui; Rosenbaum, Daniel M.; Roth, Michael G.; Rice, Luke M.; Gardner, Kevin H.My research project focuses on mechanistic studies of a new group of transcriptional coactivators (coiled-coil coactivators: TACC3, TRIP230, CoCoA), involved in cancer development and progression. Normally, these coactivators play an essential role in the HIF hypoxia response, directly interacting with the ARNT subunit of HIF in a novel and promoter-specific way. However, misregulation by overexpression or activating fusions (for example, FGFR-TACC3) is sufficient for transformation and associated with the development of glioblastoma, renal cell carcinoma and other cancers. In light of this connection between coiled-coil coactivators (CCCs) and HIF signaling, tools that inhibit HIF/CCC complex formation might present opportunities to interrogate the linkage between different CCC-containing pathways and may offer a novel route to blocking cancer formation and progression. As a member of a new group of transcription coactivators, knowing how TACC3 interacts with ARNT is critical in understanding the general role of CCCs in HIF-dependent transcription machinery. In the first half of my study, I characterized the ARNT/TACC3 complex with various biophysical and biochemical methods including solution NMR, X-ray crystallography, circular dichroism, luminescence proximity and numerous cell-based assays. A 3.15 Å ARNT/TACC3 crystal structure was solved by molecular replacement, revealing details of this protein complex and providing a structural funcation for coactivator recruitment in HIF signaling pathway. The second half of this study focuses on the search for ARNT/TACC3 inhibitors with in vitro screens to regulate ARNT/CCCs activity in a rapid and flexible way. From a fragment-based NMR screen, I identified small molecules that specifically bound within the second PER-ARNT-SIM (PAS) domain of ARNT and perturb its interaction with TACC3. However, these small molecules have drawbacks, such as low potency or unclear modes of action. To identify higher potency small molecules targeting ARNT/TACC3 complexes, I developed an AlphaScreen-based high throughput screen. Hopefully the discovery of artificial ligands with known mode-of-action that inhibit this typically "undruggable" protein complex will provide new perspectives in small molecule inhibitor development, and also serve as a very useful tool in cell biology for studying pathways utilizing ARNT/TACC3.