Browsing by Subject "GTPases"

Now showing 1 - 3 of 3
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    Assessing the Influence of Mutation on GTPase Transition States by Using X‐ray Crystallography, 19F NMR, and DFT Approaches
    (Wiley, 2017-08-07) Jin, Yi; Molt, Robert W.; Pellegrini, Erika; Cliff, Matthew J.; Bowler, Matthew W.; Richards, Nigel G. J.; Blackburn, G. Michael; Waltho, Jonathan P.; Biochemistry and Molecular Biology, School of Medicine
    We report X‐ray crystallographic and 19F NMR studies of the G‐protein RhoA complexed with MgF3 −, GDP, and RhoGAP, which has the mutation Arg85′Ala. When combined with DFT calculations, these data permit the identification of changes in transition state (TS) properties. The X‐ray data show how Tyr34 maintains solvent exclusion and the core H‐bond network in the active site by relocating to replace the missing Arg85′ sidechain. The 19F NMR data show deshielding effects that indicate the main function of Arg85′ is electronic polarization of the transferring phosphoryl group, primarily mediated by H‐bonding to O3G and thence to PG. DFT calculations identify electron‐density redistribution and pinpoint why the TS for guanosine 5′‐triphosphate (GTP) hydrolysis is higher in energy when RhoA is complexed with RhoGAPArg85′Ala relative to wild‐type (WT) RhoGAP. This study demonstrates that 19F NMR measurements, in combination with X‐ray crystallography and DFT calculations, can reliably dissect the response of small GTPases to site‐specific modifications.
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    BIOCHEMICAL CHARACTERIZATION OF SMALL MOLECULES TARGETING RAL GTPASE
    (Office of the Vice Chancellor for Research, 2012-04-13) Ishikawa, Megan; Khanna, May; Jo, Inha; Meroueh, Samy
    The Ral subfamily of GTPases consists of highly similar RalA and RalB isoforms that participate in diverse cellular functions including endocytosis, exocytosis, actin cytoskeletal dynamics, and transcription. A large body of evidence has implicated Ral GTPases with tumor cell growth, migration, and angiogenesis in bladder, prostate, lung, and pancreatic cancer. The purpose of this project was to target the activity of Ral GTPases and their association with effector proteins through the identification of small molecule inhibitors that block this interaction. In order to accomplish this, both direct binding to RalB as well as disruption of protein-protein interaction were investigated. The top 200 compounds from a larger computational library of 500,000 compounds targeting the RalBP1 binding site on RalB were tested. Differen-tial scanning fluorimetry (DSF) was used to measure the degree of direct binding between compound and protein through thermal melting shift. To measure disruption between RalB and RalBP1 by small molecules, a novel enzyme-linked immunosorbent assay (ELISA) was developed. Identification of a few key compounds binding to RalB as well as optimization of an ELISA assay for RalBP1 was accomplished. Further direction of this project would be to utilize the ELISA assay to test inhibition of the protein-protein interac-tion between RalB and RalBP1 using the top compounds from the DSF trials.
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    Rap1 is a Potential Therapeutic Target for Non-myeloablative Conditioning
    (Office of the Vice Chancellor for Research, 2013-04-05) Ghosh, Joydeep; Ramdas, Baskar; Quilliam, Lawrence; Kapur, Reuben
    various side effects including gastrointestinal mucositis. Identification of therapeutic targets and determining their role in HSC development and function is important to determine a regimen for nonmyeloablative conditioning. Previous studies have shown that GTPases play a critical role in self-renewal, engraftment and retention of HSCs. Rap1, a GTPase, is necessary for migration, adhesion as well as function of mature hematopoietic cells. To study the role of Rap1 in hematopoietic stem and progenitor cells (HSC/Ps), we have generated a mouse model in which the Rap1a and Rap1b isoforms of Rap1 are conditionally deleted in HSC/Ps (Rap1a/b -/-). Deficiency of Rap1a/b results in increased peripheral blood count as well as increase in HSCs in bone marrow along with a decrease in bone marrow cellularity. Rap1a/b deficient bone marrow HSC/Ps also have reduced adhesion capability in vitro. The self-renewal property of HSCs, in conjunction with their ability of multi-lineage reconstitution is important to repopulate the hematopoietic system of irradiated recipients of bone marrow transplant. Rap1a/b -/- HSCs show a defect in engraftment as well as multi-lineage reconstitution when they are transplanted into lethally irradiated hosts. Rap1 deficient HSCs show decreased homing into bone marrow of lethally irradiated recipients. To determine whether Rap1 can be used as a potential target for nonmyeloablative conditioning, we performed bone marrow transplant into WT and Rap1a/b -/- mice without prior irradiation. Deficiency of Rap1a/b in HSCs resulted in availability of bone marrow niche for exogenously transplanted HSCs to engraft along with subsequent multi-lineage reconstitution. Overall, our study reveals that Rap1a/b are important for homing and retention of hematopoietic cells in bone marrow and deletion of Rap1a/b in HSCs result in engraftment of exogenous HSCs within the bone marrow of non-irradiated recipients.