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BIOPHYSICAL AND PHARMACOLOGICAL CHARACTERIZATION OF CYTOPLASMIC DYNEIN HEAVY CHAIN 1

Daghestani, Hikmat (2011) BIOPHYSICAL AND PHARMACOLOGICAL CHARACTERIZATION OF CYTOPLASMIC DYNEIN HEAVY CHAIN 1. Doctoral Dissertation, University of Pittsburgh.

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    Abstract

    The cytoplasmic dynein motor protein complex transports a number of different important cargos along microtubules (MTs) in a retrograde manner. Cytoplasmic dynein plays an important role in many cellular processes and a number of diseases have been associated with defects in its activity. Despite its importance, there are no small molecules that selectively modulate cytoplasmic dynein activity, nor is its atomic structure elucidated. In an effort to identify compounds that target cytoplasmic dynein, hits from a high information content cell-based nuclear translocation assay were further evaluated biochemically. High throughput assays were developed to screen for glucocorticoid ligand competition, MT perturbation, and the ATPase activities of Hsp 70 and 90, cytoplasmic dynein heavy chain 1, and myosin. Several compounds from screening the Library of Pharmacologically Active Compounds (LOPAC1280) were identified to inhibit cytoplasmic dynein, though they had several unattractive pharmacological properties and were generally non-specific. Additional screening of the Molecular Libraries Screening Center Network >220,000-member library showed a number of compounds that specifically inhibited the ATPase activity of cytoplasmic dynein heavy chain 1 with little or no interaction with other proteins involved in cargo complex formation. A novel approach to screen for MT perturbing agents was also developed using biosensors. Thickness, mass, and density measurements from dual polarization interferometry suggested the growth process of MTs on surfaces. Resonant mirror biosensors were used to distinguish MT stabilizers from destabilizers based on rates of MT assembly on the surfaces.In addition, the structure of the cytoplasmic dynein heavy chain motor domain was characterized by computational and experimental methods. Comparative homology structural modeling was used to predict 15 surface accessible cysteines, which were then correlated experimentally by mass spectrometry. Five cysteines were matched computationally and experimentally to be surface-accessible, suggesting some inadequacy of the proposed model. Finally, attempts to reconstruct a model of cytoplasmic dynein heavy chain 1 by electron microscopy were hindered by the purification of the protein from both a Hi5/baculovirus expression system and bovine brain, although the latter appeared to provide better quality micrographs. Ultimately, structural characterization will assist with the discovery of cytoplasmic dynein heavy chain 1 modulators.


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    Item Type: University of Pittsburgh ETD
    ETD Committee:
    ETD Committee TypeCommittee MemberEmailORCID
    Committee ChairDay, Billy Wbday@pitt.edu
    Committee MemberRomero, Guillermoggr@pitt.edu
    Committee MemberConway, Jamesjxc100@pitt.edu
    Committee MemberCascio, Michaelcasciom@duq.edu
    Committee MemberWetzel, Ronaldrwetzel@pitt.edu
    Title: BIOPHYSICAL AND PHARMACOLOGICAL CHARACTERIZATION OF CYTOPLASMIC DYNEIN HEAVY CHAIN 1
    Status: Unpublished
    Abstract: The cytoplasmic dynein motor protein complex transports a number of different important cargos along microtubules (MTs) in a retrograde manner. Cytoplasmic dynein plays an important role in many cellular processes and a number of diseases have been associated with defects in its activity. Despite its importance, there are no small molecules that selectively modulate cytoplasmic dynein activity, nor is its atomic structure elucidated. In an effort to identify compounds that target cytoplasmic dynein, hits from a high information content cell-based nuclear translocation assay were further evaluated biochemically. High throughput assays were developed to screen for glucocorticoid ligand competition, MT perturbation, and the ATPase activities of Hsp 70 and 90, cytoplasmic dynein heavy chain 1, and myosin. Several compounds from screening the Library of Pharmacologically Active Compounds (LOPAC1280) were identified to inhibit cytoplasmic dynein, though they had several unattractive pharmacological properties and were generally non-specific. Additional screening of the Molecular Libraries Screening Center Network >220,000-member library showed a number of compounds that specifically inhibited the ATPase activity of cytoplasmic dynein heavy chain 1 with little or no interaction with other proteins involved in cargo complex formation. A novel approach to screen for MT perturbing agents was also developed using biosensors. Thickness, mass, and density measurements from dual polarization interferometry suggested the growth process of MTs on surfaces. Resonant mirror biosensors were used to distinguish MT stabilizers from destabilizers based on rates of MT assembly on the surfaces.In addition, the structure of the cytoplasmic dynein heavy chain motor domain was characterized by computational and experimental methods. Comparative homology structural modeling was used to predict 15 surface accessible cysteines, which were then correlated experimentally by mass spectrometry. Five cysteines were matched computationally and experimentally to be surface-accessible, suggesting some inadequacy of the proposed model. Finally, attempts to reconstruct a model of cytoplasmic dynein heavy chain 1 by electron microscopy were hindered by the purification of the protein from both a Hi5/baculovirus expression system and bovine brain, although the latter appeared to provide better quality micrographs. Ultimately, structural characterization will assist with the discovery of cytoplasmic dynein heavy chain 1 modulators.
    Date: 07 January 2011
    Date Type: Completion
    Defense Date: 10 December 2010
    Approval Date: 07 January 2011
    Submission Date: 21 December 2010
    Access Restriction: 5 year -- Restrict access to University of Pittsburgh for a period of 5 years.
    Patent pending: No
    Institution: University of Pittsburgh
    Thesis Type: Doctoral Dissertation
    Refereed: Yes
    Degree: PhD - Doctor of Philosophy
    URN: etd-12212010-113824
    Uncontrolled Keywords: biosensors; comparative homology modeling; cysteine mapping; cytoplasmic dynein; dynein modulator screen; microtubules
    Schools and Programs: School of Medicine > Molecular Biophysics and Structural Biology
    Date Deposited: 10 Nov 2011 15:11
    Last Modified: 30 May 2012 10:51
    Other ID: http://etd.library.pitt.edu/ETD/available/etd-12212010-113824/, etd-12212010-113824

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