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Department of Biochemistry & Molecular Biology

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These digital collections include theses, dissertations, and datasets from the Department of Biochemistry & Molecular Biology.

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Browsing Department of Biochemistry & Molecular Biology by Author "Bamburg, James R., committee member"

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    Dynein mutagenesis reveals the molecular basis for dynein regulation in broad spectrum neurological diseases
    (Colorado State University. Libraries, 2020) Marzo, Matthew G., author; Markus, Steven M., advisor; Bamburg, James R., committee member; DeLuca, Jennifer, committee member; Prasad, Ashok, committee member
    Eukaryotic cells rely on cytoskeletal networks to organize materials, transport organelles, give cells shape, and provide locomotion. The cytoskeleton is comprised of many diverse proteins, and three classes of polymeric protein structures are the actin, microtubule, and intermediate filament networks. The microtubule network, and its associated motors, dynein and kinesin, is of interest to the field of neurological disease, due to the prevalence of mutations in the microtubule network in human disease. To better understand the molecular basis for the diseases caused by de novo dynein mutations, we performed a screen of mutants using budding yeast dynein. The results from our experiments present a platform for the molecular dissection of dynein mutations which can be readily applied to new mutations or precisely explore known mutations. The screen-based approach allowed us to identify a new mechanism of yeast dynein regulation, which is autoinhibition of the dynein motor. We demonstrate that this mechanism regulates dynein activity in cells and functions to limit in vivo motor activity in the cytoplasm. Autoinhibition is regulated by Pac1 in yeast, a Lissencephaly-1 homolog, and we demonstrate that Pac1 operates in the dynein autoinhibition pathway by preventing the "closed" autoinhibited state, thereby promoting "open" dynein. This represents an entirely novel function of Pac1/LIS1, and allows us to further refine our model for cortical offloading.
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    Molecular characterization of the protein-protein interaction between HTLV-1 Tax and GSK-3ß
    (Colorado State University. Libraries, 2010) Wang, Guoliang, author; Nyborg, Jennifer, advisor; Bamburg, James R., committee member; Quackenbush, Sandra L., committee member; Laybourn, Paul J., committee member
    The human T-cell leukemia virus type 1 (HTLV-1) encodes a viral oncoprotein termed Tax, which plays a major role in transforming HTLV-1- infected cells. Tax is a potent transcriptional activator that stimulates HTLV-1 viral and cellular gene transcription. In addition, Tax disrupts a number of cell signaling pathways involved in cell growth and survival. Glycogen synthase kinase-33 (GSK-33) is a ubiquitously expressed serine/threonine kinase present in all eukaryotic cells, which functions as a critical regulator of a wide range of cell signaling pathways. As GSK-33 is constitutively active in resting cells, it is primarily regulated through inhibition. Ser-9 phosphorylation is inhibitory to the kinase activity of GSK-33. Deregulation of GSK-33 has been linked to many human diseases such as Alzheimer’s disease and cancers. It has been reported in Aida Ulloa’s thesis that Tax inhibits GSK-33 kinase activity toward both primed and non-primed substrates through direct association. To delineate the protein-protein interaction between Tax and GSK-33, we compared the amino acid sequence of Tax with a well-characterized short peptide deriving from the GSK-33 interacting domain (GID) of Axin, and found that Tax contains a notable amino acid sequence homology to Axin GID. The region spanning Tax amino acids 185 - 205 has 24% sequence identity and 19% similarity with Axin GID. We named this region the putative Tax GID. We characterized the putative Tax GID biochemically, and discovered that a longer peptide (Tax aa. 138 - 205) of the putative Tax GID strongly inhibits GSK-3(3 kinase activity in vitro. Bioinformatics computation was used to predict the secondary structure of the Tax GID, which was further used in our docking test to identify a potential binding interface in GSK-3p. This was tested by GST pulldown and Co-IP assays using point and deletion mutants. In addition, the effects of Tax-GSK-3(3 interaction on the downstream (3-catenin and NFAT pathways were characterized by luciferase reporter assays. However, unexpectedly, we observed that Tax expression has little effects on p-catenin and NFAT transcriptional activation.