Browsing by Subject "Ubiquitin"

Now showing 1 - 4 of 4
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    Defining how the 26S proteasome recognizes ubiquitinated substrates
    (2012-12) Randles, Leah Ann
    Regulated protein degradation in eukaryotes is performed predominantly by the ubiquitin-proteasome pathway. Prior to their degradation by the 26S proteasome, protein substrates become covalently modified with ubiquitin chains. Such ubiquitination enables the 19S regulatory particle of the proteasome to recognize the doomed protein substrate. My thesis research focuses on defining how the 19S regulatory particle of the proteasome recognizes ubiquitinated substrates. When I began my thesis research, S5a/Rpn10 was the only known proteasomal ubiquitin receptor; yet, it is not essential for degradation of ubiquitinated substrates by the proteasome. My thesis research helped establish Adrm1/ARM1/Rpn13 as the missing proteasomal ubiquitin receptor. I used NMR spectroscopy to provide mechanistic insights into how human Rpn13 binds ubiquitinated substrates. I adapted a protocol developed by the late Cecile Pickart to fine tune polyubiquitin synthesis for use in NMR structural studies. By using this approach, I selectively labeled individual subunits within polyubiquitin to determine Rpn13's selective binding to the proximal subunit of K48-linked diubiquitin (diUb). These results, along with additional results from my lab and our collaborators' labs, are described in Chapters 2 and 3.I continued to explore Rpn13's role as a ubiquitin receptor by testing whether it is able to work in cooperation with S5a/Rpn10. Along with other members of my lab, we solved the structure the S5a/K48-linked diUb complex by utilizing the method I optimized for synthesizing selectively labeled polyubiquitin. I then helped expand this work to reveal that Rpn13 and S5a are able to bind a common ubiquitin chain and thereby work cooperatively to capture ubiquitinated substrates. These results are described in Chapter 4.In Chapter 5, I describe a novel interaction of Rpn13's Pru domain to a ubiquitin processing enzyme, namely E2 ubiquitin conjugating enzyme Cdc34. NMR experiments reveal that an Rpn13 surface that neighbors its ubiquitin-binding loops binds to Cdc34's unique C-terminal tail and that this interaction does not restrict Rpn13 binding to ubiquitin. Immunoprecipitation experiments performed on HeLa cells with endogenous protein levels demonstrate Rpn13 and Cdc34 to be in complex in the cellular environment. The Rpn13:Cdc34 interaction suggests that Rpn13 may play a role in SCF-mediated ubiquitination. Proteasome dysfunction is implicated in many diseases, such as cancer, cystic fibrosis, heart disease, and neurodegenerative diseases and the ubiquitin-proteasome pathway has therefore become a major pharmaceutical target. My thesis research in trying to understand how the proteasome recognizes ubiquitinated substrates provides structural and mechanistic information that could be used to help develop new drugs and possible treatments for these diseases.
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    Functional characterization of the three isoforms of Fbw7 (F-box and WD repeat domain containing 7) in ubiquitin dependent proteolysis.
    (2010-01) Zhang, Wei
    Fbw7 is the F-box protein of SCFFbw7 E3 ubiquitin ligase, which specifically associates with the substrates to be ubiquitinated. Substrates of Fbw7 play important roles in cell cycle regulation, proliferation, signal transduction and metabolism, which are related to tumor formation, suggesting that Fbw7 functions as a tumor suppressor. Fbw7 has three splicing variants α, β and γ, and the biological function of each isoform is not well understood. Our lab is interested in how the Fbw7 isoforms regulate cyclin E proteolysis and the cell cycle. By using mammalian and insect cell culture systems, I demonstrate that the three isoforms can form homo- and heterodimers in vivo and in vitro. The dimerization domain is located immediately upstream of the F-box motif, and it is highly conserved in all Fbw7 homologues and other related F-box proteins, indicating the dimerization may be common feature of a subset of F-box proteins. Abolishment of dimerization inhibits cyclin E proteolysis and leads to a prolonged half-life of cyclin E, although it does not affect Fbw7 binding to cyclin E or to the Cul-Rbx1-Skp1 E3 catalytic module. Cyclin E accumulation can be commonly found in many primary tumors and cancer cell lines. These results suggest a novel mechanism of how F-box proteins recognize their substrates. Fbw7 isoforms show different protein stabilities, where the α isoform is stable, but the β and γ isoforms are not. The stability of the β and γ isoforms is largely controlled by their N- terminal unique region. In order to better understand the mechanism regulating their stability, we performed a yeast two hybrid screen and identified SLP1 (stomatin like protein 1) as an Fbw7γ isoform specific interacting protein. SLP1 binds to the unique region of γ isoform, and stabilizes γ. We find that Cdk2 promotes the degradation of both SLP1 and the γ isoform, and this function of Cdk2 is dependent on its kinase activity. SLP1 also physically interacts with Cdk2 through its membrane association domain. These results support a model in which Fbw7γ and SLP1 are coordinately targeted for ubiquitin mediated degradation by Cdk2.
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    Molecular Mechanism of Wolbachia Induced Cytoplasmic Incompatibility
    (2014-05) Beckmann, John Frederick
    Wolbachia are obligate intracellular endosymbionts which live in the gonads of many arthropods of economic and medical importance. In insects, Wolbachia manipulate reproduction in a way that favors the spread of their infection. Cytoplasmic Incompatibility (CI), is a particular effect induced by Wolbachia infection in mosquitoes and other insects. CI causes conditional male sterility and produces a selective pressure in mixed populations of infected and uninfected mosquitoes giving Wolbachia-infected females a reproductive advantage. CI has been proposed as a gene drive tool which could be used to replace wild arthropod disease vectors with genetically modified ones less capable of transmitting diseases. CI has been demonstrated to be an effective agent at manipulating vector populations in the wild. When I began my research on Wolbachia in 2009, a central unresolved question, which has remained unanswered since the 1950's, concerned the molecular basis of CI; my doctoral research has wholly focused on answering this basic question, "What is the Wolbachia gene/protein that induces CI in mosquitoes?"
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    Proteasome activation by the 19S regulatory particle: structural dynamics of 26S assembly and substrate recognition
    (2013-06) Ehlinger, Aaron Christopher
    Since its discovery in the late 1970s, the ubiquitin-proteasome system (UPS) has become recognized as the major pathway for regulated cellular proteolysis. Processes ranging from cell cycle control, pathogen resistance, and protein quality control rely on selective protein degradation at the proteasome for homeostatic function. Perhaps as a consequence of the importance of this pathway, and the genesis of severe diseases upon its dysregulation, protein degradation by the UPS is highly controlled from the level of substrate recognition to proteolysis. Technological advances over the last decade have created an explosion of structural and mechanistic information that has underscored the complexity of the proteasome and its upstream regulatory factors. Significant insights have come from study of the 19S proteasome regulatory particle (RP) responsible for recognition and processing of ubiquitinated substrates destined for proteolysis. Established as a highly dynamic proteasome activator, a large number of both permanent and transient RP components with specialized functional roles are critical for proteasome function. This research investigates the dynamic nature of protein-protein interactions involved in proteasome assembly and substrate recruitment, and how they provide context to our current understanding of proteasome activation by the RP.