Browsing by Subject "Proteasome"

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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    Exploiting quiescent state vulnerability to deplete cytostatic cancer cells
    (2019-12) Kim, Lisa
    Controlling cancers as chronic conditions, by arresting and sustaining tumor cells at a quiescent or non-proliferating cytostatic state, is emerging as a standard treatment approach with the increasing use of targeted therapies for cancers. While effective, persisting cytostatic tumor cells pose fundamental challenges to achieving prolonged effects as they require continual maintenance and are prone to develop resistance and recurrence. Depleting these tumor populations could improve treatment outcome, but no rational approach exists, besides monitoring, because no targeting strategy is designed for non-proliferating cells. Here, we exploited vulnerabilities evoked by oncogenic signals at quiescent states to devise targeting strategies for cytostatic tumor populations harboring AKT hyperactivation, an oncogenic change associated with treatment-tolerance and relapse. Using an organotypic model of normal quiescent mammary cells, we found that AKT hyperactivation dysregulates redox and protein homeostasis and sensitizes the quiescent cells to apoptosis upon proteasome inhibition in a p70S6K- and redox-dependent manner. Intriguingly, therapeutic exploitation of this AKT-driven quiescent-state proteasome-vulnerability showed efficacies on cytostatic cancer cells with aberrant PI3K/AKT signaling activation across different breast cancer subtypes, epithelial tissue origins, and arresting mechanisms. Moreover, transient proteasome-inhibitor treatment in spheroid and mouse xenograft models of cytostatic tumors significantly reduced recurrent growth after treatment-cessation. Our work highlights the effects of oncogenic mutations on altering homeostasis at non-proliferating states that could form the basis for devising targeted approaches for depleting genetically-predisposed cytostatic tumor subpopulations.
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    Probing cardiac calcium regulation using fluorescence spectroscopy.
    (2011-06) Lockamy, Elizabeth Lee
    Calcium (Ca2+) is stored in the sarcoplasmic reticulum (SR) in both cardiac and skeletal muscle. A Ca2+ induced Ca2+ release mechanism triggers the ryanodine receptor (RyR) to release Ca2+ from the SR into the cytoplasm. This Ca2+ discharge increases the Ca2+ concentration causing the muscle to contract. RyR is regulated by calmodulin (CaM), a Ca2+ binding protein that inhibits RyR when the [Ca2+] > mM. To relax the muscle, the Sarco-endoplasmic Reticulum Calcium Adenosine Triphosphatase (SERCA), an integral membrane enzyme, pumps Ca2+ back into the SR driven by ATP hydrolysis. In cardiac tissue, SERCA is regulated by phospholamban (PLB), an integral membrane protein that inhibits SERCA at submicromolar [Ca2+]. This inhibition is relieved either by addition of micromolar Ca2+ or by phosphorylation of PLB by cAMP-dependent protein kinase A (PKA). The goal of this research was to investigate Ca2+ regulation during muscle contraction and relaxation. The major findings included: 1) two PLB variants bind tightly to SERCA, thus competing with and displacing wild-type (WT) PLB, 2) SERCA contains a novel nucleotide binding site that is not an artifact of crystallization, and 3) oxidation of specific Met residues in CaM are vital for proteasomal degradation. Using functional co-reconstitution and fluorescence resonance energy transfer (FRET), we tested the hypothesis that the loss-of-function (LOF) mutants can compete with WT-PLB to relieve SERCA inhibition. We investigated two LOF mutants, S16E (phosphorylation mimic) and L31A, for their inhibitory potency and their ability to compete with WT-PLB. Our functional studies demonstrate that SERCA co-reconstituted with mixtures of WT-PLB and LOF PLB mutants had a lower inhibitory potency compared to SERCA and WT-PLB mixtures only. FRET experiments added further support by showing that unlabeled LOF mutants lowered the FRET between donor-labeled SERCA and acceptor-labeled WT-PLB. Thus, we have provided a convenient FRET method for screening future PLB mutants for the use in gene therapy to treat heart failure. Similarly, we used another fluorescence technique, time-resolved fluorescence resonance energy transfer (TR-FRET), to investigate nucleotide binding in SERCA. Based on biochemistry and crystallography, it has been proposed that SERCA has two distinct modes of nucleotide binding. To extend this observation from the crystal to the functional sarcoplasmic reticulum membrane, we have performed TR-FRET to measure the distance between donor-labeled SERCA and the fluorescent nucleotide TNP-ADP, in the presence and absence of inhibitors. TR-FRET experiments confirmed a novel binding site in SERCA, bringing the gamma-phosphate of ADP closer to the phosphorylation site, Asp351, compared to other crystal structures with bound nucleotide. To determine whether these modes of nucleotide binding occur in solution during SERCA enzymatic cycle, we performed transient TR-FRET ([TR]2FRET) experiments, in which a complete subnanosecond TR-FRET decay was recorded every 0.1 ms after rapid mixing of donor-labeled SERCA and TNP-ADP in a stopped-flow instrument. We clearly observed a biphasic reaction with a fast component (260 s-1) and a slower component (17 s-1). TR-FRET is a powerful technique for connecting structural dynamics of SERCA with its static crystal structures. The major focus of this research has been muscle relaxation through the interaction of SERCA and PLB utilizing fluorescence spectroscopy. However, another project with implications for muscle contraction concentrated on the signals for proteasomal degradation by using CaM as a model system. CaM variants were designed using site-directed mutagenesis in order to perform site-specific oxidation of Met residues. Utilizing circular dichroism (CD), thermodynamic stability CD experiments, and proteasomal degradation assays, it was demonstrated that oxidation of Met residues 51, 71, and 72 located in the N-terminus of CaM are essential for degradation. Functional data from ryanodine binding assays showed that oxidation of Met residues in the C-terminus of CaM completely abolished CaM's ability to bind and inhibit RyR. Accumulation of these CaM within the cell could be detrimental to CaM regulation of RyR impairing Ca2+ regulation during muscle contraction.
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    Understanding The Mechanisms Of Muscle Atrophy
    (2016-06) Liu, Haiming
    Skeletal muscle mass is regulated by protein turnover, the balance between protein synthesis and degradation. Muscle atrophy or a loss of muscle mass occurs when protein degradation exceeds protein synthesis, under conditions such as denervation and aging. Muscle atrophy is usually accompanied with reduced muscle contractility that lead to impaired physical activities and decreased quality of life. As a result, understanding the cellular and molecular mechanisms underlying the protein turnover is important to provide potential interventions and treatments for individuals suffered from muscle atrophy. In skeletal muscle the majority of the proteins are degraded by the ubiquitin-proteasome system (UPS). The core of this system is called proteasome, which works as a “garbage disposal” for the degradation of the myofibrillar proteins via specific enzymatic activities. The immunoproteasome, an inducible form of proteasome, also has a function of performing proteasome enzymatic activities primarily demonstrated in the immune system (generate peptides for antigen presentation). However, the role of the immunoproteasome during skeletal muscle protein degradation is unknown. Therefore, the purpose of the first study was to investigate the role of the standard and immuno-proteasome in denervation-induced protein degradation in skeletal muscle. In this study, wild type (WT) and the immunoproteasome deficient (lmp7-/-/mecl-1-/- double knockout, L7M1) mice were used to test the hypotheses that (1) the proteasome system is activated in denervation-induced muscle atrophy and (2) deletion of immunoproteasome subunits attenuates muscle atrophy by altering the proteasome composition and activities. Three major findings were found: following 7 and 14 days of denervation (1) an activation of the proteasome system occurs in conjunction with significant muscle atrophy in the WT mice; (2) the composition of the subunits within the 20S core appears to be influenced by deletion of the two immunoproteasome subunits; (3) however, the immunoproteasome was not essential for protein degradation induced by denervation. The purpose of the next study was to elucidate the physiological properties of skeletal muscle in response to denervation with a hypothesis that the UPS is a finely-tuned system that degrades myofibrillar proteins without impairing the contractility of the intact myosin and actin. We found an activation of the UPS accompanied with decreases in muscle size and force production post 14-day denervation. Importantly, the specific force and power were not impaired in the denervated muscles when compared to the controls. These results suggest that an activation of the UPS is associated with reductions in skeletal muscle quantity rather than quality. Frailty is a clinical syndrome, which is highly associated with sarcopenia, leads to adverse health outcomes, and increased mortalities. Animal models have the potential to tease out the cellular mechanisms underlying frailty. The purpose of the third study in this dissertation is to initiate the development of a Frailty Index in 27- to 28-month-old C57BL/6 mice that matches the established clinical frailty phenotype index in humans (weakness, slow walking speed, low activity level, poor endurance) 1. A frail or mildly-frail mouse was identified if presented ≥ three or two frailty criteria, respectively. From this study, we showed that one mouse was identified as frail and one was mildly-frail. This prevalence of 9% frailty is consistent with the prevalence of frailty in humans at the same survival age (11% frailty in human at age of 76-841). This work has been published in The Journals of Gerontology. Series A: Biological Sciences and Medical Sciences.