Browsing by Subject "Proteolysis"

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    Conformational Regulation of Cell Surface Receptor Proteolysis
    (2019-02) Hayward, Amanda
    Cell surface receptors can commonly undergo ectodomain shedding to modulate signaling pathways and cell contacts. To date, there are over 400 proteins that serve diverse functions on the cell surface that are predicted to undergo ectodomain shedding, and the shedding rates are commonly modulated in diseases such as cancer and inflammation. However, validating targets and understanding shedding regulation has been difficult due to a lack of control of proteolysis-inducing stimuli and unknown fates of cleavage products. Therefore, we built tools to help progress the field forward in understanding as well as investigated two cell surface receptors. The development of technology and tools to study proteolytically sensitive proteins is ongoing. Herein, two different tools that aid in the identification and study of mechanically sensitive proteins are described. We utilized HUH-tags to covalently link single-stranded DNA to target proteins of interest for single molecule force spectroscopy studies. We then developed an assay called SNAPS to identify switch-like proteins and screen for modulation of proteolysis by drugs. The SNAPS assay has innumerous applications in biotechnology and assessment of potential therapeutics, a few are described herein. We also investigated the biological impact of proteolysis on dystroglycan, an essential protein that forms a bridge between the extracellular matrix (ECM) and actin cytoskeleton. Proteolysis of dystroglycan is enhanced in disease states such as cancer, yet the biological impact of its proteolysis has not been elucidated. Moreover, there are muscular dystrophy disease-related mutations that map to the region where dystroglycan is cleaved by matrix metalloproteases (MMPs), but how they contribute to the mechanism of pathogenesis is not known. We have shown that disease mutations within the proteolysis domain of dystroglycan impact the overall conformation and stability of this domain, resulting in an increased in MMP cleavage. Proteolysis also has an impact on cell migration and cell morphology, suggesting that it may contribute to muscular dystrophy disease pathogenesis. Lastly, we turned our attention to Polycystin-1, a 7-pass transmembrane protein that is predicted to act as a mechanosensitive unit with Polycystin-2 in primary cilia. Its putative shedding is predicted to be functionally different than dystroglycan’s; instead of having a buried proteolysis site, its extracellular juxtamembrane region is proposed to involve dissociation of two non-covalently linked subunits. We investigated several disease-related mutations within its domain and performed initial tests for studying Polycystin-1 in the context of cultured cells.
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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.