Browsing by Subject "Proteolysis"
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Item Intramembrane Proteolysis Mediated by the gamma-Secretase Complex : Nicastrin Functions as a Substrate Receptor(2006-08-11) Shah, Sanjiv; Yu, GangThe proteolytic processing of proteins within the lipid bilayer, and release of their membrane tethered biologically active fragments, fundamentally controls a growing list of cell signaling events. The gamma -secretase, one of a small family of independently evolved proteases, performs this enigmatic hydrolysis of a peptide bond within the membrane. Remarkably atypical, gamma-secretase activity: (1) requires a complex of proteins that include presenilin, nicastrin, Aph1, and Pen-2; (2) catalyzes the intramembrane cleavage of a broad range of substrates, regulating physiology from neurodevelopment to neurodegeneration. The aim of this thesis is to elucidate the mechanism by which the gamma -secretase recognizes its substrates. I provide evidence that nicastrin, in addition to being a critical component of the complex, plays a major function in substrate recognition. The ectodomain of nicastrin binds the new amino terminus that is generated upon the prerequisite 'shedding' of substrates, thereby recruiting substrates into the gamma -secretase complex. The gamma -secretase complex has been traditionally viewed as a hub for signal transduction of substrates such as Notch and APP. The mechanism by which a broad range of substrates may be recognized and subsequently cleaved, as demonstrated in this thesis, supports a mutually inclusive function as a protease that has evolved to simply dispose transmembrane domains thus controlling the repertoire of a class of proteins present in the membrane.Item Modulation of Transcription Factor Activity by Mono-Ubiquitin(2008-09-12) Archer, Chase Tanner; Kodadek, Thomas J.The Ubiquitin-Proteasome Pathway plays both proteolytic and non-proteolytic roles in the regulation of transcription. We recently reported that the ATPases of the 26S proteasome can destabilize activator-DNA complexes in a non-proteolytic manner that requires direct interactions between the Rpt4 and 6 subunits with the activation domain of the activator. Remarkably, mono-ubiquitylation of the activator blocks this repressive activity. In this study, we probe the mechanism of this protective effect. Using novel label transfer and chemical cross-linking techniques, we show that ubiquitin contacts the ATPase complex directly, apparently via Rpn1 and/or Rpt1, and that this interaction results in the dissociation of the activation domain-ATPase complex via an allosteric process. We also provide in vivo evidence demonstrating the importance of monoubiquitylation in inhibition of activator-DNA destabilization. A model is proposed in which activator mono-ubiquitylation serves to limit the lifetime of the activator-ATPase complex interaction and thus the ability of the ATPases to unfold the activator and dissociate the protein-DNA complex.Item Neuronal Maintenance via a Neuron-Specific Degradation Pathway(2015-01-26) Schmidt, Taylor; Jin, Eugene Jennifer; Ozel, Mehmet Neset; Epstein, Daniel; Marchant, Corey; Hiesinger, RobinBACKGROUND: Neurons can survive for decades via cell maintenance and protein degradation. This process includes the general protein endolysosomal degradation pathway, an integral part of which is the Rab GTPase proteins. Recently, components of a neuron-specific protein degradation pathway were discovered, which include the neuronal vesicle ATPase component V100 and the synaptic vesicle protein neuronal Synaptobrevin (n-Syb). While this neuron-specific degradation pathway has been shown as necessary for neuronal maintenance in adult Drosophila melanogaster fruit flies, it is not known what this neuron-specific degradation pathway does, nor how it interacts with the general protein degradation pathway. Our research aimed to fill this gap in knowledge. Such research may be salient because the misregulation of protein degradation in neurons leads to neurodegenerative diseases like dementia. OBJECTIVE: We hypothesized that neurons either have an increased or a specialized need for protein degradation in comparison to other cells. METHODS: 1. The lab chose a myristoylated protein (myr) to represent general proteins found in every cell, and Synaptotagmin1 (Syt1) to represent neuron-specific proteins. The acidification-sensitive tag mCherry-pHluorin, which changes color with a decrease in pH, was placed on Syt1 and myr to visualize acidification and degradation of the two proteins. 2. The lab generated Drosophila lines to compare acidification and degradation of Syt1 and myr in wild-type versus the following three mutants: rab7 mutants to disrupt general protein degradation, v100 to disrupt the neuron-specific protein degradation, and synaptobrevin also to disrupt neuron-specific degradation. 3. We performed live imaging to visualize acidification and protein degradation at synaptic terminals. Brains of Drosophila pupae from each cross were dissected, mounted onto Petri dishes, and surrounded with a culture medium to be kept alive. A resonant confocal microscope was used to observe the brain's lamina, a layer of neurons between the eye and the brain. At the lamina, we recorded 30-minute videos showing changes in fluorescence representing protein degradation. RESULTS AND CONCLUSION: Preliminary data show that nsyb and v100 mutations may cause defects in the degradation of neuron-specific cargo. Such evidence suggests that the neuron-specific endolysosomal degradation pathway specifically degrades the synaptic vesicle protein Synaptotagmin1. Also, the experiments indicate that disruption of either the neuron-specific or the general endolysosomal degradation pathway has no effect on the acidification of the myristoylated protein. Such evidence implies that the general pathway of protein degradation occurs at synapses, but has no specificity for protein cargo. A greater sample size is needed for future experiments, as well as quantitative analysis.Item Regulation of Hepatic Cholesterol Homeostasis Through Accelerated Degradation of HMG CoA Reductase(2017-04-06) Hwang, Seonghwan; Liang, Guosheng; DeBose-Boyd, Russell A.; Bruick, Richard K.; Scherer, PhilippCholesterol biosynthesis is rigorously controlled by negative feedback regulation. This reaction occurs, in part, through sterol-accelerated degradation of HMG CoA reductase (HMGCR), which catalyzes the rate-limiting step in cholesterol biosynthesis. The molecular mechanisms for the degradation of HMGCR have been actively investigated; however, the physiological relevance of the degradative regulation in animals is unclear. The current study investigates the role of sterol-accelerated degradation of HMGCR in overall regulation of HMGCR protein and cholesterol homeostasis in the liver. This was achieved by utilizing two mouse models: (1) liver-specific transgenic mice expressing the membrane domain of HMGCR, which is necessary and sufficient for sterol-regulated degradation of HMGCR in cultured cells and (2) knock-in mice expressing mutant HMGCR that is resistant to sterol-induced ubiquitination. These models were subjected to various feeding regimens known to modulate Insig and Scap, key players in feedback regulation of HMGCR. Cholesterol replenishment accelerates degradation of HMGCR in the liver of transgenic animals, whereas deprivation of sterols by lovastatin administration suppresses degradation of HMGCR. Ubiquitination-resistant HMGCR accumulated in the liver and resulted in the elevation of hepatic cholesterol, indicating degradation plays a significant role in the in vivo regulation of the enzyme and cholesterol homeostasis. This study further explored the physiological settings other than changing cholesterol status that may modulate the degradation of HMGCR in the two mouse models. As cholesterol synthesis is an oxygen-consumptive process, I determined the link between oxygen sensing and feedback control of cholesterol synthesis. In cultured human fibroblasts, stabilization of oxygen-sensitive transcription factor, hypoxia-inducible factor-1α (HIF-1α) directly activates transcription of INSIG-2 gene. Insig-2 inhibits cholesterol synthesis by mediating sterol-induced ubiquitination and subsequent degradation of HMGCR. Hepatic levels of Insig-2 mRNA are enhanced in mouse models of hypoxia. Moreover, pharmacologic stabilization of HIF-1α in liver stimulates HMGCR degradation through a reaction that requires the protein's prior ubiquitination and the presence of Insig-2. These results indicate that HIF-mediated induction of Insig-2 and degradation of HMGCR are physiologically relevant events in the cellular adaptation to hypoxic stress. Overall, the current study provides evidence supporting the physiological significance of the accelerated degradation of HMGCR in cholesterol homeostasis.Item Sequential Actions of VCP/p97 and the Proteasome 19S Regulatory Particle in Sterol-Accelerated, ER-Associated Degradation of HMG CoA Reductase(2014-05-28) Morris, Lindsey LaChelle; Goodman, Joel M.; DeBose-Boyd, Russell A.; Lehrman, Mark A.; De Martino, GeorgeAccelerated endoplasmic reticulum (ER)-associated degradation (ERAD) of the cholesterol biosynthetic enzyme HMG CoA reductase results from its sterol-induced binding to ER membrane proteins called Insig-1 and Insig-2. This binding allows for subsequent ubiquitination of reductase by Insig-associated ubiquitin ligases. Once ubiquitinated, reductase becomes dislocated from ER membranes into the cytosol for degradation by 26S proteasomes through poorly defined reactions mediated by the AAA-ATPase VCP/p97 and augmented by the nonsterol isoprenoid geranylgeraniol. Here, we report that the oxysterol 25-hydroxycholesterol and geranylgeraniol combine to trigger extraction of reductase across ER membranes prior to its cytosolic release. This conclusion was drawn from studies utilizing a novel assay that measures membrane extraction of reductase by determining susceptibility of a lumenal epitope in the enzyme to in vitro protease digestion. Susceptibility of the lumenal epitope to protease digestion, and thus membrane extraction of reductase, was tightly regulated by 25-hydroxycholesterol and geranylgeraniol. The reaction was inhibited by RNA interference mediated knockdown of either Insigs or VCP/p97. In contrast, reductase continued to become membrane extracted, but not cytosolically dislocated, in cells deficient for AAA-ATPases of the proteasome 19S regulatory particle. These findings establish sequential roles for VCP/p97 and the 19S regulatory particle in the sterol-accelerated ERAD of reductase that may be applicable to the ERAD of other substrates.