Browsing by Subject "Prions"
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Item Enabling Structural Studies of the Yeast Prion Protein Within a Cellular Environment(2022-05) Costello, Whitney Nicole; Lin, Milo; Rizo-Rey, José; Diamond, Marc; Frederick, KendraMy motivation for the work in this thesis was to take steps towards bridging the gap in structural information between atomic models of metastable proteins in isolated and cellular environments. Most biophysical techniques are generally limited by either sample composition or resolution. One technique, Nuclear Magnetic Resonance (NMR) is not limited by sample composition and can provide atomic-level resolution. However, NMR is limited by sensitivity. Recent advancements in the field produced a sensitivity-enhanced solid-state NMR technique, namely Dynamic Nuclear Polarization (DNP) NMR. Using DNP NMR, I observed sensitivity enhancements of up to 90-fold increase in sensitivity, eliminating this barrier. Recent work on a metastable protein, Sup35, assembled in cellular lysates using DNP NMR demonstrates that the biological environment has a dramatic effect on the Sup35 protein structure. In this thesis, I sought to harness sensitivity gains from DNP NMR to identify strategies for the specific detection of isotopically labeled proteins at within a cellular lysate for structural analysis. First I present theoretical calculations, validated by experimental results, for the expected signal of detection ratio of an isotopically labeled protein within a cellular lysate. These results concluded that DNP NMR can specifically detect a 30 kDa, uniformly isotopically labeled protein at low micromolar concentrations. However, sensitivity is still a barrier to specifically detect proteins with lower molecular weights or non-uniform isotopic labeling. Therefore, I optimized sample preparation for maximum sensitivity for DNP NMR on cellular lysates. DNP sensitivity enhancement depends on sample composition. DNP NMR is performed at 100 K, and requires sample glassing, polarizing agent, and protonation for optimal DNP enhancement. Some of the first DNP NMR experiments on purified protein samples were optimized for a matrix of 60:30:10, d8-glycerol:D2O:H2O with 10 mM biradical. These matrix conditions became standard in the field, known as "DNP Juice". However, I found that these matrix conditions are not optimal for DNP NMR cellular lysate samples. In the presence of cellular lysate, sensitivity is improved by addition of lower cryoprotectant (15%) and biradical concentrations (5 mM). I also found that deuteration was unnecessary. Finally, I investigated methods to simplify DNP NMR spectra through segmental labeling of proteins. The strategies in this thesis benefit future research of structural studies on environmentally sensitive proteins, such as alpha-synuclein or tau, within their native environment at physiological concentrations.Item Functional Prions in Mammalian Innate Immune Signaling(2014-07-07) Cai, Xin; Zinn, Andrew R.; Beutler, Bruce; Chen, Zhijian J.; Goldstein, Joseph L.Pathogens and cellular danger signals activate mammalian cytosolic sensors such as RIG-I and NLRP3 which signal through respective adaptor proteins MAVS and ASC to produce robust innate immune and inflammatory responses. MAVS and ASC harbor N-terminal CARD and PYRIN domains, respectively, essential for their signaling ability. Using the Sup35 based yeast prion assay, we show that CARD and PYRIN function as bona fide prions in yeast when fused to Sup35C. In response to respective upstream sensors RIG-I and NLRP3, both CARD and PYRIN form self-perpetuating, SDS-resistant polymers that are inherited cytoplasmically through multiple cell divisions. Similar to other cases of prion switch, CARD exhibits nucleation- and polymerization-dependent prion conversion in yeast. Likewise, a yeast prion domain (NM) can functionally replace CARD and PYRIN in mammalian innate immune and inflammasome signaling. Mutations in MAVS and ASC that disrupt their prion activities in yeast also abrogate their ability to signal in mammalian cells. Furthermore, fibers of recombinant PYRIN can convert ASC into functional polymers capable of activating caspase-1. Remarkably, homologous domains from a conserved NOD- like receptor (NWD2) and classic prion (HET-s) in fungi can functionally reconstitute signaling of NLRP3 and ASC PYRINs in mammalian cells. These results indicate that prion- like polymerization is a conserved signal transduction mechanism in innate immunity and inflammation.Item Spongiform encephalopathy: a problem of prions(1990-09-27) Brown, Michael S.Item Tau Seeding in Health and Disease(2022-08-01T05:00:00.000Z) LaCroix, Michael Shane; Nam, Yunsun; Herz, Joachim; Joachimiak, Lukasz; Diamond, Marc; Rice, Luke M.An abundance of evidence supports that the protein tau adopts a wide variety of conformations with the ability to self-assemble and propagate in living systems, and that this prion behavior may drive neurodegeneration in tauopathies. However, the inciting events that lead to tau seed formation and aggregation are unknown. It remains possible that tau can act as a prion outside the context of disease, as part of its normal function, and the accumulation of tau prions in neurodegenerative diseases reflects a loss of control of this normal function. During my dissertation research, I completed a series of investigations on tau's ability to form seeds outside the context of classical tauopathies. I discovered that tau seeds are present in the cerebral cortex of healthy individuals. Tau seeds form in a region and species specific manner, being absent in the cerebellum of healthy individuals and undetectable in murine models. Seeding in healthy individuals was independent of age, implying it is not a result of emerging tauopathy but rather, that prion formation is a normal aspect of tau biology. This may be related to its interactions with RNA. I also surveyed for tau seeding in several inflammatory diseases with neurodegenerative components that have been reported to exhibit tau accumulation based on immunohistochemistry. I found seeds at levels beyond that of healthy individuals in temporal lobe epilepsy as well as multiple sclerosis. Thus, tau may be a target of many convergent pathways that lead to neurodegeneration. The work here highlights the significant role that tau plays in human health and disease. Further understanding of how normal biological processes, as well as inflammation, affect tau's prion state will be essential for the development of therapeutic strategies for the prevention and treatment of tauopathies.Item [UT Southwestern Medical Center News](2011-08-09) Wormser, Deborah