Browsing by Subject "TPP"

Now showing 1 - 3 of 3
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    Characterizing the Unfolded Protein Response by Changes in Protein Thermal Stability
    (2023-09) McCracken, Neil Andrew; Mosley, Amber; Wek, Ron; Evans-Molina, Carmella; Georgiadis, Millie; Quinney, Sara
    The Unfolded Protein Response (UPR) protects eukaryotic cells from the threat of excessive protein flux into the Endoplasmic Reticulum (ER). UPR sentries PERK, Ire1 and ATF6 detect unfolded protein in the ER and alert the cell of the condition. Downstream pathways increase translation of select responders while simultaneously decreasing the global protein load in order that toxic protein aggregates do not form in the cell. While this warning system has been characterized over several decades through extensive reporting of UPR impact on transcript and protein abundance, little is known about the biophysical changes that occur to proteins as part of the UPR in the context of the cellular environment. An understanding of how the UPR affects the folding, stability and protein oligomerization is vital for describing subtle but important changes that occur and contribute to maladaptive physiology in diseases including diabetes, cancer, and neurodegeneration. I propose that deficiencies in characterizing the UPR can be overcome by using thermal shifts assays (TSA) that quantify changes in protein stability post stimuli. Findings described herein show the utility of the biophysical thermal shift assay in characterizing the UPR. Thermal shift assays (TSA) measure susceptibility of proteins to denature upon heat treatment and consequently detect changes in protein structure, modification, and interactions in the cellular environment. Previously unobserved protein relationships related to the UPR were detected using TSA. These workflows were improved through more strategic upstream sampling and downstream data analysis through creation of the publicly available InflectSSP program. Observed UPR phenomena during N-linked glycosylation inhibition and UPR induction include protein degradation, changes in stability of N-linked glycosylation enzymes, and transcriptional targets canonical to the UPR. Stability changes in proteins downstream of PERK were also observed in experiments where PERK genetic ablation was combined with UPR induction. Finally, the thermal shift assay was used to develop a “signature” for the UPR that holistically describes the ER stress response. Results described in this dissertation provide an improved perspective of the UPR along with an approach that can be used to identify novel targets for therapeutic intervention of the UPR.
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    Investigation of the Evolutionary Aspects of Thiamin Diphosphate-Dependent Decarboxylases
    (2015) Rogers, Megan P.; McLeish, Michael J.
    Thiamin diphosphate (ThDP)-dependent enzymes catalyze a wide range of reactions including the oxidative and nonoxidative decarboxylation of 2-keto acids, carboligation reactions, the cleavage of C-C bonds, and the formation of C-S, C-N, and C-O bonds. Surprisingly, given this diversity, all ThDP-dependent enzyme catalyzed reactions proceed through essentially the same intermediate. This suggests that these enzymes share a common ancestry and have evolved to become the diverse group of enzymes seen today. Sequence alignments have revealed that all ThDP-dependent enzymes share two common ThDP binding domains, the PYR domain and the PP domain. In addition to these conserved domains, over time, other domains have been added creating further diversity in this superfamily. For instance, the TH3 domain, found in many ThDP-dependent enzymes, serves the function of binding additional cofactors such as FAD in enzymes like acetohydroxyacid synthase (AHAS) but in others, like pyruvate decarboxylase (PDC), it has lost this function completely. The work presented here focuses on ThDP-dependent decarboxylases. In this thesis, several evolutionary aspects of this group of enzymes will be examined including (i) the characterization of an evolutionary forerunner in the presence of a mechanism-based inhibitor, (ii) the characterization of the minor isozymes of pyruvate decarboxylase from Saccharomyces cerevisiae, and (iii) the development of a selection method to increase the efficiency of the site-saturation mutagenesis used to study ThDP-dependent enzyme evolution.
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    Steroids Precipitating Acute Thyrotoxic Paralysis
    (2022-03-25) Garcia, Jennifer; Pelton, Sarah; Vander Missen, Marissa; Vultorius, Daniela; Patel, Neha; Saeed, Zeb
    Case Description: A 34-year-old Black male was admitted with a new diagnosis of Graves’ disease and impending thyroid storm with a Burch-Wartofsky Score of 25. Initial labs showed undetectable TSH, total T3 of >800, free T4 of 7.21, and TrAb of 21.53. He was started on methimazole, propranolol, and hydrocortisone 100mg q8h. On day 2 of hospitalization, he presented with acute bilateral lower extremity paralysis shortly after eating lunch. His blood glucose was 231, and a stat BMP showed a potassium of 2.0. He was found to have thyrotoxic periodic paralysis (TPP). Steroids were stopped immediately, and he was given additional propranolol and potassium repletion. He received a total of 60mEq KCl and did not have recurrence after steroids were stopped. Conclusion: Steroids are very commonly used in treatment of thyroid storm but can potentially exacerbate endocrine emergencies, such as thyrotoxic periodic paralysis. Additionally, demographic factors may have also decreased the likelihood of considering the potential for TPP as the patient’s race did not correspond to the most common demographics, Asian populations. Thus, it is important to be aware of the potential effects of steroids. Clinical Significance: TPP is a rare complication of thyrotoxicosis. Excess thyroid hormones in the blood increase activity of the Na+/K+-ATPase pump, leading to intracellular shifts of potassium and consequential hypokalemia. Glucocorticoids have been shown to increase the relative amounts of Na+/K+-ATPase pumps and exacerbate hypokalemia. Steroids are one of four common treatments for thyroid storm, so their effects on transcellular ion balance must be monitored. Insulin was also found to increase Na+/K+-ATPase pump activity, explaining why episodes of TPP often correlate with carbohydrate-rich meals. Treatment of TPP, which can be done with repletion of KCl and beta blockers, must monitor for and avoid inducing a hyperkalemic state.