Protein arginine methylation is a posttranslational modification catalyzed by the family of proteins known as the protein arginine methyltransferases (PRMTs). Thousands of methylated arginines have been found in mammalian cells. Many targets of arginine regulation are involved in important cellular processes like transcription, RNA transport and processing, translation, cellular signaling, and DNA repair. Since PRMT dysregulation has been linked to a variety of disease states, understanding how the activity of the PRMTs is regulated is of paramount importance. PRMT1 is the predominant PRMT, responsible for about 85% of all arginine methylation in cells, but very little is known about how PRMT1 is regulated. Although a few methods to regulate PRMT1 activity have been reported, the details of interaction and regulatory mechanisms remain largely unknown. To better understand how PRMT1 is able to bind its substrates and how PRMT1 activity is regulated, we followed a mechanistic and structural biology approach to better understand how PRMT1 interacts with its substrates and protein regulators. In this study the regulation of Hmt1 methyltransferase activity by the Air1 and Air2 proteins was analyzed and only one was determined to affect Hmt1 activity. The posttranslational phosphorylation of Hmt1 had also been reported to affect Hmt1 activity in vivo and our preliminary studies suggest that additional factors may help influence the regulatory effect of phosphorylation. Lastly, we report a new method of PRMT regulation through the reversible oxidation of key PRMT1 cysteine residues. We are also able to show that this regulation occurs in cells and affects several PRMT isoforms.

INTRODUCTION: Inflammatory bowel disease (IBD) is a growing public health concern with a pressing need for new diagnostic and disease activity biomarkers. Recently, studies have linked IBD disease factors to an imbalance in the gut’s reductive-oxidative (redox) defensive mechanisms and the resulting oxidative stress due to reactive oxygen species. With this new paradigm, direct measurement of redox status in the body could be utilized as a novel biomarker of disease activity for patients with IBD. OBJECTIVES: The goal of this study is to determine how gastrointestinal oxidative state relates to IBD disease activity. Additionally, this study aims to establish a reproducible and accurate measurement protocol for measuring redox status in the human body. METHODS: Patients with and without IBD admitted to Boston Children’s Hospital (Boston, MA) were enrolled in the study from October 2020 to February 2021. Stool and urine samples were collected from these patients and the oxidative status in these biosamples was measured via three redox measuring systems. RESULTS: Data suggests that relative stool oxidative state is more positive in patients with active disease states compared to controls. Also, a finalized protocol for the measurement of relative redox status in stool and urine was established in this study. CONCLUSION: With a reliable and accurate method of measurement established, the potential for relative redox status in the human body to serve as a predictive biomarker for IBD state is promising. Moving forward this study will focus on expanding the study’s size and types of samples to make more significant conclusions in the future. The usefulness of oxidative state in the body as a disease biomarker is just beginning to be realized.

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第19764号 / 農博第2160号 / 新制||農||1039(附属図書館) / 学位論文||H28||N4980(農学部図書室) / 32800 / 京都大学大学院農学研究科応用生命科学専攻 / (主査)教授 阪井 康能, 教授 植田 和光, 教授 三芳 秀人 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DGAM

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Thesis advisor: Jeffery A. Byers / This dissertation covers the development of redox-switchable ring-opening polymerizations for the synthesis of copolymers of underutilized monomers. In Chapter one, the progress in the development of switchable methods for ring-opening polymerization and ring-opening copolymerizations. Chapter two describes a method for the redox-switchable copolymerization of L-lactide, propylene oxide and carbon dioxide. The benefits of this method are demonstrated through the facile synthesis of blocky and statistical copolymers of the three monomers. In Chapter three, a method for the redox-switchable polymerization of N-carboxyanhydrides is presented. A mechanistic analysis and copolymerizations of N-carboxyanhydrides and either lactones or epoxides follow the initial findings. Chapter four further expands the uses of N-carboxyanhydride redox-switchable polymerizations by immobilizing the catalysts onto semiconductor surfaces for the synthesis of surface bound polyamides. / Thesis (PhD) — Boston College, 2021. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

<p> Biological thiols are a class of labile and redox-active metabolite with significant interest to biomedical research due to their involvement in redox mechanisms of cell signaling and physiological control. As a result of oxidative stress, levels of various reduced thiols and oxidized disulfides are altered, which disrupts major cellular regulation pathways modulating protein function and gene expression. Thus, analysis of thiols in biological fluids is essential for understanding the role of oxidative stress and thiol dysregulation in aging and human diseases. However, reliable ex-vivo thiol determination is challenging due to their low abundance and susceptibility to auto-oxidation and thiol-disulfide exchange reactions. In this thesis, capillary electrophoresis-electrospray ionization-mass spectrometry (CE-ESI-MS) in conjunction with maleimide labeling is developed as an integrative strategy for comprehensive plasma thiol redox status analysis for metabolomics. Maleimide labeling helps to address both major constraints in thiol analysis by stabilizing free sulfhydryl groups as their thioether adducts while improving ionization efficiency and analytical sensitivity. This enhancement in ionization efficiency can be quantitatively predicted based on relative changes in fundamental physicochemical properties of thiols that occur upon covalent derivatization when using multivariate calibration. On-line sample preconcentration together with thiol-selective labeling using a cationic quaternary ammonium maleimide analog allowed for simultaneous analysis of reduced thiols and intact oxidized disulfides by CE-ESI-MS with low nanomolar detection limits of 8-30 nM. Improved identification of unknown low abundance thiols and other classes of polar metabolites is also demonstrated by prediction of relative migration times in CE that is complementary to ESI-MS. Comprehensive plasma thiol speciation together with untargeted profiling of polar metabolites provides a novel platform for holistic understanding of complex changes in metabolic networks associated with thiol dysregulation and/or nutritional intervention for the prevention or treatment of human disorders. </p> / Thesis / Master of Science (MSc)

Oxidative stress is commonly associated with diet-induced metabolic syndrome (MetS) and left ventricular cardiac remodeling, but much remains unknown about the role of redox signaling, sensors, and switches in mediating the effects of high fat and sugar intake. In this work, I describe and apply an optimized method for quantifying changes in reversible protein-cysteine oxidation in the heart. This method uses isobaric tagging of cysteine thiols and mass spectrometry in a modified biotin switch on whole tissue lysate. Analyzing the resulting data with systems biology approaches helped delineate redox pathways playing a role in disease development, while cysteine-specificity provided exact targets for mutation-based mechanistic studies. Initial findings in a mouse model for MetS, wherein C57Bl6J mice were fed a high fat/high sucrose diet, identified energy pathways as the primary target of changing reversible cysteine oxidation. In follow-up studies, our collaborators helped validate the pathophysiological role of two particular cysteines in complex II; their early reversible oxidation and later irreversible oxidation contributed to decreased ATP output from cardiac mitochondria. A subsequent, more robust study revealed a weakness in our original method. While investigating the role of hydrogen peroxide-induced oxidative post-translational modifications (OPTMs) in the development of MetS sequelae, analysis of four mouse groups, each with an n=5, revealed that measurements of reversibly oxidized cysteine thiols were highly variable compared to those of all available thiols. Thus, I developed a strategy to address the source of variability and, in the process, improved many additional steps in the switch protocol. Finally, in an effort to clarify the role of the most stable reversible OPTM, glutathionylation (RSSG), we characterized the HFHS diet response in mice engineered to have more or less RSSG via genetic manipulation of glutaredoxin-1 expression. Those with more RSSG suffered worsened cardiac function, making them an ideal model for future studies with the methods optimized in this work. Studying the progression from poor diet to cardiac involvement in these and other mouse models using the methods described herein will aid in the design of diagnostics and targeted therapies against the growing burden of metabolic CVD.

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The advancement of quantum dot sensitized solar cell (QDSSC) technology depends on optimizing directional charge transfer between light absorbing quantum dots, TiO2, and a redox mediator. Kinetically, reduction of oxidized quantum dots by the redox mediator should be rapid and faster than the back electron transfer between TiO2 and oxidized quantum dots to maintain photocurrent. Thermodynamically, the reduction potential of the redox mediator should be sufficiently positive to provide high photovoltages. To satisfy both criteria and enhance power conversion efficiencies, we introduced charge transfer spin-crossover MnII/III complexes as promising redox mediator alternatives in QDSSCs. High photovoltages ~ 1 V were achieved by a series of Mn poly(pyrazolyl)borates, with reduction potentials ~0.51 V vs Ag/AgCl. Back electron transfer rates were slower than Co(bpy)3, where bpy = 2,2'-bipyridine. This is indicative of a large barrier to recombination imposed by spin-crossover in these complexes. By capitalizing on these characteristics, efficient MnII/III-based QDSSCs can be achieved with more soluble Mn-complexes. In hybrid bulk heterojunction solar cells (HBHJs), light-absorbing conjugated polymers are interfaced with films of nanostructured TiO2. Photovoltaic action requires photoelectrons in the polymer to transfer into the TiO2, and therefore, polymers are designed with lowest unoccupied molecular orbital levels higher in energy than the conduction band of TiO2 for thermodynamically favorable electron transfer. Currently, the energy level values used to guide solar cell design are referenced from the separated materials, neglecting the fact that upon heterojunction formation material energetics are altered. With spectroelectrochemistry, we discovered that spontaneous charge transfer occurs upon heterojunction formation between poly(3-hexylthiophene) (P3HT) and TiO2. It was determined that deep trap states in TiO2 accept electrons from P3HT and form hole polarons in the polymer. This equilibrium charge separation alters energetics through the formation of interfacial dipoles and results in band bending that inhibits desired photoelectron injection into TiO2, limiting HBHJ solar cell performance. New guidelines for improved photocurrent are proposed by tuning the energetics of the heterojunction to reverse the direction of the interfacial dipole, enhancing photoelectron injection. / Master of Science