Investigating the dynamics of hydrogen peroxide metabolism in the peroxiredoxin system of Saccharomyces cerevisiae

Badenhorst, Melinda (2020-03)

Thesis (MSc)--Stellenbosch University, 2020.

Thesis

ENGLISH ABSTRACT: Reactive oxygen species (ROS) are derivations of molecular oxygen that can have detrimental e ects in cells. ROS can readily react with DNA, proteins and lipids often resulting in the loss of structure integrity of these essential cellular components. Living cells are exposed to a normal level of ROS produced by metabolic processes such as aerobic respiration or immune responses. During oxidative stress, however, the level of ROS increases to such an extent that the mechanisms to neutralize them become exhausted. Hydrogen peroxide (H2O2), being a ROS itself, has been associated with cancer, age-related diseases, human immunode ciency virus infection and cardiovascular diseases. Despite these undesired e ects of H2O2, it has also been recognized as a signaling molecule that functions in important cellular processes such as cell proliferation and di erentiation, immune response and apoptosis. Fortunately, the cell is equipped with an antioxidant system that can neutralize H2O2, while maintaining its levels for important signaling functions: the peroxiredoxin system. Peroxiredoxins belong to a family of redox proteins that are ubiquitously expressed across all kingdoms of life. They form part of a larger redox network that accepts reducing energy upstream from the thioredoxin system (thioredoxin, thioredoxin reductase and NADPH) to reduce H2O2 to H2O. The dynamics of the peroxiredoxin system remain poorly understood. The aim of this study was to further our understanding of the kinetic behaviour of this system and how it metabolizes H2O2 by constructing a kinetic model for the peroxiredoxin system of Saccharomyces cerevisiae. A kinetic model could enable us to determine exactly how this system is able to homeostatically maintain levels of H2O2 for signaling function and antioxidant defense. First, the proteins of the peroxiredoxin system were puri ed using recombinant protein expression techniques. The proteins were then used in spectrophotometric assays to obtain experimental data that were used to estimate the required model parameters. Once the model was constructed and evaluated, a stress condition was simulated by subjecting the system to a pulse-like H2O2 input of varying concentrations. The response of the system with regard to its capacity to ef ciently neutralize H2O2 under these conditions were then determined. The peroxiredoxin system proteins were successfully expressed to a high degree of homogeneity. The spectrophotometric assays and parameter estimations resulted in usable values for parameters to construct a simple kinetic model that could describe the system. Model simulations could further explain some discrepancies found in the experimental data for the H2O2 reduction reaction of the system. Additionally, the pulse-like simulation results demonstrated that when a certain H2O2 concentration threshold is reached, the capacity of the system to e ciently neutralize H2O2 becomes limited.

AFRIKAANSE OPSOMMING: Reaktiewe suurstofspesies (RSS) is reaktiewe molekules afgelei van molekulêre suurstof wat nadelige gevolge in selle kan hê. RSS reageer maklik met DNA, proteïene en lipiede wat kan lei tot die verlies van struktuurintegriteit van sulke noodsaaklike sellulêre komponente. Lewende selle word oor die algemeen blootgestel aan `n normale vlak van RSS deur middel van metaboliese prosesse soos aërobiese respirasie of gedurende `n immuunrespons. Tydens oksidatiewe stres neem die vlakke van RSS egter tot so `n mate toe, dat die meganismes om RSS te neutraliseer uitgeput raak. Waterstofperoksied (H2O2) is `n RSS tipe wat met kanker, ouderdomsverwante siektes, menslike immuniteitsgebrekvirusinfeksie en kardiovaskulêre siektes geassosieer word. Ten spyte van hierdie ongewenste gevolge van H2O2, word dit ook erken as `n seinmolekule wat funksioneer in belangrike sellulêre prosesse soos selproliferasie en di erensiasie, immuunrespons en apoptose. Die sel is egter toegerus met `n antioksidantsisteem wat H2O2 e ektief kan neutraliseer, terwyl die vlakke vir belangrike seinfunksies gehandhaaf word: die peroksiredoksiensisteem. Peroksiredoksiene behoort aan `n familie van redoksproteïene wat in al die koninkryke van lewe voorkom. Hulle vorm deel van `n groter redoksnetwerk wat reduserende energie ontvang van die tioredoksiensisteem (tioredoksien, tioredoksien reduktase en NADPH) om H2O2 na H2O om te skakel. Die dinamika van die peroksiredoksiensisteem word egter nie goed verstaan nie. Die doel van hierdie studie is om ons begrip van die kinetiese gedrag van hierdie sisteem en hoe dit H2O2 metaboliseer beter te verstaan deur die bou van 'n kinetiese model vir die peroksiredoksiensisteem van Saccharomyces cerevisiae. `n Kinetiese model kan ons in staat stel om presies te bepaal hoe hierdie sisteem dit regkry om H2O2-vlak homeostase te handhaaf vir seinfunksie en beskerming teen oksidante. Eerstens was die proteïene wat aan die peroksiredoksiensisteem behoort gesuiwer met behulp van rekombinante proteïenuitdrukkingstegnieke. Die proteïene was daarna inspektrofotometriese eksperimente gebruik om eksperimentele data te win wat gebruik is om die vereiste modelparameters te skat. Sodra die model gebou en geëvalueer is, was 'n strestoestand gesimuleer deur die sisteem aan `n pulsagtige H2O2 inset van wisselende konsentrasies te onderwerp. Die reaksie van die sisteem ten opsigte van sy kapasiteit om H2O2 e ektief te neutraliseer onder hierdie toestande was daarna bepaal. Die proteïene van die peroksiredoksiensisteem is suksesvol tot `n hoë mate van homogeniteit uitgedruk. Die spektrofometriese eksperimente en parameterberamings het gelei tot bruikbare waardes vir parameters om `n eenvoudige kinetiese model te bou wat die sisteem kon beskryf. Modelsimulasies kon verder enkele teenstrydighede teenwoordig in die eksperimentele data vir die H2O2 reduksie-reaksie verklaar. Daarbenewens het die resultate van die puls-simulasie getoon dat wanneer `n sekere H2O2 konsentrasiedrempel bereik word, die vermoë van die sisteem om H2O2 e ektief te neutraliseer beperkend raak.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/108258
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