Kinetic modelling of the peroxiredoxin system
| dc.contributor.advisor | Rohwer, J. M. | en_ZA |
| dc.contributor.advisor | Pillay, C. S. | en_ZA |
| dc.contributor.author | Barry, Christopher James | en_ZA |
| dc.contributor.other | Stellenbosch University. Faculty of Science. Dept. of Biochemistry. | en_ZA |
| dc.date.accessioned | 2024-03-01T13:51:56Z | |
| dc.date.accessioned | 2024-04-26T14:46:34Z | |
| dc.date.available | 2024-03-01T13:51:56Z | |
| dc.date.available | 2024-04-26T14:46:34Z | |
| dc.date.issued | 2024-03 | |
| dc.description | Thesis (PhD)--Stellenbosch University, 2024. | en_ZA |
| dc.description.abstract | ENGLISH ABSTRACT: Hydrogen peroxide is a cellular oxidant that disrupts numerous cellular systems by being readily converted to the hydroxyl radical, which indiscriminately reacts with biomolecules. However, in many contexts, it also serves crucial cellular functions and is intricately connected to maintaining normal functioning. As a result, antioxidant sys- tems find themselves in a delicate balance between two opposing roles: they must defend cells against this cellular toxin without being so effective as to disrupt the normal cellular functions of hydrogen peroxide. The peroxiredoxin protein forms the core of one such antioxidant system and, belying its significance, is found across all domains of life. This versatile and vital protein can switch between several levels of hydrogen peroxide metabolism and possesses its own signalling and chaperone properties. The switch between roles is effectuated through a combination of redox configuration transitions and quaternary structure transformations. The launching-point for this project was a 100-fold change in the ability of peroxire- doxin to neutralise hydrogen peroxide when transitioning from a dimer to a decamer. While this relationship between oligomeric form and peroxidase activity had already been thoroughly established, it had yet to be explored in any dynamic sense. The first major outcome of the research in this dissertation was the formulation of a theoretical framework for the transition between dimers and decamers. This was closely followed by a characterisation of the kinetics for the association and dissociation of a re- duced peroxiredoxin decamer. Armed with these data, the dimer-decamer transition was explored through simulation and found to have an inhibition-like effect on peroxiredoxin activity. Impressively, integrating this process into an established in vivo model resolved an outstanding discrepancy between the experimental and simulated responses of the peroxiredoxin oxidation state to hydrogen peroxide insult. In the course of researching the kinetics of peroxiredoxin, a bias was discovered in the data of multiple independent reports of horse radish peroxidase competition assays. Investigating this bias led to the discovery of a flaw in the data analysis methodology of this assay. This issue was resolved by developing a method of fitting the target rate constant directly to assay trace data instead of through the established fractional inhibition method. We provide tools for researchers to easily apply the improved analysis method in their own work. The final major outcome of this project was the formulation of a model of hydrogen peroxide neutralisation in a Saccharomyces cerevisiae cell culture. The model was parame- terised by deriving several new parameters and the parameter sensitivities of the model were assessed using a Fourier amplitude sensitivity test and metabolic control analysis. This model is capable of simulating the dynamic responses of the peroxidase systems of a S. cerevisiae culture in media exposed to hydrogen peroxide stimulation—a widely used experimental system in redox biology. This dissertation is focused on the exploration of the peroxiredoxin protein using a systems biology approach. This work has culminated in multiple novel findings which contribute significantly to our understanding of the dynamics of hydrogen peroxide metabolism by peroxiredoxin and to redox biology more broadly. | en_ZA |
| dc.description.abstract | AFRIKAANSE OPSOMMING: Waterstofperoksied is ’n oksidant wat in selle voorkom en talle stelsels kan ontwrig deur omgeskakel te word na die hidroksiel-radikaal, wat willekeurig en geredelik met biomolekule te reageer. In baie kontekste vervul dit egter ook noodsaaklike sellulêre funksies en is dit nou betrokke by die normale handhawing van hierdie funksies. As gevolg hiervan is daar ’n fyn balans tussen twee teenstrydige rolle van antioksidantstelsels: hulle moet selle teen hierdie selgif beskerm sonder om só doeltreffend te wees dat die normale sel-funksies van waterstofperoksied ontwrig word. Die peroksiredoksien-proteïen vorm die kern van een so ’n antioksidantstelsel en dit kom regdeur alle domeine van die lewe voor, wat die belang daarvan beklemtoon. Hierdie veelsydige proteïen kan skakel tussen verskeie vlakke van waterstofperoksied- metabolisme en besit verder ook seinoordrag- en chaperoon-eienskappe. Die oorskake- ling tussen hierdie verskillende rolle word bewerkstellig deur ’n kombinasie van redoks- konfigurasie oorgange en kwaternêre struktuur-transformasies. Die beginpunt vir hierdie projek was ’n 100-voudige verandering in peroksiredoksien se vermoë om waterstofperoksied te neutraliseer wanneer die struktuur van ’n dimeer na ’n dekameer verander. Terwyl hierdie verwantskap tussen die oligomeriese vorm en peroksidase-aktiwiteit reeds etlike jare bekend was, is die dinamika van die proses tot dusver nog nie ondersoek nie. In die eerste deel van die navorsing in hierdie proefskrif is ’n teoretiese raamwerk vir die oorgang tussen peroksiredoksien dimere en dekamere ontwikkel. Voorts is die kinetika vir die assosiasie en dissosiasie van ’n gereduseerde peroksiredoksien dekameer gekarakteriseer. Met behulp van hierdie data is die dimeer-dekameer oorgang deur sim- ulasie van ’n kinetiese model verken. Die teenwoordigheid van die dimeer-dekameer ewewig het gelei tot ’n waargenome inhibisie van die aktiwiteit van peroksiredoksien, en hierdie proses is in ’n bestaande in vivo kinetiese model geïntegreer. Dit het tot die indrukwekkende uitkoms gelei dat ons ’n teenstrydigheid tussen eksperimentele en ges- imuleerde resultate met betrekking tot die respons van peroksiredoksien se oksidasiestoe- stand teenoor waterstofperoksied-toediening kon oplos. Tydens die bestudering van peroksiredoksien se kinetika is ’n sistematiese afwyking in die data van verskeie onafhanklike berigte van peperwortel peroksidase (HRP) kompetisie essai’s ontdek. Deur hierdie afwyking verder te ondersoek, het ons ’n fout in die data- analise metode van die HRP essai opgetel. Hierdie probleem is opgelos deur ’n metode te ontwikkel om die reaksie se snelheidskonstante direk op die tydsafhanklike data van die essai te pas, eerder as om dit met die gevestigde fraksionele inhibisie metode te verwerk. As deel van hierdie werk verskaf ons gereedskap vir navorsers om die verbeterde analisemetode maklik self toe te pas in hul eie werk. Die finale groot uitkoms van hierdie projek was die ontwikkeling en formulering van ’n kinetiese model van waterstofperoksied afbraak in Saccharomyces cerevisiae selkulture. Verskeie nuwe parameters moes afgelei word om die model te parametriseer. Die gevoe- ligheid van die model vir die verskillende parameters is geassesseer met behulp van ’n “Fourier Amplitude Sensitivity Test” (FAST) en metaboliese kontrole-analise. Die model is in staat om die dinamiese respons van die peroksidase sisteme in ’n S. cerevisiae kultuur in media wat blootgestel is aan waterstofperoksied, te simuleer. Hierdie eksperimentele sisteem word wyd gebruik in redoksbiologie. In hierdie proefskrif is ’n aantal aspekte van die peroksiredoksien proteïen deur middel van ’n sisteembiologie-benadering verken. Daar word berig oor verskeie nuwe bevindings wat ’n beduidende bydrae kan lewer tot ons begrip van die dinamika van waterstofperoksied-metabolisme deur peroksiredoksien spesifiek en redoksbiologie in die breë. | af_ZA |
| dc.description.version | Doctoral | en_ZA |
| dc.format.extent | xv, 143 pages : illustrations (some color) | en_ZA |
| dc.identifier.uri | https://scholar.sun.ac.za/handle/10019.1/130360 | |
| dc.language.iso | en_ZA | en_ZA |
| dc.language.iso | en_ZA | en_ZA |
| dc.publisher | Stellenbosch : Stellenbosch University | en_ZA |
| dc.rights.holder | Stellenbosch University | en_ZA |
| dc.subject.lcsh | Hydrogen peroxide -- Reactivity | en_ZA |
| dc.subject.lcsh | Peroxiredoxins -- Oxidation | en_ZA |
| dc.subject.lcsh | Saccharomyces cerevisiae | en_ZA |
| dc.subject.lcsh | Oxidative stress | en_ZA |
| dc.subject.lcsh | Hydroxyl radical | en_ZA |
| dc.subject.lcsh | Radicals (Chemistry) -- Reactivity | en_ZA |
| dc.subject.lcsh | Proteins -- Effect of stress on | en_ZA |
| dc.subject.name | UCTD | en_ZA |
| dc.title | Kinetic modelling of the peroxiredoxin system | en_ZA |
| dc.type | Thesis | en_ZA |
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