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dc.contributor.author
Nguyen, Jade
dc.contributor.supervisor
Wennemers, Helma
dc.contributor.supervisor
Altmann, Karl-Heinz
dc.contributor.supervisor
Rivera Fuentes, Pablo
dc.date.accessioned
2021-11-25T06:55:34Z
dc.date.available
2021-11-24T18:08:14Z
dc.date.available
2021-11-25T06:55:34Z
dc.date.issued
2021
dc.identifier.uri
http://hdl.handle.net/20.500.11850/516749
dc.identifier.doi
10.3929/ethz-b-000516749
dc.description.abstract
Intracellular redox balance is crucial for cell function and is primarily regulated by the relative concentrations of glutathione (GSH) and its oxidized dimer (GSSG). Multiple physiological processes, ranging from cell signaling to protein folding, depend on redox homeostasis. Therefore, several pathological conditions such as cancer, diabetes, and neurodegenerative diseases have been associated with redox imbalances. In eukaryotes, this homeostasis is controlled at the subcellular level, and each organelle possesses its own redox environment. Whereas the effects of oxidative stress have been thoroughly investigated, reductive stress has remained significantly underexplored. One way to understand this essential physiological process is to develop biocompatible probes to induce reductive stress and study the triggered responses. We envisioned that the GSH/GSSG ratio could be manipulated by direct reduction of the disulfide bond in GSSG to GSH using trialkylphosphine derivatives. We aim to achieve subcellular targeting by taking advantage of the local enzymatic activity of organelle-resident enzymes, triggering the release of tributylphosphine and a fluorescent reporter from a masked precursor. In this Thesis, we report the development of a mitochondria-targeted tributylphosphine probe to induce reductive stress in live cells and study the mitochondria-specific reductive stress response. A series of live-cell experiments, including fluorescence imaging and biological assays, showed that reductive stress is eventually transformed into oxidative stress through superoxide production. It activates the ATF4-ATF3-CHOP cascade, part of the integrated stress response, which upregulates the CHAC1 gene. CHAC1 plays a role in the regulation of glutathione and elevated expression of this gene promotes GSH depletion. Regulation of gene expression plays a crucial role in stress-response pathways. Commonly used reporter gene luminescent or colorimetric methods for use in live cells include assays based on secreted enzymes, luciferase, and GFP. These techniques often require exogenous substrates or lack sensitivity due to endogenous activity of isoenzymes or non-amplified GFP signal. We developed a HaloTag fluorescence reporter system for gene expression monitoring in live cells to complement these methods and address some of their limitations. The expression of the self-labeling HaloTag protein is correlated to that of the target gene through co-expression of both genes as non-altered separate proteins. Two fluorogenic HaloTag ligands with different spectral properties and binding kinetics label the HaloTag protein and result in a ratiometric fluorescence output. We envisioned that by adjusting the concentrations of the two fluorogenic ligands, the system could be tuned to reflect differential expression with a large dynamic range. As proof-of-principle, we chose to monitor the expression of the chaperone BiP upon induction of the unfolded protein response of the endoplasmic reticulum. Here, we describe the gene reporter design, the establishment of the reporter cell line using CRISPR-Cas9 gene-editing, the synthesis of fluorogenic HaloTag ligands, and preliminary live cells experiments for the reporter system validation. Building on the design of our original mitochondria-targeted probe, we also developed a series of tributylphosphine probes to modulate redox homeostasis in the endoplasmic reticulum. Here, we report the systematic approach to optimize the design of enzymatically activated ER-targeted tributylphosphines, their synthesis, and preliminary fluorescence imaging results. Altogether, this work demonstrated the importance of trialkylphosphines as chemical probes to modulate redox biology by developing strategies to tune their reactivity and target them to specific organelles. These probes potentially impact new therapies' development and enrich our understanding of the subcellular compartmentalization of redox signaling and organelle-specific stress responses.
en_US
dc.format
application/pdf
en_US
dc.language.iso
en
en_US
dc.publisher
ETH Zurich
en_US
dc.rights.uri
http://rightsstatements.org/page/InC-NC/1.0/
dc.title
Development of Chemical Biology Tools to Modulate Redox Homeostasis and Study Subcellular Stress Responses
en_US
dc.type
Doctoral Thesis
dc.rights.license
In Copyright - Non-Commercial Use Permitted
dc.date.published
2021-10-25
ethz.size
268 p.
en_US
ethz.code.ddc
DDC - DDC::5 - Science::540 - Chemistry
en_US
ethz.identifier.diss
27892
en_US
ethz.publication.place
Zurich
en_US
ethz.publication.status
published
en_US
ethz.leitzahl
ETH Zürich::00002 - ETH Zürich::00012 - Lehre und Forschung::00007 - Departemente::02020 - Dep. Chemie und Angewandte Biowiss. / Dep. of Chemistry and Applied Biosc.::02514 - Laboratorium für Organische Chemie / Laboratory of Organic Chemistry::03940 - Wennemers, Helma / Wennemers, Helma
en_US
ethz.leitzahl
ETH Zürich::00002 - ETH Zürich::00012 - Lehre und Forschung::00007 - Departemente::02020 - Dep. Chemie und Angewandte Biowiss. / Dep. of Chemistry and Applied Biosc.::02514 - Laboratorium für Organische Chemie / Laboratory of Organic Chemistry::03940 - Wennemers, Helma / Wennemers, Helma
ethz.leitzahl.certified
ETH Zürich::00002 - ETH Zürich::00012 - Lehre und Forschung::00007 - Departemente::02020 - Dep. Chemie und Angewandte Biowiss. / Dep. of Chemistry and Applied Biosc.::02514 - Laboratorium für Organische Chemie / Laboratory of Organic Chemistry::03940 - Wennemers, Helma / Wennemers, Helma
en_US
ethz.leitzahl.certified
ETH Zürich::00002 - ETH Zürich::00012 - Lehre und Forschung::00007 - Departemente::02020 - Dep. Chemie und Angewandte Biowiss. / Dep. of Chemistry and Applied Biosc.::02514 - Laboratorium für Organische Chemie / Laboratory of Organic Chemistry::03940 - Wennemers, Helma / Wennemers, Helma
ethz.tag
redox
en_US
ethz.tag
stress
en_US
ethz.tag
redox stress
en_US
ethz.tag
mitochondria
en_US
ethz.tag
chemical biology
en_US
ethz.tag
redox homeostasis
en_US
ethz.tag
stress response
en_US
ethz.tag
reductive stress
en_US
ethz.tag
phosphine
en_US
ethz.date.deposited
2021-11-24T18:08:31Z
ethz.source
FORM
ethz.eth
yes
en_US
ethz.availability
Open access
en_US
ethz.rosetta.installDate
2021-11-25T06:55:42Z
ethz.rosetta.lastUpdated
2022-03-29T16:08:17Z
ethz.rosetta.versionExported
true
ethz.COinS
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