Project

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Chemical probes to control redox biology with subcellular precision

Applicant Rivera Fuentes Pablo Marcelo
Number 186862
Funding scheme Eccellenza grant
Research institution Institut für Chemie Universität Zürich
Institution of higher education University of Zurich - ZH
Main discipline Organic Chemistry
Start/End 01.01.2020 - 31.12.2024
Approved amount 1'496'422.00
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All Disciplines (4)

Discipline
Organic Chemistry
Cellular Biology, Cytology
Molecular Biology
Biochemistry

Keywords (5)

Organelle targeting; Chemical probes; Chemical biology; Bio-orthogonal chemistry; Redox biology

Lay Summary (French)

Lead
Les cellules doivent maintenir un environnement chimique spécifique pour rester en bonne santé. Bien que nous sachions que cet équilibre doit être préservé, nous ne connaissons pas les mécanismes exacts par lesquels les cellules détectent et réagissent aux perturbations, par exemple, de l'environnement.
Lay summary

Contenu et objectifs des travaux de recherche

Dans ce projet, nous testerons, dans des conditions contrôlées, comment les cellules détectent et réagissent aux perturbations de l'équilibre entre molécules oxydées et réduites. Cet équilibre, appelé homéostasie redox, est établi dans les cellules vivantes par l'action de l'oxygène et de plusieurs enzymes spécialisées. Les cellules humaines, cependant, ont des compartiments internes qui doivent maintenir chacun leur homéostasie redox individuelle. Nous développerons des composés chimiques qui ciblent spécifiquement certaines zones de la cellule et induisent une modification de l'homéostasie redox, contrecarrant ainsi l'action oxydante de l'oxygène. Après avoir induit cette perturbation, nous évaluerons comment la cellule réagit et comment cette réponse est liée aux pathologies humaines.

Contexte social et scientifique du travail de recherche

Ce projet est principalement orienté vers la découverte des aspects fondamentaux de la chimie biologique. Cependant, une meilleure compréhension des mécanismes de l'homéostasie redox dans les cellules humaines peut mener au développement de meilleurs traitements pour des maladies comme le diabète, le cancer, l'infarctus et les maladies neurodégénératives.
Direct link to Lay Summary Last update: 20.11.2019

Responsible applicant and co-applicants

Employees

Associated projects

Number Title Start Funding scheme
180541 NCCR AntiResist (phase I) 01.08.2020 National Centres of Competence in Research (NCCRs)
165551 Development of Intracellular Photoactivatable Probes 01.04.2016 Project funding

Abstract

The chemical environment of subcellular compartments largely defines the biological processes that occur in eukaryotic cells. Indirect observations suggest that shifts in these chemical equilibria induce pathologies, but the molecular mechanisms of how these changes affect physiology remain largely unknown. In this project, our goal is to modify selectively the redox balance in three selected organelles: mitochondria, endoplasmic reticulum, and Golgi apparatus. Redox homeostasis is largely controlled by the relative concentrations of reduced glutathione (GSH) and its oxidized, disulfide bonded form (GSSG). The ratios of GSH to GSSG are different in every subcellular compartment, and deviations from these optimal ratios lead to disrupted physiology. We are specifically interested in investigating the effects of increasing the relative concentrations of GSH, leading to reductive stress. This condition has been indirectly studied, and a number of human pathologies, including neurodegenerative and metabolic diseases, have been circumstantially associated with reductive stress. This project aims at providing molecular details of how organelles sense reductive stress, how the cell is affected by this disruption, and how it tries to solve it.The ratio of GSH to GSSG can be modulated by breaking the disulfide bond in GSSG to form GSH. Our own preliminary results demonstrate that this reaction can be carried out efficiently and selectively in live cells by trialkylphosphines. These potent reducing agents, however, must be activated only within the organelle in which they are needed. To meet this challenge, we propose two classes of trialkylphosphine small molecule probes with two activation modes: photochemical (fast and acute) and chemical/enzymatic (slow and sustained). We recently developed a photoremovable carrier that brings phosphines in an unreactive form into the cell. We will use this platform to develop probes that can be targeted to subcellular locations, with mitochondria and the endoplasmic reticulum as the first targets. We plan to use these probes to test whether reductive stress can be transmitted between cells, a proposed mechanism by which cancer cells acquire malignant and drug-resistant phenotypes within tumors.In addition to these photocleavable probes, we will explore strategies to activate phosphine-containing probes using chemical and enzymatic reactions. We have already discovered that nitroreductases present in mitochondria can release phosphines by reducing nitroaromatic probes. Based on this concept, we will develop phosphine probes that can be activated by carboxylesterases in the endoplasmic reticulum. The biological effects of these probes will be evaluated by a combination of fluorescence microscopy, immunoblotting analyses, reverse transcription polymerase chain reaction, and global transcriptomics.There is a lack of appropriate endogenous enzymes for the activation of phosphines in the Golgi apparatus. Therefore, to induce reductive stress in this important organelle, we plan to develop a new orthogonal enzyme/substrate activation system based on bacterial isonitrile hydratase and isonitriles. In this way, the isonitrile hydratase will be expressed ectopically in the Golgi apparatus to enable activation of the isonitrile-phosphine probe in that organelle. The biological evaluation of these probes will follow the workflow developed for mitochondrial and endoplasmic reticulum phosphine probes.With probes in hand that can induce acute or sustained reductive stress in three different organelles, we will evaluate the differences, similarities, synergies, and cross-talk between the response programs of the three separate organelles. These studies will provide a comprehensive picture of cellular reductive stress at an unprecedented level of spatial and chemical specificity, with potential applications in the development of new therapies for diseases associated with disruption of redox homeostasis.
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