Periodic Reporting for period 4 - Redox Relays (Detecting, understanding and exploiting intracellular redox signaling relays)
Berichtszeitraum: 2022-04-01 bis 2024-03-31
In all forms of life, metabolism fundamentally depends on redox (reduction-oxidation) reactions, i.e. chemical transformations in which electrons are transferred between molecules. For example, photosynthesis, respiration and fermentation all depend on the reshuffling of electrons between donors (reductants) and recipients (oxidants). Organisms and their cells need to constantly adapt to changes in metabolism, as for example caused by variations in the availability of oxidants (e.g. oxygen) and reductants (e.g. sugars). It is now recognized that many, if not most, proteins, the executioners of almost all cell functions, can ‘feel’ the presence of key redox molecules (oxidants and reductants), to change their activity in an adaptive manner, so that cellular behavior (e.g. growth) matches metabolically imposed opportunities or restrictions. However, it has been difficult to explain how proteins are specifically regulated by redox reactions, at the right time and place inside the cell, a process called ‘redox regulation’ or ‘redox signaling’. The emerging solution to this conundrum is that oxidants and reductants are channeled towards individual target proteins through protein-protein contacts acting in a highly specific and localized manner. This project aimed to systematically uncover, monitor and manipulate the localized protein interactions that give specificity and efficiency to redox signaling. The project discovered that metabolism-driven intracellular redox changes are acting on individual protein complexes, rather than affecting all proteins within a subcellular compartment. These findings show that protein redox control is much more localized, precise, and dynamic than previously appreciated. These insights are expected to enhance our understanding of biological redox regulation which has long been recognized to be deregulated in disease, including cancer and diabetes.
In this project, we tagged all proteins with genetically encoded redox biosensors to monitor their local redox environment inside living yeast cells. Specifically, we generated yeast libraries in which redox probes for H2O2 and thiol oxidation, respectively, were attached to all protein-coding open reading frames. The libraries were screened to identify proteins located within oxidizing microenvironments. Measuring the libraries under different conditions of nutrient availability allowed us to identify individual proteins and protein complexes undergoing metabolism-induced oxidation or reduction. In general, the redox state of protein-tethered probes differs substantially between individual proteins located within the same subcellular compartment. These protein context-dependent redox differences could not be observed with conventional probes and procedures. We identified both known and unknown intracellular hotspots of H2O2 generation. Changes in nutrient availability caused changes in the redox environment for highly distinct sets of proteins, which are often metabolic enzymes. We also observed that opposing redox changes occur in parallel within the same subcellular compartment. By crossing the fusion protein library with a deletion library, we identified oxidant-generating and consuming enzymes controlling context-specific protein oxidation and reduction. Taken together, these findings strongly support the notion that nanoscale redox domains exist on the level of individual protein complexes, which potentially are part of biomolecular condensates. These findings show that intracellular thiol oxidation generation is much more regulated, localized and functionally differentiated, i.e. protein-specific, than previously recognized. The project also involved the development of new genetically encoded redox probes providing specific advantages over previous probes. The tools developed in this project, i.e. libraries, strains, fusion constructs and probes, are available to the community upon request. The project also undertook step towards transferring these strategies to mammalian cells. The role of peroxiredoxins as transmitters of oxidation was further investigated and consolidated. Based on screening results, we studied individual redox-regulated proteins in more detail. One example is the sulfur transferase MPST which was identified as an enzyme mediating redox changes in other proteins. This involves a distinct kind of thiol modification, called persulfidation. Another key example is asparagine synthetase which was revealed to undergo thiol oxidation while undergoing condensation, suggesting a causal connection between localized redox regulation and phase separation. This is an example of how the project has opened new and unexpected research directions. Finally, the project contributed to the training of a new generation of redox biologists.