Periodic Reporting for period 1 - PRO-CLEAR (Mechanisms for the selective clearance of chemically damaged proteins in mammalian cells)
Reporting period: 2021-09-01 to 2023-08-31
This project addresses the fundamental question of how cells identify and selectively remove individual damaged proteins.
Degradation of damaged proteins is essential for the survival of all cells. Impaired protein turnover can lead to diverse disorders such as neurodegeneration. In human cells, a system of quality control factors can surveil the proteome for damage. For example, misfolded proteins can be recognized and selectively degraded to prevent the build-up of toxic aggregates throughout life and in response to stress. In addition, proteins can experience any of several hundred chemical modifications throughout their life-cycle. How cells monitor the proteome for these types of modifications and potential signs of damage remains poorly understood.
In this project, we aimed to identify chemical modifications that can act as marks of protein damage, to induce their selective removal. More specifically, we asked: How can we measure the impact of individual protein modifications on protein turnover? Which known forms of chemical damage could serve as triggers of selective protein degradation in human cells? What is the underlying cellular machinery and how can it recognize chemically damaged proteins at the molecular level? And lastly, how does this chemical protein surveillance contribute to maintaining an intact proteome under physiological conditions and proteotoxic stress.
This project was designed to test the long-standing hypothesis that chemical protein modifications could provide a generic signal, that allows human cells to broadly survey the proteome for damage. In addition to its contribution to our fundamental understanding of cell homeostasis, these questions are central to understanding the mechanisms at play when protein homeostasis is perturbed, for example during ageing or neurodegeneration. In addition, the machinery that mediates selective protein degradation can be reprogrammed to target other proteins in a cell by pharmacological means. For example, the class of IMiD drugs mimics a chemical modification recognized by the ubiquitin ligase CUL4/CRBN and redirects its activity towards therapeutically relevant proteins, such as IKZF1. This mechanism underlies the action of the drug thalidomide, which has been highly successful in the treatment of multiple myeloma. Identifying other such chemical protein surveillance factors could therefore also open up avenues for designing new targeted protein therapeutics in future projects.
Degradation of damaged proteins is essential for the survival of all cells. Impaired protein turnover can lead to diverse disorders such as neurodegeneration. In human cells, a system of quality control factors can surveil the proteome for damage. For example, misfolded proteins can be recognized and selectively degraded to prevent the build-up of toxic aggregates throughout life and in response to stress. In addition, proteins can experience any of several hundred chemical modifications throughout their life-cycle. How cells monitor the proteome for these types of modifications and potential signs of damage remains poorly understood.
In this project, we aimed to identify chemical modifications that can act as marks of protein damage, to induce their selective removal. More specifically, we asked: How can we measure the impact of individual protein modifications on protein turnover? Which known forms of chemical damage could serve as triggers of selective protein degradation in human cells? What is the underlying cellular machinery and how can it recognize chemically damaged proteins at the molecular level? And lastly, how does this chemical protein surveillance contribute to maintaining an intact proteome under physiological conditions and proteotoxic stress.
This project was designed to test the long-standing hypothesis that chemical protein modifications could provide a generic signal, that allows human cells to broadly survey the proteome for damage. In addition to its contribution to our fundamental understanding of cell homeostasis, these questions are central to understanding the mechanisms at play when protein homeostasis is perturbed, for example during ageing or neurodegeneration. In addition, the machinery that mediates selective protein degradation can be reprogrammed to target other proteins in a cell by pharmacological means. For example, the class of IMiD drugs mimics a chemical modification recognized by the ubiquitin ligase CUL4/CRBN and redirects its activity towards therapeutically relevant proteins, such as IKZF1. This mechanism underlies the action of the drug thalidomide, which has been highly successful in the treatment of multiple myeloma. Identifying other such chemical protein surveillance factors could therefore also open up avenues for designing new targeted protein therapeutics in future projects.
We first set out to identify protein modifications that serve as triggers selective degradation. To this end, we generated semi-synthetic model proteins carrying different chemical modifications found in human cells. Measuring their turnover in cells revealed that C-terminal amides mark proteins for removal in diverse human cells. A genetic screen revealed that the ubiquitin ligase SCF/FBXO31 can recognize C-terminal amide bearing proteins (CTAPs) and hand them over to the degradation machinery. Biochemical and genetic approaches identified the underlying molecular mechanisms. Mass-spectrometry revealed a set of endogenous proteins that are recognized by FBXO31, specifically following oxidative stress. A mutation in the substrate binding pocket of FBXO31 was previously found to recur in patients with spastic diplegic cerebral palsy. We found that this D334N mutation impairs CTAP recognition and redirects the activity of FBXO31 to a range of other essential proteins in cells.
The results of this project were already presented to a wider public before its conclusion. To the scientific community, the study was presented in talks at the "Cold Spring Harbor Meeting on Ubiquitin, Autophagy and Disease 2023" and the Workshop "Protein quality control: From molecular mechanisms to therapeutic intervention 2023" organized by the European Molecular Biology Organization (EMBO). It was also presented and discussed publicly in a freely accessible online event organized by the Dana-Farber Cancer Institute as part of its Targeted Protein Degradation Webinar Series. Some of the work presented here inspired patent applications and the founding of a new start-up company by members of the host institution. The potential of this work was also highlighted by a nomination for the Spark Award for the most promising inventions by ETH Zürich.
The results of this project were already presented to a wider public before its conclusion. To the scientific community, the study was presented in talks at the "Cold Spring Harbor Meeting on Ubiquitin, Autophagy and Disease 2023" and the Workshop "Protein quality control: From molecular mechanisms to therapeutic intervention 2023" organized by the European Molecular Biology Organization (EMBO). It was also presented and discussed publicly in a freely accessible online event organized by the Dana-Farber Cancer Institute as part of its Targeted Protein Degradation Webinar Series. Some of the work presented here inspired patent applications and the founding of a new start-up company by members of the host institution. The potential of this work was also highlighted by a nomination for the Spark Award for the most promising inventions by ETH Zürich.
Altogether, this project revealed a novel chemical mark that triggers selective degradation of individual proteins and the underlying cellular machinery. The importance of this mechanism to human health is highlighted by the fact, that mutations impairing FBXO31 function were previously identified to cause some cases of heritable intellectual disability. This study also revealed how a mutation in the substrate binding pocket of FBXO31 can give rise to a new toxic ubiquitin ligase activity in some cases of spastic diplegic cerebral palsy, thereby shedding light on the patho-mechanisms of these cases of innate neurodevelopmental disorders.
Understanding client recognition of a ubiquitin ligase like FBXO31 provides the first step for exploiting it as a mediator of targeted protein degradation. This class of therapeutics holds great promise as it could allow to selectively remove disease-related proteins within human cells. This approach is currently being pursued by others to redirect ubiquitin ligases such as CUL4/CRBN and CUL2/VHL for the treatment of diverse cancers and neurodegenerative disorders. Since each ubiquitin ligase can only be targeted to a limited set of privileged clients, this project has garnered interest by the scientific community seeking to also exploit other ligases, such as FBXO31, for drug development. The success of this project has also spurred follow-up projects at the host institution, aiming to identify additional such regulatory protein modifications and their respective reader proteins.
Understanding client recognition of a ubiquitin ligase like FBXO31 provides the first step for exploiting it as a mediator of targeted protein degradation. This class of therapeutics holds great promise as it could allow to selectively remove disease-related proteins within human cells. This approach is currently being pursued by others to redirect ubiquitin ligases such as CUL4/CRBN and CUL2/VHL for the treatment of diverse cancers and neurodegenerative disorders. Since each ubiquitin ligase can only be targeted to a limited set of privileged clients, this project has garnered interest by the scientific community seeking to also exploit other ligases, such as FBXO31, for drug development. The success of this project has also spurred follow-up projects at the host institution, aiming to identify additional such regulatory protein modifications and their respective reader proteins.