Periodic Reporting for period 1 - MSCA_dk_2021 (Identification and characterization of cellular DeUrmylases)
Período documentado: 2022-07-01 hasta 2024-06-30
Significance and overall goals of the proposal
Ubiquitin-related modifier 1 (Urm1) is known for its dual role as sulfur carrier protein (SCP) in tRNA
thiolation and as an ubiquitin-like protein (UBL) that leads to an oxidant-induced posttranslational protein
modification (PTM). We recently, managed to recapitulate the Urm1-conjugation reaction for various targets
in vitro and characterize the underlying molecular principles. In most ubiquitin (Ub) and ubiquitin-like
conjugation systems the active removal of the covalently attached PTM constitutes a key feature of the
regulatory mechanisms. Although, various targets of urmylation have been reported, not a single enzyme that
could reverse the modification has been identified, yet.
In this project, I plan to incorporate different complementary de novo discovery strategies to
unambiguously identify possible “deurmylation” enzymes in yeast and human, understand their
biochemical mode of action and assess their biological significance.
This project aims to uncover the guiding principles
that regulate the evolutionary most ancient UBL system in
eukaryotes. Hence, the expected results will not only answer
one of the most intriguing questions related to Urm1, but have
far reaching consequences for our understanding of all UBL
systems in eukaryotes. Foremost, the constant removal of
Urm1-conjugation sites, leads to a drastic underestimation of
the actual numbers of genuine Urm1 target and target sites in
vivo. Therefore, the identification and specific
inhibition/deletion of cellular deurmylation activities will
facilitate the detection of urmylation in living cells and enable
future studies of these dynamic processes in organisms. Last
but not least, several studies hint at the critical function of
Urm1 conjugation in oxidative stress response and at its direct
involvement in severe human diseases. As various
deubiquitylating enzymes (DUBs) are successfully developed as
potent targets for small-molecule based drug therapies, the identified enzyme(s) could be well suited for novel
therapeutic and diagnostic strategies.
In the proposed project I aim to address and answer the following two main research questions
• How can we identify active “deurmylating” enzyme(s) in yeast and human cells?
• How do “deurmylating” enzymes work and how specific are they?
I designed and developed a comprehensive research plan to experimentally address these questions within
the requested funding period. I have performed preliminary experiments and introduced complementary
approaches to mitigate the identified technical risks in each individual task. The project will be carried out in
one of the leading structural biology labs in the field, which provides an ideal scientific environment to
conduct this ambitious project and to support the development of my scientific career.
Ubiquitin-related modifier 1 (Urm1) is known for its dual role as sulfur carrier protein (SCP) in tRNA
thiolation and as an ubiquitin-like protein (UBL) that leads to an oxidant-induced posttranslational protein
modification (PTM). We recently, managed to recapitulate the Urm1-conjugation reaction for various targets
in vitro and characterize the underlying molecular principles. In most ubiquitin (Ub) and ubiquitin-like
conjugation systems the active removal of the covalently attached PTM constitutes a key feature of the
regulatory mechanisms. Although, various targets of urmylation have been reported, not a single enzyme that
could reverse the modification has been identified, yet.
In this project, I plan to incorporate different complementary de novo discovery strategies to
unambiguously identify possible “deurmylation” enzymes in yeast and human, understand their
biochemical mode of action and assess their biological significance.
This project aims to uncover the guiding principles
that regulate the evolutionary most ancient UBL system in
eukaryotes. Hence, the expected results will not only answer
one of the most intriguing questions related to Urm1, but have
far reaching consequences for our understanding of all UBL
systems in eukaryotes. Foremost, the constant removal of
Urm1-conjugation sites, leads to a drastic underestimation of
the actual numbers of genuine Urm1 target and target sites in
vivo. Therefore, the identification and specific
inhibition/deletion of cellular deurmylation activities will
facilitate the detection of urmylation in living cells and enable
future studies of these dynamic processes in organisms. Last
but not least, several studies hint at the critical function of
Urm1 conjugation in oxidative stress response and at its direct
involvement in severe human diseases. As various
deubiquitylating enzymes (DUBs) are successfully developed as
potent targets for small-molecule based drug therapies, the identified enzyme(s) could be well suited for novel
therapeutic and diagnostic strategies.
In the proposed project I aim to address and answer the following two main research questions
• How can we identify active “deurmylating” enzyme(s) in yeast and human cells?
• How do “deurmylating” enzymes work and how specific are they?
I designed and developed a comprehensive research plan to experimentally address these questions within
the requested funding period. I have performed preliminary experiments and introduced complementary
approaches to mitigate the identified technical risks in each individual task. The project will be carried out in
one of the leading structural biology labs in the field, which provides an ideal scientific environment to
conduct this ambitious project and to support the development of my scientific career.
WP1 achievements
1.1 Urm1 Activity-based Probes design and generation Fully achieved Now due to the collaboration more AB probes are available
1.1 Substrate-based probes design and generation Fully achieved
1.1 Probe coupling to Nickel resin optimisation Fully achieved
1.1 Pull down, followed by MS identification Fully achieved
1.2 Reagents for the fractionation-based identification preparation Fully achieved
1.2 Fractionation-based identification Not fully achieved Requires more optimization
WP2 achievements
2.1 Validation of the results from the Task 1.1 Fully achieved None of the identified DUBs was active toward urmylated substartes
2.1 Expression and purification of the recombinant DUB candidates Fully achieved
2.2 Specificity test Fully achieved None of the identified DUBs was active toward urmylated substartes
2.2 Measurements of the enzyme activity NA
1.1 Urm1 Activity-based Probes design and generation Fully achieved Now due to the collaboration more AB probes are available
1.1 Substrate-based probes design and generation Fully achieved
1.1 Probe coupling to Nickel resin optimisation Fully achieved
1.1 Pull down, followed by MS identification Fully achieved
1.2 Reagents for the fractionation-based identification preparation Fully achieved
1.2 Fractionation-based identification Not fully achieved Requires more optimization
WP2 achievements
2.1 Validation of the results from the Task 1.1 Fully achieved None of the identified DUBs was active toward urmylated substartes
2.1 Expression and purification of the recombinant DUB candidates Fully achieved
2.2 Specificity test Fully achieved None of the identified DUBs was active toward urmylated substartes
2.2 Measurements of the enzyme activity NA
The project undertook a comprehensive exploration of various tools and methodologies to identify enzymes capable of reversing urmylation, a specific post-translational modification. Although the precise de-urmylating enzyme was not pinpointed, the project successfully generated a wealth of valuable data and resources that significantly enhanced the scientific community's understanding of urmylation within cellular contexts.
A key objective was to map the conjugation of Urm1 within cells, a process crucial for advancing our knowledge in this field. To achieve this, the project developed a range of innovative activity-based probes designed to improve the detection of urmylated substrates, particularly under conditions of cellular stress. These probes represent an important alternative approach for characterizing Urm1 targets, opening up new avenues for future research. Additionally, the creation of human overexpression cell lines and other tools during the project has provided researchers with versatile resources to continue studying this process in greater detail.
The project's findings are also supported by recent reports, such as the study by Cairo et al. in Cell (2024), which links urmylation to the formation of stress-dependent condensates in yeast, further highlighting the significance of this research area. The collaborative efforts and interdisciplinary approach of this project have laid a solid foundation for future studies, with the long-term goal of understanding how Urm1-targets involved in persulfidation contribute to aging and other cellular processes.
A key objective was to map the conjugation of Urm1 within cells, a process crucial for advancing our knowledge in this field. To achieve this, the project developed a range of innovative activity-based probes designed to improve the detection of urmylated substrates, particularly under conditions of cellular stress. These probes represent an important alternative approach for characterizing Urm1 targets, opening up new avenues for future research. Additionally, the creation of human overexpression cell lines and other tools during the project has provided researchers with versatile resources to continue studying this process in greater detail.
The project's findings are also supported by recent reports, such as the study by Cairo et al. in Cell (2024), which links urmylation to the formation of stress-dependent condensates in yeast, further highlighting the significance of this research area. The collaborative efforts and interdisciplinary approach of this project have laid a solid foundation for future studies, with the long-term goal of understanding how Urm1-targets involved in persulfidation contribute to aging and other cellular processes.