Periodic Reporting for period 2 - AMP-Alarm (Diadenosine Polyphosphate Alarmones as Drivers for Protein AMPylation)
Reporting period: 2023-01-01 to 2024-06-30
It is puzzling that the human genome has only slightly more genes than the fruit fly and only half the number of cauliflower. Obviously, the sheer number of genes alone cannot determine the complexity of human development and the sophisticated signalling systems that maintain homeostasis. In fact, the complexity of information from genome to proteome is greatly increased. Drivers for the complexity of the proteome are posttranslational modifications (PTMs), i.e. covalent modifications of polypeptides after translation.
The aim of this project is to shine light on a scientific mystery known for decades. We will elucidate the cellular roles and functions of diadenosine polyphosphates (ApnAs), which are formed in response to stress and are therefore called "alarmones", and their interactions with the PTM processes "AMPylation", i.e. the covalent modification of the target protein by adenosine monophosphate.
Our initial findings have unveiled the interaction between ApnAs and AMPylation for the first time. We are now embarking on a quest to discover, identify, and delineate new key players in AMPylation, elucidating their interplay with ApnAs. To achieve this, we will craft and employ novel chemical tools in proteome-wide studies within living cells.
We will further explore the molecular role of a previously identified protein using ApnA-based probes. This protein, now known as Rlig1 (previously C12orf29), has been identified as a "5'-3' RNA ligase"—the first of its kind discovered in human cells. Emerging evidence suggests that Rlig1 plays a critical role in RNA repair mechanisms within human cells, a process that remains inadequately examined and understood. Consequently, our research will focus on comprehensively understanding the molecular dynamics, functionalities, and the specific contributions of Rlig1 to RNA repair processes.
Overall, this project will bring significant new advances in this under-researched field and provide a guide for future translational research in the fight against disease
The aim of this project is to shine light on a scientific mystery known for decades. We will elucidate the cellular roles and functions of diadenosine polyphosphates (ApnAs), which are formed in response to stress and are therefore called "alarmones", and their interactions with the PTM processes "AMPylation", i.e. the covalent modification of the target protein by adenosine monophosphate.
Our initial findings have unveiled the interaction between ApnAs and AMPylation for the first time. We are now embarking on a quest to discover, identify, and delineate new key players in AMPylation, elucidating their interplay with ApnAs. To achieve this, we will craft and employ novel chemical tools in proteome-wide studies within living cells.
We will further explore the molecular role of a previously identified protein using ApnA-based probes. This protein, now known as Rlig1 (previously C12orf29), has been identified as a "5'-3' RNA ligase"—the first of its kind discovered in human cells. Emerging evidence suggests that Rlig1 plays a critical role in RNA repair mechanisms within human cells, a process that remains inadequately examined and understood. Consequently, our research will focus on comprehensively understanding the molecular dynamics, functionalities, and the specific contributions of Rlig1 to RNA repair processes.
Overall, this project will bring significant new advances in this under-researched field and provide a guide for future translational research in the fight against disease
In the project we have a) developed new synthetic probes for investigating AMPylation and found that ApnAs are co-substrates for AMPylation (see DOI: 10.1002/anie.202213279) b) applied those in chemical proteomics studies in order to identify protein targets of AMPylation and c) investigated a protein of unknown function (formerly termed C12orf29) that we identified in the chemical proteomics studies. This protein turned out to be the first 5’-3’ RNA ligase to be identified in humans and is now termed Rlig1. Investigation on biochemical and cellular level indicate the involvement of this enzyme to be involved in RNA repair (see DOI: 10.1038/s41467-023-36451-x).
The ERC project "AMP-Alarm" has made several significant advancements:
1.) Identification of ApnAs as Co-Substrates for Protein AMPylation: The project discovered that diadenosine polyphosphates (ApnAs) can serve as substrates for AMPylation, altering cellular signaling during stress. This finding alters the proteome-wide AMPylation pattern, a process not reported before.
2.) Development of New Probes for Cellular Studies: The project developed new probes for studying AMPylation in living cells, overcoming previous limitations and enabling the identification of AMPylation targets. We expect that these probes will allow the identification of targets that are AMPylated in living cells and might eventually identify new AMPylators.
3.) Discovery of Human RNA Ligase: Through Ap3A-based proteomic studies, a previously unknown human RNA ligase (Rlig1) was discovered. This enzyme appears to play a crucial role in RNA repair mechanisms, particularly under oxidative stress conditions. The discovery of Rlig1 garnered significant attention within the scientific community, ranking highly in article metrics and attracting thousands of views shortly after publication.
In future, we expect that we will gain a deeper insight into the mechanism of RNA repair and its involvement in cellular homeostasis.
1.) Identification of ApnAs as Co-Substrates for Protein AMPylation: The project discovered that diadenosine polyphosphates (ApnAs) can serve as substrates for AMPylation, altering cellular signaling during stress. This finding alters the proteome-wide AMPylation pattern, a process not reported before.
2.) Development of New Probes for Cellular Studies: The project developed new probes for studying AMPylation in living cells, overcoming previous limitations and enabling the identification of AMPylation targets. We expect that these probes will allow the identification of targets that are AMPylated in living cells and might eventually identify new AMPylators.
3.) Discovery of Human RNA Ligase: Through Ap3A-based proteomic studies, a previously unknown human RNA ligase (Rlig1) was discovered. This enzyme appears to play a crucial role in RNA repair mechanisms, particularly under oxidative stress conditions. The discovery of Rlig1 garnered significant attention within the scientific community, ranking highly in article metrics and attracting thousands of views shortly after publication.
In future, we expect that we will gain a deeper insight into the mechanism of RNA repair and its involvement in cellular homeostasis.