Periodic Reporting for period 2 - nbPTMs (A multifaceted platform for exploring nucleotide-based post-translational modifications)
Berichtszeitraum: 2022-10-01 bis 2024-03-31
Post-translational modifications (PTMs) act as key cellular signals that are involved in virtually all processes that govern the fate of cells and organisms. By altering the chemical state of proteins dynamically, they not only enormously expand the functional repertoire of the proteome but also do it in a time-dependent manner as part of a biological system’s response to its environment. PTMs come in different varieties, ranging from small chemical moieties, such as phosphorylation and acetylation, to entire proteins like ubiquitin, and extending to the attachment of nucleotides. Nucleotide-based post-translational modifications (nbPTMs) play key roles in health and disease, from bacterial pathogenesis to cancer. The study of nbPTMs has far-reaching implications for society, particularly in healthcare and medical research. Understanding nbPTMs is pivotal in comprehending the molecular basis of various diseases and developing novel therapeutic strategies and diagnostic tools. However, technical challenges of these versatile, but chemically complex protein modifications have constrained our fundamental understanding of even the most intensely studied nbPTMs, including ADP-ribosylation (ADPr) and AMPylation, for decades.
The overarching aim of the EU-funded project ‘nbPTMs’ is to establish, apply and disseminate a methodology to transform the study of nbPTMs. A key goal is generating nbPTM-specific recombinant antibodies by converting our basic discoveries of Serine ADPr by the writer complex HPF1/PARP1 into a technology for preparing challenging peptide antigens. By combining these novel tools with unambiguous and unbiased mass spectrometry-based proteomics, the project aims to detect known nbPTMs with unprecedented sensitivity and specificity, as well as unveil novel forms of nbPTMs. Particularly intriguing and significant nbPTMs will be selected for extensive biological characterization. The resulting tools, materials, methods, discoveries and proteomic datasets will be made publicly available, enabling investigations of nbPTMs by the scientific community.
As a project at the intersection of biology, chemistry, and technology, 'nbPTMs' is uniquely poised to make significant contributions to biomedical research. By integrating state-of-the-art technological advances with biological inquiries, the project is expected to yield fundamental biological insights and tools that could transform our approach to disease treatment and diagnosis.
The overarching aim of the EU-funded project ‘nbPTMs’ is to establish, apply and disseminate a methodology to transform the study of nbPTMs. A key goal is generating nbPTM-specific recombinant antibodies by converting our basic discoveries of Serine ADPr by the writer complex HPF1/PARP1 into a technology for preparing challenging peptide antigens. By combining these novel tools with unambiguous and unbiased mass spectrometry-based proteomics, the project aims to detect known nbPTMs with unprecedented sensitivity and specificity, as well as unveil novel forms of nbPTMs. Particularly intriguing and significant nbPTMs will be selected for extensive biological characterization. The resulting tools, materials, methods, discoveries and proteomic datasets will be made publicly available, enabling investigations of nbPTMs by the scientific community.
As a project at the intersection of biology, chemistry, and technology, 'nbPTMs' is uniquely poised to make significant contributions to biomedical research. By integrating state-of-the-art technological advances with biological inquiries, the project is expected to yield fundamental biological insights and tools that could transform our approach to disease treatment and diagnosis.
Since the beginning of the ‘nbPTMs’ project, we have made significant progress in generating innovative nbPTM-specific antibodies through a sophisticated chemical biology strategy. This pivotal development has enabled us to uncover elusive nbPTMs and conduct detailed biological characterization of a particularly intriguing form of nbPTM, shedding new light on its role in the DNA damage response. Additionally, our team is advancing proteomic approaches aimed at preserving labile nbPTMs, further enhancing our ability to study these complex modifications.
To generate anti-nbPTMs antibodies, we have successfully circumvented the challenge of chemically synthesizing peptides. By employing a chemoenzymatic strategy, inspired by our foundational biological discoveries (Leidecker et al. Nature Chemical Biology 2016; Bonfiglio et al. Molecular Cell 2017; Bonfiglio et al. Cell 2020), we succeeded in preparing a range of precisely-modified, pure peptides in a scalable and rapid manner. This innovative approach was combined with phage display recombinant antibody technology and the SpyTag/SpyCatcher protein system (Dauben et al. Trends Biochem Sci. 2023). The multiple ADPr peptides we prepared proved to be instrumental not only in antibody selection but also in determining their specificities. This has led to the generation of modular antibodies capable of detecting nbPTMs with unprecedented specificity and sensitivity (Longarini et al. Molecular Cell 2023), marking a significant advancement in the study of nbPTMs.
These new tools have already yielded key insights, enabling us to uncover chromatin mono-ADPr as a second wave of PARP1 signaling. This discovery is particularly significant considering the biological and clinical importance of PARP1. Through detailed biological characterization, we have elucidated how mono-ADPr is orchestrated by the writer complex HPF1/PARP1 and modulated by the eraser ARH3. Moreover, we have identified several readers of chromatin mono-ADPr, such as the ubiquitin E3 ligase RNF114 (Longarini et al. Molecular Cell 2023).
These research and technological achievements underscore the 'nbPTMs' project's pivotal role in advancing our understanding of nbPTMs and establishing their significance in critical biological processes. A prime example of this is our key finding, facilitated by our innovative tools, that mono-ADPr serves as a crucial information carrier in the DNA damage response.
To generate anti-nbPTMs antibodies, we have successfully circumvented the challenge of chemically synthesizing peptides. By employing a chemoenzymatic strategy, inspired by our foundational biological discoveries (Leidecker et al. Nature Chemical Biology 2016; Bonfiglio et al. Molecular Cell 2017; Bonfiglio et al. Cell 2020), we succeeded in preparing a range of precisely-modified, pure peptides in a scalable and rapid manner. This innovative approach was combined with phage display recombinant antibody technology and the SpyTag/SpyCatcher protein system (Dauben et al. Trends Biochem Sci. 2023). The multiple ADPr peptides we prepared proved to be instrumental not only in antibody selection but also in determining their specificities. This has led to the generation of modular antibodies capable of detecting nbPTMs with unprecedented specificity and sensitivity (Longarini et al. Molecular Cell 2023), marking a significant advancement in the study of nbPTMs.
These new tools have already yielded key insights, enabling us to uncover chromatin mono-ADPr as a second wave of PARP1 signaling. This discovery is particularly significant considering the biological and clinical importance of PARP1. Through detailed biological characterization, we have elucidated how mono-ADPr is orchestrated by the writer complex HPF1/PARP1 and modulated by the eraser ARH3. Moreover, we have identified several readers of chromatin mono-ADPr, such as the ubiquitin E3 ligase RNF114 (Longarini et al. Molecular Cell 2023).
These research and technological achievements underscore the 'nbPTMs' project's pivotal role in advancing our understanding of nbPTMs and establishing their significance in critical biological processes. A prime example of this is our key finding, facilitated by our innovative tools, that mono-ADPr serves as a crucial information carrier in the DNA damage response.
The ‘nbPTMs’ project has already made key advancements beyond the state of the art, significantly broadening the scope of nbPTM research. As the most notable achievement so far, we have unlocked the ability to explore all forms of mono-ADPr, a particularly prevalent class of nbPTMs (Longarini et al. Molecular Cell 2023). Such progress is remarkable considering the historic oversight of mono-ADPr in the initial decades of research. It is now known that mono-ADPr is the primary product of the vast majority of ADPr writers. Due to our insights, mono-ADPr is now recognized as the prevalent outcome of PARP1, the most studied and clinically important nbPTM writer. Specifically, the new antibodies have enabled us to elucidate DNA damage-induced mono-ADPr as a second wave of PARP1 signaling (Longarini et al. Molecular Cell 2023). This new concept is resonating broadly in the field and challenges the existing paradigm that poly-ADPr is the exclusive outcome of PARP1. This reevaluation highlights the project’s substantial contribution to redefining our understanding of nbPTMs. The significance of our methodology has been acknowledged by a highlight in Nature Reviews Molecular Cell Biology, a Cell Press 'Spotlight', and an invitation to contribute a ‘Technology of the Month’ article (Trends Biochem Sci. 2023, including the front cover). This technological leap now provides both academic and biotech laboratories with innovative tools to delve deep into how mono-ADPr orchestrates many biological and disease processes.
Regarding the future milestones of the 'nbPTMs' project, by building upon the early successes our focus is set on extending our innovative strategy to encompass a broader range of overlooked or yet-to-be-discovered nbPTMs. A key aspect of this endeavor involves development of nbPTM-tailored proteomics strategies to preserve even the most labile nbPTMs. Similarly to what we have achieved for mono-ADPr, we aim to unearth and biologically characterize elusive nbPTMs, starting with the new nbPTMs we have already discovered. The expectation is that by the end of the project, we will have identified a number of important nbPTMs, providing deeper insights into their diverse roles and mechanisms in cellular processes. This expansion of knowledge on nbPTMs is anticipated to have profound implications for various fields, from fundamental biology to translational research, potentially uncovering new pathways for therapeutic interventions and biomarker discovery.
Regarding the future milestones of the 'nbPTMs' project, by building upon the early successes our focus is set on extending our innovative strategy to encompass a broader range of overlooked or yet-to-be-discovered nbPTMs. A key aspect of this endeavor involves development of nbPTM-tailored proteomics strategies to preserve even the most labile nbPTMs. Similarly to what we have achieved for mono-ADPr, we aim to unearth and biologically characterize elusive nbPTMs, starting with the new nbPTMs we have already discovered. The expectation is that by the end of the project, we will have identified a number of important nbPTMs, providing deeper insights into their diverse roles and mechanisms in cellular processes. This expansion of knowledge on nbPTMs is anticipated to have profound implications for various fields, from fundamental biology to translational research, potentially uncovering new pathways for therapeutic interventions and biomarker discovery.