Periodic Reporting for period 4 - inCITe (Seeing Citrulline: A Molecular Toolbox for Peptidyl Arginine Deiminases)
Reporting period: 2023-07-01 to 2024-08-31
Roughly 1% of the world’s population is affected by rheumatoid arthritis (RA); a devastating autoimmune disease causing cartilage destruction and bone erosion. Recent evidences suggest that dysregulation of Peptidyl Arginine Deiminase (PAD) levels are associated with the onset of the disease, leading to the production of antibodies targeting the citrullinated neoepitopes. The exact role of each of the PAD isotypes in these pathological processes is unknown and fundamental questions on the intracellular activation mechanism and substrate specificity remain unanswered. Moreover, isoform specific and high affinity enzyme inhibitors are lacking thereby not only hampering fundamental research towards each PAD isotype, but also excluding PAD as a potential therapeutic target for these diseases.
In the inCITe action we aimed to generating a chemical platform for next-generation drug discovery and therapeutic approaches for citrulline-related autoimmune diseases (e.g. rheumatoid arthritis; RA) as well as developing highly innovative chemical biology tools to study Peptidyl Arginine Deiminases (PAD) and citrullinated protein biology in neutrophils. The action covers 5 workpackages in chemistry and biology. More specifically, we aim to address five major unanswered but fundamentally important questions in the field namely 1) What are the intracellular interacting proteins that can activate PAD, 2) What are the intracellular PAD substrates, 3) What is the role of PAD in NETosis, 4) What is the role of (extracellular) PAD on disease progression and 5) Is citrullination a reversible post translational modification?
The workpackages described in this ambitious and highly interdisciplinary proposal deliver high-end molecules and methods that can be used to answer fundamental (conflicting) questions on citrullination and PAD biology. All WPs are conceptually novel and beyond the state of the art. Besides the fundamental advances, I expect to deliver molecular leads and PAD-targeting strategies for RA treatment to improve patients wellbeing.
In the inCITe action we aimed to generating a chemical platform for next-generation drug discovery and therapeutic approaches for citrulline-related autoimmune diseases (e.g. rheumatoid arthritis; RA) as well as developing highly innovative chemical biology tools to study Peptidyl Arginine Deiminases (PAD) and citrullinated protein biology in neutrophils. The action covers 5 workpackages in chemistry and biology. More specifically, we aim to address five major unanswered but fundamentally important questions in the field namely 1) What are the intracellular interacting proteins that can activate PAD, 2) What are the intracellular PAD substrates, 3) What is the role of PAD in NETosis, 4) What is the role of (extracellular) PAD on disease progression and 5) Is citrullination a reversible post translational modification?
The workpackages described in this ambitious and highly interdisciplinary proposal deliver high-end molecules and methods that can be used to answer fundamental (conflicting) questions on citrullination and PAD biology. All WPs are conceptually novel and beyond the state of the art. Besides the fundamental advances, I expect to deliver molecular leads and PAD-targeting strategies for RA treatment to improve patients wellbeing.
This proposal aims to develop innovative chemical biology and molecular tools to study PAD functioning and protein citrullination in health and disease. It reflects my interdisciplinary expertise and my interest in chemical immunology, as well as my ambition to enhance patient well-being. Specifically, I aim to: 1) Identify unknown PAD modulators, 2) Discover PAD substrates, 3) Develop selective, high-affinity PAD inhibitors using enzyme-templated inhibitor evolution as a novel lead discovery strategy, 4) Explore multifunctional targeted PAD ‘nanosponges’ as an advanced avidity-based nanomedicine approach, and 5) Investigate novel citrulline ‘eraser’ enzymes through innovative chemical biology strategies.
To address these goals, we constructed PAD fusion proteins with engineered enzymes for proximity-dependent labeling (PDL) of proteins in living cells. The PDL methodology has advanced significantly in recent years. We further refined PDL technology using hNAT1, an enzyme developed in our lab that converts hydroxamic acid substrates into reactive electrophilic nitrenium ions, which react with nearby nucleophilic residues. A key advantage of hNAT1 in PDL experiments is that it does not require additional activation triggers (e.g. hydrogen peroxide) and allows flexibility in the reactive handle used. We generated PAD2-hNAT1 and PAD4-hNAT1 constructs, achieving good localization with commercially available PAD antibodies. We are finalizing pulldown experiments to obtain proteomic data on PAD2 and PAD4 interacting proteins, which we will compare to published datasets.
A significant breakthrough in the inCITe action is the development of a novel metabolic residue displacement technology, THRONCAT. This method replaces threonine residues with unnatural analogues, such as β-ethynyl serine (published in Nature Communications, 2023), during protein synthesis. This technology enables profiling PAD lifetime dynamics in living cells, providing insights into its regulation and function. We are preparing additional non-natural threonine analogues containing photocrosslinkers to identify PAD interacting proteins and substrates. Our work on THRONCAT has been presented at several conferences and has spurred new collaborations in the field.
To discover novel probes and inhibitors for PAD, we introduced 'turn-on' fluorophores to existing chloroamidine activity-based probes. These probes enable imaging of active PAD during neutrophil extracellular trap (NET) formation in primary neutrophils. Our profiling has provided new insights into PAD biology during NETosis and demonstrated that high probe concentrations inhibit this process. A manuscript on these findings is in preparation.
We envisioned creating an elastin-like peptide (ELP)-based nanoparticle platform to target and image PAD activity. Large-scale bacterial expression successfully yielded ELP monomers, but the resulting nanoparticles required stabilization. We established a crosslinking and purification system to produce highly stable, well-defined particles that can be modified with targeting ligands. This toolbox now facilitates a plug-and-play approach for targeting, imaging, and drug delivery applications. We are actively seeking further funding to expand this work.
Finally, we synthesized citrullinated histone probes containing photocrosslinkers to identify potential citrulline-modulating enzymes. Protocols for pulldown experiments are in place to obtain sufficient material for proteomic analysis, and these experiments are ongoing.
To address these goals, we constructed PAD fusion proteins with engineered enzymes for proximity-dependent labeling (PDL) of proteins in living cells. The PDL methodology has advanced significantly in recent years. We further refined PDL technology using hNAT1, an enzyme developed in our lab that converts hydroxamic acid substrates into reactive electrophilic nitrenium ions, which react with nearby nucleophilic residues. A key advantage of hNAT1 in PDL experiments is that it does not require additional activation triggers (e.g. hydrogen peroxide) and allows flexibility in the reactive handle used. We generated PAD2-hNAT1 and PAD4-hNAT1 constructs, achieving good localization with commercially available PAD antibodies. We are finalizing pulldown experiments to obtain proteomic data on PAD2 and PAD4 interacting proteins, which we will compare to published datasets.
A significant breakthrough in the inCITe action is the development of a novel metabolic residue displacement technology, THRONCAT. This method replaces threonine residues with unnatural analogues, such as β-ethynyl serine (published in Nature Communications, 2023), during protein synthesis. This technology enables profiling PAD lifetime dynamics in living cells, providing insights into its regulation and function. We are preparing additional non-natural threonine analogues containing photocrosslinkers to identify PAD interacting proteins and substrates. Our work on THRONCAT has been presented at several conferences and has spurred new collaborations in the field.
To discover novel probes and inhibitors for PAD, we introduced 'turn-on' fluorophores to existing chloroamidine activity-based probes. These probes enable imaging of active PAD during neutrophil extracellular trap (NET) formation in primary neutrophils. Our profiling has provided new insights into PAD biology during NETosis and demonstrated that high probe concentrations inhibit this process. A manuscript on these findings is in preparation.
We envisioned creating an elastin-like peptide (ELP)-based nanoparticle platform to target and image PAD activity. Large-scale bacterial expression successfully yielded ELP monomers, but the resulting nanoparticles required stabilization. We established a crosslinking and purification system to produce highly stable, well-defined particles that can be modified with targeting ligands. This toolbox now facilitates a plug-and-play approach for targeting, imaging, and drug delivery applications. We are actively seeking further funding to expand this work.
Finally, we synthesized citrullinated histone probes containing photocrosslinkers to identify potential citrulline-modulating enzymes. Protocols for pulldown experiments are in place to obtain sufficient material for proteomic analysis, and these experiments are ongoing.
On the basis of many autoimmune diseases, including RA, lies protein citrullination; an emerging and yet highly understudied post translational modification. We are only at the beginning to understand the role in epigenetics and chromatin organization. Moreover, a continuous number of literature reports appear on citrulline-dependent protein activity and protein-protein interactions, suggesting that citrullination is more specific and a more common post translational modification than currently anticipated. It is not surprising that PAD-mediated citrullination has not only been linked to RA but also to various other autoimmune diseases such as multiple sclerosis, Alzheimer’s disease and lupus as well as several types of cancer. Understanding the exact role of each PAD isotype in health and disease is fundamentally important and provides openings to consider PAD as therapeutic target. In this action, we have created a toolset and molecular probes to gain better insight on the intracellular role of PAD and how these enzymes and citrulline biology are associated with autoimmune diseases, such as RA. The work has offered a strong foundation for further research that we will continue in my laboratory.