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.
