Chemical Biology of Protein Citrullination
In this seminar, I’ll discuss my postdoctoral research on protein citrullination, a post-translational modification associated with multiple autoimmune disorders, and my research proposals.
Protein citrullination by protein arginine deiminases (PADs – PAD1, 2, 3 and 4) plays pivotal roles in several physiological processes, such as epigenetic regulation of gene expression, neutrophil extracellular trap (NET) formation and DNA-damage induced apoptosis. However, strong links between aberrant protein citrullination and multiple autoimmune disorders as well as certain forms of cancer have established PADs as potential therapeutic targets. As PADs are cysteine hydrolases and contain a cysteine residue in the active site, cysteine-targeted haloacetamidine warheads installed on suitable small molecule scaffolds are generally used to irreversibly inhibit PADs. Since photoactivation of small molecule drugs at the target tissue can significantly reduce their off-target toxicity, we incorporated an azobenzene photoswitch that undergoes trans-cis isomerization in the presence of light in a known PAD inhibitor scaffold, BB-Cl-amidine. This led to the development of a PAD2 inhibitor that exhibits 10-fold higher potency upon irradiation with 350 nm light. This inhibitor can be activated in HEK293TPAD2 cells with light for the inhibition of histone H3 citrullination.
Citrullination has remarkable effects on the structure and activity of proteins. For example, citrullination of serine protease inhibitors (SERPINs) and nicotinamide N-methyl transferase (NNMT) dramatically abolishes their activity. Interestingly, autocitrullination of PAD4 is proposed to regulate its enzymatic activity. Although numerous proteins are known to be citrullinated at various positions, the downstream implications of citrullination at each of these positions in a given protein remain elusive. To aid this, we developed, for the first time, a photocaged-citrulline for site-specific incorporation into proteins and subsequent conversion into citrulline (Cit) with light. Using amber codon suppression technique and an engineered leucyl-tRNA synthetase (LeuRS)/tRNALeu pair, we incorporated citrulline into enhanced green fluorescent protein (GFP) at 39 position and into PAD4 at two known autocitrullination sites, 372 and 374. Using various enzyme kinetic assays, we have shown that the R372Cit and R374Cit mutants of PAD4 are 292- and 10-fold, respectively, less active than the wild-type enzyme, indicating that citrullination has remarkable effect on the activity of PAD4.
In addition to the aforementioned topics, I’ll also discuss my future plans for research on the covalent modification of proteins and photochemical control of the bioactivity of small molecules.
1. S. Mondal, P. R. Thompson, Acc. Chem. Res. 2019, 52, 818.
2. S. Mondal, S. S. Parelkar, M. Nagar, P. R. Thompson, ACS Chem. Biol. 2018, 13, 1057.
3. S. Sen, S. Mondal, L. Zheng, A. Salinger, W. Fast, E. Weerapana, P. R. Thompson ACS Chem. Biol., 2019, 14, 613-618.
4. S. Mondal,† S. Wang,† Y. Zheng, A. Chatterjee, P. R. Thompson, Unpublished results