Chemical Proteomic Platform To Identify Citrullinated Proteins.

Anti-citrullinated protein antibodies (ACPAs) are a hallmark of rheumatoid arthritis (RA) and are routinely used for disease diagnosis. Protein citrullination is also increased in cancer and other autoimmune disorders, suggesting that citrullinated proteins may serve as biomarkers for diseases beyond RA. To identify these citrullinated proteins, we developed biotin-conjugated phenylglyoxal (biotin-PG). Using this probe and our platform technology, we identified >50 intracellular citrullinated proteins. More than 20 of these are involved in RNA splicing, suggesting, for the first time, that citrullination modulates RNA biology. Overall, this chemical proteomic platform will play a key role in furthering our understanding of protein citrullination in rheumatoid arthritis and potentially a wider spectrum of inflammatory diseases.

Mechanistic studies of protein arginine deiminase 2: evidence for a substrate-assisted mechanism.

Citrullination, which is catalyzed by protein arginine deiminases (PADs 1-4 and 6), is a post-translational modification (PTM) that effectively neutralizes the positive charge of a guanidinium group by its replacement with a neutral urea. Given the sequence similarity of PAD2 across mammalian species and the genomic organization of the PAD2 gene, PAD2 is predicted to be the ancestral homologue of the PADs. Although PAD2 has long been known to play a role in myelination, it has only recently been linked to other cellular processes, including gene transcription and macrophage extracellular trap formation. For example, PAD2 deiminates histone H3 at R26, and this PTM leads to the increased transcription of more than 200 genes under the control of the estrogen receptor. Given that our understanding of PAD2 biology remains incomplete, we initiated mechanistic studies on this enzyme to aid the development of PAD2-specific inhibitors. Herein, we report that the substrate specificity and calcium dependence of PAD2 are similar to those of PADs 1, 3, and 4. However, unlike those isozymes, PAD2 appears to use a substrate-assisted mechanism of catalysis in which the positively charged substrate guanidinium depresses the pKa of the nucleophilic cysteine. By contrast, PADs 1, 3, and 4 use a reverse-protonation mechanism. These mechanistic differences will aid the development of isozyme-specific inhibitors.

A FluoPol-ABPP PAD2 high-throughput screen identifies the first calcium site inhibitor targeting the PADs.

The protein arginine deiminases (PADs) catalyze the post-translational hydrolysis of peptidyl-arginine to form peptidyl-citrulline in a process termed deimination or citrullination. PADs likely play a role in the progression of a range of disease states because dysregulated PAD activity is observed in a host of inflammatory diseases and cancer. For example, recent studies have shown that PAD2 activates ERα target gene expression in breast cancer cells by citrullinating histone H3 at ER target promoters. To date, all known PAD inhibitors bind directly to the enzyme active site. PADs, however, also require calcium ions to drive a conformational change between the inactive apo-state and the fully active calcium bound holoenzyme, suggesting that it would be possible to identify inhibitors that bind the apoenzyme and prevent this conformational change. As such, we set out to develop a screen that can identify PAD2 inhibitors that bind to either the apo or calcium bound form of PAD2. Herein, we provide definitive proof of concept for this approach and report the first PAD inhibitor, ruthenium red (Ki of 17 µM), to preferentially bind the apoenzyme.

Inhibiting AMPylation: a novel screen to identify the first small molecule inhibitors of protein AMPylation.

Enzymatic transfer of the AMP portion of ATP to substrate proteins has recently been described as an essential mechanism of bacterial infection for several pathogens. The first AMPylator to be discovered, VopS from Vibrio parahemolyticus, catalyzes the transfer of AMP onto the host GTPases Cdc42 and Rac1. Modification of these proteins disrupts downstream signaling events, contributing to cell rounding and apoptosis, and recent studies have suggested that blocking AMPylation may be an effective route to stop infection. To date, however, no small molecule inhibitors have been discovered for any of the AMPylators. Therefore, we developed a fluorescence-polarization-based high-throughput screening assay and used it to discover the first inhibitors of protein AMPylation. Herein we report the discovery of the first small molecule VopS inhibitors (e.g., calmidazolium, GW7647, and MK886) with Ki's ranging from 6 to 50 µM and upward of 30-fold selectivity versus HYPE, the only known human AMPylator.

Probing adenylation: using a fluorescently labelled ATP probe to directly label and immunoprecipitate VopS substrates.

The bacterial effector VopS from Vibrio parahaemolyticus modifies host Rho GTPases to prevent downstream signalling, which leads to cell rounding and eventually apoptosis. While previous studies have used [α-(32)P] ATP for studying this enzyme, we sought to develop a non-radioactive chemical probe of VopS function. To guide these studies, the kinetic parameters were determined for a variety of nucleotides and the results indicated that the C6 position of adenosine was amenable to modification. Since Fl-ATP is a commercially available ATP analogue that is fluorescently tagged at the C6 position, we tested it as a VopS substrate, and the results show that VopS uses Fl-ATP to label Cdc42 in vitro and in MCF7 whole cell extracts. The utility of this probe was further demonstrated by immunoprecipitating Fl-ATP labeled Cdc42 as well as several novel substrate proteins. The proteins, which were identified by LC-MS/MS, include the small GTPases Rac1 and Cdc42 as well as several proteins that are potential VopS substrates and may be important for V. parahaemolyticus pathology. In total, these studies identify Fl-ATP as a valuable chemical probe of protein AMPylation.

Identification and characterization of the carbohydrate ligands recognized by pertussis toxin via a glycan microarray and surface plasmon resonance.

Binding of pertussis toxin (PTx) was examined by a glycan microarray; 53 positive hits fell into four general groups. One group represents sialylated biantennary compounds with an N-glycan core terminating in alpha2-6-linked sialic acid. The second group consists of multiantennary compounds with a canonical N-glycan core, but lacking terminal sialic acids, which represents a departure from the previous understanding of PTx binding to N-glycans. The third group consists of Neu5Acalpha2-3(lactose or N-acetyllactosamine) forms that lack the branched mannose core found in N-glycans; thus, their presentation is more similar to that of O-linked glycans and glycolipids. The fourth group of compounds consists of Neu5Acalpha2-8Neu5Acalpha2-8Neu5Ac, which is seen in the c series gangliosides and some N-glycans. Quantitative analysis by surface plasmon resonance of the relative affinities of PTx for terminal Neu5Acalpha2-3 versus Neu5Acalpha2-6, as well as the affinities for the trisaccharide Neu5Acalpha2-8Neu5Acalpha2-8Neu5Ac versus disaccharide, revealed identical global affinities, even though the amount of bound glycan varied by 4-5-fold. These studies suggest that the conformational space occupied by a glycan can play an important role in binding, independent of affinity. Characterization of N-terminal and C-terminal binding sites on the S2 and S3 subunits by mutational analysis revealed that binding to all sialylated compounds was mediated by the C-terminal binding sites, and binding to nonsialylated N-linked glycans is mediated by the N-terminal sites present on both the S2 and S3 subunits. A detailed understanding of the glycans recognized by pertussis toxin is essential to understanding which cells are targeted in clinical disease.

Detection of intact influenza viruses using biotinylated biantennary S-sialosides.

We report the modular synthesis of robust, biotinylated biantennary sialylglycoconjugates and their ability to differentiate between two type A influenza strains. This is the first demonstration of glycoconjugate-based discriminatory capture and detection of two strains of intact influenza virus, in the presence of the innate enzymatic activity of viral neuraminidases. We also demonstrate a "carboassay" using glycoconjugates as capture and reporter elements, which therefore, does not require antibodies. The capture of intact influenza viruses is of potential benefit for clinical diagnostics.