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Peptidyl arginine deiminases: detection and functional analysis of protein citrullination.

ABSTRACT: Citrullination is a post-translational modification of arginine that is catalyzed by the protein arginine deiminases (PADs). Abnormal citrullination is observed in many autoimmune diseases and cancers. Anti-citrullinated protein antibodies (ACPA) are hallmarks of RA and used as diagnostic markers for disease diagnosis. Even though citrullination is associated with many different pathologies, its role remains unclear due to the challenges associated with the detection of citrullinated proteins since the mass change is only 0.984?Da. Moreover, the functional effects of protein citrullination remain mostly unknown. Herein, we discuss a brief overview of PAD structure and function, recent advances in the detection of citrullinated proteins in complex biological systems and the functional consequences of protein citrullination.

SUBMITTER: Tilvawala R

PROVIDER: S-EPMC6717693 | biostudies-literature | 2019 Dec

REPOSITORIES: biostudies-literature

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Peptidyl arginine deiminases: detection and functional analysis of protein citrullination.

Tilvawala Ronak R Thompson Paul R PR

Current opinion in structural biology 20190301

Citrullination is a post-translational modification of arginine that is catalyzed by the protein arginine deiminases (PADs). Abnormal citrullination is observed in many autoimmune diseases and cancers. Anti-citrullinated protein antibodies (ACPA) are hallmarks of RA and used as diagnostic markers for disease diagnosis. Even though citrullination is associated with many different pathologies, its role remains unclear due to the challenges associated with the detection of citrullinated proteins si ...[more]

PMID: 30833201

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Project description:Proteins are well-known to undergo a variety of post-translational modifications (PTMs). One such PTM is citrullination, an arginine modification that is catalyzed by a group of hydrolases called protein arginine deiminases (PADs). Hundreds of proteins are known to be citrullinated and hypercitrullination is associated with autoimmune diseases including rheumatoid arthritis (RA), lupus, ulcerative colitis (UC), Alzheimer's disease, multiple sclerosis (MS), and certain cancers. In this Account, we summarize our efforts to understand the structure and mechanism of the PADs and to develop small molecule chemical probes of protein citrullination. PAD activity is highly regulated by calcium. Structural studies with PAD2 revealed that calcium-binding occurs in a stepwise fashion and induces a series of dramatic conformational changes to form a catalytically competent active site. These studies also identified the presence of a calcium-switch that controls the overall calcium-dependence and a gatekeeper residue that shields the active site in the absence of calcium. Using biochemical and site-directed mutagenesis studies, we identified the key residues (two aspartates, a cysteine, and a histidine) responsible for catalysis and proposed a general mechanism of citrullination. Although all PADs follow this mechanism, substrate binding to the thiolate or thiol form of the enzyme varies for different isozymes. Substrate-specificity studies revealed that PADs 1-4 prefer peptidyl-arginine over free arginine and certain citrullination sites on a peptide substrate. Using high-throughput screening and activity-based protein profiling (ABPP), we identified several reversible (streptomycin, minocycline, and chlorotetracycline) and irreversible (streptonigrin, NSC 95397) PAD-inhibitors. Screening of a DNA-encoded library and lead-optimization led to the development of GSK199 and GSK484 as highly potent PAD4-selective inhibitors. Furthermore, use of an electrophilic, cysteine-targeted haloacetamidine warhead to mimic the guanidinium group in arginine afforded several mechanism-based pan-PAD-inhibitors including Cl-amidine and BB-Cl-amidine. These compounds are highly efficacious in various animal models, including those mimicking RA, UC, and lupus. Structure-activity relationships identified numerous covalent PAD-inhibitors with different bioavailability, in vivo stability, and isozyme-selectivity (PAD1-selective: D-Cl-amidine; PAD2-selective: compounds 16-20; PAD3-selective: Cl4-amidine; and PAD4-selective: TDFA). Finally, this Account describes the development of PAD-targeted and citrulline-specific chemical probes. While PAD-targeted probes were utilized for identifying off-targets and developing high-throughput inhibitor screening platforms, citrulline-specific probes enabled the proteomic identification of novel diagnostic biomarkers of hypercitrullination-related autoimmune diseases.

Dataset Information

Peptidyl arginine deiminases: detection and functional analysis of protein citrullination.

Publications

Peptidyl arginine deiminases: detection and functional analysis of protein citrullination.

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