Trigger...Generate an experimental model

Immunizing mice with citrullinated human fibrinogen (CHF) induces the production of anti-CHF antibodies, citrulline-specific T cell activation, and RA-like pathophysiology. By promoting the production of antibodies that recognize citrullinated epitopes, this model is thought to more closely approximate the pathophysiology of the human disease compared to other mouse models of arthritis, including collagen-induced arthritis.

Detect...Identify and test

In the clinic, detection is important for early diagnosis and course of therapy. In animal models, detection is used to correlate degree of inflammation, PAD expression, citrullination, and autoantibody production with experimental variables. A fluorescent probe that detects any citrullinated protein and an antibody that detects citrullinated fibrinogen are available in the catalog. Also available is an assay to help accurately analyze the anti-citrulline response induced in mice by immunization with CHF by distinguishing it from an antibody response to unmodified human fibrinogen. An active recombinant PAD4 can be used for functional studies of the enzyme, and an additional assay identifies autoantibodies generated against PAD4 itself.

Inhibit...Develop therapeutics and screen for efficacy

Reduction of citrullination is one therapeutic approach to ameliorate RA. However, cautionary considerations must be taken with this approach since the physiological roles of PAD enzymes also involve apoptosis, formation of neutrophil extracellular traps, altering chemokine function, and (particularly for PAD4) regulating DNA transcription. Pan-PAD inhibitors and a potent PAD4-specific inhibitor are available in the catalog, and a screening assay has been developed to identify candidate inhibitors specific to PAD4.

PAD4 is the only PAD enzyme with a nuclear localization signal. Thus, its substrates also include histones, whose post-translational modifications play a significant role in regulating chromatin structure and gene expression. Recent studies have found that histone citrullination counteracts the effect of histone arginine methylation and functions as a repressive marker to turn off gene expression. PAD4 is also essential for chromatin decondensation during neutrophil extracellular trap (NET) formation in the innate immune process of NETosis. Roles for PAD4 in tumor progression, tumor-associated inflammation, neurological disorders, as well as regulation of stem cell pluripotency have also been suggested.

Cayman offers the following products to support research on these additional roles of PAD4-mediated citrullination:

Learn More: Background Reading on PAD4

Protein Arginine Deiminase 4 (PAD4) catalyzes the post-translational modification of arginine to citrulline within peptides or large proteins.¹ By removal of the terminal guanidine group, arginine loses a positive charge, resulting in the disruption of ionic interactions with negatively charged macromolecules, including DNA. This is thought to be the mechanism whereby PAD4 promotes chromatin decondensation. This process must be tightly regulated. Unrestrained PAD4 activation leading to widespread arginine deimination could disrupt intramolecular interactions within individual proteins, promoting protein unfolding and loss of function and increasing susceptibility to proteolytic degradation. Furthermore, by transforming positively charged arginine to a charge neutral citrulline, PAD4 alters the ability of processed peptides containing citrulline to bind to the major histocompatibility Class II proteins on antigen presenting cells.² This effect could result in the misidentification of "self" peptides as "non-self" by T lymphocytes, leading to a T cell-dependent autoimmune response directed towards the citrullinated protein.

The normal function of PAD in the body is to citrullinate proteins to change their function. Citrullination occurs as a post-translational modification in which the amino acid arginine is deiminated to a non-standard residue, citrulline, in the presence of calcium. The end gain of this process results in a positively charged arginine molecule changed to a neutral citrulline molecule whose intramolecular interactions are dissolved, allowing the protein to unfold. The modified protein is now more susceptible to proteolytic cleavage by other enzymes and/or the formation of new intermolecular interactions.

There are five different isotypes of PAD, each with a specific expression pattern and target protein. All are dependent upon calcium for their enzymatic activity.

Of these five isotypes, PAD2 and PAD4 are most likely pathogenic in RA, a systemic autoimmune disease characterized by chronic inflammation of the joints that results in bone erosion and progressive joint destruction. PAD2-positive macrophages have been found in inflamed synovium and one of its target proteins is vimentin, a known RA autoantigen (see below). PAD2 and citrullinated proteins are also elevated in the lungs of smokers.³ As such, smoking is strongly associated with RA, and thought to be an environmental factor in the development of the autoimmune response. An antigen response mounted against citrullinated proteins can be detected long before the physical presentation of RA symptoms. Most attention in RA has focused on PAD4, which is found in monocytes and macrophages in hematological and rheumatoid synovial tissue and is genetically associated with development of RA in certain ethnic populations.

[Note: The collection of PAD products in the Cayman catalog is centered on PAD4, which is studied mainly for its role in RA but also for its role in histone modification and gene regulation. PAD2 (human recombinant) will soon be available. PAD1 (human recombinant) (Item No. 10784) , which is thought to have a role in skin hydration, is also available in the Cayman catalog, though its function is outside the realm of arthritis research.]

In RA, proteins modified by PAD4 and enzymes like it, generate an immune response in the inflamed tissues. T cells are activated and B cells produce autoantibodies specifically against citrullinated peptides (and not their arginine counterparts). The accumulation of these complexes further drives inflammation, ultimately leading to clinical manifestations of RA. Prime immune targets (antigens) related to RA include citrullinated filaggrin, vimentin, and fibrin. Detection of antibodies that recognize a cyclic citrullinated peptide (CCP) is now used as a specific biomarker in diagnosing RA even before its pathogenesis is clinically apparent.

PAD4 autoimmune activity (that is, citrullination-triggered production of autoantibodies) perpetuates the ongoing bone destruction in RA. The more active the enzyme is, the worse the disease. Currently, the factors that activate PAD4 are not well understood. Intracellular calcium is a required cofactor for PAD activity. In vitro cells expressing PAD enzymes will not produce citrulline-modified antigens unless an exogenous stimulus capable of markedly increasing calcium is applied. Recently, a unique autoantibody has been identified that binds to PAD4 and increases its catalytic activity.⁴ This PAD4-activating antibody occupies a key calcium-binding region and most likely substitutes for some of the calcium that is usually required to modify proteins. This ability to activate an enzyme that itself generates citrullinated autoantigens creates a significant feed-forward loop that would further drive the erosive consequences of the disease. Furthermore, PAD4 itself can undergo autocitrullination at several sites, which inhibits its enzymatic activity and may play an important role in regulating citrullination in cells.⁵

References

1. Shirai, H., et al. Trends Biochem. Sci. 26(8), 465-468 (2001).

2. Hill, J.A., et al. J. Immunol. 171, 538-541 (2003).

3. Makrygiannakis, D., et al. Ann. Rheum. Dis. 67(10), 1488-1492 (2008).

4. Darrah, E., et al. Sci. Transl. Med. 5(186), 186ra65 (2013).

5. Andrade, F., et al. Arthritis Rheum. 62(6), 1630-1640 (2010).