Citrullination: Its Role in Health and Disease

Proteins in our bodies are constantly altered to perform their designated functions. One such alteration is citrullination, a chemical modification that occurs after a protein has been built. This process involves the conversion of the amino acid arginine into another non-standard amino acid, citrulline. Understanding citrullination is providing new insights into normal biological activities and the development of several human diseases, reshaping how researchers view the dynamic nature of proteins.

The Molecular Mechanism of Citrullination

Citrullination is a precise biochemical reaction catalyzed by a family of enzymes known as Peptidylarginine Deiminases, or PADs. The process targets arginine, an amino acid that carries a positive electrical charge at physiological pH. These enzymes hydrolyze a part of the arginine molecule called the guanidinium group, replacing it with a ureido group to form citrulline.

This chemical transformation neutralizes the positive charge of the original arginine residue. The loss of charge can alter the protein’s three-dimensional structure, or folding, affecting how it interacts with other molecules like DNA or other proteins. The PAD enzyme family in mammals consists of five main types, including PAD1, PAD2, PAD3, and PAD4, each with distinct functions.

PAD enzyme activity requires high concentrations of calcium ions. In healthy cells, intracellular calcium levels are kept very low, meaning PAD enzymes are normally inactive. They are only switched on during events that cause a significant, localized increase in calcium, such as programmed cell death or in response to specific cellular signals. This tight regulation ensures that citrullination only occurs when and where it is needed.

Essential Roles of Citrullination in Health

Citrullination is a regulated process that supports several bodily functions, including the formation and maintenance of the skin. In the epidermis, the outermost layer of skin, PAD enzymes like PAD1 and PAD3 citrullinate structural proteins such as keratin and filaggrin. This modification is part of the terminal differentiation of skin cells, helping to create a robust and protective barrier against the environment.

The structural integrity of hair follicles also relies on this modification. PAD3 is active in hair follicles, where it targets a protein called trichohyalin. The citrullination of trichohyalin mechanically strengthens the inner root sheath of the hair, contributing to healthy hair growth and structure.

In the nervous system, citrullination contributes to forming the myelin sheath, the insulating layer surrounding nerve fibers that enables rapid electrical signaling. The enzyme PAD2 modifies myelin basic protein (MBP), a major component of this sheath. Appropriate levels of MBP citrullination are needed for the compaction and stability of the myelin layer.

Citrullination also helps regulate gene expression. Histones are proteins that act like spools for DNA, and modifications to them control which genes are active. PAD2 and PAD4 can enter the cell nucleus and citrullinate histones, such as histone H3, which alters chromatin structure and influences the accessibility of DNA for gene transcription.

The innate immune system uses citrullination as a defense mechanism. When neutrophils encounter bacteria, they can undergo a form of cell death called NETosis. During this process, PAD4 hypercitrullinates histones, causing chromatin to decondense and be expelled from the cell as a web-like Neutrophil Extracellular Trap (NET). These NETs physically trap and kill invading microbes, helping to contain infections.

Citrullination’s Link to Disease Pathogenesis

While beneficial in controlled amounts, dysregulated citrullination is linked to the development of diseases, particularly autoimmune disorders. In these conditions, the immune system mistakenly attacks the body’s own tissues. Citrullinated proteins can become the target of this attack because the modification can make a “self” protein appear “foreign” to immune cells, breaking the body’s tolerance.

A prominent example is rheumatoid arthritis (RA), a chronic inflammatory disease affecting the joints. In the inflamed synovium of RA patients, proteins such as fibrin and vimentin become citrullinated. The immune system then generates anti-citrullinated protein antibodies (ACPAs) that recognize these modified proteins. The binding of ACPAs to citrullinated proteins in the joints forms immune complexes that perpetuate inflammation and contribute to tissue destruction.

The connection to autoimmunity extends beyond RA. In multiple sclerosis (MS), abnormal citrullination of myelin basic protein (MBP) occurs in the central nervous system. While some citrullination of MBP is normal, excessive modification by PAD2 and PAD4 is associated with the destabilization and demyelination of the myelin sheath. This process exposes the modified MBP to the immune system, triggering an autoimmune response against the nerve’s protective covering.

Other autoimmune conditions are also associated with this process. Autoantibodies targeting citrullinated proteins are sometimes found in patients with systemic lupus erythematosus and Sjögren’s syndrome. In these cases, inflammation and cell death can increase the availability of PAD enzymes and citrullinated proteins, creating a cycle that fuels the autoimmune response.

Beyond autoimmunity, aberrant citrullination is implicated in other conditions. In some cancers, PAD enzymes are overexpressed and may promote tumor growth by modifying proteins involved in cell signaling. There is also evidence linking citrullination to neurodegenerative diseases like Alzheimer’s, where citrullinated proteins are found in the cellular debris associated with neuronal damage.

Diagnostic and Therapeutic Perspectives

The discovery of ACPAs in rheumatoid arthritis has led to practical applications in diagnostics. The test for antibodies to cyclic citrullinated peptides (anti-CCP) is a standard blood test used by clinicians. It is highly specific for RA, with a specificity of 88–96%, helping to distinguish it from other forms of arthritis.

The presence of these antibodies is also prognostic. Patients who test positive for anti-CCP antibodies often have a more aggressive disease course with a higher likelihood of joint erosion. This information allows doctors to plan intensive therapeutic strategies early. The anti-CCP test can detect these antibodies long before symptoms appear, opening a window for early intervention.

From a therapeutic standpoint, PAD enzymes have become attractive targets for drug development. The goal is to create molecules, known as PAD inhibitors, that block these enzymes to reduce the abnormal citrullination that drives disease. Several pan-PAD inhibitors, which block multiple PAD isozymes, and more selective inhibitors targeting specific enzymes like PAD2 or PAD4, have been developed. Compounds like Cl-amidine are examples of such inhibitors that have shown effectiveness in animal models.

These PAD inhibitors may be useful for treating a range of conditions. In autoimmune diseases like RA, lupus, and MS, blocking PADs could prevent the formation of citrullinated autoantigens that trigger the immune response. There is also interest in their potential for treating certain cancers where PAD activity is elevated. This is an active area of research to develop inhibitors safe for clinical use.