Novel inhibitors of protein arginine deiminase with potential activity in multiple sclerosis animal model.

In multiple sclerosis (MS), myelin basic protein (MBP), critical for the maintenance of myelin compaction and protecting against degradation, is known to contain concentrations of the noncoded amino acid, "citrulline", in abnormal proportions. Peptidyl arginine deiminase (PAD) catalyzes the post-translational citrullination of proteins via the deimination of Arg residues. In the central nervous system, specifically PAD2 and PAD4, are the enzymes responsible for the citrullination.

Interrogation of the Active Sites of Protein Arginine Deiminases (PAD1, -2, and -4) Using Designer Probes.

Protein arginine deiminases (PADs) are involved in a number of cellular pathways, and they catalyze the transformation of peptidyl arginine residue into a citrulline as part of post-translational modifications. To understand ligand preferences, a group of probe molecules were investigated against PAD1, PAD2, and PAD4. These probe molecules carried a well-known covalent modifier of the catalytic cysteine residue, 2-chloroacetamidine moiety, which was tethered to an α-amino acid via a carbon linker.

Inhibition of peptidyl-arginine deiminases reverses protein-hypercitrullination and disease in mouse models of multiple sclerosis.

Multiple sclerosis (MS) is the most common CNS-demyelinating disease of humans, showing clinical and pathological heterogeneity and a general resistance to therapy. We first discovered that abnormal myelin hypercitrullination, even in normal-appearing white matter, by peptidylarginine deiminases (PADs) correlates strongly with disease severity and might have an important role in MS progression. Hypercitrullination is known to promote focal demyelination through reduced myelin compaction.

Myelin basic protein undergoes a broader range of modifications in mammals than in lower vertebrates.

Myelin basic protein (MBP) is an important component of the myelin sheath surrounding neurons, and it is directly affected in demyelinating diseases. MBP contains a relatively large number of post-translational modifications (PTMs), which have been reported to play a role in multiple sclerosis, while MBPs from lower vertebrates have been reported to be incapable of inducing multiple sclerosis or allergic encephalitis. This study reveals the extent of differences in PTM patterns for mammalian and nonmammalian MBPs.

Peptidylarginine deiminase 2 (PAD2) overexpression in transgenic mice leads to myelin loss in the central nervous system.

Demyelination in the central nervous system is the hallmark feature in multiple sclerosis (MS). The mechanism resulting in destabilization of myelin is a complex multi-faceted process, part of which involves deimination of myelin basic protein (MBP). Deimination, the conversion of protein-bound arginine to citrulline, is mediated by the peptidylarginine deiminase (PAD) family of enzymes, of which the PAD2 and PAD4 isoforms are present in myelin.

Myelin localization of peptidylarginine deiminases 2 and 4: comparison of PAD2 and PAD4 activities.

An understanding of the structure and composition of the myelin sheath is essential to understand the pathogenesis of demyelinating diseases such as multiple sclerosis (MS). The presence of citrulline in myelin proteins in particular myelin basic protein (MBP) causes an important change in myelin structure, which destabilizes myelin. The peptidylarginine deiminases (PADs) are responsible for converting arginine in proteins to citrulline.

Peptidyl argininedeiminase 2 CpG island in multiple sclerosis white matter is hypomethylated.

In previous studies, we documented increased citrullinated myelin basic protein (MBP) was present in MBP isolated from multiple sclerosis (MS) normal appearing white matter (NAWM). This increase was due to the myelin enzyme peptidyl argininedeiminase 2 (PAD2). In this study, we show that methylation of cytosine of the PAD2 promoter in DNA from MS NAWM was decreased to one-third of the level of that in DNA from normal white matter.

Increased citrullination of histone H3 in multiple sclerosis brain and animal models of demyelination: a role for tumor necrosis factor-induced peptidylarginine deiminase 4 translocation.

Modification of arginine residues by citrullination is catalyzed by peptidylarginine deiminases (PADs), of which five are known, generating irreversible protein structural modifications. We have shown previously that enhanced citrullination of myelin basic protein contributed to destabilization of the myelin membrane in the CNS of multiple sclerosis (MS) patients. We now report increased citrullination of nucleosomal histones by PAD4 in normal-appearing white matter (NAWM) of MS patients and in animal models of demyelination.

The role of citrullinated proteins suggests a novel mechanism in the pathogenesis of multiple sclerosis.

The pathogenesis of MS is unknown. In our studies, we have demonstrated an important role for citrullinated myelin basic protein (MBP). The accompanying loss of positive charge compromises the ability of MBP to interact with the lipid bilayer.

Differences in susceptibility of MBP charge isomers to digestion by stromelysin-1 (MMP-3) and release of an immunodominant epitope.

Charge microheterogeneity of myelin basic protein is known to affect its conformation and function. Here, the citrullinated myelin basic protein charge isomer, component-8, was shown to be more susceptible to stromelysin-1 cleavage than myelin basic protein component-1. Since levels of component-8 are increased in multiple sclerosis brain, the increased susceptibility of component-8 to proteolytic digestion may play a role in the pathogenesis of multiple sclerosis.

Autocatalytic cleavage of myelin basic protein: an alternative to molecular mimicry.

Although multiple sclerosis (MS) is thought to be an autoimmune disease, the mechanisms by which immunodominant epitopes are generated and lymphocytes are activated are not known. Here, myelin basic protein-component 1 (MBP-C1) from MS tissue was shown to undergo autocatalytic cleavage at slightly alkaline pH. Importantly, one of the major peptides released contained the immunodominant epitope 84-89.

Molecules affecting myelin stability: a novel hypothesis regarding the pathogenesis of multiple sclerosis.

In this Mini-Review we present a new hypothesis in support of the neurodegenerative theory as a mechanism for the pathogenesis of multiple sclerosis (MS). The pathogenesis of MS results from changes in two distinct CNS compartments. These are the "myelin" and "nonmyelin" compartments.

Attenuation of experimental autoimmune encephalomyelitis and nonimmune demyelination by IFN-beta plus vitamin B12: treatment to modify notch-1/sonic hedgehog balance.

Interferon-beta is a mainstay therapy of demyelinating diseases, but its effects are incomplete in human multiple sclerosis and several of its animal models. In this study, we demonstrate dramatic improvements of clinical, histological, and laboratory parameters in in vivo mouse models of demyelinating disease through combination therapy with IFN-beta plus vitamin B(12) cyanocobalamin (B(12)CN) in nonautoimmune primary demyelinating ND4 (DM20) transgenics, and in acute and chronic experimental autoimmune encephalomyelitis in SJL mice. Clinical improvement (p values <0.

The amount of sonic hedgehog in multiple sclerosis white matter is decreased and cleavage to the signaling peptide is deficient.

We have demonstrated that sonic hedgehog (Shh), vital for oligodendrocyte development, is present in both gray and white matter of normal human brain. Both the 45 kDa precursor protein and the 20 kDa N-terminal sonic hedgehog signaling portion (ShhN) were demonstrated by immunoblot and a partial purification has been achieved. In gray matter from brains of multiple sclerosis (MS) victims, the total amount of Shh was less than normals and the signaling 20 kDa protein was greatly reduced.

Expression and properties of the recombinant murine Golli-myelin basic protein isoform J37.

A recombinant form of the murine Golli-myelin basic protein (MBP) isoform J37 (rmJ37) has been expressed in Escherichia coli and isolated to 95% purity via metal chelation and ion exchange chromatography. The protein did not aggregate lipid vesicles containing acidic phospholipids, unlike the 18.5 kDa isoform of MBP.

An Arg/Lys-->Gln mutant of recombinant murine myelin basic protein as a mimic of the deiminated form implicated in multiple sclerosis.

The degree of post-translational enzymatic deimination (conversion of arginyl to citrullinyl residues) of myelin basic protein (MBP) is correlated with the severity of the human autoimmune disease multiple sclerosis (MS). It is difficult to obtain large quantities of deiminated MBP from natural sources (autopsy material), and in vitro deimination using peptidylarginine deiminase (EC 3.5.

The up-regulation of stromelysin-1 (MMP-3) in a spontaneously demyelinating transgenic mouse precedes onset of disease.

The matrix metalloproteinases (MMPs) are a family of endoproteinases that degrade various components of the extracellular matrix and have been implicated in the pathogenesis of multiple sclerosis. To determine whether up-regulation of MMP-3, or stromelysin-1, was a causative factor during the development of demyelination, we have examined the expression of MMP-3 mRNA and protein in brain tissue of a spontaneously demyelinating mouse model overexpressing DM20 (ND4 line) prior to and during the progression of disease. Stromelysin-1, but not other MMP mRNA was elevated approximately 10-fold in transgenic mice between 5 days and 1 month of age, more than 2 months before the onset of disease, and was coordinately expressed with the DM20 transgene.