RESUMO
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. Histone citrullination was attributable to increased levels and activity of nuclear PAD4. PAD4 translocation into the nucleus was attributable to elevated tumor necrosis factor-alpha (TNF-alpha) protein. The elevated TNF-alpha in MS NAWM was not associated with CD3+ or CD8+ lymphocytes, nor was it associated with CD68+ microglia/macrophages. GFAP, a measure of astrocytosis, was the only cytological marker that was consistently elevated in the MS NAWM, suggesting that TNF-alpha may have been derived from astrocytes. In cell cultures of mouse and human oligodendroglial cell lines, PAD4 was predominantly cytosolic but TNF-alpha treatment induced its nuclear translocation. To address the involvement of TNF-alpha in targeting PAD4 to the nucleus, we found that transgenic mice overexpressing TNF-alpha also had increased levels of citrullinated histones and elevated nuclear PAD4 before demyelination. In conclusion, high citrullination of histones consequent to PAD4 nuclear translocation is part of the process that leads to irreversible changes in oligodendrocytes and may contribute to apoptosis of oligodendrocytes in MS.
Assuntos
Encéfalo/metabolismo , Citrulina/metabolismo , Modelos Animais de Doenças , Histonas/metabolismo , Hidrolases/metabolismo , Esclerose Múltipla/metabolismo , Fator de Necrose Tumoral alfa/fisiologia , Transporte Ativo do Núcleo Celular/fisiologia , Animais , Encéfalo/patologia , Doenças Desmielinizantes/genética , Doenças Desmielinizantes/metabolismo , Doenças Desmielinizantes/patologia , Feminino , Histonas/genética , Humanos , Hidrolases/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Esclerose Múltipla/genética , Esclerose Múltipla/patologia , Proteína-Arginina Desiminase do Tipo 4 , Desiminases de Arginina em ProteínasRESUMO
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. Here we report that 2-chloroacetamidine (2CA), a small-molecule, PAD active-site inhibitor, dramatically attenuates disease at any stage in independent neurodegenerative as well as autoimmune MS mouse models. 2CA reduced PAD activity and protein citrullination to pre-disease status. In the autoimmune models, disease induction uniformly induced spontaneous hypercitrullination with citrulline+ epitopes targeted frequently. 2CA rapidly suppressed T cell autoreactivity, clearing brain and spinal cord infiltrates, through selective removal of newly activated T cells. 2CA essentially prevented disease when administered before disease onset or before autoimmune induction, making hypercitrullination, and specifically PAD enzymes, a therapeutic target in MS models and thus possibly in MS.
Assuntos
Citrulina/metabolismo , Hidrolases/antagonistas & inibidores , Esclerose Múltipla/enzimologia , Esclerose Múltipla/patologia , Amidinas/química , Amidinas/farmacologia , Amidinas/uso terapêutico , Animais , Encéfalo/enzimologia , Encéfalo/patologia , Complexo CD3/metabolismo , Doenças Desmielinizantes/enzimologia , Doenças Desmielinizantes/patologia , Modelos Animais de Doenças , Encefalomielite Autoimune Experimental/tratamento farmacológico , Encefalomielite Autoimune Experimental/enzimologia , Encefalomielite Autoimune Experimental/patologia , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/uso terapêutico , Humanos , Hidrolases/metabolismo , Linfócitos/efeitos dos fármacos , Linfócitos/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Esclerose Múltipla/tratamento farmacológico , Nervo Óptico/efeitos dos fármacos , Nervo Óptico/patologia , Nervo Óptico/ultraestrutura , Desiminases de Arginina em Proteínas , Baço/efeitos dos fármacos , Baço/metabolismo , Baço/patologia , Análise de SobrevidaRESUMO
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. The conversion of arginine to citrulline in brain is carried out by an enzyme peptidyl arginine deiminase (PAD) 2. The amount of PAD 2 in brain was increased in MS normal-appearing white matter. The mechanism responsible for this increase involved hypomethylation of the promoter region in the PAD 2 gene in MS, but no change (compared to normal) was found in thymus tissue DNA from the same MS patients. In addition, no change was observed in other neurological diseases, including Alzheimer's, Parkinson's, and Huntington's. We propose that citrullinated MBP, resulting from elevated levels of PAD 2 represents an important biochemical pathway in the pathogenesis of MS.
Assuntos
Citrulina/fisiologia , Esclerose Múltipla/etiologia , Proteína Básica da Mielina/metabolismo , Apoptose , Humanos , Hidrolases/metabolismo , Metilação , Esclerose Múltipla/enzimologia , Oligodendroglia/fisiologia , Conformação Proteica/efeitos dos fármacos , Processamento de Proteína Pós-Traducional , Desiminases de Arginina em ProteínasRESUMO
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. The PAD2 promoter in DNA from thymus obtained from the same MS patients and white matter DNA from Alzheimer's, Huntington's, and Parkinson's was not hypomethylated. DNA demethylase activity in supernatants prepared from NAWM of MS patients was 2-fold higher than the DNA demethylase from normal, Alzheimer's, Huntington's and Parkinson's disease white matter. The amount of PAD2 enzyme and citrullinated MBP was increased in MS NAWM. The decreased methylation of cytosines in the PAD2 promoter may explain the increased synthesis of PAD2 protein that is responsible for the increased amount of citrullinated MBP, which in turn results in loss of myelin stability in MS brain.
Assuntos
Encéfalo/enzimologia , Ilhas de CpG/fisiologia , Hidrolases/metabolismo , Esclerose Múltipla/enzimologia , 5-Metilcitosina/metabolismo , Western Blotting , Citrulina/metabolismo , DNA/biossíntese , DNA/genética , DNA de Cadeia Simples/metabolismo , Imunofluorescência , Humanos , Metilação , Proteína Básica da Mielina/metabolismo , Regiões Promotoras Genéticas/genética , Proteína-Arginina Desiminase do Tipo 2 , Desiminases de Arginina em Proteínas , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sulfitos/farmacologia , Timo/metabolismoRESUMO
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. Interestingly, MBP isolated from MS patients showed a faster time course of cleavage and a more robust release of epitope 84-89 than MBP isolated from normal individuals. The cleavage reaction was not inhibited by protease inhibitors, except for phenylmethanesulfonyl fluoride (PMSF), a serine protease inhibitor. Since PMSF inhibition suggested a role for a serine residue in the cleavage, we labeled myelin basic protein with diisopropyl fluorophosphate (DFP), known to bind active site serine residues. Mass spectrometry was used to identify the labeled peptide, which consisted of residues 140-152. Since this peptide contained a single serine residue, we concluded it to be the active serine. The importance of this cleavage mechanism is that it provides for a ready source of the immunodominant peptide for sensitization of T-cells. It is not necessary to invoke other mechanisms such as molecular mimicry.
Assuntos
Esclerose Múltipla/imunologia , Proteína Básica da Mielina/química , Proteína Básica da Mielina/imunologia , Animais , Catálise , Humanos , Concentração de Íons de Hidrogênio , Epitopos Imunodominantes/química , Isoflurofato/farmacologia , Lipídeos/química , Camundongos , Mimetismo Molecular , Proteína Básica da Mielina/metabolismo , Fragmentos de Peptídeos/química , Inibidores de Proteases/farmacologia , Proteínas Recombinantes/metabolismo , Serina/análise , TemperaturaRESUMO
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 3.5.3.15) is both non-specific and irreproducible. Since there is no known codon for citrulline, we have constructed a mutant form of recombinant murine MBP (rmMBP) in which 5 Arg and 1 Lys residues have been replaced by Gln as the most reasonable analogue of Cit. The residues were chosen to correspond to the 6 Arg residues in human MBP which are most commonly deiminated in chronic MS. The mutant species, rmMBP-qCit(6) where the "q" represents "quasi-," was probed by numerous biochemical and biophysical techniques. Highly homogeneous protein preparations were obtained using a modified expression system which minimised spurious misincorporation of Lys for Arg, as ascertained by electrospray ionisation mass spectrometry. The mutant form rmMBP-qCit(6) had a reduced ability to aggregate lipid vesicles, a slightly greater susceptibility to digestion by cathepsin D, a greater proportion of random secondary structure, and different conformational responses to lipids, compared with the unmodified rmMBP. Overall, the mutant protein's properties were consistent with the effects of deimination and support its use as a model for evaluating the effects of this modification.
Assuntos
Glutamina/genética , Lisina/genética , Mimetismo Molecular , Esclerose Múltipla/genética , Mutação de Sentido Incorreto/genética , Proteína Básica da Mielina/química , Proteína Básica da Mielina/genética , Sequência de Aminoácidos , Animais , Arginina/genética , Western Blotting , Catepsina D/metabolismo , Cromatografia Líquida de Alta Pressão , Dicroísmo Circular , Cinética , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Peso Molecular , Proteína Básica da Mielina/isolamento & purificação , Proteína Básica da Mielina/metabolismo , Ligação Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/isolamento & purificação , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
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. This result is consistent with J37 having a functional role prior to the assembly of compact myelin. Circular dichroic spectroscopy showed that rmJ37 had a large proportion of random coil in aqueous solution but gained alpha-helix and beta-sheet in the presence of monosialoganglioside G(M1) and PI(4)P. Thus, like "classic" MBP, J37 is intrinsically unstructured, and its conformation depends on its environment and bound ligands. Analyses of the amino acid sequence of rmJ37 predicted an N-terminal calmodulin (CaM)-binding site. It was determined via a gel-shift assay and fluorescence spectroscopy that rmJ37 and CaM interacted in a 1:1 ratio in a Ca(2+)-dependent manner. However, the interaction was weak compared with 18.5 kDa MBP.