RESUMEN
Neutrophils can form neutrophil extracellular traps (NETs) to capture microbes and facilitate their clearance. NETs consist of decondensed chromatin decorated with anti-microbial proteins. Here, we describe the effect of neutrophil proteases on the protein content of NETs. We show that the neutrophil serine proteases degrade several neutrophil proteins associated with NETs. Interestingly, the anti-bacterial proteins associated with NETs, such as myeloperoxidase, calgranulin B and neutrophil elastase (NE), seem to be less susceptible to proteolytic degradation than other NET proteins, such as actin and MNDA. NETs have been proposed to play a role in autoimmune reactions. Our data demonstrate that a large number of the autoepitopes of NET proteins that are recognized by autoantibodies produced by systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA) patients are also removed by the proteases. In conclusion, neutrophil serine proteases have a major impact on the NET proteome and the proteolytic changes of NET-associated proteins may counteract autoimmune reactions to NET components.
Asunto(s)
Artritis Reumatoide/inmunología , Trampas Extracelulares/inmunología , Lupus Eritematoso Sistémico/inmunología , Neutrófilos/inmunología , Péptido Hidrolasas/inmunología , Artritis Reumatoide/patología , Femenino , Humanos , Lupus Eritematoso Sistémico/patología , Masculino , Neutrófilos/patologíaRESUMEN
Small non-coding RNAs have emerged as possible biomarkers for various diseases including autoimmune diseases. A number of studies have demonstrated that the expression of specific microRNAs (miRNAs) is dysregulated in rheumatoid arthritis (RA). So far, all studies on miRNAs in RA patients have been performed using either microarray or reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analyses. Compared to RT-qPCR and microarray analyses, next-generation sequencing (NGS) allows the genome-wide analysis of small RNAs and the differentiation between miRNAs that differ by a single nucleotide. The application of NGS to the analysis of small RNAs circulating in sera of RA patients has not been reported. This study provides a global overview of the circulating small RNAs in the sera of RA patients and healthy subjects and identifies differences between these groups using NGS. Several classes of small RNAs, including hY RNA-derived fragments, tRNA-derived fragments and miRNAs, were determined. Differentially expressed individual small RNAs were verified by RT-qPCR. The levels of two miRNAs, miR-223-3p and miR-16-5p, were significantly lower in the sera from early RA patients than in those from established RA patients and healthy controls. In contrast, the serum level of miR-16-5p was higher in patients with established RA than in healthy control samples. These miRNAs may not only serve as biomarkers, but may also shed more light on the pathophysiology of RA.
Asunto(s)
Artritis Reumatoide/diagnóstico , MicroARNs/genética , Biomarcadores/metabolismo , Progresión de la Enfermedad , Diagnóstico Precoz , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Análisis por MicromatricesRESUMEN
OBJECTIVES: Autoantibodies directed against cytosolic 5'-nucleotidase 1A have been identified in many patients with inclusion body myositis. This retrospective study investigated the association between anticytosolic 5'-nucleotidase 1A antibody status and clinical, serological and histopathological features to explore the utility of this antibody to identify inclusion body myositis subgroups and to predict prognosis. MATERIALS AND METHODS: Data from various European inclusion body myositis registries were pooled. Anticytosolic 5'-nucleotidase 1A status was determined by an established ELISA technique. Cases were stratified according to antibody status and comparisons made. Survival and mobility aid requirement analyses were performed using Kaplan-Meier curves and Cox proportional hazards regression. RESULTS: Data from 311 patients were available for analysis; 102 (33%) had anticytosolic 5'-nucleotidase 1A antibodies. Antibody-positive patients had a higher adjusted mortality risk (HR 1.89, 95% CI 1.11 to 3.21, p=0.019), lower frequency of proximal upper limb weakness at disease onset (8% vs 23%, adjusted OR 0.29, 95% CI 0.12 to 0.68, p=0.005) and an increased prevalence of excess of cytochrome oxidase deficient fibres on muscle biopsy analysis (87% vs 72%, adjusted OR 2.80, 95% CI 1.17 to 6.66, p=0.020), compared with antibody-negative patients. INTERPRETATION: Differences were observed in clinical and histopathological features between anticytosolic 5'-nucleotidase 1A antibody positive and negative patients with inclusion body myositis, and antibody-positive patients had a higher adjusted mortality risk. Stratification of inclusion body myositis by anticytosolic 5'-nucleotidase 1A antibody status may be useful, potentially highlighting a distinct inclusion body myositis subtype with a more severe phenotype.
Asunto(s)
5'-Nucleotidasa/inmunología , Autoanticuerpos/sangre , Fibras Musculares Esqueléticas/patología , Miositis por Cuerpos de Inclusión/sangre , Miositis por Cuerpos de Inclusión/diagnóstico , Edad de Inicio , Anciano , Anciano de 80 o más Años , Biomarcadores/sangre , Citosol , Complejo IV de Transporte de Electrones/análisis , Femenino , Humanos , Estimación de Kaplan-Meier , Masculino , Persona de Mediana Edad , Fibras Musculares Esqueléticas/química , Debilidad Muscular/etiología , Miositis por Cuerpos de Inclusión/patología , Pronóstico , Modelos de Riesgos Proporcionales , Estudios Retrospectivos , Dispositivos de Autoayuda/estadística & datos numéricos , Tasa de Supervivencia , Factores de TiempoRESUMEN
Testing for autoantibodies (AABs) is becoming more and more relevant, not only for diagnosing autoimmune diseases (AIDs) but also for the differentiation of defined AID subtypes with different clinical manifestations, course and prognosis as well as the very early diagnosis for adequate management in the context of personalized medicine. A major challenge to improve diagnostic accuracy is to harmonize or even standardize AAB analyses. This review presents the results of the 12th Dresden Symposium on Autoantibodies that focused on several aspects of improving autoimmune diagnostics. Topics that are addressed include the International Consensus on ANA Patterns (ICAP) and the International Autoantibody Standardization (IAS) initiatives, the optimization of diagnostic algorithms, the description and evaluation of novel disease-specific AABs as well as the development and introduction of novel assays into routine diagnostics. This review also highlights important developments of recent years, most notably the improvement in diagnosing and predicting the course of rheumatoid arthritis, systemic sclerosis, idiopathic inflammatory myopathies, and of autoimmune neurological, gastrointestinal and liver diseases; the potential diagnostic role of anti-DFS70 antibodies and tumor-associated AABs. Furthermore, some hot topics in autoimmunity regarding disease pathogenesis and management are described.
Asunto(s)
Autoanticuerpos/sangre , Enfermedades Autoinmunes/diagnóstico , Autoinmunidad , Diagnóstico Precoz , Congresos como Asunto , Alemania , HumanosRESUMEN
Three sisters who carried the BRCA-1 gene mutation had a preventive mastectomy and were reconstructed with silicone breast implants. After the reconstruction all three patients developed fatigue, arthralgia, myalgia and sleep disturbances within a period of four years. Because the complaints were thought to be related to the silicone breast implants, they were advised to have the implants replaced by non-silicone gel containing Monobloc Hydrogel breast implants. After this replacement operation, all complaints improved as evaluated 2.5 years later. Since the complaints developed during the presence of silicone implants and since the reversal was observed after replacement by hydrogel implants we postulate that our patients suffered from ASIA due to silicone implants, i.e. Silicone Implant Incompatibility Syndrome (SIIS). The generation of this syndrome in three sisters suggests that the susceptibility to the development of SIIS may be genetically determined.
Asunto(s)
Enfermedades Autoinmunes , Implantación de Mama/efectos adversos , Implantes de Mama/efectos adversos , Mastectomía/rehabilitación , Complicaciones Posoperatorias , Geles de Silicona/efectos adversos , Adulto , Enfermedades Autoinmunes/etiología , Enfermedades Autoinmunes/inmunología , Enfermedades Autoinmunes/fisiopatología , Enfermedades Autoinmunes/cirugía , Autoinmunidad/efectos de los fármacos , Implantación de Mama/métodos , Implantes de Mama/clasificación , Susceptibilidad a Enfermedades/etiología , Femenino , Genes BRCA1 , Humanos , Mastectomía/métodos , Mutación , Cuidados Posoperatorios/efectos adversos , Cuidados Posoperatorios/métodos , Complicaciones Posoperatorias/etiología , Complicaciones Posoperatorias/inmunología , Complicaciones Posoperatorias/fisiopatología , Complicaciones Posoperatorias/cirugía , Medicina Preventiva/métodos , Falla de Prótesis , Reoperación/métodos , Hermanos , Geles de Silicona/uso terapéutico , Resultado del TratamientoRESUMEN
OBJECTIVE: In classic studies on the genetic background of antibody production, the major histocompatibility complex (MHC) has been shown to act as the most prominent immune response gene that controls the magnitude and the specificity of antibody production. The strongest genetic risk factor for rheumatoid arthritis (RA), the human MHC HLA-DRB1 shared epitope (SE) alleles, predisposes for antibodies against citrullinated proteins (ACPAs). ACPA levels are higher in SE-positive patients with RA than in SE-negative patients with RA. The aim of the present study was to determine whether SE influences not only the magnitude but also the specificity of the ACPA response. METHODS: In 2 cohorts of anti-citrullinated peptide 2-positive patients with RA, one from a study of recent-onset arthritis (n = 206) and the other from a treatment strategy study (n = 141), serum antibodies against a citrullinated peptide derived from vimentin (cVim) and antibodies against a citrullinated fibrinogen peptide (cFibr) were determined by enzyme-linked immunosorbent assay. HLA-DRB1 genotyping was performed. RESULTS: In the first cohort, SE alleles were significantly associated with the presence of antibodies against cVim (odds ratio [OR] 4.95, 95% confidence interval [95% CI] 1.87-15.3) and were not significantly associated with the presence of antibodies against cFibr (OR 1.71, 95% CI 0.70-4.14). These results were replicated in the second cohort (OR 5.05, 95% CI 1.92-13.6 and OR 1.19, 95% CI 0.30-3.97, respectively). CONCLUSION: In 2 cohorts of ACPA-positive patients with RA, SE alleles predisposed for the development of antibodies against cVim but not for the development of antibodies against cFibr. These data indicate that SE alleles act as "classic" immune response genes in the ACPA response, because they influence both the magnitude and the specificity of this RA-specific antibody response.
Asunto(s)
Alelos , Anticuerpos Antiidiotipos/metabolismo , Especificidad de Anticuerpos/genética , Citrulina/metabolismo , Epítopos/genética , Fibrinógeno/inmunología , Vimentina/inmunología , Anticuerpos Antiidiotipos/genética , Anticuerpos Antiidiotipos/inmunología , Especificidad de Anticuerpos/inmunología , Artritis Reumatoide/genética , Artritis Reumatoide/inmunología , Estudios de Cohortes , Predisposición Genética a la Enfermedad/genética , Antígenos HLA-DR/genética , Antígenos HLA-DR/inmunología , Cadenas HLA-DRB1 , HumanosRESUMEN
BACKGROUND: Antibodies directed against citrullinated proteins (eg anti-cyclic citrullinated peptide (CCP)) have excellent diagnostic and good prognostic potential for rheumatoid arthritis. Type 1 autoimmune hepatitis (AIH-1) is a chronic liver disease characterised by a variety of serum autoantibodies. Recently, in a large group of patients with AIH-1 without clear rheumatoid arthritis overlap, a relatively high percentage (9%) of anti-CCP2 positivity was scored. OBJECTIVES: To characterise the citrulline-dependence of the observed anti-CCP2 positivity in AIH-1 sera as well as in other groups of patients without rheumatoid arthritis (mainly rheumatic diseases). METHODS: Serum samples of 57 patients with AIH-1 and 66 patients without rheumatoid arthritis, most of them reported as anti-CCP positive, were tested for citrulline-specific reactivity with a second generation anti-CCP kit, with the citrullinated and the corresponding non-citrullinated (arginine-containing) antigen. A subset of AIH-1 sera was also tested with a CCP1 ELISA (and arginine control). RESULTS: The anti-CCP2 reactivity of most non-rheumatoid arthritis rheumatic diseases samples (87-93%) was citrulline-specific, whereas a relatively high percentage of AIH-1 samples (42-50%) turned out to be reactive in a citrulline-independent manner. The use of citrullinated and non-citrullinated CCP1 peptides confirmed a high occurrence of citrulline-independent reactivity in AIH-1 samples. CONCLUSIONS: In rheumatoid arthritis and most non-rheumatoid arthritis rheumatologic disease sera, anti-CCP positivity is citrulline-dependent. However in some patients, particularly patients with AIH-1, citrulline-independent reactivity in the anti-CCP2 test can occur. A positive CCP test in a non-rheumatic disease (eg liver disease) should therefore be interpreted with care, and preferably followed by a control ELISA with a non-citrullinated antigen.
Asunto(s)
Artritis/inmunología , Autoanticuerpos/sangre , Citrulina/inmunología , Hepatitis Autoinmune/inmunología , Péptidos Cíclicos/inmunología , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Artritis/diagnóstico , Artritis Reumatoide/diagnóstico , Artritis Reumatoide/inmunología , Autoantígenos/inmunología , Biomarcadores/sangre , Niño , Ensayo de Inmunoadsorción Enzimática/métodos , Epítopos , Femenino , Humanos , Masculino , Persona de Mediana Edad , Juego de Reactivos para Diagnóstico , Enfermedades Reumáticas/inmunologíaAsunto(s)
Artritis Reumatoide/diagnóstico , Autoanticuerpos/análisis , Péptidos Cíclicos/inmunología , Artritis Reumatoide/tratamiento farmacológico , Artritis Reumatoide/inmunología , Autoanticuerpos/inmunología , Biomarcadores/análisis , Predicción , Humanos , Pronóstico , Sensibilidad y EspecificidadRESUMEN
Recent studies have implicated the dying cell as a potential reservoir of modified autoantigens that might initiate and drive systemic autoimmunity in susceptible hosts. The spliceosomal Sm proteins are recognized by the so-called anti-Sm autoantibodies, an antibody population found exclusively in patients suffering from systemic lupus erythematosus. We have studied the effects of apoptosis on the Sm proteins and demonstrate that one of the Sm proteins, the Sm-F protein, is proteolytically cleaved in apoptotic cells. Cleavage of the Sm-F protein generates a 9-kDa apoptotic fragment, which remains associated with the U snRNP complexes in apoptotic cells. Sm-F cleavage is dependent on caspase activation and the cleavage site has been located near the C-terminus, EEED(81) downward arrow G. Use of different caspase inhibitors suggests that besides caspase-8 other caspases are implicated in Sm-F cleavage. A C-terminally truncated mutant of the Sm-F protein, representing the modified form of the protein, is capable of forming an Sm E-F-G complex in vitro that is recognized by many anti-Sm patient sera.
Asunto(s)
Apoptosis , Caspasas/metabolismo , Ribonucleoproteína Nuclear Pequeña U1/metabolismo , Ribonucleoproteínas Nucleares Pequeñas/metabolismo , Anticuerpos Monoclonales/farmacología , Anticuerpos Monoclonales de Origen Murino , Apoptosis/efectos de los fármacos , Apoptosis/inmunología , Autoanticuerpos/sangre , Autoantígenos , Western Blotting , Inhibidores de Caspasas , Línea Celular Tumoral , Inhibidores de Cisteína Proteinasa/farmacología , Electroforesis en Gel de Poliacrilamida , Células HeLa , Humanos , Células Jurkat , Lupus Eritematoso Sistémico/sangre , Lupus Eritematoso Sistémico/inmunología , Ribonucleoproteínas Nucleares Pequeñas/genética , Ribonucleoproteínas Nucleares Pequeñas/inmunología , Factores de Tiempo , Receptor fas/inmunología , Proteínas Nucleares snRNPRESUMEN
Sera from patients suffering from systemic autoimmune diseases such as systemic lupus erythematosus (SLE) and systemic sclerosis (SSc) have been shown to contain reactivities to nuclear components. Autoantibodies specifically targeting nucleolar antigens are found most frequently in patients suffering from SSc or SSc overlap syndromes. We determined the prevalence and clinical significance of autoantibodies directed to nucleolar RNA-protein complexes, the so-called small nucleolar ribonucleoprotein complexes (snoRNPs). A total of 172 patient sera with antinucleolar antibodies were analysed by immunoprecipitation. From 100 of these patients clinical information was obtained by chart review. Autoantibodies directed to snoRNPs were detected not only in patients suffering from SSc and primary Raynaud's phenomenon (RP), but also in patients suffering from SLE, rheumatoid arthritis (RA) and myositis (PM/DM). Antibodies against box C/D small snoRNPs can be subdivided in antifibrillarin positive and antifibrillarin negative reactivity. Antifibrillarin-positive patient sera were associated with a poor prognosis in comparison with antifibrillarin negative (reactivity with U3 or U8 snoRNP only) patient sera. Anti-Th/To autoantibodies were associated with SSc, primary RP and SLE and were found predominantly in patients suffering from decreased co-diffusion and oesophagus motility and xerophthalmia. For the first time autoantibodies that recognize box H/ACA snoRNPs are described, identifying this class of snoRNPs as a novel autoantigenic activity. Taken together, our data show that antinucleolar patient sera directed to small nucleolar ribonucleoprotein complexes are found frequently in other diseases than SSc and that categorization of diagnoses and clinical manifestations based on autoantibody profiles seems particularly informative in patient sera recognizing box C/D snoRNPs.
Asunto(s)
Autoanticuerpos/análisis , Lupus Eritematoso Sistémico/inmunología , Ribonucleoproteínas Nucleares Pequeñas/inmunología , Esclerodermia Sistémica/inmunología , Autoanticuerpos/clasificación , Northern Blotting , Western Blotting , HumanosRESUMEN
The RNase MRP and RNase P ribonucleoprotein particles both function as endoribonucleases, have a similar RNA component, and share several protein subunits. RNase MRP has been implicated in pre-rRNA processing and mitochondrial DNA replication, whereas RNase P functions in pre-tRNA processing. Both RNase MRP and RNase P accumulate in the nucleolus of eukaryotic cells. In this report we show that for three protein subunits of the RNase MRP complex (hPop1, hPop4, and Rpp38) basic domains are responsible for their nucleolar accumulation and that they are able to accumulate in the nucleolus independently of their association with the RNase MRP and RNase P complexes. We also show that certain mutants of hPop4 accumulate in the Cajal bodies, suggesting that hPop4 traverses through these bodies to the nucleolus. Furthermore, we characterized a deletion mutant of Rpp38 that preferentially associates with the RNase MRP complex, giving a first clue about the difference in protein composition of the human RNase MRP and RNase P complexes. On the basis of all available data on nucleolar localization sequences, we hypothesize that nucleolar accumulation of proteins containing basic domains proceeds by diffusion and retention rather than by an active transport process. The existence of nucleolar localization sequences is discussed.
Asunto(s)
Autoantígenos/metabolismo , Proteínas Portadoras , Endonucleasas/metabolismo , Endorribonucleasas/metabolismo , ARN Catalítico/metabolismo , Ribonucleasas , Ribonucleoproteínas/metabolismo , Proteínas Reguladoras de la Apoptosis , Autoantígenos/genética , Sitios de Unión , Nucléolo Celular/metabolismo , Endonucleasas/genética , Humanos , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Ribonucleasa P , Ribonucleoproteínas/genética , Células Tumorales CultivadasRESUMEN
Inherently unstable mammalian mRNAs contain AU-rich elements (AREs) within their 3' untranslated regions. Although found 15 years ago, the mechanism by which AREs dictate rapid mRNA decay is not clear. In yeast, 3'-to-5' mRNA degradation is mediated by the exosome, a multisubunit particle. We have purified and characterized the human exosome by mass spectrometry and found its composition to be similar to its yeast counterpart. Using a cell-free RNA decay system, we demonstrate that the mammalian exosome is required for rapid degradation of ARE-containing RNAs but not for poly(A) shortening. The mammalian exosome does not recognize ARE-containing RNAs on its own. ARE recognition requires certain ARE binding proteins that can interact with the exosome and recruit it to unstable RNAs, thereby promoting their rapid degradation.
Asunto(s)
Regiones no Traducidas 3'/genética , Silenciador del Gen/fisiología , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/fisiología , Transactivadores , Secuencia de Aminoácidos , Animales , Emparejamiento Base , Secuencia de Bases , Sistema Libre de Células , Genes fos , Humanos , Interleucina-2/genética , Células Jurkat/efectos de los fármacos , Células Jurkat/metabolismo , Mamíferos/genética , Datos de Secuencia Molecular , Poli A/metabolismo , ARN Neoplásico/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Nucleótidos de Timina/farmacologíaRESUMEN
The RNase MRP and RNase P particles both function as endoribonucleases. RNase MRP has been implicated in the processing of precursor-rRNA, whereas RNase P has been shown to function in the processing of pre-tRNA. Both ribonucleoprotein particles have an RNA component that can be folded into a similar secondary structure and share several protein components. We have identified human, rat, mouse, cow, and Drosophila homologues of the Pop5p protein subunit of the yeast RNase MRP and RNase P complexes. The human Pop5 cDNA encodes a protein of 163 amino acids with a predicted molecular mass of 18.8 kDa. Polyclonal antibodies raised against recombinant hPop5 identified a 19-kDa polypeptide in HeLa cells and showed that hPop5 is associated with both RNase MRP and RNase P. Using affinity-purified anti-hPop5 antibodies, we demonstrated that the endogenous hPop5 protein is localized in the nucleus and accumulates in the nucleolus, which is consistent with its association with RNase MRP and RNase P. Catalytically active RNase P was partially purified from HeLa cells, and hPop5 was shown to be associated with it. Finally, the evolutionarily conserved acidic C-terminal tail of hPop5 appeared to be required neither for complex formation nor for RNase P activity.
Asunto(s)
Endorribonucleasas/química , ARN Catalítico/química , Ribonucleasa P/química , Secuencia de Aminoácidos , Secuencia de Bases , Catálisis , Clonación Molecular , ADN Complementario , Endorribonucleasas/genética , Endorribonucleasas/metabolismo , Humanos , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Pruebas de Precipitina , ARN/metabolismo , ARN Catalítico/genética , ARN Catalítico/metabolismo , Proteínas Recombinantes/química , Ribonucleasa P/genética , Ribonucleasa P/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
Ro RNPs are small cytoplasmic RNA-protein complexes of unknown function that have been found in all metazoan cells studied so far. In human cells, Ro RNPs consist of one of four small RNA molecules, termed hY RNAs and at least two well-characterized proteins, Ro60 and La. In previous Xenopus laevis oocyte microinjection studies, we showed that an intact Ro60 binding site (Stem-loop 1) is a prerequisite for efficient nuclear export of hY1 RNA, whereas an intact La-binding site promotes nuclear retention (Simons et al. RNA, 1996, 2:264-273). Here we present evidence that the distal half (Stem 2) of the conserved base-paired stem structure found in all hY RNAs also plays a critical role in the export process. A minimal RNA molecule containing this region, L1S2 RNA, competes effectively for the export of full-length hY1 RNAs and is itself exported very rapidly in a Ro60-independent and RanGTP-dependent manner. Mutational analyses of this RNA shows that a 5'/3' terminal double-stranded stem structure (>10 bp) of no specific nucleotide sequence constitutes a novel nuclear export element (NEE). Cross-competition studies indicate that this type of NEE may also be involved in export of other classes of RNAs. Like full-length hY1 RNA, L1S2 RNA also competes for export of ET-202 RNA, an RNA that was selected for its efficient nuclear export in the presence of the nuclear transport inhibitor, VSV Matrix protein (Grimm et al. Proc Natl Acad Sci USA, 1997, 94:10122-10127). However, export of L1S2 RNA is strongly inhibited by VSV-M protein, showing that these RNAs use partially overlapping, but not identical export pathways. We propose that export of Y RNAs is mediated by two contiguous cis-acting elements in the 5'/3' double-stranded stem region that is conserved between different Y RNAs.
Asunto(s)
Núcleo Celular/metabolismo , ARN/metabolismo , Transporte Activo de Núcleo Celular , Animales , Secuencia de Bases , Sitios de Unión , Humanos , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , ARN/química , Xenopus laevisRESUMEN
The hY RNAs are a group of four small cytoplasmic RNAs of unknown function that are stably associated with at least two proteins, Ro60 and La, to form Ro ribonucleoprotein complexes. Here we show that the heterogeneous nuclear ribonucleoproteins (hnRNP) I and K are able to associate with a subset of hY RNAs in vitro and demonstrate these interactions to occur also in vivo in a yeast three-hybrid system. Experiments performed in vitro and in vivo with deletion mutants of hY1 RNA revealed its pyrimidine-rich central loop to be involved in interactions with both hnRNP I and K and clearly showed their binding sites to be different from the Ro60 binding site. Both hY1 and hY3 RNAs coprecipitated with hnRNP I in immunoprecipitation experiments performed with HeLa S100 extracts and cell extracts from COS-1 cells transiently transfected with VSV-G-tagged hnRNP-I, respectively. Furthermore, both anti-Ro60 and anti-La antibodies coprecipitated hnRNP I, whereas coprecipitation of hnRNP K was not observed. Taken together, these data strongly suggest that hnRNP I is a stable component of a subpopulation of Ro RNPs, whereas hnRNP K may be transiently bound or interact only with (rare) Y RNAs that are devoid of Ro60 and La. Given that functions related to translation regulation have been assigned to both proteins and also to La, our findings may provide novel clues toward understanding the role of Y RNAs and their respective RNP complexes.
Asunto(s)
Autoantígenos , ARN Citoplasmático Pequeño/metabolismo , Ribonucleoproteínas/metabolismo , Animales , Secuencia de Bases , Células COS , Cartilla de ADN , Células HeLa , Ribonucleoproteína Heterogénea-Nuclear Grupo K , Ribonucleoproteínas Nucleares Heterogéneas , Humanos , Técnicas In Vitro , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Unión Proteica , ARN Citoplasmático Pequeño/química , Ribonucleoproteínas/químicaRESUMEN
The anti-PM/Scl autoantibodies are known to characterize a subset of autoimmune patients with myositis, scleroderma (Scl), and the PM/Scl overlap syndrome. The major autoantigens that are recognized by anti-PM/Scl autoantibodies are designated PM/Scl-100 and PM/Scl-75. These autoantigens have been reported to associate into a large complex consisting of 11 to 16 proteins and to play a role in ribosome synthesis. Recently, it was discovered that the PM/Scl complex is the human counterpart of the yeast (Saccharomyces cerevisiae) exosome, which is an RNA-processing complex consisting of 11 3' --> 5' exoribonucleases. To date, 10 human exosome components have been identified, although only some of these were studied in more detail. In this review, we discuss some recent advances in the characterization of the PM/Scl complex.
Asunto(s)
Autoantígenos/inmunología , Exorribonucleasas/inmunología , Complejos Multienzimáticos/inmunología , Polimiositis/enzimología , Polimiositis/inmunología , Esclerodermia Sistémica/enzimología , Esclerodermia Sistémica/inmunología , Autoantígenos/química , Exorribonucleasas/química , Complejo Multienzimático de Ribonucleasas del Exosoma , Humanos , Complejos Multienzimáticos/químicaRESUMEN
The small nuclear ribonucleoprotein 70K (snRNP 70K; U1-70 kDa) is an integral part of the spliceosome, a large RNA-protein complex catalyzing the removal of introns from nuclear pre-mRNA. snRNP is one of the best-studied essential subunits of snRNPs, is highly conserved and its inactivation was shown to result in complete inhibition of splicing. Applying subtractive hybridization, we found a sequence with 100% identity to snRNP absent in fetal Down syndrome (DS) brain. This observation made us determine snRNP-mRNA steady-state levels and protein levels in brains of adult patients with DS. snRNP-mRNA and protein levels of five individual brain regions of DS and controls each, were determined by blotting techniques. snRNP-mRNA steady state levels were significantly decreased in DS brain. Performing Western blots with monoclonal and human antibodies, snRNP protein levels were decreased in several regions of DS brain, although one monoclonal antibody did not reveal different snRNP-immunoreactivity. Although decreased snRNP-protein could be explained by decreased mRNA-steady state levels, another underlying mechanism might be suggested: snRNP is one of the death substrates rapidly cleaved during apoptosis by interleukin-1-beta-converting enzyme-like (ICE) proteases, which was well-documented by several groups. As apoptosis is unrequivocally taking place in DS brain leading to permanent cell loses, decreased snRNP-protein levels may therefore reflect decreased synthesis and increased apoptosis-related proteolytic cleavage.
Asunto(s)
Encéfalo/metabolismo , Síndrome de Down/metabolismo , Ribonucleoproteína Nuclear Pequeña U1/deficiencia , Adulto , Encéfalo/patología , Síndrome de Down/patología , Humanos , Hibridación de Ácido Nucleico/métodos , ARN Mensajero , Ribonucleoproteína Nuclear Pequeña U1/genética , Ribonucleoproteína Nuclear Pequeña U1/metabolismoRESUMEN
The yeast exosome is a complex of 3' --> 5' exoribonucleases. Sequence analysis identified putative human homologues for exosome components, although several were found only as expressed sequence tags. Here we report the cloning of full-length cDNAs, which encode putative human homologues of the Rrp40p, Rrp41p, and Rrp46p components of the exosome. Recombinant proteins were expressed and used to raise rabbit antisera. In Western blotting experiments, these decorated HeLa cell proteins of the predicted sizes. All three human proteins were enriched in the HeLa cells nucleus and nucleolus, but were also clearly detected in the cytoplasm. Size exclusion chromatography revealed that hRrp40p, hRrp41p, and hRrp46p were present in a large complex. This cofractionated with the human homologues of other exosome components, hRrp4p and PM/Scl-100. Anti-PM/Scl-positive patient sera coimmunoprecipitated hRrp40p, hRrp41p, and hRrp46p demonstrating their physical association. The immunoprecipitated complex exhibited 3' --> 5' exoribonuclease activity in vitro. hRrp41p was expressed in yeast and shown to suppress the lethality of genetic depletion of yeast Rrp41p. We conclude that hRrp40p, hRrp41p, and hRrp46p represent novel components of the human exosome complex.
Asunto(s)
Exorribonucleasas/análisis , Animales , Secuencia de Bases , Núcleo Celular/química , Clonación Molecular , Citoplasma/química , Exorribonucleasas/genética , Complejo Multienzimático de Ribonucleasas del Exosoma , Células HeLa , Humanos , Datos de Secuencia Molecular , Peso Molecular , Pruebas de Precipitina , Proteínas de Unión al ARN , ConejosRESUMEN
A new autoantibody activity, which is almost 100% specific for rheumatoid arthritis (RA), has been found. The essential part of the B-cell epitope is a modified form of arginine (ie citrulline). The conversion of protein-contained arginine to citrulline is an enzymatic process that is carried out by peptidylarginine deiminase (PAD), an enzyme that appears to be hormonally controlled. Because of its remarkable specificity, citrullination and related processes might open new possibilities for studying the aetiology of RA.
Asunto(s)
Artritis Reumatoide/inmunología , Artritis Reumatoide/metabolismo , Autoanticuerpos/metabolismo , Citrulina/metabolismo , Animales , Apoptosis/fisiología , Arginina/metabolismo , Autoanticuerpos/inmunología , Autoantígenos/inmunología , Autoantígenos/metabolismo , Citrulina/inmunología , HumanosRESUMEN
In the past decade, important advances have been made in our knowledge of the composition of human RNase MRP and RNase P complexes. Both ribonucleoprotein particles function as endonucleases and contain RNA components that are structurally related. RNase MRP has been suggested to be involved in the processing of precursor rRNA; RNase P, in the maturation of tRNA. Here we give an overview of current data on the structure and function of human RNase MRP and RNase P particles, with emphasis on their molecular composition. At present, seven protein subunits, probably all associated with both ribonucleoprotein particles, have been isolated and their corresponding cDNAs cloned. Although no known structural motifs can be identified in the amino acid sequences of these proteins, the majority is clearly rich in basic residues. For two protein subunits, a cluster of basic amino acids have been shown to be involved in nucleolar accumulation, whereas another protein, which lacks such a region, probably enters the nucleolus by way of a piggyback mechanism. The binding regions for several of the protein subunits on the RNA have been identified, and the data have been used to create a putative structural model for the RNase MRP particle. The rather obscure situation concerning the association of the autoantigenic Th-40 protein and its possible relationship with one of the subunits, Rpp38, is discussed.