Oxidative stress-induced modifications of histidyl-tRNA synthetase affect its tRNA aminoacylation activity but not its immunoreactivity.

The aminoacyl-tRNA synthetases are ubiquitously expressed enzymes that catalyze the esterification of amino acids to their cognate tRNAs. Autoantibodies against several aminoacyl-tRNA synthetases are found in autoimmune polymyositis and dermatomyositis patients. Because necrosis is often found in skeletal muscle biopsies of these patients, we hypothesized that cell-death-induced protein modifications may help in breaking immunological tolerance. Since cell death is associated with oxidative stress, the effect of oxidative stress on the main myositis-specific autoantibody target Jo-1 (histidyl-tRNA synthetase; HisRS) was studied in detail. The exposure of Jurkat cells to hydrogen peroxide resulted in the detection of several oxidized methionines and one oxidized tryptophan residue in the HisRS protein, as demonstrated by mass spectrometry. Unexpectedly, the tRNA aminoacylation activity of HisRS appeared to be increased upon oxidative modification. The analysis of myositis patient sera did not lead to the detection of autoantibodies that are specifically reactive with the modified HisRS protein. The results of this study demonstrate that the Jo-1/HisRS autoantigen is modified under oxidative stress conditions. The consequences of these modifications for the function of HisRS and its autoantigenicity are discussed.

Autoantigen microarrays for multiplex characterization of autoantibody responses.

We constructed miniaturized autoantigen arrays to perform large-scale multiplex characterization of autoantibody responses directed against structurally diverse autoantigens, using submicroliter quantities of clinical samples. Autoantigen microarrays were produced by attaching hundreds of proteins, peptides and other biomolecules to the surface of derivatized glass slides using a robotic arrayer. Arrays were incubated with patient serum, and spectrally resolvable fluorescent labels were used to detect autoantibody binding to specific autoantigens on the array. We describe and characterize arrays containing the major autoantigens in eight distinct human autoimmune diseases, including systemic lupus erythematosus and rheumatoid arthritis. This represents the first report of application of such technology to multiple human disease sera, and will enable validated detection of antibodies recognizing autoantigens including proteins, peptides, enzyme complexes, ribonucleoprotein complexes, DNA and post-translationally modified antigens. Autoantigen microarrays represent a powerful tool to study the specificity and pathogenesis of autoantibody responses, and to identify and define relevant autoantigens in human autoimmune diseases.

Cartilage-hair hypoplasia-associated mutations in the RNase MRP P3 domain affect RNA folding and ribonucleoprotein assembly.

Cartilage-hair hypoplasia (CHH) is caused by mutations in the gene encoding the RNA component of RNase MRP. Currently it is unknown how these mutations affect the function of this endoribonuclease. In this study we investigated the effect of mutations in the P3 domain on protein binding and RNA folding. Our data demonstrate that a number of P3 nucleotide substitutions reduced the efficiency of its interaction with Rpp25 and Rpp20, two protein subunits binding as a heterodimer to this domain. The CHH-associated 40G>A substitution, as well as the replacement of residue 47, almost completely abrogated Rpp25 and Rpp20 binding in different assays. Also other CHH-associated P3 mutations reduced the efficiency by which the RNase MRP RNA is bound by Rpp25-Rpp20. These data demonstrate that the most important residues for binding of the Rpp25-Rpp20 dimer reside in the apical stem-loop of the P3 domain. Structural analyses by NMR not only showed that this loop may adopt a pseudo-triloop structure, but also demonstrated that the 40G>A substitution alters the folding of this part of the P3 domain. Our data are the first to provide insight into the molecular mechanism by which CHH-associated mutations affect the function of RNase MRP.

Role of pre-rRNA base pairing and 80S complex formation in subnucleolar localization of the U3 snoRNP.

In the nucleolus the U3 snoRNA is recruited to the 80S pre-rRNA processing complex in the dense fibrillar component (DFC). The U3 snoRNA is found throughout the nucleolus and has been proposed to move with the preribosomes to the granular component (GC). In contrast, the localization of other RNAs, such as the U8 snoRNA, is restricted to the DFC. Here we show that the incorporation of the U3 snoRNA into the 80S processing complex is not dependent on pre-rRNA base pairing sequences but requires the B/C motif, a U3-specific protein-binding element. We also show that the binding of Mpp10 to the 80S U3 complex is dependent on sequences within the U3 snoRNA that base pair with the pre-rRNA adjacent to the initial cleavage site. Furthermore, mutations that inhibit 80S complex formation and/or the association of Mpp10 result in retention of the U3 snoRNA in the DFC. From this we propose that the GC localization of the U3 snoRNA is a direct result of its active involvement in the initial steps of ribosome biogenesis.

Rabip4' is an effector of rab5 and rab4 and regulates transport through early endosomes.

We describe the characterization of an 80-kDa protein cross-reacting with a monoclonal antibody against the human La autoantigen. The 80-kDa protein is a variant of rabip4 with an N-terminal extension of 108 amino acids and is expressed in the same cells. For this reason, we named it rabip4'. rabip4' is a peripheral membrane protein, which colocalized with internalized transferrin and EEA1 on early endosomes. Membrane association required the presence of the FYVE domain and was perturbed by the phosphatidylinositol 3-kinase inhibitor wortmannin. Expression of a dominant negative rabip4' mutant reduced internalization and recycling of transferrin from early endosomes, suggesting that it may be functionally linked to rab4 and rab5. In agreement with this, we found that rabip4' colocalized with the two GTPases on early endosomes and bound specifically and simultaneously to the GTP form of both rab4 and rab5. We conclude that rabip4' may coordinate the activities of rab4 and rab5, regulating membrane dynamics in the early endosomal system.

Experimental autoimmune encephalomyelitis induction in peptidylarginine deiminase 2 knockout mice.

During the development of multiple sclerosis the destruction of the myelin sheath surrounding the neurites is accompanied by citrullination of several central nervous system (CNS) proteins, including myelin basic protein and glial fibrillary acidic protein. In experimental autoimmune encephalomyelitis (EAE), a disease induced in animals by immunization with proteins or peptides from the CNS, the animals develop symptoms similar to multiple sclerosis (MS). The increased levels of citrullinated CNS proteins associated with MS are also observed during the development of EAE. To study the role of CNS protein citrullination in EAE development, we induced EAE with a peptide derived from myelin oligodendrocyte glycoprotein (MOG(35-55)) in mice lacking the peptidylarginine deiminase 2 (PAD2) protein, because this enzyme was the most likely candidate to be involved in catalyzing CNS protein citrullination in the diseased state. Even though the PAD2 knockout mice displayed a dramatic reduction in the amount of citrullination present in the CNS, indicating that PAD2 is indeed responsible for the majority of detectable citrullination observed in EAE, the development of EAE was not impaired by genetic deletion of PAD2, suggesting that PAD2 catalyzed citrullination is not essential to the development of EAE.

Autoantibodies to citrullinated antigens in (early) rheumatoid arthritis.

Rheumatoid arthritis (RA) is a common, systemic autoimmune disease characterized by chronic inflammation of the synovium, that can lead to progressive joint destruction and in many cases results in severe disability and poor quality of life. With the availability of more sophisticated and effective therapies and with increasing evidence that the first few months of disease represent an unique therapeutic opportunity and that such early therapeutic intervention is crucial in preventing irreversible joint damage, it is widely accepted that early and accurate diagnosis of RA is critical in disease management. Within the last three years a growing number of publications have reported that the second generation anti-CCP (cyclic citrullinated peptide) test may become the marker of choice for diagnosing early RA as it appears to be highly specific for the disease with a sensitivity comparable to the widely used but less specific rheumatoid factor test. Additionally, anti-CCP2 positivity can predict future development of RA in both asymptomatic individuals and in patients with undifferentiated arthritis. Furthermore, antibody levels at presentation can correlate with progression to erosive disease.

Mutual interactions between subunits of the human RNase MRP ribonucleoprotein complex.

The eukaryotic ribonuclease for mitochondrial RNA processing (RNase MRP) is mainly located in the nucleoli and belongs to the small nucleolar ribonucleoprotein (snoRNP) particles. RNase MRP is involved in the processing of pre-rRNA and the generation of RNA primers for mitochondrial DNA replication. A closely related snoRNP, which shares protein subunits with RNase MRP and contains a structurally related RNA subunit, is the pre-tRNA processing factor RNase P. Up to now, 10 protein subunits of these complexes have been described, designated hPop1, hPop4, hPop5, Rpp14, Rpp20, Rpp21, Rpp25, Rpp30, Rpp38 and Rpp40. To get more insight into the assembly of the human RNase MRP complex we studied protein-protein and protein-RNA interactions by means of GST pull-down experiments. A total of 19 direct protein-protein and six direct protein-RNA interactions were observed. The analysis of mutant RNase MRP RNAs showed that distinct regions are involved in the direct interaction with protein subunits. The results provide insight into the way the protein and RNA subunits assemble into a ribonucleoprotein particle. Based upon these data a new model for the architecture of the human RNase MRP complex was generated.

The human Imp3 and Imp4 proteins form a ternary complex with hMpp10, which only interacts with the U3 snoRNA in 60-80S ribonucleoprotein complexes.

Ribosome biogenesis requires a vast number of trans-acting factors many of which are required for the chemical modification and processing of the pre-rRNA component. The U3 snoRNP complex is required for the early cleavage steps in pre-rRNA processing. We have cloned cDNAs encoding the human and mouse homologs of the yeast U3 snoRNP-associated proteins Imp3 and Imp4. Both human proteins localize to nucleoli and interact with the U3 snoRNA. The results of complementation experiments show that, in contrast to mouse Imp4, mouse Imp3 can partially alleviate the growth defect of the corresponding yeast null strain, indicating that the role of Imp3 in pre-rRNA processing is evolutionarily conserved. The results of density gradient centrifugation experiments show that, in contrast to hU3-55K, the human Imp3 and Imp4 proteins predominantly interact with the U3 snoRNA in 60-80S ribonucleoprotein complexes. In addition, we have found that hImp3, hImp4 and hMpp10 can form a stable hetero-trimeric complex in vitro, which is generated by direct interactions of both hImp3 and hImp4 with hMpp10. The analysis of hImp3 and hImp4 mutants indicated that their binding to hMpp10 correlates with their nucleolar accumulation, strongly suggesting that the formation of the ternary complex of hImp3, hImp4 and hMpp10 is required for their association with nucleolar components.

Citrullination of central nervous system proteins during the development of experimental autoimmune encephalomyelitis.

Immunization of mammals with central nervous system (CNS)-derived proteins or peptides induces experimental autoimmune encephalomyelitis (EAE), a disease resembling the human autoimmune disease multiple sclerosis (MS). Both diseases are accompanied by destruction of a part of the of the myelin sheaths, which surround neurites in the CNS. Previous studies in MS have described alterations in the citrullination of myelin basic protein, one of the main protein constituents of the myelin sheath. Here, we show that, also during the development of EAE in mice, hypercitrullination occurs in the areas of the spinal cord that show the highest degree of inflammation and that myelin basic protein and glial fibrillary acidic protein are among the hypercitrullinated proteins. We conclude that hypercitrullination of myelin proteins in the CNS is a common phenomenon in demyelinating disease. Hypercitrullination may cause conformational changes in proteins, so the affected proteins may be involved in the pathogenesis of CNS autoimmune disease by acting as autoreactive T-cell epitopes. This is the first report in which hypercitrullination of CNS proteins in EAE is described and in which proteins other than myelin basic protein are reported to be citrullinated during autoimmune-mediated CNS inflammation.

Protein-protein interactions between human exosome components support the assembly of RNase PH-type subunits into a six-membered PNPase-like ring.

The exosome is a complex of 3'-->5' exoribonucleases, which functions in a variety of cellular processes, all requiring the processing or degradation of RNA. Here we present a model for the assembly of the six human RNase PH-like exosome subunits into a hexameric ring structure. In part, this structure is on the basis of the evolutionarily related bacterial degradosome, the core of which consists of three copies of the PNPase protein, each containing two RNase PH domains. In our model three additional exosome subunits, which contain S1 RNA-binding domains, are positioned on the outer surface of this ring. Evidence for this model was obtained by the identification of protein-protein interactions between individual exosome subunits in a mammalian two-hybrid system. In addition, the results of co-immunoprecipitation assays indicate that at least two copies of hRrp4p and hRrp41p are associated with a single exosome, suggesting that at least two of these ring structures are present in this complex. Finally, the identification of a human gene encoding the putative human counterpart of the bacterial PNPase protein is described, which suggests that the exosome is not the eukaryotic equivalent of the bacterial degradosome, although they do share similar functional activities.

Protein-protein interactions of hCsl4p with other human exosome subunits.

The exosome is a complex of 3'-->5' exoribonucleases, which functions in a variety of cellular processes, all requiring the processing or degradation of RNA. We demonstrate that the two human proteins hCsl4p and hRrp42p, which have been identified on the basis of their sequence homology with Saccharomyces cerevisiae proteins, are associated with the human exosome. By mammalian two-hybrid and GST pull-down assays, we show that the hCsl4p protein interacts directly with two other exosome proteins, hRrp42p and hRrp46p. Mutants of hCsl4p that fail to interact with either hRrp42p or hRrp46p are also not able to associate with exosome complexes in vivo. These results indicate that the association of hCsl4p with the exosome is mediated by protein-protein interactions with hRrp42p and hRrp46p.

Nucleolin associates with a subset of the human Ro ribonucleoprotein complexes.

Ro RNPs are evolutionarily conserved, small cytoplasmic RNA-protein complexes with an unknown function. In human cells, Ro RNPs consist of one of the four hY RNAs and two core proteins: Ro60 and La. Recently, the association of hnRNP I and hnRNP K with particles containing hY1 and hY3 RNAs has been described. The association of three other proteins, namely calreticulin, Ro52 and RoBPI, with (subsets of) the Ro RNPs is still controversial. To gain more insight into the composition and function of the Ro RNPs, we have immunopurified these particles from HeLa cell extracts using monoclonal antibodies against Ro60 and La. Using this approach, we have identified the RNA-binding protein nucleolin as a novel subunit of Ro RNP particles containing hY1 or hY3 RNA, but not hY4 and hY5 RNA. Using an in vitro hY RNA-binding assay we established that the internal pyrimidine-rich loop of hY1 and hY3 RNA is essential for the association of nucleolin. The binding is critically dependent on the presence of all four RNP motifs of nucleolin, but not of the C-terminal RGG-box. Moreover, we demonstrate that, in contrast to nucleolin and hnRNP K, nucleolin and hnRNP I can bind simultaneously to the internal pyrimidine-rich loop of hY1 RNA. We postulate that nucleolin functions in the biogenesis and/or trafficking of hY1 and hY3 RNPs through the nucleolus and subsequent transport to the cytoplasm.

Human recombinant anti-La (SS-B) autoantibodies demonstrate the accumulation of phosphoserine-366-containing la isoforms in nucleoplasmic speckles.

Using the recombinant La (SS-B) protein or a phosphorylated peptide derived thereof 27 La-specific human recombinant autoantibodies were selected from anti-La-positive systemic lupus erythematosus and systemic sclerosis patient-derived combinatorial phage display antibody libraries. Binding of these anti-La antibodies to various isoforms of the La protein present in normal and apoptotic cell extracts was analysed by Western blotting. Twenty-four of the selected antibodies recognize most, if not all isoforms of La, whereas three are exclusively reactive with the protein phosphorylated at serine-366. Sequence analysis of the selected antibodies showed a restricted spectrum of diversity in their VH germline gene usage. Remarkably, the recombinant antibodies recognizing exclusively the phosphoserine-366-containing isoform of La displayed a spleckled nucleoplasmic staining pattern in immunofluorescence analysis of HeLa and HEp-2 cells. This pattern differed markedly from those obtained with anti-La antibodies recognizing all isoforms of the La protein. Colocalization experiments with marker antibodies for spliceosomal UsnRNPs and RNA polymerase III subunits revealed that the anti-phosphorylated La antibodies stain the same nucleoplasmic speckles as anti-UsnRNP antibodies. In contrast to anti-UsnRNP antibodies the anti-phosphorylated La antibodies did not stain the Cajal bodies. In addition, no colocalization of phosphorylated La with RNA polymerase III was observed. Potential functional implications of the accumulation of phosphorylated La in nucleoplasmic speckles are discussed.

The fate of the U1 snRNP autoantigen during apoptosis: implications for systemic autoimmunity.

Recent studies have implicated the dying cell as a potential reservoir of modified autoantigens that may initiate and drive systemic autoimmunity in susceptible hosts. The uridine-rich small nuclear ribonucleoprotein complex is a common target for autoantibodies present in the serum of patients with systemic lupus erythematosus and SLE-overlap syndromes. Four modifications occurring in this complex during apoptosis have been described to date: the caspase-mediated cleavage of the U1-70K protein, the U1 RNA and the Sm-F protein, and the association with hyperphosphorylated SR proteins. In addition, the U1 snRNP complex has been shown to translocate from its normal subcellular localization to apoptotic bodies near the surface of cells undergoing apoptosis. This redistribution might facilitate exposure of the modified components of the U1 snRNP complex to the immune system when the clearance of apoptotic cell remnants is somehow disturbed. The modifications in the U1 snRNP components during apoptosis might represent the initial epitopes to which an immune response is generated and may be the trigger for the production of autoantibodies to this complex in patients with SLE or SLE-overlap syndromes. Therefore, it can be hypothesized that the exposure of elevated levels of apoptotically modified U1 snRNP to the immune system of a genetically susceptible individual might lead to the breaking of immunologic tolerance towards the U1 snRNP complex.

How citrullination invaded rheumatoid arthritis research.

Citrullination and the immune response to citrullinated proteins have been fundamental for the early recognition of rheumatoid arthritis by serological tests and a better understanding of its pathophysiology. In the first years after the initial publications, the focus was on the antibodies directed to citrullinated proteins. It is now realized that citrullinating enzymes and citrullinated proteins may have important roles in the maintenance of the inflammatory processes in the joints. There is also accumulating evidence for a direct role of citrullination in tissue destruction in the rheumatoid synovium. Here we will discuss the development and importance of anti-citrullinated protein antibodies in rheumatoid arthritis as well as recent findings implicating citrullination in the pathophysiology of rheumatoid arthritis.

Myositis-specific autoantibodies: detection and clinical associations.

In recent years, the detection and characterization of (novel) autoantibodies is becoming increasingly important for the early diagnosis of autoimmune diseases. The idiopathic inflammatory myopathies (IIM, also indicated with myositis) are a group of systemic autoimmune disorders that involve inflammation and weakness of skeletal muscles. One of the hallmarks is the infiltration of inflammatory cells in muscle tissues. A number of myositis-specific autoantibodies have been identified and these may be associated with distinct IIM subclasses and clinical symptoms. Here, we review all myositis-specific autoantibodies identified today as well as their target proteins, together with their clinical associations in IIM patients. Post-translational modifications that might be associated with the generation of autoantibodies and the development of the disease are discussed as well. In addition, we describe well established autoantibody detection techniques that are currently being used in diagnostic laboratories, as well as novel multiplexed methods. The latter techniques provide great opportunities for the simultaneous detection of distinct autoantibodies, but may also contribute to the identification of novel autoantibody profiles, which may have additional diagnostic and prognostic value. The ongoing characterization of novel autoantibody specificities emphasizes the complexity of processes involved in the development of such autoimmune diseases.

Anti-CCP antibodies: the past, the present and the future.

Rheumatoid arthritis (RA) is an autoimmune disease characterized by autoantibodies against citrullinated antigens. The importance of citrulline for the epitopes bound by these autoantibodies, referred to as ACPA (anti-citrullinated peptide/protein antibodies), was first described in 1998. In addition to citrullinated proteins, cyclic citrullinated peptides (CCP) can also be used as test substrates for detecting ACPA. The standard test for these antibodies is the second-generation CCP (CCP2) test, which is one of the best in terms of sensitivity and specificity. The generation of ACPA is an early event in the disease course, and is dependent on the presence of certain MHC class II alleles. ACPA in the inflamed synovium have been shown to associate with citrullinated antigens to form immune complexes, resulting in progression of the inflammatory process. The involvement of ACPA in the chronicity of RA is probably the reason why ACPA-positive patients have a more erosive disease course than ACPA-negative patients. The presence of ACPA has been included in the 2010 RA classification criteria. Thus, it is important to further standardize ACPA testing, for example by including an internal serum standard, which may lead to a better distinction between low and high ACPA levels.