Search

Autoimmune diseases are numerous and heterogeneous, with a broad spectrum of clinical presentations and unpredictable courses. Treatment options include such medications as analgesics and nonsteroidal antiinflammatory drugs, disease-modifying antirheumatic drugs, biological agents, and glucocorticoids. If the disease is diagnosed early, the major treatment goal is remission with no active inflammation and no functional deterioration. Numerous mouse and human studies have improved our understanding of the contribution of different immune mediators to the pathogenesis of autoimmune diseases. Despite these advances, the main causes of the breakdown of immune tolerance that lead to the development of autoimmune diseases remain largely unknown. A large proportion of the risk for developing an autoimmune disease is attributable to genetic factors. For example, HLA regions contribute to approximately half of the genetic susceptibility for rheumatoid arthritis (RA).7 Genomewide association studies have identified variants in potentially pathogenic genes in non-HLA regions, including PTPN22 8 [protein tyrosine phosphatase, non-receptor type 22 (lymphoid)], the TRAF1–C5 locus [ TRAF1 (TNF receptor-associated factor 1) to C5 (complement component 5)], PADI4 (peptidyl arginine deiminase, type IV), and STAT4 (signal transducer and activator of transcription 4). Other polymorphisms located in genes coding for the cytokines tumor necrosis factor (TNF), interleukin-1 (IL-1), IL-6, IL-4, and IL-5 seem to be related to an aggressive disease phenotype. Epigenetics and microRNAs are receiving increasing attention as mechanisms that interact with the genome in leading to the persistent inflammatory response in autoimmune disease. The clinical management of autoimmune diseases presents a considerable challenge to healthcare providers. The lack of definition of disease subsets in individuals with early autoimmune disease is one of the key gaps in the field. In this Q&A, 3 experts discuss recent developments in the area of early diagnosis of autoimmune diseases and their implications for improved treatment strategies. Can you highlight some recent …

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.

The most specific biomarker associated with the diagnosis of rheumatoid arthritis (RA) is autoantibodies to citrullinated peptides/proteins (ACPA). Though recognized as an important marker of progressive erosive disease its use has been hampered by doubt about what is a positive versus a negative reaction in the several assays that have become available commercially. This review intends to indicate that the CCP2 assay has the highest specificity and sensitivity in stratified studies that encompass sera from RA patients and non-RA inflammatory controls compared to other ACPA tests. Still, larger and strictly stratified studies are highly warranted to substantiate this conclusion.

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.

To identify peripheral blood autoantibody and cytokine profiles that characterise clinically relevant subgroups of patients with early rheumatoid arthritis using arthritis antigen microarrays and a multiplex cytokine assay.Serum samples from 56 patients with a diagnosis of rheumatoid arthritis of6 months' duration were tested. Cytokine profiles were also determined in samples from patients with psoriatic arthritis (PsA) and ankylosing spondylitis (n = 21), and from healthy individuals (n = 19). Data were analysed using Kruskal-Wallis test with Dunn's adjustment for multiple comparisons, linear correlation tests, significance analysis of microarrays (SAM) and hierarchical clustering software.Distinct antibody profiles were associated with subgroups of patients who exhibited high serum levels of tumour necrosis factor (TNF)alpha, interleukin (IL)1beta, IL6, IL13, IL15 and granulocyte macrophage colony-stimulating factor. Significantly increased autoantibody reactivity against citrullinated epitopes was observed in patients within the cytokine "high" subgroup. Increased levels of TNFalpha, IL1alpha, IL12p40 and IL13, and the chemokines eotaxin/CCL11, monocyte chemoattractant protein-1 and interferon-inducible protein 10, were present in early rheumatoid arthritis as compared with controls (p0.001). Chemokines showed some of the most impressive differences. Only IL8/CXCL8 concentrations were higher in patients with PsA/ankylosing spondylitis (p = 0.02).Increased blood levels of proinflammatory cytokines are associated with autoantibody targeting of citrullinated antigens and surrogate markers of disease activity in patients with early rheumatoid arthritis. Proteomic analysis of serum autoantibodies, cytokines and chemokines enables stratification of patients with early rheumatoid arthritis into molecular subgroups.

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.

Rheumatoid arthritis (RA) is a common systemic autoimmune disease with a prevalence of about 1% worldwide [1]. The American College of Rheumatology (ACR) criteria for the classification of RA [2] are not very well suited to diagnose RA at an early stage of the disease [3, 4], because these criteria rely heavily on the expression of clinical symptoms of RA. In early RA these clinical parameters are often not (yet) manifest. Therefore, a specific and sensitive (serological) marker, which is present very early in the disease, is needed. A good marker should ideally not only indicate the development of the disease, but also be able to predict its erosive or non-erosive progression. The serological parameter that meets these requirements for a good and useful marker for early RA is the anti-citrullinated protein antibody. The sensitivity of this antibody is comparable to that of the rheumatoid factor (RF) (approximately 80%), but its specificity is much higher, about 98%. Several assays have been developed to detect this class of autoantibodies, which are termed anti-CCP because the most sensitive test is based upon cyclic citrullinated peptides. This review will discuss the potential of this autoantibody system for the diagnosis and prognosis of RA.

Item does not contain fulltext 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.

Rheumatoid arthritis (RA) is a chronic, destructive autoimmune disease affecting the joints. With more sophisticated and effective therapies becoming available and with the understanding that early intervention is crucial in preventing irreversible joint damage, it is more and more important to diagnose RA at a very early stage in the disease. To facilitate diagnosis during the early stages of the disease, when often not all clinical symptoms are manifest, a good serological marker is needed. Antibodies directed to citrullinated proteins provide this ability. The most sensitive assay to detect these antibodies is the so-called anti-cyclic citrullinated peptide (CCP) enzyme-linked immunosorbent assay (ELISA) assay. In this review, the diagnostic and prognostic potential and the general utility in clinical practice of anti-CCP antibodies are discussed. Furthermore, we elaborate on the mechanisms involved in the generation of citrullinated autoantigens and the possible role of the anti-CCP antibodies and their antigens in the disease.

Introduction Autoimmune serology has become a potentially powerful tool for experienced clinicians. However, it currently is not being used optimally due to lack of widely used standardized assays, local working conditions, traditions, different technology, cost constraints by medical insurers, and ability on the part of clinicians to correctly interpret the results of certain assays. New serologic technologies and assays are developed by laboratory scientists and the commercial industry at a very fast pace and may eventually lead to better reproducibility in confirming a diagnosis and estimating prognosis, ultimately improving the quality of clinical care. To achieve this goal, the accuracy of such testing must be demonstrated in studies involving sufficiently large patient populations before a new assay can be accepted for clinical use. In the past, there have been very few studies with a prospective, unbiased, multicenter design. Thus, the clinical accuracy of certain laboratory diagnostic studies is still unknown. To be useful in a clinical day-to-day diagnostics setting, the differential diagnostic potential and performance characteristics of a test must be known so that clinical misinterpretation, false diagnoses, and potentially harmful treatment can be avoided. The degree to which an assay will give false-positive or false-negative results is important. To aid in developing a followup and therapeutic strategy in a given patient, the prognostic significance of identifying a certain autoantibody in a patient with clinical findings of an autoimmune disease should be known. Presence of autoantibodies indicating a poor prognosis in the clinical setting of early signs of disease should lead to appropriate followup and monitoring of risk manifestations. Such findings should also evoke early awareness of the potential need for effective treatment, before irreversible organ damage takes place. Factual knowledge about these matters necessitates close collaboration between patients, experienced and motivated clinicians, and laboratory scientists, as well as cooperation with the diagnostics industry. Once a diagnostic assay has been developed, standardized postmarketing surveillance and quality assurance by manufacturers and laboratories alike should be mandatory (1).

Contains fulltext : 58699.pdf (Publisher’s version ) (Open Access) 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.

Contains fulltext : 57913.pdf (Publisher’s version ) (Open Access) 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.

Item does not contain fulltext 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.

Item does not contain fulltext The human La (SS-B) autoantigen is an abundantly expressed putative RNA chaperone, functioning in various intracellular processes involving RNA. To further explore the molecular mechanisms by which La functions in these processes, we performed large-scale immunoprecipitations of La from HeLa S100 extracts using the anti-La monoclonal antibody SW5. La-associated proteins were subsequently identified by sequence analysis. This approach allowed the identification of DDX15 as a protein interacting with La. DDX15, the human ortholog of yeast Prp43, is a member of the superfamily of DEAH-box RNA helicases that appeared to interact with La both in vivo and in vitro. The region needed for the interaction with La partly overlaps the DEAH-box domain of DDX15. Immunofluorescence data indicated that endogenous DDX15 accumulates in U snRNP containing nuclear speckles in HEp-2 cells. Surprisingly DDX15 also accumulates in the nucleoli of HEp-2 cells. Moreover, DDX15 and La seem to colocalize in the nucleoli. Regions of DDX15 involved in nuclear, nuclear speckle, and nucleolar localization are located within the N- and C-terminal regions flanking the DEAH-box. RNA coprecipitation experiments indicated that DDX15 is associated with spliceosomal U small nuclear RNAs in HeLa cell extracts. The possible functional implications of the interaction between La and DDX15 are discussed.

SUMMARY Coimmunoprecipitation studies with SW5, a frequently used and specific mouse monoclonal antibody (mAb) directed against the human La autoantigen, led to the identification of a functionally unrelated 80 000 MW protein, designated early endosome antigen 2 (EEA2). EEA2 appeared to be directly targeted by mAb SW5. Because an RNA-binding domain, a structural element of La containing the SW5-epitope, was not discernable in the primary structure of EEA2, the SW5-epitope on EEA2 was determined. Coiled-coil region 3 of EEA2 appeared to contain the epitope recognized by SW5. The SW5 epitope regions of La and EEA2 share a limited sequence homology and probably share a higher degree of structural similarity at the tertiary level. Most likely, the most critical determinants for recognition by SW5 reside in elements adopting alpha-helical conformations. These data indicate that the application of specific mAbs to purify and characterize (functionally) interacting proteins can be severely obscured by the cross-reactivity of mAbs with structurally, but not functionally, similar proteins.

Item does not contain fulltext 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.

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.

Objective To understand the regulation of anti–citrulline-containing peptide antibody (anti-CCP) production in rheumatoid arthritis (RA), production of anti-CCP by B cells derived from peripheral blood (PB), bone marrow (BM), and synovial fluid (SF) was examined. Methods Purified PB and SF B cells were isolated by negative selection and then cultured in the absence or presence of L–CD40 ligand cells and interleukin-10 or anti-CD3–activated T cells. Total IgM and IgM–anti-CCP were detected after 14 days of culture by enzyme-linked immunosorbent assay. Enzyme-linked immunospot assays were performed to analyze the frequency of cells that spontaneously produced IgM–anti-CCP in BM and SF B cells. Results IgM–anti-CCP autoantibodies were induced in PB B cells from healthy controls and RA patients following coculture with activated T cells or application of the CD40 activation system, whereas no production could be detected when PB B cells were cultured in the absence of a stimulus. SF and BM B cells from anti-CCP–seropositive RA patients, but not anti-CCP–seronegative patients, actively produced IgM–anti-CCP without stimulation. The frequency of spontaneous production of IgM–anti-CCP among the IgM-secreting cells ranged from 2.2% to 25%. Conclusion These results indicate the presence of B cell precursors for anti-CCP autoantibodies that are able to produce antibodies upon stimulation in the PB B cell repertoire of healthy controls and patients with RA. In contrast, B cells that actively secreted anti-CCP were specifically present in the BM and SF compartment of anti-CCP–seropositive RA patients. The local presence of anti-CCP–secreting cells in the inflamed joints provides evidence for an antigen-driven maturation of CCP-specific B cells at the site of inflammation in RA.

Contains fulltext : 216407.pdf (Publisher’s version ) (Open Access) We report the first construction of two combinatorial human phage display libraries derived from malaria-immune patients. Specific single-chain Fv fragments (scFv) against Pfs48/45, a gamete surface protein of the sexual stages of Plasmodium falciparum, were selected and analyzed extensively. The selected scFv reacted with the surface of extracellular sexual forms of the parasite and showed Pfs48/45 reactivity on immunoblot. The scFv inhibit binding of human malaria sera to native Pfs48/45 from gametocytes. Moreover, the scFv bind to target epitopes of Pfs48/45 exposed in natural infections. Sequence analysis of eight scFv clones specific for epitope III of Pfs48/45 revealed that these clones could be divided into one V(H) family-derived germ-line gene (V(H)1) and two V(L) family segments (V(L)2 and V(K)I).

Pre-rRNA processing in eukaryotic cells requires participation of several snoRNPs. These include the highly conserved and abundant U3 snoRNP, which is essential for synthesis of 18S rRNA. Here we report the characterization of Rrp9p, a novel yeast U3 protein, identified via its homology to the human U3-55k protein. Epitope-tagged Rrp9p specifically precipitates U3 snoRNA, but Rrp9p is not required for the stable accumulation of this snoRNA. Genetic depletion of Rrp9p inhibits the early cleavages of the primary pre-rRNA transcript at A0, A1, and A2 and, consequently, production of 18S, but not 25S and 5.8S, rRNA. The hU3-55k protein can partially complement a yeast rrp9 null mutant, indicating that the function of this protein has been conserved. Immunoprecipitation of extracts from cells that coexpress epitope-tagged Rrp9p and various mutant forms of U3 snoRNA limits the region required for association of Rrp9p to the U3-specific box B/C motif. Box C is essential, whereas box B plays a supportive role.

The U1-70K protein is specifically bound to stemloop I of the U1 small nuclear RNA contained in the U1 small nuclear ribonucleoprotein complex (U1 snRNP), which is involved in the splicing of pre-mRNA. All components of the U1 snRNP complex, including the U1-70K protein, are important autoantigens in patients with systemic lupus erythematosus (SLE) and mixed connective tissue disease (MCTD). Here we describe for the first time the selection and characterization of recombinant human anti-U1-70K single chain autoantibody fragments (anti-hU1-70K scFv) from autoimmune patient-derived phage display antibody libraries. All scFv specifically recognize parts of the hU1-70K protein and its apoptotic 40-kDa cleavage product. In Western blotting assays a number of scFv preferentially recognize the 40-kDa apoptotic cleavage fragment of the U1-70K protein, suggesting a possible involvement of this apoptotic cleavage product in the autoimmune response of patients. The germ-line gene usage of these recombinant autoantibodies was also determined. Using several U1-70K deletion and point mutants of both human (h) and Drosophila melanogaster (Dm) origin, it was established that the U1-70K epitope that is recognized by the anti-hU1-70K scFv is located within the RNA binding domain.

Objective. Proteins that are phosphorylated during apoptosis are commonly precipitated by autoantibodies found in the sera of patients with systemic lupus erythematosus. We sought to determine whether scleroderma autoantigens such as small nucleolar RNPs (snoRNP) also associate with phosphoproteins in response to various cellular stressors. Methods. We screened a panel of monoclonal antibodies derived from mice exposed to mercury, a well-characterized murine model of the anti-snoRNP autoimmune response, for the ability to selectively precipitate phosphoproteins from radiolabeled lysates prepared from Jurkat T cells subjected to stressful stimuli. Results. Monoclonal antibodies reactive with snoRNPs precipitated a phosphoprotein complex (pp42, pp34, and pp23) from lysates prepared from apoptotic cells. Several novel phosphoproteins (pp62 and pp18) were also observed. The phosphorylation and/or recruitment of these proteins to the snoRNP complex is induced by multiple apoptotic stimuli (e.g., Fas ligation, anisomycin, or ultraviolet irradiation), an effect that is blocked by overexpression of Bcl-2. We were unable to demonstrate an association of the phosphoprotein complex with snoRNPs in cells treated with the xenobiotic agent mercury. The snoRNP-associated phosphoprotein complex is composed of serine/arginine (SR) splicing factors, including SRp40. Conclusion. The association of phosphorylated SR proteins with snoRNPs in cells undergoing apoptosis suggests that the immune response to fibrillarin that characterizes a subset of patients with scleroderma may be related to cell death induced by apoptotic stimuli (e.g., Fas ligation, irradiation, or chemical toxins), or by exposure to mercury.

The U1 small nuclear ribonucleoprotein (snRNP) particle is the most abundant member of the spliceosomal snRNPs. Human U1 snRNP is comprised of 10 proteins and the 164-nucleotide U1 small nuclear RNA (U1RNA) and is required for splicing of pre-mRNA (38). One of the U1 snRNP-specific proteins, the U1A protein, contains two evolutionarily conserved RNA recognition motifs (RRMs) characteristic of a large family of proteins involved in the biosynthesis of cellular RNA (reviewed in reference 37). The signature motifs for the RRM family consist of two ribonucleoprotein (RNP) sequences, RNP1 and RNP2, which are the most conserved features of this family. The N-terminal RRM of U1A is, together with some flanking amino acids, necessary and sufficient for binding to the loop part of stem-loop 2 (SL2) sequence AUUGCAC of U1RNA (22, 27, 28). The structure of the N-terminal RRM of the U1A protein (amino acids [aa] 2 to 95) has been solved both by X-ray crystallography and by nuclear magnetic resonance (NMR) and consists of a β1α1β2β3α2β4 structure in which the β strands form a sheet with the highly conserved RNP1 and RNP2 motifs located in the two central β strands, β3 and β1, respectively (14, 23). An additional α helix (helix 3; hereafter referred to as helix C) is present when a longer fragment of the U1A protein is analyzed (aa 2 to 102; reference 15). Using both NMR and X-ray crystallography, the structure of U1A aa 2 to 98 in complex with SL2 of U1RNA has also been solved (1, 2, 15, 24). In this structure, the RNA loop lies across the β sheet, fitting into a groove formed between loop 3 (connecting β2 and β3) and the C-terminal portion of the RRM domain. In spite of intensive investigation, the C-terminal RRM (aa 202 to 283) of U1A does not seem to have any affinity for RNA (21). The U1 snRNP particle is involved in the first step of spliceosome formation, in which it binds to the 5′ splice site of the pre-mRNA (reviewed in reference 18). It is possible that U1A is not essential for the splicing reaction, since in vitro splicing can still proceed in the absence of U1A. It has been suggested, however, that the U1A protein might play an important role in 5′ and 3′ splice site communication (33). In vertebrates, the U1A protein is able to regulate the polyadenylation of U1A pre-mRNA, thereby regulating its own expression level (4). The 3′ untranslated region (UTR) of the human U1A pre-mRNA contains a 50-nucleotide region, designated the polyadenylation-inhibitory element (PIE) RNA, whose sequence and structure are conserved in vertebrates. Located within the PIE RNA are two stretches of seven unpaired nucleotides designated loops 1 and 2, each being able to bind one molecule of U1A protein. Although one of the loops, when studied in isolation, has 27-fold lower affinity for U1A than the other, it was demonstrated that two molecules of U1A bind with high affinity (Kd, ∼0.1 nM) to PIE RNA, indicative of cooperative RNA binding (4, 35). The resulting (U1A)2-PIE RNA complex inhibits addition of the poly(A) tail to the U1A pre-mRNA by specifically inhibiting the enzyme poly(A) polymerase (PAP) (10). Inhibition of polyadenylation requires both the C-terminal 20 residues of PAP and aa 103 to 115 of U1A. A model has been proposed in which the U1A autoregulatory complex inhibits PAP by bringing two copies of U1A aa 103 to 115 into close proximity (11, 34). In support of this model, it was found that two molecules, but not one molecule, of U1A bound to PIE RNA inhibit PAP. Likewise, a monomeric peptide consisting of U1A aa 103 to 115 is unable to inhibit PAP; however, upon increasing its local concentration by conjugation to bovine serum albumin (BSA), this same peptide becomes a potent inhibitor of PAP (11). PAP inhibition by the BSA-peptide conjugate does not require PIE RNA, suggesting that the main role of PIE RNA in PAP inhibition is to bring the two U1A proteins into close proximity. Indeed, the unusual secondary structure of PIE RNA is not essential for inhibition (11, 34). Independent of the biochemical analysis, the determination of the structure by NMR of one molecule of U1A (aa 2 to 98) bound to PIE RNA (1) has also led to the proposal (based on modeling) that the two PIE-RNA-bound U1A proteins make extensive protein-protein interactions throughout the N-terminal 100 residues (16). Here we show, by using both the yeast two-hybrid system and in vitro assays, that U1A is able to homodimerize in the absence of RNA sequences that specifically bind U1A (i.e., SL2 of U1RNA and PIE RNA). Dimerization requires two regions, both located in the N-terminal 115 residues. Mutations of the second region (aa 103 to 115) which abolish dimerization also result in either reduction or complete loss of cooperative binding to PIE RNA but with no effect on U1A binding as a monomer to PIE RNA. These same mutations also result in loss of U1A's ability to inhibit polyadenylation. A dimeric form of a peptide containing these residues also inhibits polyadenylation. A model integrating these results will be presented explaining how the U1A autoregulatory complex functions.

Systemic lupus erythematosus (SLE) is an autoimmune disease that is characterized by the presence of high immunoglobulin serum titers, but the mechanism by which these arise remains unclear. It has been suggested that the disease is associated with specific antibody features, including variable gene use, the presence of charged complementarity-determining region residues and/or an aberrant process of secondary light chain rearrangement. To study this in more detail, we compared variable, diversity and joining gene segment use, somatic mutation, and heavy and light chain pairings in single peripheral IgG(+) B cells between one normal (209 B cells) and two SLE (156 B cells) donors. In contrast to others, we found no systematic differences, indicating that the memory B cell repertoires in normal and SLE donors are shaped in a similar way.

We have sequenced the heavy and light chain genes from 365 IgG+ B cells and found that 24 (6.5 %) contain somatically introduced insertions or deletions. These insertions and deletions are clustered at “hot-spots” in the antigen-binding site and frequently result in the creation of new combinations of canonical loop structures or entirely new loops that are not present in the human germline repertoire, but are similar to those seen in other species. Somatic insertion and deletion therefore provides a further mechanism for introducing structural diversity into antibodies in addition to somatic point mutation and receptor editing, which have small (single amino acid changes) and large (chain replacement) impacts on structural diversity, respectively.

Abs to U1 RNA are frequently found in patients suffering from systemic lupus erythematosus overlap syndromes and Ab titers correlate with disease activity. We describe the isolation of the first human anti-U1 RNA autoantibodies from a combinatorial IgG library made from the bone marrow of a systemic lupus erythematosus patient. With the use of phage display technology, two anti-U1 RNA single-chain variable fragment (scFv) Abs were selected. Both high affinity anti-U1 RNA Ab fragments (Kd ∼ 1 nM) recognize stem II of U1 RNA and were derived from the same heavy chain gene (VH3–11) and the same λ (3r) light chain gene although somatic mutations, predominantly present in the complementarity-determining regions, are different. Experiments, in which the heavy chain genes of both anti-U1 RNA scFvs were reshuffled with the original light chain repertoire of the patient resulted, after selection on stem loop II, in a large number of RNA-binding Ab fragments. All these stem loop II-specific RNA binding clones used a similar, but not identical, 3r λ light chain. When scFvs were selected from the reshuffled libraries by stem loop IV, representing the other autoantigenic site of U1 RNA, most selected Ab clones did react with stem loop IV, but no longer with stem loop II. The stem loop IV-reactive Ab clones contained different, not 3r-related, light chains. These results point to a major role for the light chain in determining the sequence specificity of these disease-related anti-U1 RNA Abs. The possibility that secondary light chain rearrangements are involved in this autoimmune response is discussed.

Contains fulltext : 272864.pdf (Publisher’s version ) (Closed access) RNase MRP is a ribonucleoprotein particle involved in the processing of pre-rRNA. The RNase MRP particle is structurally highly related to the RNase P particle, which is involved in pre-tRNA processing. Their RNA components fold into a similar secondary structure and they share several protein subunits. We have identified and characterised human and mouse cDNAs that encode proteins homologous to yPop4p, a protein subunit of both the yeast RNase MRP and RNase P complexes. The human Pop4 cDNA encodes a highly basic protein of 220 amino acids. Transfection experiments with epitope-tagged hPop4 protein indicated that hPop4 is localised in the nucleus and accumulates in the nucleolus. Immunoprecipitation assays using extracts from transfected cells expressing epitope-tagged hPop4 revealed that this protein is associated with both the human RNase MRP and RNase P particles. Polyclonal rabbit antibodies raised against recombinant hPop4 recognised a 30 kDa protein in total HeLa cell extracts and specifically co-immunoprecipitated the RNA components of the RNase MRP and RNase P complexes. Finally we showed that anti-hPop4 immunoprecipitates possess RNase P enzymatic activity. Taken together, these data show that we have identified a protein that represents the human counterpart of the yeast Pop4p protein.

Objective To determine the performance characteristics of enzyme-based immunoassay (EIA) kits for the detection of antinuclear and other autoantibodies of defined specificities. Methods Nine manufacturers of EIA kits to detect antibodies of defined specificities participated in a study in which they received coded sera from the Centers for Disease Control and Prevention. These coded sera contained different dilutions of antibody of one specificity mixed with sera containing antibodies of other specificities. The manufacturers were asked to use their standard technology to determine antibody content and send the data to a committee of the International Union of Immunological Societies for analysis. The data were analyzed for sensitivity and specificity in the detection of anti–double-stranded DNA (anti-dsDNA), anti–single-stranded DNA, antihistone, anti-Sm, anti–U1 RNP, anti-SSA/Ro, anti-SSB/La, anti–Scl-70 (DNA topoisomerase I), anticentromere, and anti–Jo-1 antibodies. In addition, replicate samples were included in the coded sera to evaluate the precision of each EIA method. Results Lack of sensitivity and specificity was most evident in the anti-dsDNA and anti-Sm kits, although 2 kits for anti-dsDNA achieved acceptable sensitivity and specificity. Generally, anti-SSA/Ro, anti-SSB/La, anti–Scl-70, anticentromere, and anti–Jo-1 kits performed well. Many false-positive results were obtained with a multiple myeloma serum containing cryoprecipitates, but multiple myeloma sera without cryoprecipitates presented no problem in the EIA system. Precision, based on evaluation of replicate samples, varied from very good to poor. Conclusion No single manufacturer was clearly superior to others in terms of their products' overall sensitivity, specificity, and precision. Areas that needed improvement were in kits for the detection of antibodies to dsDNA and to Sm antigen. Some EIA kits achieved good sensitivity and specificity. Individual manufacturers were informed of the performance of their respective kits so they could take measures to correct perceived deficiencies and thus improve the reliability of a group of important diagnostic assays used in the evaluation of systemic rheumatic diseases.

Proteins cleaved by apoptotic caspases are commonly recognized by autoantibodies found in the serum of patients with rheumatic disease. We report that the 72-kDa signal recognition particle (SRP) protein, a rare target of autoantibodies found in the serum of patients with dermatomyositis and systemic lupus erythematosus, is rapidly cleaved in Jurkat T cells treated with apoptotic (i.e. Fas ligation, treatment with gamma or ultraviolet radiation, or co-culture with anisomycin or staurosporine) but not proliferative (CD3 cross-linking) stimuli. Cleavage of SRP 72 produces a 66-kDa amino-terminal fragment and a 6-kDa carboxyl-terminal fragment that is selectively phosphorylated on serine residues. Cleavage of SRP 72 is prevented by chemical and peptide caspase inhibitors, and by overexpression of bcl-2, an inhibitor of apoptotic cell death. Analysis of the carboxyl terminus of SRP 72 has identified a putative cleavage site (SELD/A) for group III caspases, and carboxyl-terminal serine residues that are highly conserved in phylogeny. Both serine phosphorylation and caspase cleavage of SRP 72 are observed in cells derived from human, dog, rat, and mouse. Canine SRP 72 is cleaved in vitro by recombinant caspase 3 but retains the ability to mediate transport of a signal peptide-containing protein into the endoplasmic reticulum lumen. The 72-kDa component of the SRP joins a growing list of autoantigens that undergo post-translational modifications during programmed cell death.

The eukaryotic nucleolus contains a large number of small RNA molecules (snoRNAs) which, in the form of small nucleolar ribonucleoprotein complexes (snoRNPs), are involved in the processing and modification of pre-rRNA. The most abundant and one of the best-conserved snoRNAs is the U3 RNA. So far, only one human U3 snoRNA-associated protein, fibrillarin, has been characterized. Previously, the U3 snoRNPwas purified from CHO cells, and three proteins of 15, 50, and 55 kDa were found to copurify with the U3 snoRNA (B. Lübben, C. Marshallsay, N. Rottmann, and R. Lührmann, Nucleic Acids Res. 21:5377–5385, 1993). Here we report the cDNA cloning and characterization of the human U3 snoRNP-associated 55-kDa protein. The isolated cDNA codes for a novel nucleolar protein which is specifically associated with the U3 snoRNA. This protein, referred to as hU3-55k, is the first characterized U3 snoRNP-specific protein from humans. hU3-55k is a new member of the family of WD-40 repeat proteins and is conserved throughout evolution. It appears that the C-terminal end of hU3-55k is required for nucleolar localization and U3 snoRNA binding.

Telomerase, a ribonucleoprotein complex, adds hexameric repeats called “telomeres” to the growing ends of chromosomal DNA. Characterization of mammalian telomerase has been elusive because of its low level of expression. We describe a bioinformatics approach to enrich and characterize the human telomerase complex. Using local sequence homology search methods, we detected similarity of the Tetrahymena p80 subunit of telomerase with the autoantigen Ro60. Antibodies to Ro60 immunoprecipitated the telomerase activity. Ro60 and p80 proteins were cross-recognizable by antibodies to either protein. Telomerase activity and the RNA component of telomerase complex were localized to a doublet in a native gel from the Ro60 antibody-precipitated material. The enriched material showed specific binding to a TTA GGG probe in vitro in an RNA template-dependent manner. Polyclonal antibodies to the doublet also immunoprecipitated the telomerase activity. These results suggest an evolutionary conservation of the telomerase proteins.

Objective. To define the fine specificity of the 10 reference sera used for determination of antinuclear antibodies (ANA) and ANA subsets which are available from the Arthritis Foundation (AF) and from the Centers for Disease Control and Prevention (CDC). Methods. AF/CDC sera were assessed by experienced laboratory staff, using indirect immunofluorescence and Western blotting. Results. The original assignment of fluorescence patterns to 4 reference sera was confirmed, and the fluorescence intensities were determined using reference fluorescent beads. On Western blots, sera AF/CDC2 (anti—SS-B/La) and AF/CDC7 (anti—SS-A/Ro) did not detect Ro antigens, sera AF/CDC9 and AF/CDC10 appeared to be monospecific anti—Scl-70 and anti—Jo-1 sera, respectively, serum AF/CDC4 (anti—U1 small nuclear RNP) recognized the 70-kd band, and serum AF/CDC5 recognized the Sm antigen with its multiple bands. Semiquantitative analyses revealed that AF/CDC5, AF/CDC2, and AF/CDC10 were strongly reactive sera, whereas AF/CDC4 and AF/CDC9 were much weaker and should be used at lower dilutions on Western blots. Conclusion. The AF/CDC ANA reference sera, originally described as reference reagents for indirect immunofluorescence and double immunodiffusion techniques, are also useful for Western blotting. The data presented herein further support the use of these sera for reference purposes.

Genes for the snRNP proteins U1-70K, U1-A, Sm-B′/B, Sm-D1 and Sm-E have been isolated from various metazoan species. The genes for Sm-D1 and Sm-E, which were isolated from a murine and human source respectively, appear to belong to a multigene family. It has been suggested that also for the mammalian U1-C protein such a multigene family exists. With the human U1-C cDNA as a probe, two genes containing sequences homologous to the probe sequence were isolated from a mouse genomic library. Simultaneously, a murine U1-C cDNA was isolated from a mouse cDNA library. This 0.74 kb cDNA contains an open reading frame (ORF) of 477 bp encoding a polypeptide of 159 amino acids (aa) which differs at only one position (position 65) from the human U1-C protein. One of the isolated U1-C genes contains an ORF as well and shares 92% nucleotide sequence identity with the mouse U1-C cDNA. The features of this gene, in particular the absence of introns, the acquisition of a 3′ poly(A) tail and flanking direct repeats, indicate that it represents a processed pseudogene. At the predicted aa sequence level, substitutions of conserved residues at functionally important positions are observed, strongly suggesting that expression of this gene would not lead to a functional polypeptide. The second U1-C gene appeared to be a pseudogene as well because it is also intronless and contains a frameshift mutation compared to the ORF in the mouse U1-C cDNA. The characterization of these two pseudogenes points to the existence of a U1-C multigene family in mice. Furthermore, comparison of aa sequences of the murine, human and Xenopus U1-C shows that the protein is highly conserved through evolution. Since the Xenopus U1-C differs from the two mammalian counterparts solely at a number of positions in the C-terminal region, it can be concluded that aa changes are less well tolerated in the N-terminal region of U1-C than in the rest of the protein.

Y RNAs are small cytoplasmic RNAs which are components of the Ro ribonucleoprotein complexes in higher eukaryotes. These complexes are frequently recognized by antibodies present in autoimmune sera. In this study we analysed the occurrence of Y RNAs in various mammalian and human cell lines and erythrocytes by means of hybridization with human Y RNA probes. Y RNAs homologous to their human counterparts, both in length and in sequence, were detected in all mammalian cells analysed. While hY1 and hY3 analogues were found in all cells, Y4 and Y5 RNA could not be detected in rodent cells. In addition, Y5 RNA was absent from bovine cells. Attempts to determine the sequence of rat Y RNAs by genomic cloning resulted in the isolation of a presumptive Y1 RNA pseudogene. Analysis of the hY RNA content of various human cell lines showed that all four human Y RNAs were present in all cell lines examined. However, the relative levels to which these RNAs were expressed showed marked differences.

Antibodies against naked U1RNA can be found in sera from patients with overlap syndromes of systemic lupus erythematosus (SLE) in addition to antibodies directed to the proteins of U1 ribonucleoproteins (U1RNP). We investigated the reactivity of these U1RNA specific autoantibodies with the native U1RNP particle both in vitro and inside the cell. For this purpose a method was developed to purify human autoantibodies directed to specific regions of U1RNA. The antibodies are specifically directed to either stemloop II or stemloop IV of U1RNA and do not crossreact with protein components of U1RNP. Both types of antibody are able to precipitate from cell extracts native U1snRNPs containing most, if not all, protein components. Immunofluorescence patterns indicate that the antigenic sites on the RNA, i.e. the stem of stemloop II and the loop of stemloop IV, are also available after fixation of the cells. Immunoelectron microscopy employing anti-stemloop IV antibodies and purified, complete U1snRNP particles showed that stemloop IV is located within the body of the U1RNP complex, which also comprises the Sm site and the common Sm proteins. The anti-U1RNA autoantibodies described in this paper recognize native U1RNP particles within the cell and can therefore be used as tools to study mechanisms involved in splicing of pre-mRNA.

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.

Rheumatoid arthritis (RA) is characterized by chronic inflammation of the joints and the presence of anti-citrullinated protein autoantibodies (ACPA). ACPA are very specific for RA and are involved in its pathophysiology. Five steps, all of which are supported by experimental evidence, can be distinguished during the development of the chronic inflammation in RA. Step 1: During inflammation a large influx of inflammatory cells takes place. These cells will ultimately die via apoptosis. When the dying cells are not cleared efficiently, citrullinated proteins and citrullinating enzymes are released into the extracellular space. Step 2: Extracellular proteins are citrullinated by these enzymes. Step 3: Only individuals with a certain genetic background produce ACPA. Step 4: Arthritis is induced by the formation of immune complexes of ACPA and citrullinated proteins. Step 5: These immune complexes stimulate the inflammation, which leads to the recruitment of new inflammatory cells. This establishes a vicious cycle, the RA cycle.

Following infection of HeLa cells with adenovirus type 5 the cellular La protein becomes predominantly associated with the virally encoded RNA polymerase III products VAI, and VAII, while most of the host RNA polymerase II (e.g. U1, U2, U4, U5 and mRNA) and RNA polymerase III transcription (e.g. U6 and pre-tRNAs) ceases. Other RNA polymerase III products such as the cellular Ro RNAs continue to be transcribed and assembled into ribonucleoprotein complexes containing the Ro (SS-A) antigens. Using a 32P-pulse chase-labeled, adenovirus-infected HeLa cellular extract as a source of antigen, anti-La (SS-B) and anti-Ro (SS-A) antibodies can be detected simultaneously using an immunoprecipitation assay. In the present study this method was found to be more sensitive in detecting anti-La antibodies then counter immunoelectrophoresis and immunoblotting. In studies of sera from patients suffering from rheumatic diseases the percentage positive for anti-La antibody was significantly elevated using this method, especially in patients with systemic lupus erythematosus.

The U1 small nuclear ribonucleoprotein (snRNP) contains three specific proteins denoted 70K, A and C, in addition to the common proteins. Specific functions of these proteins are not known although recently protein C was shown to be involved in the binding of U1 snRNP to the 5' splice site of a pre-mRNA. Unlike proteins A and 70K, U1-C lacks an RNA binding domain (RNP-80 motif) and does not appear to bind directly to U1 snRNA. However, at the amino terminal end protein C contains a zinc finger-like structure of the CC-HH type found in transcription factor TF IIIA. Several lines of evidence indicate that the zinc finger-like structure is essential for the binding of protein C to U1 snRNP particles: i) deletion analysis of protein C showed that the N-terminal 45 amino acids are sufficient for binding to U1 snRNPs, ii) modification of the cysteine residues in the N-terminal domain with N-ethylmaleimide and iii) single point mutations of the cysteines and histidines contributing to the putative zinc finger abolished binding of protein C to U1 snRNPs. Interestingly, unlike the proteins U1-A and U1-70K the U1-C protein is unable to bind to naked U1 snRNA. On the other hand it is shown that protein C does not bind to the known protein constituents of the U1 particle without the U1 snRNA being present. These data indicate that the binding of protein C to U1 snRNP is dependent on the presence of both the U1 snRNA and one or more of the U1 snRNP proteins.

The literature of the last 4 years confirms that the anti-CCP2 test is a very useful marker for the early and specific diagnosis of rheumatoid arthritis (RA). The anti-CCP2 test is very specific for RA (95-99%) and has sensitivity comparable to that of the rheumatoid factor (70-75%). The antibodies can be detected very early in the disease and can be used as an indicator for the progression and prognosis of RA. In this review, these interesting properties and some future possibilities of this diagnostic test are discussed.

Members of the family of peptidylarginine deiminases (PADs, EC 3.5.3.15) catalyze the posttranslational modification of peptidylarginine into peptidylcitrulline. Citrulline-containing epitopes have been shown to be major and specific targets of autoantibodies produced by rheumatoid arthritis patients. Recently, the citrullination of histone proteins by PAD enzyme was reported to influence gene expression levels. These findings greatly increase the interest in the PAD enzymes and their activities. A few procedures to monitor PAD activity in biological samples have been described previously. However, these assays either have low sensitivity or are rather laborious. Here we describe a reliable and reproducible method for the determination of PAD activity in both purified and crude samples. The method is based on the quantification of PAD-dependent citrullination of peptides, immobilized in microtiter plates, using antibodies that are exclusively reactive with the reaction product(s). Our results demonstrate that this antibody-based assay for PAD activity, called ABAP, is very sensitive and can be applied to monitor PAD activity in biological samples.

Rpp20 and Rpp25 are subunits of the human RNase MRP and RNase P endoribonucleases belonging to the Alba superfamily of nucleic acid binding proteins. These proteins, which bind very strongly to each other, transiently associate with RNase MRP. Here, we show that the Rpp20-Rpp25 heterodimer is resistant to both high concentrations of salt and a nonionic detergent. The interaction of Rpp20 and Rpp25 with the P3 domain of the RNase MRP RNA appeared to be strongly enhanced by their heterodimerization. Coimmunoprecipitation experiments demonstrated that only a single copy of each of these proteins is associated with the RNase MRP and RNase P particles in HEp-2 cells. Both proteins accumulate in the nucleoli, which in case of Rpp20 is strongly dependent on its interaction with Rpp25. Finally, the results of overexpression and knock-down experiments indicate that their expression levels are codependent. Taken together, these data indicate that the Rpp20-Rpp25 heterodimerization regulates their RNA-binding activity, subcellular localization, and expression, which suggests that their interaction is also crucial for their role in RNase MRP/P function.

Recent studies have implicated the dying cell as a potential reservoir of modified autoantigens that might initiate and drive systemic autoimmunity in susceptible hosts. A number of subunits of the exosome, a complex of 3'--5' exoribonucleases that functions in a variety of cellular processes, are recognized by the so-called anti-PM/Scl autoantibodies, found predominantly in patients suffering from an overlap syndrome of myositis and scleroderma. Here we show that one of these subunits, PM/Scl-75, is cleaved during apoptosis. PM/Scl-75 cleavage is inhibited by several different caspase inhibitors. The analysis of PM/Scl-75 cleavage by recombinant caspase proteins shows that PM/Scl-75 is efficiently cleaved by caspase-1, to a smaller extent by caspase-8, and relatively inefficiently by caspase-3 and caspase-7. Cleavage of the PM/Scl-75 protein occurs in the C-terminal part of the protein at Asp369 (IILD369 [see text] G), and at least a fraction of the resulting N-terminal fragments of PM/Scl-75 remains associated with the exosome. Finally, the implications of PM/Scl-75 cleavage for exosome function and the generation of anti-PM/Scl-75 autoantibodies are discussed.

RNase MRP is a eukaryotic endoribonuclease involved in nucleolar and mitochondrial RNA processing events. RNase MRP is a ribonucleoprotein particle, which is structurally related to RNase P, an endoribonuclease involved in pre-tRNA processing. Most of the protein components of RNase MRP have been reported to be associated with RNase P as well. In this study we determined the association of these protein subunits with the human RNase MRP and RNase P particles by glycerol gradient sedimentation and coimmunoprecipitation. In agreement with previous studies, RNase MRP sedimented at 12S and 60–80S. In contrast, only a single major peak was observed for RNase P at 12S. The analysis of individual protein subunits revealed that hPop4 (also known as Rpp29), Rpp21, Rpp20, and Rpp25 only sedimented in 12S fractions, whereas hPop1, Rpp40, Rpp38, and Rpp30 were also found in 60–80S fractions. In agreement with their cosedimentation with RNase P RNA in the 12S peak, coimmunoprecipitation with VSV-epitope-tagged protein subunits revealed that hPop4, Rpp21, and in addition Rpp14 preferentially associate with RNase P. These data show that hPop4, Rpp21, and Rpp14 may not be associated with RNase MRP. Furthermore, Rpp20 and Rpp25 appear to be associated with only a subset of RNase MRP particles, in contrast to hPop1, Rpp40, Rpp38, and Rpp30 (and possibly also hPop5), which are probably associated with all RNase MRP complexes. Our data are consistent with a transient association of Rpp20 and Rpp25 with RNase MRP, which may be inversely correlated to its involvement in pre-rRNA processing.

Objective. Because rheumatoid arthritis (RA) is a heterogeneous autoimmune disease in terms of disease manifestations, clinical outcomes, and therapeutic responses, we developed and applied a novel antigen microarray technology to identify distinct serum antibody profiles in patients with RA. Methods. Synovial proteome microarrays, containing 225 peptides and proteins that represent candidate and control antigens, were developed. These arrays were used to profile autoantibodies in randomly selected sera from 2 different cohorts of patients: the Stanford Arthritis Center inception cohort, comprising 18 patients with established RA and 38 controls, and the Arthritis, Rheumatism, and Aging Medical Information System cohort, comprising 58 patients with a clinical diagnosis of RA of