Apoptosis-linked changes in the phosphorylation status and subcellular localization of the spliceosomal autoantigen U1-70K.

Apoptosis consists of highly regulated pathways involving post-translational modifications and cleavage of proteins leading to sequential inactivation of the main cellular processes. Here, we focused on the apoptotic processing of one of the essential components of the mRNA splicing machinery, the U1-70K snRNP protein. We found that at an early stage of apoptosis, before the cleavage of the C-terminal part of the protein by caspase-3, the basal phosphorylation of the Ser140 residue located within the RNA recognition motif, increases very significantly. A caspase-dependent, PP1-mediated dephosphorylation of other serine residues takes place in a subset of U1-70K proteins. The U1-70K protein phosphorylated at Ser140 is clustered in heterogeneous ectopic RNP-derived structures, which are finally extruded in apoptotic bodies. The elaborate processing of the spliceosomal U1-70K protein we identified might play an important role in the regulated breakdown of the mRNA splicing machinery during early apoptosis. In addition, these specific changes in the phosphorylation/dephosphorylation balance and the subcellular localization of the U1-70K protein might explain why the region encompassing the Ser140 residue becomes a central autoantigen during the autoimmune disease systemic lupus erythematosus.

[The spliceosome and its interest for lupus therapy]. / Le splicéosome et son intérêt dans la recherche thérapeutique sur le lupus.

INTRODUCTION: The spliceosome, which is a particle containing a molecule of U-RNA and proteins that are specific to each U ribonuclear particle (U-snRNP) or common to every U-snRNPs, is one of the numerous nuclear targets recognized by the antibodies (Abs) and CD4+ T cells from patients with systemic lupus erythematosus and lupus mice. EXEGESIS: We recently characterized a peptide from the spliceosomal protein U1-70K (sequence 131-151), which is recognized by the Abs and CD4+ T cells from lupus mice and patients. This peptide contains a conserved RNP1 motif, which is also present in other spliceosomal proteins targeted by the Abs from individuals with lupus. We further showed that peptide 131-151 containing a phosphoserine at position 140 (peptide P140) possessed tolerogenic properties in lupus mice and was recognized by the Abs and CD4+ T cells from lupus patients. CONCLUSION: Thanks to its RNP1 motif, the peptide P140 might play an important role in the initiation and perpetuation steps of the humoral and cellular immune response diversification in lupus individuals. Therapeutic and particularly immunomodulating properties of P140 peptide are being evaluated in humans (a phase III clinical trial will be undertaken in the next weeks).

Laboratory protocols for the identification of Th cell epitopes on self-antigens in mice with systemic autoimmune diseases.

T cells play a critical role in both the immunological and clinical manifestations of systemic autoimmune diseases such as systemic lupus erythematosus (SLE). Although in normal mice multiple T cell epitopes have been characterized in several self-proteins, there is little information on the fine specificity of autoreactive T cells in lupus model mice and humans. In SLE-prone mice and humans, the only Th cell epitopes identified at the molecular level in self-antigens concern histones and nucleosomes, and the 70-kD U1-snRNP protein. T cell characterization in certain autoimmune mice such as MRL lpr/lpr and NZB/NZW mice has been largely impaired by their hyporesponsiveness in response to mitogen and minimal IL-2 secretion. In addition, MRL lpr/lpr mice also develop lymphadenopathy characterized by the progressive accumulation of functionally immature CD4(-) CD8(-) T cells. It is therefore important to optimize the methods used to measure T cell proliferation and cytokine production ex vivo in order to identify minimal activation in the presence of appropriate antigen. The protocol described in this article has been used for identifying in young MRL lpr/lpr and NZB/NZW mice a CD4(+) T cell epitope in the murine 70-kD U1-RNP protein.

Key sequences involved in the spreading of the systemic autoimmune response to spliceosomal proteins.

Immune spreading to multiple intracellular antigens is likely to be of primary importance in organ-specific and systemic autoimmune diseases. A number of mechanisms by which immune spreading may occur from only a single autoreactive epitope have been proposed. Search for an initiator or early epitope thus represents an important area of investigation. For example, many studies have focused on the identification of epitopes recognized by the antibodies from both patients with systemic lupus erythematosus (SLE) and lupus-prone mice. Recently, an autoepitope present in the 70K U1 ribonucleo protein (RNP) and recognized by CD4+ T cells from lupus mice has also been identified. Here, we analyze the results of B- and T-cell-epitope mapping studies of several RNPs present in the spliceosome and propose a model of epitope spreading. In this model, a consensus sequence (the RNP motif) conserved in many nuclear, nucleolar and cytoplasmic antigens, might play a role as 'driver' epitope. This hypothesis is based on the observation that this sequence is recognized by CD4+ T cells from lupus mice and is often targeted by autoantibodies, very early during the course of the disease. Targeting this region that is repeated in different self-antigens, might represent an interesting strategy to interfere with the continuous T-cell stimulation and exposure to specific antigens.

B and T cell immune response to small nuclear ribonucleoprotein particles in lupus mice: autoreactive CD4(+) T cells recognize a T cell epitope located within the RNP80 motif of the 70K protein.

Systemic lupus erythematosus is characterized by the presence of high titers of autoantibodies reacting with various components of the U1 small nuclear ribonucleoprotein particle (snRNP). It has been suggested that these antibodies are produced by an antigen-driven mechanism under the dependence of antigen-specific T cells. To investigate the role of T cell help in this process, we sought, with 20 overlapping peptides, the Th epitopes on the U1-70K snRNP in unprimed H-2(k) MRL / lpr lupus mice and immunized CBA normal mice. The peptide 131 - 151 was recognized by both IgG autoantibodies and CD4(+) T cells from 7 - 9-week-old MRL / lpr mice. In this test, antigen-presenting cells (APC) from MRL / lpr mice were required; APC from naive CBA mice failed to stimulate CD4(+) cells from MRL / lpr mice. The potential role of MRL / lpr B cells as APC, the expression of MHC class II molecules at their surface and their activation state (expression of CD69, CD80 / B7-1 and CD86 / B7-2 molecules) were studied. Peptide 131 - 151 bound both I-A(k) and I-E(k) class II molecules and favored an IL-2-positive T cell response but not IFN-gamma, IL-6 and IL-10 secretion. Segment 131 - 151 is localized within the RNP80 motif and contains residues that are highly conserved in many nuclear, nucleolar and cytoplasmic RNA binding proteins.

Murine models of systemic lupus erythematosus: B and T cell responses to spliceosomal ribonucleoproteins in MRL/Fas(lpr) and (NZB x NZW)F(1) lupus mice.

(NZB x NZW)F(1) and MRL/Fas(lpr) lupus mice present a similar phenotype with a spectrum of autoantibodies associated with very severe nephritis. It is thought, however, that in contrast to other lupus-prone mice such as MRL/Fas(lpr) mice, (NZB x NZW)F(1) mice do not generate autoantibodies to ribonucleoproteins (RNP) Sm/RNP. In this study, we demonstrate that contrary to previous reports, the autoimmune response directed against Sm/RNP antigens also occurs in NZB x NZW mice. CD4(+) T cells from unprimed 10-week-old NZB x NZW mice proliferate and secrete IL-2 in response to peptide 131-151 of the U1-70K protein, which is known to contain a T(h) epitope recognized by CD4(+) T cells from MRL/Fas(lpr) mice. Peptide 131-151, which was found to bind I-A(k) and I-E(k) class II MHC molecules, also bound both I-A(d) and I-E(d) molecules. This result led us to also re-evaluate longitudinally the anti-Sm/RNP antibody response in NZB x NZW mice. We found that 25-week-old mice do produce antibodies reacting with several small nuclear and heterogeneous nuclear (hn) RNP proteins, such as SmD1, U1-70K and hnRNP A2/B1 proteins. The fine specificity of these antibodies was studied with overlapping synthetic peptides. The same antigenically positive and negative peptides were characterized in MRL/Fas(lpr) and NZB x NZW mice in the three proteins. This new finding can help to understand the mechanisms involved in the development of the anti-Sm/RNP antibody response and, particularly, the role played by non-MHC genes in this autoimmune response.

B and T cell responses to the spliceosomal heterogeneous nuclear ribonucleoproteins A2 and B1 in normal and lupus mice.

Autoantibodies directed against spliceosomal heterogeneous nuclear ribonucleoproteins (hnRNPs) are a typical feature of rheumatoid arthritis, systemic lupus erythematosus, and mixed-connective tissue disease. With the aim of investigating a potential pathogenic role of these Abs, we have studied the Ab response to A2/B1 hnRNPs in different murine models of lupus. The specificity of anti-A2/B1 Abs was tested with a series of 14 overlapping synthetic peptides covering the region 1-206 of A2 that contains most of the epitopes recognized by patients' Abs. A major epitope recognized very early during the course of the disease by Abs from most of MRL lpr/lpr mice but not from other lupus mice and from mice of different MHC haplotypes immunized against B1 was identified in residues 50-70. This peptide contains a highly conserved sequence RGFGFVTF also present in other hnRNPs and small nuclear ribonucleoproteins. Abs reacting with a second A2 epitope identified in residues 35-55 were detectable several weeks later, suggesting an intramolecular B cell epitope spreading during the course of the disease. We identified several T cell epitopes within the region 35-175 that generated an effective Th cell response with IL-2 and IFN-gamma secretion in nonautoimmune CBA/J mice sharing the same MHC haplotype H-2k as MRL/lpr mice. None of the peptides stimulated T cells primed in vivo with B1. Because Abs to peptide 50-70 were detected significantly earlier than Abs reacting with other A2 peptides and the protein itself, it is possible that within the protein, this segment contains residues playing an initiator role in the induction of the anti-A2/B1 and antispliceosome Ab response.