Truncation of C-mip (Tc-mip), a New Proximal Signaling Protein, Induces c-maf Th2 Transcription Factor and Cytoskeleton Reorganization

Several arguments suggest that minimal change nephrotic syndrome (MCNS) results from yet unknown systemic disorder of T cell function. By screening a cDNA library from T cell relapse, we identified a new pleckstrin homology (PH) domain-containing protein encoded by a gene located on chromosome 16q24. Two alternative transcripts were identified. The first species (c-mip) was expressed in fetal liver, kidney, and peripheral blood mononuclear cells (PBMCs), but weakly detected in PBMCs from MCNS patients. The second form (Tc-mip, standing for truncated c-maf inducing protein), corresponds to subtracted transcript and lacks the NH(2)-terminal PH domain. The expression of Tc-mip was restricted to fetal liver, thymus, and MCNS PBMCs where it was specifically recruited in CD4(+) T cells subset. Overexpression of Tc-mip in T cell Jurkat induced c-maf, transactivated the interleukin 4 gene and down-regulated the interferon γ expression, characteristic of a Th2 commitment. Moreover, the overexpression of Tc-mip induced Src phosphorylation, T cell clustering, and a cellular redistribution of the cytoskeleton-associated L-plastin, by a PI3 kinase independent pathway. Tc-mip represents therefore the first identified protein, which links proximal signaling to c-maf induction

Étude des gènes surexprimés au cours du syndrome néphrotique à lésions glomérulaires minimes

Le syndrome néphrotique à lésions glomérulaires minimes (SNLGM) est une glomérulopathie fréquente dont l étiologie est inconnue. Afin d isoler les gènes activés au cours des phases actives de la maladie, nous avons entrepris la construction et le criblage différentiel d une banque d ADN complémentaire réalisé à partir des cellules mononuclées du sang périphérique d un patient. L étude de certains des gènes ainsi isolés a permis l identification d une voie de signalisation recrutée dans les lymphocytes T des patients en poussée de SNLGM impliquant une nouvelle protéine adaptatrice que nous avons appelé Tc-mip (c-maf inducing protéin). Nous avons également mis en évidence une anomalie de maturation de certains ARN messagers dans les lymphocytes T des patients liée à un défaut d expression des SR protéines SRp75 et SRp 40. Ces travaux permettent, grâce à la stratégie utilisée, une progression significative dans notre compréhension de l immunopathologie du SNLGM.Minimal change nephrotic syndrome (MCNS) is a giomerulopathy of unknown origin. In order to identify gene involved in the disase, we performed a substractive cDNA library from peripheral blood mononuclear cells of MCNS patient. This strategy Iead us to identify a new signaling pathway in T lymphocyte from MCNS patient including a new cytosolic adaptor protein we named Tc-mip (c-maf inducing protein). We also identify abnomalities in mRNA maturation during MCNS relapse, related to a lack of expression of two SR proteins (SRp4O and SRp75) involved in mRNA splicing. This study highlight the immunopathogeny of MCNS.PARIS12-CRETEIL BU Multidisc. (940282102) / SudocSudocFranceF

Approches moléculaires des dysfonctions lymphocytaires T au cours du syndrome néphrotique à lésions glomérulaires minimes

Des arguments cliniques et expérimentaux suggèrent que le syndrome néphrotique à lésions glomérulaires minimes (SNLGM) implique des dysfonctions lymphocytaires T. Nous avons identifié les voies de régulation NF-kB(p65/p50) et c-maf. Nous avons montré que l induction de c-maf, un facteur de transcription Th2, est associée aux poussées du SNLGM. Cette particularité est liée à l induction spécifique de Tc-mip (Truncated c-maf inducing protein) dans les cellules T des malades. La surexpression de Tc-mip dans les cellules T Jurkat simule les signaux normalement induits par l activation du TCR en entraînant une réorganisation du cytosquelette et un capping, de même qu une expression de c-maf et la transactivation du gène de l IL-4. En revanche, la surexpression de c-mip n entraîne pas ce phénomène et l induction de c-maf reste faible. Les résultats obtenus dans ce travail apportent des éléments nouveaux qui permettent une approche intégrée de la physiopathologie du SNLGM.Clinical and experimental observations suggest that Minimal Change Nephrotic Syndrome (MCNS) results from T lymphocytes dysfunctions. We identified the pathways that regulates NF-KB(p65/p50) and c-maf. We demonstrated that the expression of c-maf, a Th2 trancription factor, is associated with the relapse of MCNS. This distinctive feature correlated with the selective induction of Tc-mip (Truncated c inducing protein) in T cells from MCNS. The overexpression of Tc-mip in Jurkat T cells suggests that Tc-mip simulates the TCR-mediated signals and therefore induces a cytoskeletal reorganization and as well as a capping phenomenon. Also. in these cells Tc-mip, but flot c-mip, activates the expression of c-maf and the transactivation of the IL-4 gene. The results of this work bring new elements to a more integrated approach in the physiopathology of MCNS.PARIS12-CRETEIL BU Multidisc. (940282102) / SudocSudocFranceF

Meiotic human sperm cells express a leucine-rich homologue of Caenorhabditis elegans early embryogenesis gene, Zyg-11.

We cloned a human protein (Hzyg) homologue to Caenorhabditis elegans Zyg-11, an essential protein for cell division at the initial developmental stages of this species, and to a Drosophila melanogaster gene product (Mei-1) which is likely to be involved in meiosis. Hzyg mRNA encodes a protein of 766 amino acids (88 kDa), 14% of which are leucine residues, with some being arranged in four leucine rich repeat motives usually involved in protein-protein interactions. Hzyg is encoded by a single gene, located on chromosome 9q32-q34.1, and transcribed as two mRNA: a 5 kb transcript strongly expressed in testis and skeletal muscle and barely detectable in other human tissues, and an abundant 3.1 kb mRNA detected only in the testis. By using in-situ hybridization and immunohistochemistry, we clearly established the presence of Hzyg expression in pachytene spermatocytes (stage V) and spermatids (stage I and/or II) around the time of meiosis. The cell specific expression of Hzyg transcripts in testis, and the conservation of this gene among distant species, suggest that this protein may have an important role during meiosis

Chromosomal localization of three human poly(A)-binding protein genes and four related pseudogenes.

In humans, the poly(A)-binding proteins (PABPs) comprise a small nuclear isoform and a conserved gene family that displays at least three functional proteins: PABP1, inducible PABP (iPABP), and PABP3, plus four pseudogenes (1, 2, 3, and PABP4). In situ hybridization of PABP3 cDNA as the probe on metaphasic chromosomes have revealed five possible loci for this gene family at 2q21-q22, 13q11-q12, 12q13.3-q15, 8q22, and 3q24-q25. Amplifications of specific DNA fragments from a human-rodent somatic cell hybrid panel have allowed us to associate PABP1 and PABP3 with 8q22 and 13q11-q12, respectively. The iPABP gene has been assigned to chromosome 1. This result, compared with radiation hybrid database information, strengthens the location of this gene to 1p32-p36. The pseudogenes PABP4, 1, and 2 have been assigned to chromosomes 15, 4, and 14, respectively. Three loci detected on chromosome spreads are not associated with any amplified fragment. They might represent other related PABP genes not yet identified

h-Goliath, paralog of GRAIL, is a new E3 ligase protein, expressed in human leukocytes.

In Drosophila, the RING finger protein d-Goliath was originally identified as a transcription factor involved in the embryo mesoderm formation [Bouchard, M.L., Cote, S., 1993. The Drosophila melanogaster developmental gene g1 encodes a variant zinc-finger-motif protein. Gene 125, 205-209]. In mouse, the m-Goliath mRNA level was shown to be increased in growth factor withdrawal-induced apoptosis of myeloid cells [Baker, S.J., Reddy, E.P., 2000. Cloning of murine G1RP, a novel gene related to Drosophila melanogaster g1. Gene 248, 33-40]. Due to its putative function of transcription factor in apoptosis, we cloned the human cDNA for h-Goliath and characterized the expression of the protein in blood and bone marrow cells. The human protein of 419 aa (44 kDa) contains a protease-associated domain, a transmembrane domain and a RING-H2 motif. This structure classifies h-Goliath as a new member of a human family of ubiquitin ligases with GRAIL (gene related to anergy in lymphocytes) as founder. This E3 ligase controls the development of T cell clonal anergy by ubiquitination [Anandasabapathy, N., Ford, G.S., Bloom, D., Holness, C., Paragas, V., Seroogy, C., Skrenta, H., Hollenhorst, M., Fathman, C.G., Soares, L., 2003. GRAIL: an E3 ubiquitin ligase that inhibits cytokine gene transcription is expressed in anergic CD4+ T cells. Immunity 18, 535-547]. In vitro ubiquitination studies support the E3 ubiquitin ligase activity of h-Goliath. In human, the protein is expressed under 3 isoforms, a major one at 28 kDa and two others at 46 and 55 kDa. These proteins come from a common precursor (44 kDa) as we observed using in vitro transcription-translation. Using immunohistochemistry on blood or bone marrow smears, of healthy or leukemia samples, we found that the protein expression was restricted to the cytoplasm of progenitors and fully differentiated leukocyte populations. We did not observe any modification of h-Goliath expression or localization in leukemia. In these cells, this new E3 ubiquitin ligase protein does not seem associated with a differentiation state of the cell or with apoptosis

hH-Rev107, a class II tumor suppressor gene, is expressed by post-meiotic testicular germ cells and CIS cells but not by human testicular germ cell tumors.

By systematic analysis of a human testis library, we have isolated the hH-Rev107-3 cDNA, identical to hH-Rev107-1 cDNA, which was previously described as a class II tumor suppressor gene. In this study, two transcripts (1 and 0.8 kb) were detected by Northern blot in all human tissues, excepted in thymus. The strongest expression was found in testis, skeletal muscle and heart. These two mRNA are probably transcribed from only one gene that we mapped to the q12-q13 region of the chromosome 11. In human testis, hH-Rev107 gene expression was localized, by in situ hybridization, within the round spermatids. To investigate a possible role for hH-Rev107 protein in testicular malignant growth, we examined the expression of this gene in germ cell tumors. A strong hH-Rev107 gene expression was observed in normal testis as well as in samples with preinvasive carcinoma in situ but was completely absent in overt tumors, both seminomas and non-seminomas. By in situ hybridization, CIS was found hH-Rev107 positive and tumor negative. A semi-quantitative assessment of hH-Rev107 mRNA level in testicular germ cell tumors, by RT-PCR, exhibited a ninefold decrease in the gene expression. No gross structural aberrations of hH-Rev107 gene were detected in these human primary tumors. The results suggest that down-regulation of hH-Rev107 may be associated with invasive progression of testicular germ cell tumors

c-mip down-regulates NF-κB activity and promotes apoptosis in podocytes.

International audienceThe mechanisms of podocyte disorders in cases of idiopathic nephrotic syndrome (INS) are complex and remain incompletely elucidated. The abnormal regulation of NF-κB may play a key role in the pathophysiology of these podocyte diseases, but at present, NF-κB has not been thoroughly investigated. In this study, we report that induction of c-mip in podocytes of patients with INS is associated with a down-regulation of RelA, a potent antiapoptotic factor that belongs to the NF-κB family. Overexpression of c-mip in differentiated podocytes promotes apoptosis by inducing caspase-3 activity and up-regulating the proapoptotic protein Bax, whereas the overall levels of the antiapoptotic protein Bcl-2 was concomitantly decreased. The associated overexpression of RelA prevented the proapoptotic effects of c-mip. In addition, the targeted induction of c-mip in podocytes in vivo inhibited the expression of the RelA protein and increased the Bax/Bcl-2 ratio. The expression of both c-mip and active caspase-3 increased in focal and segmental glomerulosclerosis biopsies, and both proteins displayed a close spatial relationship. These results suggest that alterations in NF-κB activity might result from the up-regulation of c-mip and are likely to contribute to podocyte disorders in cases of INS

Absence of the Adaptor Protein PEA-15 Is Associated with Altered Pattern of Th Cytokines Production by Activated CD4+ T Lymphocytes In Vitro, and Defective Red Blood Cell Alloimmune Response In Vivo

International audienceTCR-dependent and costimulation signaling, cell division, and cytokine environment are major factors driving cytokines expression induced by CD4+ T cell activation. PEA-15 15 (Protein Enriched in Astrocyte / 15kDa) is an adaptor protein that regulates death receptor-induced apoptosis and proliferation signaling by binding to FADD and relocating ERK1/2 to the cytosol, respectively. By using PEA-15-deficient mice, we examined the role of PEA-15 in TCR-dependent cytokine production in CD4+ T cells. TCR-stimulated PEA-15-deficient CD4+ T cells exhibited defective progression through the cell cycle associated with impaired expression of cyclin E and phosphoRb, two ERK1/2-dependent proteins of the cell cycle. Accordingly, expression of the division cycle-dependent cytokines IL-2 and IFNγ, a Th1 cytokine, was reduced in stimulated PEA-15-deficient CD4+ T cells. This was associated with abnormal subcellular compartmentalization of activated ERK1/2 in PEA-15-deficient T cells. Furthermore, in vitro TCR-dependent differentiation of naive CD4+ CD62L+ PEA-15-deficient T cells was associated with a lower production of the Th2 cytokine, IL-4, whereas expression of the Th17-associated molecule IL4I1 was enhanced. Finally, a defective humoral response was shown in PEA-15-deficient mice in a model of red blood cell alloimmunization performed with Poly IC, a classical adjuvant of Th1 response in vivo. Collectively, our data suggest that PEA-15 contributes to the specification of the cytokine pattern of activated Th cells, thus highlighting a potential new target to interfere with T cell functional polarization and subsequent immune response