Association of the Epstein-Barr virus latent membrane protein 1 with lipid rafts is mediated through its N-terminal region.

The latent membrane protein 1 (LMP1) encoded by the Epstein-Barr virus acts like a constitutively activated receptor of the tumor necrosis factor receptor (TNFR) family and is enriched in lipid rafts. We showed that LMP1 is targeted to lipid rafts in transfected HEK 293 cells, and that the endogenous TNFR-associated factor 3 binds LMP1 and is recruited to lipid rafts upon LMP1 expression. An LMP1 mutant lacking the C-terminal 55 amino acids (Cdelta55) behaves like the wild-type (WT) LMP1 with respect to membrane localization. In contrast, a mutant with a deletion of the 25 N-terminal residues (Ndelta25) does not concentrate in lipid rafts but still binds TRAF3, demonstrating that cell localization of LMP1 was not crucial for TRAF3 localization. Moreover, Ndelta25 inhibited WT LMP1-mediated induction of the transcription factors NF-kappaB and AP-1. Morphological data indicate that Ndelta25 hampers WT LMP1 plasma membrane localization, thus blocking LMP1 function.

CHARACTERIZATION OF EPSTEIN-BARR VIRUS-ENCODED LATENT MEMBRANE PROTEIN 1

Le virus d'Epstein-Barr (EBV), un virus de la famille des gammaherpesvirus, infecte plus de 95% de la population adulte mondiale. EBV est associé à plusieurs types de cancers dont le lymphome de Hodgkin, le lymphome de Burkitt et le carcinome nasopharyngé. La protéine membranaire de latence 1 (LMP1), l'oncogène principal d'EBV, est une protéine membranaire intégrale composée d'une petite extrémité N-terminale cytoplasmique, six segments transmembranaires (TMs) lié par de petites boucles et un long domaine C-terminale cytoplasmique. Le gène de LMP1, BNLF-1, est très polymorphe et plusieurs variants de la protéine LMP1 ont été décrits. Parmi les variants de LMP1 la majeure différence décrite est leur capacité à activer le facteur de transcription NF-κB. Nous avons défini des polymorphismes permettant aux variants d'avoir une activation accrue de NF-κB comparé au prototype B95-8 LMP1. Tous les polymorphismes cruciaux identifiés dans notre étude se trouvent dans les TMs 4 et 5 de LMP1. Nous avons étudié l'implication de chaque paire de TMs dans l'association à la membrane, l'auto-agrégation, la liaison aux partenaires cellulaires de LMP1 TRAF3 et β-TrCP, ainsi que pour NF-κB. De plus, nous avons décrit un nouveau rôle pour LMP1 consistant à inhiber l'activation contrôlée par MAVS de ISRE et du promoteur d'IFNβ. En résumé, nous avons observé que les différentes paires de TMs, ainsi que les deux boucles intracellulaires, ne sont pas équivalents. Dans l'ensemble, notre étude a montré que les TMs jouent un rôle clé dans les interactions protéine-protéine et la signalisation et qu'ils peuvent être considérés comme des régulateurs essentiels des activités de LMP1.

Ubiquitination of the Epstein-Barr virus-encoded latent membrane protein 1 depends on the integrity of the TRAF binding site.

The latent membrane protein 1 (LMP1) encoded by the Epstein-Barr virus functions as a constitutively activated receptor of the tumor necrosis factor receptor family. LMP1 is a short-lived protein that is ubiquitinated and degraded by the proteasome. We have previously shown that LMP1 recruits the adapter protein tumor necrosis factor receptor-associated factor 3 (TRAF3) to lipid rafts. To test if TRAFs are involved in LMP1's ubiquitination, we have mutated the LMP1 CTAR1 site that has been identified as a TRAF binding site. We show that the CTAR1 mutant (CTAR1(-)) is expressed after transfection at a similar level to wild-type LMP1, and behaves as wild-type LMP1 with respect to membrane localization. However, CTAR1(-) does not bind TRAF3. We demonstrate that ubiquitination of CTAR1(-) is significantly reduced when compared to wild-type LMP1. In addition, the expression of wild-type LMP1 induces the ubiquitination, an effect that is significantly reduced when the CTAR1(-) is expressed. Taken together, our results suggest that TRAF proteins are involved in the ubiquitination of LMP1, and that their binding to LMP1 may facilitate their own ubiquitination.

Vaccination of melanoma patients with Melan-A/Mart-1 peptide and Klebsiella outer membrane protein p40 as an adjuvant.

Toll-like receptor ligands are potentially useful adjuvants for the development of clinical T cell vaccination. Here we investigated the novel Toll-like receptor2 ligand P40, the outer membrane protein A derived from Klebsiella pneumoniae. Seventeen human leukocyte antigen-A*0201 positive stage III/IV melanoma patients were vaccinated with P40 and Melan-A/Mart-1 peptide subcutaneously in monthly intervals. Adverse reactions were mild-to-moderate. Fourteen patients received at least 8 vaccinations and were thus evaluable for clinical tumor and immune responses. Seven patients experienced progressive disease, whereas 2 patients had stable disease throughout the trial period, 1 of them with regression of multiple skin metastases. The remaining 5 patients had no measurable disease. Melan-A/Mart-1 specific CD8 T cells were analyzed ex vivo, with positive results in 6 of 14 evaluable patients. Increased percentages of T cells were found in three patients, memory/effector T cell differentiation in 4 patients, and a positive interferon-gamma Elispot assay in 1 patient. Antibody responses to P40 were observed in all patients. We conclude that vaccination with peptide and P40 was feasible and induced ex vivo detectable tumor antigen specific T cell responses in 6 of 14 patients with advanced melanoma.

Cell biological characterization of the malaria vaccine candidate trophozoite exported protein 1.

In a genome-wide screen for alpha-helical coiled coil motifs aiming at structurally defined vaccine candidates we identified PFF0165c. This protein is exported in the trophozoite stage and was named accordingly Trophozoite exported protein 1 (Tex1). In an extensive preclinical evaluation of its coiled coil peptides Tex1 was identified as promising novel malaria vaccine candidate providing the rational for a comprehensive cell biological characterization of Tex1. Antibodies generated against an intrinsically unstructured N-terminal region of Tex1 and against a coiled coil domain were used to investigate cytological localization, solubility and expression profile. Co-localization experiments revealed that Tex1 is exported across the parasitophorous vacuole membrane and located to Maurer's clefts. Change in location is accompanied by a change in solubility: from a soluble state within the parasite to a membrane-associated state after export to Maurer's clefts. No classical export motifs such as PEXEL, signal sequence/anchor or transmembrane domain was identified for Tex1.

Cell biological characterization of the malaria vaccine candidate trophozoite exported protein 1.

In a genome-wide screen for alpha-helical coiled coil motifs aiming at structurally defined vaccine candidates we identified PFF0165c. This protein is exported in the trophozoite stage and was named accordingly Trophozoite exported protein 1 (Tex1). In an extensive preclinical evaluation of its coiled coil peptides Tex1 was identified as promising novel malaria vaccine candidate providing the rational for a comprehensive cell biological characterization of Tex1. Antibodies generated against an intrinsically unstructured N-terminal region of Tex1 and against a coiled coil domain were used to investigate cytological localization, solubility and expression profile. Co-localization experiments revealed that Tex1 is exported across the parasitophorous vacuole membrane and located to Maurer's clefts. Change in location is accompanied by a change in solubility: from a soluble state within the parasite to a membrane-associated state after export to Maurer's clefts. No classical export motifs such as PEXEL, signal sequence/anchor or transmembrane domain was identified for Tex1.

Sorting mechanisms regulating membrane protein traffic in the apical transcytotic pathway of polarized MDCK cells.

The transcytotic pathway followed by the polymeric IgA receptor (pIgR) carrying its bound ligand (dIgA) from the basolateral to the apical surface of polarized MDCK cells has been mapped using morphological tracers. At 20 degreesC dIgA-pIgR internalize to interconnected groups of vacuoles and tubules that comprise the endosomal compartment and in which they codistribute with internalized transferrin receptors (TR) and epidermal growth factor receptors (EGFR). Upon transfer to 37 degreesC the endosome vacuoles develop long tubules that give rise to a distinctive population of 100-nm-diam cup-shaped vesicles containing pIgR. At the same time, the endosome gives rise to multivesicular endosomes (MVB) enriched in EGFR and to 60-nm-diam basolateral vesicles. The cup-shaped vesicles carry the dIgA/pIgR complexes to the apical surface where they exocytose. Using video microscopy and correlative electron microscopy to study cells grown thin and flat we show that endosome vacuoles tubulate in response to dIgA/pIgR but that the tubules contain TR as well as pIgR. However, we show that TR are removed from these dIgA-induced tubules via clathrin-coated buds and, as a result, the cup-shaped vesicles to which the tubules give rise become enriched in dIgA/pIgR. Taken together with the published information available on pIgR trafficking signals, our observations suggest that the steady-state concentrations of TR and unoccupied pIgR on the basolateral surface of polarized MDCK cells are maintained by a signal-dependent, clathrin-based sorting mechanism that operates along the length of the transcytotic pathway. We propose that the differential sorting of occupied receptors within the MDCK endosome is achieved by this clathrin-based mechanism continuously retrieving receptors like TR from the pathways that deliver pIgR to the apical surface and EGFR to the lysosome.

The Candida albicans plasma membrane protein Rch1p, a member of the vertebrate SLC10 carrier family, is a novel regulator of cytosolic Ca(2+) homoeostasis.

Candida albicans RCH1 (regulator of Ca(2+) homoeostasis 1) encodes a protein of ten TM (transmembrane) domains, homologous with human SLC10A7 (solute carrier family 10 member 7), and Rch1p localizes in the plasma membrane. Deletion of RCH1 confers hypersensitivity to high concentrations of extracellular Ca(2+) and tolerance to azoles and Li(+), which phenocopies the deletion of CaPMC1 (C. albicans PMC1) encoding the vacuolar Ca(2+) pump. Additive to CaPMC1 mutation, lack of RCH1 alone shows an increase in Ca(2+) sensitivity, Ca(2+) uptake and cytosolic Ca(2+) level. The Ca(2+) hypersensitivity is abolished by cyclosporin A and magnesium. In addition, deletion of RCH1 elevates the expression of CaUTR2 (C. albicans UTR2), a downstream target of the Ca(2+)/calcineurin signalling. Mutational and functional analysis indicates that the Rch1p TM8 domain, but not the TM9 and TM10 domains, are required for its protein stability, cellular functions and subcellular localization. Therefore Rch1p is a novel regulator of cytosolic Ca(2+) homoeostasis, which expands the functional spectrum of the vertebrate SLC10 family.

Activation/division of lymphocytes results in increased levels of cytoplasmic activation/proliferation-associated protein-1: prototype of a new family of proteins.

We purified from activated T lymphocytes a novel, highly conserved, 116-kDa, intracellular protein that occurred at high levels in the large, dividing cells of the thymus, was up-regulated when resting T or B lymphocytes or hemopoietic progenitors were activated, and was down-regulated when a monocytic leukemia, M1, was induced to differentiate. Expression of the protein was highest in the thymus and spleen and lowest in tissues with a low proportion of dividing cells such as kidney or muscle, although expression was high in the brain. The protein was localized to the cytosol and was phosphorylated, which is consistent with a previous report that the Xenopus laevis ortholog was phosphorylated by a mitotically activated kinase (1 ). The cDNA was previously mischaracterized as encoding p137, a 137-kDa GPI-linked membrane protein (2 ). We propose that the authentic protein encoded by this cDNA be called cytoplasmic activation/proliferation-associated protein-1 (caprin-1), and show that it is the prototype of a novel family of proteins characterized by two novel protein domains, termed homology regions-1 and -2 (HR-1, HR-2). Although we have found evidence for caprins only in urochordates and vertebrates, two insect proteins exhibit well-conserved HR-1 domains. The HR-1 and HR-2 domains have no known function, although the HR-1 of caprin-1 appeared necessary for formation of multimeric complexes of caprin-1. Overexpression of a fusion protein of enhanced green fluorescent protein and caprin-1 induced a specific, dose-dependent suppression of the proliferation of NIH-3T3 cells, consistent with the notion that caprin-1 plays a role in cellular activation or proliferation.

Tumor necrosis factor (TNF) signaling, but not TWEAK (TNF-like weak inducer of apoptosis)-triggered cIAP1 (cellular inhibitor of apoptosis protein 1) degradation, requires cIAP1 RING dimerization and E2 binding.

Cellular inhibitor of apoptosis (cIAP) proteins, cIAP1 and cIAP2, are important regulators of tumor necrosis factor (TNF) superfamily (SF) signaling and are amplified in a number of tumor types. They are targeted by IAP antagonist compounds that are undergoing clinical trials. IAP antagonist compounds trigger cIAP autoubiquitylation and degradation. The TNFSF member TWEAK induces lysosomal degradation of TRAF2 and cIAPs, leading to elevated NIK levels and activation of non-canonical NF-kappaB. To investigate the role of the ubiquitin ligase RING domain of cIAP1 in these pathways, we used cIAP-deleted cells reconstituted with cIAP1 point mutants designed to interfere with the ability of the RING to dimerize or to interact with E2 enzymes. We show that RING dimerization and E2 binding are required for IAP antagonists to induce cIAP1 degradation and protect cells from TNF-induced cell death. The RING functions of cIAP1 are required for full TNF-induced activation of NF-kappaB, however, delayed activation of NF-kappaB still occurs in cIAP1 and -2 double knock-out cells. The RING functions of cIAP1 are also required to prevent constitutive activation of non-canonical NF-kappaB by targeting NIK for proteasomal degradation. However, in cIAP double knock-out cells TWEAK was still able to increase NIK levels demonstrating that NIK can be regulated by cIAP-independent pathways. Finally we show that, unlike IAP antagonists, TWEAK was able to induce degradation of cIAP1 RING mutants. These results emphasize the critical importance of the RING of cIAP1 in many signaling scenarios, but also demonstrate that in some pathways RING functions are not required.

Cardiac Lineage Protein-1 (CLP-1) Regulates Cardiac Remodeling via Transcriptional Modulation of Diverse Hypertrophic and Fibrotic Responses and Angiotensin II-transforming Growth Factor β (TGF-β1) Signaling Axis.

It is well known that the renin-angiotensin system contributes to left ventricular hypertrophy and fibrosis, a major determinant of myocardial stiffness. TGF-β1 and renin-angiotensin system signaling alters the fibroblast phenotype by promoting its differentiation into morphologically distinct pathological myofibroblasts, which potentiates collagen synthesis and fibrosis and causes enhanced extracellular matrix deposition. However, the atrial natriuretic peptide, which is induced during left ventricular hypertrophy, plays an anti-fibrogenic and anti-hypertrophic role by blocking, among others, the TGF-β-induced nuclear localization of Smads. It is not clear how the hypertrophic and fibrotic responses are transcriptionally regulated. CLP-1, the mouse homolog of human hexamethylene bis-acetamide inducible-1 (HEXIM-1), regulates the pTEFb activity via direct association with pTEFb causing inhibition of the Cdk9-mediated serine 2 phosphorylation in the carboxyl-terminal domain of RNA polymerase II. It was recently reported that the serine kinase activity of Cdk9 not only targets RNA polymerase II but also the conserved serine residues of the polylinker region in Smad3, suggesting that CLP-1-mediated changes in pTEFb activity may trigger Cdk9-dependent Smad3 signaling that can modulate collagen expression and fibrosis. In this study, we evaluated the role of CLP-1 in vivo in induction of left ventricular hypertrophy in angiotensinogen-overexpressing transgenic mice harboring CLP-1 heterozygosity. We observed that introduction of CLP-1 haplodeficiency in the transgenic α-myosin heavy chain-angiotensinogen mice causes prominent changes in hypertrophic and fibrotic responses accompanied by augmentation of Smad3/Stat3 signaling. Together, our findings underscore the critical role of CLP-1 in remodeling of the genetic response during hypertrophy and fibrosis.

A pea plasma membrane protein exhibiting blue light-induced phosphorylation retains photosensitivity following triton solubilization.

Phosphorylation of a polypeptide of approximately 120 kD in pea (Pisum sativum L.) plasma membranes in response to blue light has been shown to be involved in phototropic curvature, but the relationship of this protein to the kinase and photoreceptor acting upon it is uncertain. Using two-phase aqueous partitioning to isolate right-side-out plasma membrane vesicles, we have obtained evidence suggesting that the photoreceptor, kinase, and substrate are localized to the plasma membrane fraction. Latent phosphorylation accessible through Triton X-100 or freeze/thaw treatments of purified plasma membrane vesicles indicates that at least the kinase moiety is present on the internal face of the plasma membrane. Effects of solubilization of vesicles on fluence-response characteristics and on phosphorylation levels provide evidence that the receptor, kinase, and protein substrate are present together in individual mixed detergent micelles, either as a stable complex or as domains of a single polypeptide. In vivo blue-light irradiation results in a small but significant decrease in mobility of the 120-kD phosphorylated protein on sodium dodecylsulfate gel electrophoresis. This mobility shift is evident on Coomassie-stained gels and on western blots probed with polyclonal antibodies raised against the 120-kD protein. Among the plasma membrane proteins bound to the reactive nucleotide analog fluorosulfonylbenzoyladenine (FSBA), a distinct protein band at 120 kD can be detected on blots probed with anti-FSBA antibodies. This band exhibits an in vivo light-dependent mobility shift identical to that observed for the protein band and antibodies specific for the 120-kD protein, implying that the 120-kD protein has an integral nucleotide binding site and consistent with the possibility that the substrate protein is also a kinase.

Mutations in the SPARC-Related Modular Calcium-Binding Protein 1 Gene, SMOC1, Cause Waardenburg Anophthalmia Syndrome.

Waardenburg anophthalmia syndrome, also known as microphthalmia with limb anomalies, ophthalmoacromelic syndrome, and anophthalmia-syndactyly, is a rare autosomal-recessive developmental disorder that has been mapped to 10p11.23. Here we show that this disease is heterogeneous by reporting on a consanguineous family, not linked to the 10p11.23 locus, whose two affected children have a homozygous mutation in SMOC1. Knockdown experiments of the zebrafish smoc1 revealed that smoc1 is important in eye development and that it is expressed in many organs, including brain and somites.

Genomic organization, fine-mapping, and expression of the human islet-brain 1 (IB1)/c-Jun-amino-terminal kinase interacting protein-1 (JIP-1) gene.

Islet-brain 1 (IB1), a regulator of the pancreatic beta-cell function in the rat, is homologous to JIP-1, a murine inhibitor of c-Jun amino-terminal kinase (JNK). Whether IB1 and JIP-1 are present in humans was not known. We report the sequence of the 2133-bp human IB1 cDNA, the expression, structure, and fine-mapping of the human IB1 gene, and the characterization of an IB1 pseudogene. Human IB1 is 94% identical to rat IB1. The tissue-specific expression of IB1 in human is similar to that observed in rodent. The IB1 gene contains 12 exons and maps to chromosome 11 (11p11.2-p12), a region that is deleted in DEFECT-11 syndrome. Apart from an IB1 pseudogene on chromosome 17 (17q21), no additional IB1-related gene was found in the human genome. Our data indicate that the sequence and expression pattern of IB1 are highly conserved between rodent and human and provide the necessary tools to investigate whether IB1 is involved in human diseases.