VP22 light controlled delivery of oligonucleotides to ocular cells in vitro and in vivo.

PURPOSE: To study VP22 light controlled delivery of antisense oligonucleotide (ODN) to ocular cells in vitro and in vivo. METHODS: The C-terminal half of VP22 was expressed in Escherichia coli, purified and mixed with 20 mer phosphorothioate oligonucleotides (ODNs) to form light sensitive complex particles (vectosomes). Uptake of vectosomes and light induced redistribution of ODNs in human choroid melanoma cells (OCM-1) and in human retinal pigment epithelial cells (ARPE-19) were studied by confocal and electron microscopy. The effect of vectosomes formed with an antisense ODN corresponding to the 3'-untranslated region of the human c-raf kinase gene on the viability and the proliferation of OCM-1 cells was assessed before and after illumination. Cells incubated with vectosomes formed with a mismatched ODN, a free antisense ODN or a free mismatched ODN served as controls. White light transscleral illumination was carried out 24 h after the intravitreal injection of vectosomes in rat eyes. The distribution of fluorescent vectosomes and free fluorescent ODN was evaluated on cryosections by fluorescence microscopy before, and 1 h after illumination. RESULTS: Overnight incubation of human OCM-1 and ARPE-19 cells with vectosomes lead to intracellular internalization of the vectosomes. When not illuminated, internalized vectosomes remained stable within the cell cytoplasm. Disruption of vectosomes and release of the complexed ODN was induced by illumination of the cultures with a cold white light or a laser beam. In vitro, up to 60% inhibition of OCM-1 cell proliferation was observed in illuminated cultures incubated with vectosomes formed with antisense c-raf ODN. No inhibitory effect on the OCM-1 cell proliferation was observed in the absence of illumination or when the cells are incubated with a free antisense c-raf ODN and illuminated. In vivo, 24 h after intravitreal injection, vectosomes were observed within the various retinal layers accumulating in the cytoplasm of RPE cells. Transscleral illumination of the injected eyes with a cold white light induced disruption of the vectosomes and a preferential localization of the "released" ODNs within the cell nuclei of the ganglion cell layer, the inner nuclear layer and the RPE cells. CONCLUSIONS: In vitro, VP22 light controlled delivery of ODNs to ocular cells nuclei was feasible using white light or laser illumination. In vivo, a single intravitreal injection of vectosomes, followed by transscleral illumination allowed for the delivery of free ODNs to retinal and RPE cells.

Design of new tools to improve the efficacy of DNA-damaging drugs used in cancer therapy

Résumé Les tumeurs sont diverses et hétérogènes, mais toutes partagent la capacité de proliférer sans contrôle. Une prolifération dérégulée de cellules couplée à une insensibilité à une réponse apoptotique constitue une condition minimale pour que l'évolution d'une tumeur se produise. Un des traitements les plus utilisés pour traité le cancer à l'heure actuelle sont les chimiothérapies, qui sont fréquemment des composés chimiques qui induisent des dommages dans l'ADN. Les agents anticancéreux sont efficaces seulement quand les cellules tumorales sont plus aisément tuées que le tissu normal environnant. L'efficacité de ces agents est en partie déterminée par leur capacité à induire l'apoptose. Nous avons récemment démontré que la protéine RasGAP est un substrat non conventionnel des caspases parce elle peut induire à la fois des signaux anti et pro-apoptotiques, selon l'ampleur de son clivage par les caspases. A un faible niveau d'activité des caspases, RasGAP est clivé, générant deux fragments (le fragment N et le fragment C). Le fragment N semble être un inhibiteur général de l'apoptose en aval de l'activation des caspases. À des niveaux plus élevés d'activité des caspases, la capacité du fragment N de contrecarrer l'apoptose est supprimée quand il est clivé à nouveau par les caspases. Ce dernier clivage produit deux nouveaux fragments, N 1 et N2, qui contrairement au fragment N sensibilisent efficacement des cellules cancéreuses envers des agents chimiothérapeutiques. Dans cette étude nous avons prouvé qu'un peptide, appelé par la suite TAT-RasGAP317-326, qui est dérivé du fragment N2 de RasGAP et est rendu perméable aux cellules, sensibilise spécifiquement des cellules cancéreuses à trois génotoxines différentes utilisées couramment dans des traitements anticancéreux, et cela dans des modèles in vitro et in vivo. Il est important de noté que ce peptide semble ne pas avoir d'effet sur des cellules non cancéreuses. Nous avons également commencé à caractériser les mécanismes moléculaires expliquant les fonctions de sensibilisation de TAT-RasGAP317-326. Nous avons démontré que le facteur de transcription p53 et une protéine sous son activité transcriptionelle, nommée Puma, sont indispensables pour l'activité de TAT-RasGAP317-326. Nous avons également prouvé que TAT-RasGAP317-326 exige la présence d'une protéine appelée G3BP1, une protéine se liant a RasGAP, pour potentialisé les effets d'agents anticancéreux. Les données obtenues dans cette étude montrent qu'il pourrait être possible d'augmenter l'efficacité des chimiothérapies à l'aide d'un composé capable d'augmenter la sensibilité des tumeurs aux génotoxines et ainsi pourrait permettre de traiter de manière plus efficace des patients sous traitement chimiothérapeutiques. Summary Tumors are diverse and heterogeneous, but all share the ability to proliferate without control. Deregulated cell proliferation coupled with suppressed apoptotic sensitivity constitutes a minimal requirement upon which tumor evolution occurs. One of the most commonly used treatments is chemotherapy, which frequently uses chemical compounds that induce DNA damages. Anticancer agents are effective only when tumors cells are more readily killed than the surrounding normal tissue. The efficacy of these agents is partly determined by their ability to induce apoptosis. We have recently demonstrated that the protein RasGAP is an unconventional caspase substrate because it can induce both anti- and pro-apoptotic signals, depending on the extent of its cleavage by caspases. At low levels of caspase activity, RasGAP is cleaved, generating an N-terminal fragment (fragment N) and a C-terminal fragment (fragment C). Fragment N appears to be a general Mocker of apoptosis downstream of caspase activation. At higher levels of caspase activity, the ability of fragment N to counteract apoptosis is suppressed when it is further cleaved. This latter cleavage event generates two fragments, N1 and N2, which in contrast to fragment N potently sensitizes cancer cells toward DNA-damaging agents induced apoptosis. In the present study we show that a cell permeable peptide derived from the N2 fragment of RasGAP, thereafter called TAT-RasGAP317-326, specifically sensitizes cancer cells to three different genotoxins commonly used in chemotherapy in vitro and in vivo models. Importantly this peptide seems not to have any effect on non cancer cells. We have also started to characterize the molecular mechanisms underlying the sensitizing functions of TAT-RasGAP317-326. We have demonstrated that the p53 transcription factor and a protein under its transcriptional activity, called Puma, are required for the activity of TATRasGAP317-326. We have also showed that TAT-RasGAP317-326 requires the presence of a protein called G3BP1, which have been shown to interact with RasGAP, to increase the effect of the DNA-damaging drug cisplatin. The data obtained in this study showed that it is possible to increase the efficacy of current used chemotherapies with a compound able to increase the efficacy of genotoxins which could be beneficial for patients subjected to chemotherapy.

T cell recognition of hapten. Anatomy of T cell receptor binding of a H-2kd-associated photoreactive peptide derivative.

To elucidate the structural basis of T cell recognition of hapten-modified antigenic peptides, we studied the interaction of the T1 T cell antigen receptor (TCR) with its ligand, the H-2Kd-bound Plasmodium berghei circumsporozoite peptide 252-260 (SYIPSAEKI) containing photoreactive 4-azidobenzoic acid (ABA) on P. berghei circumsporozoite Lys259. The photoaffinity-labeled TCR residue(s) were mapped as Tyr48 and/or Tyr50 of complementary determining region 2beta (CDR2beta). Other TCR-ligand contacts were identified by mutational analysis. Molecular modeling, based on crystallographic coordinates of closely related TCR and major histocompatibility complex I molecules, indicated that ABA binds strongly and specifically in a cavity between CDR3alpha and CDR2beta. We conclude that TCR expressing selective Vbeta and CDR3alpha sequences form a binding domain between CDR3alpha and CDR2beta that can accommodate nonpeptidic moieties conjugated at the C-terminal portion of peptides binding to major histocompatibility complex (MHC) encoded proteins.

An Ion Transport-Independent Role for the Cation-Chloride Cotransporter KCC2 in Dendritic Spinogenesis In Vivo.

The neuron-specific K-Cl cotransporter, KCC2, is highly expressed in the vicinity of excitatory synapses in pyramidal neurons, and recent in vitro data suggest that this protein plays a role in the development of dendritic spines. The in vivo relevance of these observations is, however, unknown. Using in utero electroporation combined with post hoc iontophoretic injection of Lucifer Yellow, we show that premature expression of KCC2 induces a highly significant and permanent increase in dendritic spine density of layer 2/3 pyramidal neurons in the somatosensory cortex. Whole-cell recordings revealed that this increased spine density is correlated with an enhanced spontaneous excitatory activity in KCC2-transfected neurons. Precocious expression of the N-terminal deleted form of KCC2, which lacks the chloride transporter function, also increased spine density. In contrast, no effect on spine density was observed following in utero electroporation of a point mutant of KCC2 (KCC2-C568A) where both the cotransporter function and the interaction with the cytoskeleton are disrupted. Transfection of the C-terminal domain of KCC2, a region involved in the interaction with the dendritic cytoskeleton, also increased spine density. Collectively, these results demonstrate a role for KCC2 in excitatory synaptogenesis in vivo through a mechanism that is independent of its ion transport function.

The adaptor complex 2 directly interacts with the alpha 1b-adrenergic receptor and plays a role in receptor endocytosis.

Using the yeast two-hybrid system, we identified the mu 2 subunit of the clathrin adaptor complex 2 as a protein interacting with the C-tail of the alpha 1b-adrenergic receptor (AR). Direct association between the alpha 1b-AR and mu 2 was demonstrated using a solid phase overlay assay. The alpha 1b-AR/mu 2 interaction occurred inside the cells, as shown by the finding that the transfected alpha 1b-AR and the endogenous mu 2 could be coimmunoprecipitated from HEK-293 cell extracts. Mutational analysis of the alpha 1b-AR revealed that the binding site for mu 2 does not involve canonical YXX Phi or dileucine motifs but a stretch of eight arginines on the receptor C-tail. The binding domain of mu 2 for the receptor C-tail involves both its N terminus and the subdomain B of its C-terminal portion. The alpha 1b-AR specifically interacted with mu 2, but not with the mu 1, mu 3, or mu 4 subunits belonging to other AP complexes. The deletion of the mu 2 binding site in the C-tail markedly decreased agonist-induced receptor internalization as demonstrated by confocal microscopy as well as by the results of a surface receptor biotinylation assay. The direct association of the adaptor complex 2 with a G protein-coupled receptor has not been reported so far and might represent a common mechanism underlying clathrin-mediated receptor endocytosis.

Molecular characterization of the Carma1-related protein card9

ABSTRACT : Fungal infections have become a major source of diseases in immuncompromised patients, but are quite benign in healthy individuals. As fungi are eukaryotes, and share many biological processes with humans, many antifungal drugs can cause toxicity in the patients. Therefore, the characterization of signaling pathways specific to the anti-fungal immune response is relevant for the better understanding of the disease and the development of new therapeutic approaches. Dectin-1 is the major mammalian pattern recognition receptor for the fungal component zymosan. Dectin-1 is an innate non-Toll-like receptor containing immunoreceptor tyrosine-based activation motifs (ITAMs). Card9, Bc110 and Maltl are proteins that have been shown to play a key role in the Dectin-l-induced signaliñg pathway by controlling Dectin-l-mediated cell activation, cytokine production and innate anti-fungal immunity in mice. Here we investigate the role of the Card9-Bc110-Maltl complex in humans using the monocytic cell line THP-1. We show that Card9 interacts with Bc110 through a CARD-CARD interaction and that interaction of Card9 with Bc110 is required for NF-xB activation. We further demonstrate that Card9 is phosphorylated in its C-terminal part on serine residues. The phosphorylation status of Card9 can influence its ability to active NF-xB, since mutation of the phosphorylation sites increases its ability to activate NF-xB. We find that Card9 is expressed in myeloid derived cells, such as the human monocytic cell lines THP1 and U937, and in human monocyte-enriched PBLs and monocyte-derived DCs. Our findings demonstrate that Card9 is implicated in anti-fungal responses, since silencing of Card9 as well as of Bc110 and Maltl diminishes the capacity of THP1 cells to produce TNF-a in response to zymosan. Interestingly, activation of the NF-xB and MAPK pathway remained normal and levels of TNF-a mRNA produced were also not affected in THP 1 cells silenced for the expression of Card9, Bc110 or Malt1. Using a Malt1 inhibitor, we provide evidence that the proteolytic activity of Malt1 is needed for zymosan-induced TNF-a production in THP 1 cells and bone marrow-derived macrophages of mice, but further experiments are required to confirm these findings and identify the substrate(s) of Malt1. In conclusion, our results reveal an important role for Card9 in the innate immune response of human macrophages to fungi. RÉSUMÉ : Les infections fongiques sont une source majeure de maladie chez les patients immunodéprimés, alors qu'elles sont plutôt bénignes chez les individus sains. Comme les champignons sont des eucaryotes et partagent beaucoup de processus biologiques avec les humains, les médicaments antifongiques peuvent être source de toxicité chez les patients. Il est donc important de mieux caractériser les voies de signalisation intracellulaire des réponses anti-fongiques pour pouvoir développer de nouvelles approches thérapeutiques. La protéine Dectin-1 est le récepteur principal du composé fongique zymosan. Les protéines Card9, Bc110 et Maltl ont été décrites comme jouant un rôle primordial dans les signaux d'activation induits par Dectin-l, en contrôlant l'activité cellulaire, la production de cytokines et la défense anti-fongique dans les souris. Dans cette étude, nous investiguons le rôle du complexe Card9-Bc110-Maltl dans la lignée monocytaire humaine THP1. Nous montrons que Card9 interagit avec Bc110 par une interaction CARD-CARD et que cette interaction est requise pour activer le facteur de transcription NF-xB. Nous observons que Card9 est phosphorylé dans sa partie C-terminale sur des résidus serine et que l'état de phosphorylation de Card9 influence sa capacité à activer NF-xB. En effet, sa capacité à activer NF-xB est augmentée, après mutation des sites de phosphorylation. La génération d'un anticorps spécifique dirigé contre Card9 nous a permis de démontrer que Card9 est exprimé dans des cellules myéloïdes comme les lignées cellulaires monocytiques THP-1 et U-937, ainsi que dans les cellules dendritiques humaines. Nos résultats démontrent que Card9 est impliqué dans la réponse immunitaire antifongique puisque la réduction de l'expression de Card9 ainsi que de Bc110 et de Malt1 diminue la capacité des THP-1 à produire du TNF-a en réponse au zymosan. Par contre, les voies de signalisation NF-xB et MAPK ainsi que les niveaux de mRNA de TNF-a produits en réponse au zymosan ne sont pas affectés dans ces cellules. En utilisant un inhibiteur de Malt1, nous montrons que l'activité protéolytique de Malt1 est nécessaire pour la production de TNF-a induite par le zymosan dans les cellules THP-1 ainsi que dans les macrophages de souris, mais d'autres expériences seront nécessaires pour confirmer cette observation et identifier le(s) substrat(s) de Malt1 responsables de cet effet. En conclusion, nos résultats révèlent un rôle important de la protéine Card9 dans la réponse immunitaire innée antifongique dans les macrophages humains.

Pob1 participates in the Cdc42 regulation of fission yeast actin cytoskeleton.

Rho GTPases regulate the actin cytoskeleton in all eukaryotes. Fission yeast Cdc42 is involved in actin cable assembly and formin For3 regulation. We isolated cdc42-879 as a thermosensitive strain with actin cable and For3 localization defects. In a multicopy suppressor screening, we identified pob1(+) as suppressor of cdc42-879 thermosensitivity. Pob1 overexpression also partially restores actin cables and localization of For3 in the mutant strain. Pob1 interacts with Cdc42 and this GTPase regulates Pob1 localization and/or stability. The C-terminal pleckstrin homology (PH) domain of Pob1 is required for Cdc42 binding. Pob1 also binds to For3 through its N-terminal sterile alpha motif (SAM) domain and contributes to the formin localization at the cell tips. The previously described pob1-664 mutant strain (Mol. Biol. Cell. 10, 2745-2757, 1999), which carries a mutation in the PH domain, as well as pob1 mutant strains in which Pob1 lacks the N-terminal region (pob1DeltaN) or the SAM domain (pob1DeltaSAM), have cytoskeletal defects similar to that of cdc42-879 cells. Expression of constitutively active For3DAD* partially restores actin organization in cdc42-879, pob1-664, pob1DeltaN, and pob1DeltaSAM. Therefore, we propose that Pob1 is required for For3 localization to the tips and facilitates Cdc42-mediated relief of For3 autoinhibition to stimulate actin cable formation.

In vitro characterization of PlySK1249, a novel phage lysin, and assessment of its antibacterial activity in a mouse model of Streptococcus agalactiae bacteremia.

Beta-hemolytic Streptococcus agalactiae is the leading cause of bacteremia and invasive infections. These diseases are treated with β-lactams or macrolides, but the emergence of less susceptible and even fully resistant strains is a cause for concern. New bacteriophage lysins could be promising alternatives against such organisms. They hydrolyze the bacterial peptidoglycan at the end of the phage cycle, in order to release the phage progeny. By using a bioinformatic approach to screen several beta-hemolytic streptococci, a gene coding for a lysin was identified on a prophage carried by Streptococcus dysgalactiae subsp. equisimilis SK1249. The gene product, named PlySK1249, harbored an original three-domain structure with a central cell wall-binding domain surrounded by an N-terminal amidase and a C-terminal CHAP domain. Purified PlySK1249 was highly lytic and bactericidal for S. dysgalactiae (2-log10 CFU/ml decrease within 15 min). Moreover, it also efficiently killed S. agalactiae (1.5-log10 CFU/ml decrease within 15 min) but not several streptococcal commensal species. We further investigated the activity of PlySK1249 in a mouse model of S. agalactiae bacteremia. Eighty percent of the animals (n = 10) challenged intraperitoneally with 10(6) CFU of S. agalactiae died within 72 h, whereas repeated injections of PlySK1249 (45 mg/kg 3 times within 24 h) significantly protected the mice (P < 0.01). Thus, PlySK1249, which was isolated from S. dysgalactiae, demonstrated high cross-lytic activity against S. agalactiae both in vitro and in vivo. These encouraging results indicated that PlySK1249 might represent a good candidate to be developed as a new enzybiotic for the treatment of systemic S. agalactiae infections.

Identification and mutational analyses of phosphorylation sites of the calcineurin-binding protein CbpA and the identification of domains required for calcineurin binding in Aspergillus fumigatus.

Calcineurin is a key protein phosphatase required for hyphal growth and virulence in Aspergillus fumigatus, making it an attractive antifungal target. However, currently available calcineurin inhibitors, FK506 and cyclosporine A, are immunosuppressive, limiting usage in the treatment of patients with invasive aspergillosis. Therefore, the identification of endogenous inhibitors of calcineurin belonging to the calcipressin family is an important parallel strategy. We previously identified the gene cbpA as the A. fumigatus calcipressin member and showed its involvement in hyphal growth and calcium homeostasis. However, the mechanism of its activation/inhibition through phosphorylation and its interaction with calcineurin remains unknown. Here we show that A. fumigatus CbpA is phosphorylated at three distinct domains, including the conserved SP repeat motif (phosphorylated domain-I; PD-I), a filamentous fungal-specific domain (PD-II), and the C-terminal CIC motif (Calcipressin Inhibitor of Calcineurin; PD-III). While mutation of three phosphorylated residues (Ser208, Ser217, Ser223) in the PD-II did not affect CbpA function in vivo, mutation of the two phosphorylated serines (Ser156, Ser160) in the SP repeat motif caused reduced hyphal growth and sensitivity to oxidative stress. Mutational analysis in the key domains in calcineurin A (CnaA) and proteomic interaction studies confirmed the requirement of PxIxIT motif-binding residues (352-NIR-354) and the calcineurin B (CnaB)-binding helix residue (V371) for the binding of CbpA to CnaA. Additionally, while the calmodulin-binding residues (442-RVF-444) did not affect CbpA binding to CnaA, three mutations (T359P, H361L, and L365S) clustered between the CnaA catalytic and the CnaB-binding helix were also required for CbpA binding. This is the first study to analyze the phosphorylation status of calcipressin in filamentous fungi and identify the domains required for binding to calcineurin.

KLF2 mutation is the most frequent somatic change in splenic marginal zone lymphoma and identifies a subset with distinct genotype.

To characterise the genetics of splenic marginal zone lymphoma (SMZL), we performed whole exome sequencing of 16 cases and identified novel recurrent inactivating mutations in Kruppel-like factor 2 (KLF2), a gene whose deficiency was previously shown to cause splenic marginal zone hyperplasia in mice. KLF2 mutation was found in 40 (42%) of 96 SMZLs, but rarely in other B-cell lymphomas. The majority of KLF2 mutations were frameshift indels or nonsense changes, with missense mutations clustered in the C-terminal zinc finger domains. Functional assays showed that these mutations inactivated the ability of KLF2 to suppress NF-κB activation by TLR, BCR, BAFFR and TNFR signalling. Further extensive investigations revealed common and distinct genetic changes between SMZL with and without KLF2 mutation. IGHV1-2 rearrangement and 7q deletion were primarily seen in SMZL with KLF2 mutation, while MYD88 and TP53 mutations were nearly exclusively found in those without KLF2 mutation. NOTCH2, TRAF3, TNFAIP3 and CARD11 mutations were observed in SMZL both with and without KLF2 mutation. Taken together, KLF2 mutation is the most common genetic change in SMZL and identifies a subset with a distinct genotype characterised by multi-genetic changes. These different genetic changes may deregulate various signalling pathways and generate cooperative oncogenic properties, thereby contributing to lymphomagenesis.

4B.05: Plasma Lasma copeptin is associated with insulin resistance in a Swiss population-based study

OBJECTIVE: Previous studies suggest that arginine vasopressin may have a role in metabolic syndrome (MetS) and diabetes by altering liver glycogenolysis, insulin, and glucagon secretion and pituitary ACTH release. We tested whether plasma copeptin, the stable C-terminal fragment of arginine vasopressin prohormone, was associated with insulin resistance and MetS in a Swiss population-based study. DESIGN AND METHOD: We analyzed data from the population-based Swiss Kidney Project on Genes in Hypertension. Copeptin was assessed by an immunoluminometric assay. Insulin resistance was derived from the HOMA model and calculated as follows: (FPI x FPG)/22.5, where FPI is fasting plasma insulin concentration (mU/L) and FPG fasting plasma glucose (mmol/L). Subjects were classified as having the MetS according to the National Cholesterol Education Program Adult Treatment Panel III criteria. Mixed multivariate linear regression models were built to explore the association of insulin resistance with copeptin. In addition, multivariate logistic regression models were built to explore the association between MetS and copeptin. In the two analyses, adjustment was done for age, gender, center, tobacco and alcohol consumption, socioeconomic status, physical activity, intake of fruits and vegetables and 24 h urine flow rate. Copeptin was log-transformed for the analyses. RESULTS: Among the 1,089 subjects included in this analysis, 47% were male. Mean (SD) age and body mass index were 47.4 (17.6) years 25.0 (4.5) kg/m2. The prevalence of MetS was 10.5%. HOMA-IR was higher in men (median 1.3, IQR 0.7-2.1) than in women (median 1.0, IQR 0.5-1.6,P < 0.0001). Plasma copeptin was higher in men (median 5.2, IQR 3.7-7.8 pmol/L) than in women (median 3.0, IQR 2.2-4.3 pmol/L), P < 0.0001. HOMA-IR was positively associated with log-copeptin after full adjustment (β (95% CI) 0.19 (0.09-0.29), P < 0.001). MetS was not associated with copeptin after full adjustment (P = 0.92). CONCLUSIONS: Insulin resistance, but not MetS, was associated with higher copeptin levels. Further studies should examine whether modifying pharmacologically the arginine vasopressin system might improve insulin resistance, thereby providing insight into the causal nature of this association.

Development of an epidermal growth factor derivative with EGFR blocking activity.

The members of the epidermal growth factor (EGF)/ErbB family are prime targets for cancer therapy. However, the therapeutic efficiency of the existing anti-ErbB agents is limited. Thus, identifying new molecules that inactivate the ErbB receptors through novel strategies is an important goal on cancer research. In this study we have developed a shorter form of human EGF (EGFt) with a truncated C-terminal as a novel EGFR inhibitor. EGFt was designed based on the superimposition of the three-dimensional structures of EGF and the Potato Carboxypeptidase Inhibitor (PCI), an EGFR blocker previously described by our group. The peptide was produced in E. coli with a high yield of the correctly folded peptide. EGFt showed specificity and high affinity for EGFR but induced poor EGFR homodimerization and phosphorylation. Interestingly, EGFt promoted EGFR internalization and translocation to the cell nucleus although it did not stimulate the cell growth. In addition, EGFt competed with EGFR native ligands, inhibiting the proliferation of cancer cells. These data indicate that EGFt may be a potential EGFR blocker for cancer therapy. In addition, the lack of EGFR-mediated growth-stimulatory activity makes EGFt an excellent delivery agent to target toxins to tumours over-expressing EGFR.

Oncogenic K-Ras segregates at spatially distinct plasma membrane signaling platforms according to its phosphorylation status

Activating mutations in the K-Ras small GTPase are extensively found in human tumors. Although these mutations induce the generation of a constitutively GTP-loaded, active form of K-Ras, phosphorylation at Ser181 within the C-terminal hypervariable region can modulate oncogenic K-Ras function without affecting the in vitro affinity for its effector Raf-1. In striking contrast, K-Ras phosphorylated at Ser181 shows increased interaction in cells with the active form of Raf-1 and with p110α, the catalytic subunit of PI 3-kinase. Because the majority of phosphorylated K-Ras is located at the plasma membrane, different localization within this membrane according to the phosphorylation status was explored. Density-gradient fractionation of the plasma membrane in the absence of detergents showed segregation of K-Ras mutants that carry a phosphomimetic or unphosphorylatable serine residue (S181D or S181A, respectively). Moreover, statistical analysis of immunoelectron microscopy showed that both phosphorylation mutants form distinct nanoclusters that do not overlap. Finally, induction of oncogenic K-Ras phosphorylation - by activation of protein kinase C (PKC) - increased its co-clustering with the phosphomimetic K-Ras mutant, whereas (when PKC is inhibited) non-phosphorylated oncogenic K-Ras clusters with the non-phosphorylatable K-Ras mutant. Most interestingly, PI 3-kinase (p110α) was found in phosphorylated K-Ras nanoclusters but not in non-phosphorylated K-Ras nanoclusters. In conclusion, our data provide - for the first time - evidence that PKC-dependent phosphorylation of oncogenic K-Ras induced its segregation in spatially distinct nanoclusters at the plasma membrane that, in turn, favor activation of Raf-1 and PI 3-kinase.

Complex regulation of CREB-binding protein by homeodomain-interacting protein kinase 2.

CREB-binding protein (CBP) and p300 are transcriptional coactivators involved in numerous biological processes that affect cell growth, transformation, differentiation, and development. In this study, we provide evidence of the involvement of homeodomain-interacting protein kinase 2 (HIPK2) in the regulation of CBP activity. We show that HIPK2 interacts with and phosphorylates several regions of CBP. We demonstrate that serines 2361, 2363, 2371, 2376, and 2381 are responsible for the HIPK2-induced mobility shift of CBP C-terminal activation domain. Moreover, we show that HIPK2 strongly potentiates the transcriptional activity of CBP. However, our data suggest that HIPK2 activates CBP mainly by counteracting the repressive action of cell cycle regulatory domain 1 (CRD1), located between amino acids 977 and 1076, independently of CBP phosphorylation. Our findings thus highlight a complex regulation of CBP activity by HIPK2, which might be relevant for the control of specific sets of target genes involved in cellular proliferation, differentiation and apoptosis.

Anti-MPER antibodies with heterogeneous neutralization capacity are detectable in most untreated HIV-1 infected individuals

Background The MPER region of the HIV-1 envelope glycoprotein gp41 is targeted by broadly neutralizing antibodies. However, the localization of this epitope in a hydrophobic environment seems to hamper the elicitation of these antibodies in HIV infected individuals. We have quantified and characterized anti-MPER antibodies by ELISA and by flow cytometry using a collection of mini gp41-derived proteins expressed on the surface of 293T cells. Longitudinal plasma samples from 35 HIV-1 infected individuals were assayed for MPER recognition and MPER-dependent neutralizing capacity using HIV-2 viruses engrafted with HIV-1 MPER sequences. Results Miniproteins devoid of the cysteine loop of gp41 exposed the MPER on 293T cell membrane. Anti-MPER antibodies were identified in most individuals and were stable when analyzed in longitudinal samples. The magnitude of the responses was strongly correlated with the global response to the HIV-1 envelope glycoprotein, suggesting no specific limitation for anti-MPER antibodies. Peptide mapping showed poor recognition of the C-terminal MPER moiety and a wide presence of antibodies against the 2F5 epitope. However, antibody titers failed to correlate with 2F5-blocking activity and, more importantly, with the specific neutralization of HIV-2 chimeric viruses bearing the HIV-1 MPER sequence; suggesting a strong functional heterogeneity in anti-MPER humoral responses. Conclusions Anti-MPER antibodies can be detected in the vast majority of HIV-1 infected individuals and are generated in the context of the global anti-Env response. However, the neutralizing capacity is heterogeneous suggesting that eliciting neutralizing anti-MPER antibodies by immunization might require refinement of immunogens to skip nonneutralizing responses.