SLX4 interacts with RTEL1 to prevent transcription-mediated DNA replication perturbations.

The SLX4 tumor suppressor is a scaffold that plays a pivotal role in several aspects of genome protection, including homologous recombination, interstrand DNA crosslink repair and the maintenance of common fragile sites and telomeres. Here, we unravel an unexpected direct interaction between SLX4 and the DNA helicase RTEL1, which, until now, were viewed as having independent and antagonistic functions. We identify cancer and Hoyeraal-Hreidarsson syndrome-associated mutations in SLX4 and RTEL1, respectively, that abolish SLX4-RTEL1 complex formation. We show that both proteins are recruited to nascent DNA, tightly co-localize with active RNA pol II, and that SLX4, in complex with RTEL1, promotes FANCD2/RNA pol II co-localization. Importantly, disrupting the SLX4-RTEL1 interaction leads to DNA replication defects in unstressed cells, which are rescued by inhibiting transcription. Our data demonstrate that SLX4 and RTEL1 interact to prevent replication-transcription conflicts and provide evidence that this is independent of the nuclease scaffold function of SLX4.

Early prognostic factors in septic shock cancer patients: a prospective study with a proteomic approach.

BACKGROUND: Organ failures are the main prognostic factors in septic shock. The aim was to assess classical clinico-biological parameters evaluating organ dysfunctions at intensive care unit admission, combined with proteomics, on day-30 mortality in critically ill onco-hematology patients admitted to the intensive care unit for septic shock. METHODS: This was a prospective monocenter cohort study. Clinico-biological parameters were collected at admission. Plasma proteomics analyses were performed, including protein profiling using isobaric Tag for Relative and Absolute Quantification (iTRAQ) and subsequent validation by ELISA. RESULTS: Sixty consecutive patients were included. Day-30 mortality was 47%. All required vasopressors, 32% mechanical ventilation, 33% non-invasive ventilation and 13% renal-replacement therapy. iTRAQ-based proteomics identified von Willebrand factor as a protein of interest. Multivariate analysis identified four factors independently associated with day-30 mortality: positive fluid balance in the first 24 h (odds ratio = 1.06, 95% CI = 1.01-1.12, P = 0.02), severe acute respiratory failure (odds ratio = 6.14, 95% CI = 1.04-36.15, P = 0.04), von Willebrand factor plasma level > 439 ng/ml (odds ratio = 9.7, 95% CI = 1.52-61.98, P = 0.02), and bacteremia (odds ratio = 6.98, 95% CI = 1.17-41.6, P = 0.03). CONCLUSION: Endothelial dysfunction, revealed by proteomics, appears as an independent prognostic factor on day-30 mortality, as well as hydric balance, acute respiratory failure and bacteremia, in critically ill cancer patients admitted to the intensive care unit. Endothelial failure is underestimated in clinical practice and represents an innovative therapeutic target.

[Publication of biological samples collections catalogues by tumor banks]. / La publication de catalogues de collections d'échantillons biologiques par les tumorothèques.

Biobanks in general, and specifically tumour banks, are considered as essential tools for the development of translational and clinical research in biology and oncology. Biobank tasks include the collection and preservation of biological samples, and their association with information that will be essential for further scientific use ("annotations" that allow for the "qualification" of biological samples in biological resource). A collection is made of a series of biological resource that are representative of a homogeneous group of individuals or patients that are defined on the basis of clinical or biological information. Collections are used by scientists that are aware of their existence. In the absence of a published catalogue, this awareness is most often limited to research teams that are geographically close, or to investigators who already established collaborative projects with medical teams within the hospital that operates the tumour bank. Publications of catalogues, especially digitalized and online catalogues, should foster the development of high-level, large-scale and multicentric scientific projects. In addition, tumour banks will formalize rules that allow publication of collections, and upstream, rules that are used to qualify biological samples in biological resource: this should translate in an improved overall quality of samples and annotations. Tumour bank catalogues remain relatively few; however, some recent achievements established the "proof of concept" and already raise questions regarding rules for publication. It will be important to demonstrate that these high expectations translate into measurable benefits.

Postoperative serum proteomic profiles may predict metastatic relapse in high-risk primary breast cancer patients receiving adjuvant chemotherapy.

A total of 30-50% of early breast cancer (EBC) patients considered as high risk using standard prognostic factors develop metastatic recurrence despite standard adjuvant systemic treatment. A means to better predict clinical outcome is needed to optimize and individualize therapeutic decisions. To identify a protein signature correlating with metastatic relapse, we performed surface-enhanced laser desorption/ionization-time of flight mass spectrometry profiling of early postoperative serum from 81 high-risk EBC patients. Denatured and fractionated serum samples were incubated with IMAC30 and CM10 ProteinChip arrays. Several protein peaks were differentially expressed according to clinical outcome. By combining partial least squares and logistic regression methods, we built a multiprotein model that correctly predicted outcome in 83% of patients. The 5-year metastasis-free survival in 'good prognosis' and 'poor prognosis' patients as defined using the multiprotein index were strikingly different (83 and 22%, respectively; P<0.0001, log-rank test). In a multivariate Cox regression including conventional pathological factors and multiprotein index, the latter retained the strongest independent prognostic significance for metastatic relapse. Major components of the multiprotein index included haptoglobin, C3a complement fraction, transferrin, apolipoprotein C1 and apolipoprotein A1. Therefore, postoperative serum protein pattern may have an important prognostic value in high-risk EBC.

[hScrib: a potential novel tumor suppressor]. / hScrib: un nouveau suppresseur de tumeur à l'horizon.

Establishment and maintenance of epithelial cell polarity rely on finely tuned protein networks comprising cell surface molecules, cytoplasmic adaptors, and enzymes connected to the actin cytoskeleton. Oncogenes and tumor suppressors promote cell proliferation and resistance to apoptosis and, in many cases, alter some of these molecular scaffolds, and profoundly affect the epithelial cytoarchitecture. Reciprocally, loss of central actors of epithelial polarity unleashes normally repressed signaling pathways and perturb the shape and functions of epithelial tissues. Among the newcomers impacting on epithelial integrity, Scribble is a scaffold protein of a remarkable importance that furthermore displays a tumor suppressing activity in Drosophila melanogaster. Together with Discs Large (Dlg) and Lethal Giant Larvae (Lgl), two known tumor suppressors, Scribble acts on the correct positioning of epithelial junctions required to organize functional epithelial sheets. Scribble, Dlg and Lgl proteins are well conserved during evolution at the molecular and subcellular level implying their potential role in cell polarity and tumorigenesis in humans. Recent findings on hScrib, the human orthologue of Scribble, are discussed here.

8p12 stem cell myeloproliferative disorder: the FOP-fibroblast growth factor receptor 1 fusion protein of the t(6;8) translocation induces cell survival mediated by mitogen-activated protein kinase and phosphatidylinositol 3-kinase/Akt/mTOR pathways.

The FOP-fibroblast growth factor receptor 1 (FGFR1) fusion protein is expressed as a consequence of a t(6;8) (q27;p12) translocation associated with a stem cell myeloproliferative disorder with lymphoma, myeloid hyperplasia and eosinophilia. In the present report, we show that the fusion of the leucine-rich N-terminal region of FOP to the catalytic domain of FGFR1 results in conversion of murine hematopoietic cell line Ba/F3 to factor-independent cell survival via an antiapoptotic effect. This survival effect is dependent upon the constitutive tyrosine phosphorylation of FOP-FGFR1. Phosphorylation of STAT1 and of STAT3, but not STAT5, is observed in cells expressing FOP-FGFR1. The survival function of FOP-FGFR1 is abrogated by mutation of the phospholipase C gamma binding site. Mitogen-activated protein kinase (MAPK) is also activated in FOP-FGFR1-expressing cells and confers cytokine-independent survival to hematopoietic cells. These results demonstrate that FOP-FGFR1 is capable of protecting cells from apoptosis by using the same effectors as the wild-type FGFR1. Furthermore, we show that FOP-FGFR1 phosphorylates phosphatidylinositol 3 (PI3)-kinase and AKT and that specific inhibitors of PI3-kinase impair its ability to promote cell survival. In addition, FOP-FGFR1-expressing cells show constitutive phosphorylation of the positive regulator of translation p70S6 kinase; this phosphorylation is inhibited by PI3-kinase and mTOR (mammalian target of rapamycin) inhibitors. These results indicate that translation control is important to mediate the cell survival effect induced by FOP-FGFR1. Finally, FOP-FGFR1 protects cells from apoptosis by survival signals including BCL2 overexpression and inactivation of caspase-9 activity. Elucidation of signaling events downstream of FOP-FGFR1 constitutive activation provides insight into the mechanism of leukemogenesis mediated by this oncogenic fusion protein.

Lano, a novel LAP protein directly connected to MAGUK proteins in epithelial cells.

Protein networks asymetrically distributed to basolateral and apical epithelial membranes maintain cell polarity and homeostasis of epithelial tissues. Genetic studies in non-vertebrates assigned two families of basolateral proteins, MAGUK (membrane-associated and guanylate kinase) and LAP (leucine-rich repeats and PDZ) proteins, to a common pathway crucial for the epithelial architecture and acting as a gatekeeper to malignancy. In mammals, three LAP proteins have been described, Densin-180, Erbin, and hScribble. Here, we identify a protein called Lano (LAP and no PDZ) only present in vertebrates and presenting strong identities with LAP proteins. Despite the lack of PDZ domain, Lano is located at the basolateral side of epithelial cells in a similar manner to Erbin and hScribble. Using in vitro and in vivo experiments, we demonstrate that Lano directly interacts with the PDZ domains of MAGUK proteins, including hDLG (human disc large), in epithelial cells. A second pool of Lano is complexed to Erbin. These LAP-MAGUK protein complexes coexist at the basolateral side of epithelial cells. We provide evidence for a direct interaction between LAP and MAGUK proteins, and we propose that various LAP-MAGUK networks targeted to the basolateral side of epithelial cells participate to homeostasis of epithelial tissues and tumor growth.

The ERBB2/HER2 receptor differentially interacts with ERBIN and PICK1 PSD-95/DLG/ZO-1 domain proteins.

Identification of protein complexes associated with the ERBB2/HER2 receptor may help unravel the mechanisms of its activation and regulation in normal and pathological situations. Interactions between ERBB2/HER2 and Src homology 2 or phosphotyrosine binding domain signaling proteins have been extensively studied. We have identified ERBIN and PICK1 as new binding partners for ERBB2/HER2 that associate with its carboxyl-terminal sequence through a PDZ (PSD-95/DLG/ZO-1) domain. This peptide sequence acts as a dominant retention or targeting basolateral signal for receptors in epithelial cells. ERBIN belongs to the newly described LAP (LRR and PDZ) protein family, whose function is crucial in non vertebrates for epithelial homeostasis. Whereas ERBIN appears to locate ERBB2/HER2 to the basolateral epithelium, PICK1 is thought to be involved in the clustering of receptors. We show here that ERBIN and PICK1 bind to ERBB2/HER2 with different mechanisms, and we propose that these interactions are regulated in cells. Since ERBIN and PICK1 tend to oligomerize, further complexity of protein networks may participate in ERBB2/HER2 functions and specificity.

Human nectin3/PRR3: a novel member of the PVR/PRR/nectin family that interacts with afadin.

We have isolated nectin3/PRR3, the fourth human member of the nectin/PRR family, also described as the alpha herpes virus receptor family. Nectin/PRR members are adhesion molecules expressed at intercellular junctions. Nectin3/PRR3 is a transmembrane protein, whose extracellular region contains three Ig-like domains (V, C and C) and shares approximately 30% identity with the other members. It is mainly expressed in testis and placental tissues. SDS-PAGE analyses demonstrate that nectin3/PRR3 has a molecular weight of 83kDa. Nectin1/PRR1L and nectin2/PRR2S and L were found to be specifically expressed at the intercellular junctions. This localization is in part due to the interaction of the C-terminal part of these receptors (ended by the consensus sequence A/EXYV) and the PDZ domain of afadin. In this report we demonstrate that the nectin3/PRR3 receptor carries the A/EXYV consensus sequence and interacts in vivo with both long and short isoforms of afadin. These results suggest that the human nectin3/PRR3 is a new afadin-associated molecule.

ERBIN: a basolateral PDZ protein that interacts with the mammalian ERBB2/HER2 receptor.

The ERBB receptors have a crucial role in morphogenesis and oncogenesis. We have identified a new PDZ protein we named ERBIN (ERBB2 interacting protein) that acts as an adaptor for the receptor ERBB2/HER2 in epithelia. ERBIN contains 16 leucine-rich repeats (LRRs) in its amino terminus and a PDZ (PSD-95/DLG/ZO-1) domain at its carboxy terminus, and belongs to a new PDZ protein family. The PDZ domain directly and specifically interacts with ERBB2/HER2. ERBIN and ERBB2/HER2 colocalize to the lateral membrane of human intestinal epithelial cells. The ERBIN-binding site in ERBB2/HER2 has a critical role in restricting this receptor to the basolateral membrane of epithelial cells, as mutation of the ERBIN-binding site leads to the mislocalization of the receptor in these cells. We suggest that ERBIN acts in the localization and signalling of ERBB2/HER2 in epithelia.

The function of PTB domain proteins.

Phosphotyrosine binding (PTB) domains have been identified in a large number of proteins. In proteins like Shc and IRS-1, the PTB domain binds in a phosphotyrosine-dependent fashion to peptides that form a b turn. In these proteins, PTB domains play an important role in signal transduction by growth factor receptors. However, in several other proteins, the PTB domains have been found to participate in phosphotyrosine-independent interactions. The X11 family of proteins contains a PTB domain that binds peptides in a phosphotyrosine-independent fashion. The homologue of X11 in C. elegans is the lin-10 gene, a gene crucial for receptor targeting to the basolateral surface of body wall epithelia. The X11/Lin-10 proteins are found in a complex with two other proteins, Lin-2 and Lin-7, which have also been implicated in basolateral targeting in worm epithelia. This protein complex is also likely to be important in the targeting of cell surface proteins in mammalian neurons and epithelia. The ability of the PTB domain to bind peptides in a phosphotyrosine-dependent and -independent fashion allows this domain to be involved in diverse cellular functions.

SHC and SHIP phosphorylation and interaction in response to activation of the FLT3 receptor.

The FLT3 receptor tyrosine kinase and its ligand, FL, regulate the development of hematopoietic stem cells and early B lymphoid progenitors. FL has a strong capacity to boost production of dendritic and natural killer cells in vivo, thereby providing a new and promising tool for anti-cancer immunotherapy. Intracellular FLT3 signaling involves tyrosine phosphorylation of several cytoplasmic proteins including SHC. We have found that upon FLT3 activation SHC phosphorylation occurs at tyrosine 239/240 and 313. SHC possesses two phosphotyrosine-binding domains: an amino-terminal phosphotyrosine binding (PTB) and a carboxy-terminal Src Homology 2 (SH2) domain. Neither is required for SHC phosphorylation, but the PTB domain is necessary and sufficient for SHC binding to the SH2 containing inositol phosphatase (SHIP). Overexpression of SHC increases the level of SHIP phosphorylation on tyrosines in response to FLT3 activation, suggesting that SHC availability is a limiting step for SHIP phosphorylation. This effect is observed only if the SHC PTB domain is functional. Interestingly, SHC overexpression in FLT3-activatable Ba/F3 cells limits FLT3-dependent cell growth and this effect requires tyrosine 313. Taken together, the present data show that SHC can antagonize cell proliferation induced by FLT3 stimulation and regulate phosphorylation of the SHIP negative regulator. In addition, our study provides the structural bases for SHC phosphorylation and formation of the SHC/SHIP complex.

Role of tyrosine residues and protein interaction domains of SHC adaptor in VEGF receptor 3 signaling.

The VEGFR3/FLT4 receptor, which is involved in vasculogenesis and angiogenesis, binds and phosphorylates SHC proteins on tyrosine residues. SHC contains two phosphotyrosine interaction domains: a PTB (Phosphotyrosine Binding) and a SH2 (Src Homology 2) domain. Previous studies have shown that SHC proteins are phosphorylated on Y239/Y240 and Y313 (Y317 in humans) by tyrosine kinases such as the EGF and IL3 receptors. We have investigated which of the SHC tyrosine residues are targeted by the VEGFR3/ FLT4 kinase and the role of the SHC PTB and SH2 domains in this process. Our results show that Y239/ Y240 and Y313 are simultaneously phosphorylated by the kinase, creating GRB2 binding sites. Mutation of SHC PTB, but not SH2, domain interferes with the SHC phosphorylation by VEGFR3/FLT4. Soft agar assay experiments revealed that the VEGFR3/FLT4 transforming capacity is increased by the mutation of Y239/Y240 to phenylalanines in SHC, suggesting that these two residues mediate an inhibitory signal for cell growth. Mutation of the two phosphorylation sites increases this effect, suggesting that they have a synergistic role.

The role of the Shc phosphotyrosine interaction/phosphotyrosine binding domain and tyrosine phosphorylation sites in polyoma middle T antigen-mediated cell transformation.

The phosphotyrosine interaction (PI)/phosphotyrosine binding (PTB) domain of Shc binds specific tyrosine-phosphorylated motifs found on activated growth factor receptors and proteins such as polyoma virus middle T antigen (MT). Phenylalanine 198 (Phe198) has been identified as a crucial residue involved in the interaction of the Shc PI/PTB with phosphopeptides. In NIH 3T3 cells expressing MT, p52 Shc carrying the F198V mutation is weakly phosphorylated and does not bind MT or Grb2. Overexpression of the PI/PTB domain alone as Shc amino acids 1-238 acted in a dominant interfering fashion blocking MT-induced transformation. However, expression of a slightly longer construct, Shc 1-260, which encompasses Tyr239/Tyr240, a novel Shc tyrosine phosphorylation site, did not block transformation. This was found to be due to the ability of Shc 1-260 to become tyrosine-phosphorylated and bind Grb2. Furthermore, full-length Shc in which Tyr239/Tyr240 had been mutated to phenylalanine did not become tyrosine-phosphorylated or bind Grb2 but did inhibit colony formation in soft agar. Conversely, p52 Shc carrying a mutation in the other tyrosine phosphorylation site, Tyr317, became heavily tyrosine-phosphorylated, bound Grb2, and gave rise to colonies in soft agar.

[Receptors for factors of the VEGF (Vascular Endothelial Growth Family)]. / Les récepteurs pour les facteurs de la famille du VEGF.

Growth factors of the VEGF (vascular endothelial growth factor) family comprises 4 well characterized members that play a crucial role in the biology of blood vessels. They interact with 3 high affinity tyrosine kinase receptors (FLT1/VEGFR1, FLK1/KDR/VEGFR2, FLT4/VEGFR3). VEGF/VEGFR interactions have essential functions in blood vessel formation during development, specific phases of adult life, and in some pathological processes with neo-vascularization such as tumor growth.

The phosphotyrosine interaction domains of X11 and FE65 bind to distinct sites on the YENPTY motif of amyloid precursor protein.

The phosphotyrosine interaction (PI) domains (also known as the PTB, or phosphotyrosine binding, domains) of Shc and IRS-1 are recently described domains that bind peptides phosphorylated on tyrosine residues. The PI/PTB domains differ from Src homology 2 (SH2) domains in that their binding specificity is determined by residues that lie amino terminal and not carboxy terminal to the phosphotyrosine. Recently, it has been appreciated that other cytoplasmic proteins also contain PI domains. We now show that the PI domain of X11 and one of the PI domains of FE65, two neuronal proteins, bind to the cytoplasmic domain of the amyloid precursor protein ((beta)APP). (beta)APP is an integral transmembrane glycoprotein whose cellular function is unknown. One of the processing pathways of (beta)APP leads to the secretion of A(beta), the major constituent of the amyloid deposited in the brain parenchyma and vessel walls of Alzheimer's disease patients. We have found that the X11 PI domain binds a YENPTY motif in the intracellular domain of (beta)APP that is strikingly similar to the NPXY motifs that bind the Shc and IRS-1 PI/PTB domains. However, unlike the case for binding of the Shc PI/PTB domain, tyrosine phosphorylation of the YENPTY motif is not required for the binding of (beta)APP to X11 or FE65. The binding site of the FE65 PI domain appears to be different from that of X11, as mutations within the YENPTY motif differentially affect the binding of X11 and FE65. Using site-directed mutagenesis, we have identified a crucial residue within the PI domain involved in X11 and FE65 binding to (beta)APP. The binding of X11 or FE65 PI domains to residues of the YENPTY motif of (beta)APP identifies PI domains as general protein interaction domains and may have important implications for the processing of (beta)APP.

Mutation at tyrosine residue 1337 abrogates ligand-dependent transforming capacity of the FLT4 receptor.

In humans, the FLT4 gene encodes two isoforms of a tyrosine kinase receptor, which differ in their carboxy terminal regions. As compared to the short form, the long form has an additional stretch of 65 amino acids containing three tyrosine residues (Y1333, Y1337 and Y1363). Once expressed in fibroblast cells, only the long form is able to elicit both anchorage-independent growth in a soft agar assay and tumors in nude mice, and thus appears endowed with a potential ligand-dependent transforming capacity. Replacement of tyrosine 1337 by phenylalanine abrogates the transforming capacity of the long form. This residue was identified as a potential autophosphorylation site, and a docking site for a substrate important in the signal transduction specific of the long FLT4 isoform. We demonstrate that the GRB2 and SHC cytoplasmic substrates are involved in FLT4 signal transduction. SHC interaction could be crucial to FLT4-mediated transforming activity associated with the long isoform. Finally, trancripts for the two forms are detected in tissues positive for FLT4 gene expression.

Biochemical characterization of two isoforms of FLT4, a VEGF receptor-related tyrosine kinase.

The FLT4 gene encodes a tyrosine kinase receptor related to the two identified receptors for vascular endothelial growth factor (VEGF), FLT1 and FLK1/KDR. Two isoforms of FLT4, differing by their C-terminal ends, have been identified. The long form has 65 additional amino acid residues. We have shown that FLT4 is a highly glycosylated, relatively stable, cell surface associated kinase of approximately 180 kDa. In order to study the signal transduction molecules associated with the FLT4 pathway, and in the absence of a known ligand, we constructed two chimeric molecules (FF4S and FF4L) made of the extracellular region of the CSF1 receptor (Fms gene product) and of the transmembrane and intracellular regions of either form of FLT4. These two chimeric forms were expressed in Rat 2 transfectants. We assayed the ligand-induced capacity of the FF4 short and long forms to sustain growth of Rat 2 cells in semisolid medium. In a soft agar assay, only the long form was able to induce the growth of Rat 2 cells upon ligand treatment. The two forms of FLT4 therefore have different functional capacities. We looked for association and/or phosphorylation of phospholipase C gamma (PLC gamma) and phosphatidylinositol-3'-phosphate (PI3K), after stimulation of the FF4 molecules by CSF1. Finally, we have studied the expression of the Flt4 gene in mouse embryos and in the adult by in situ hybridization. Flt4 transcripts were found at day 12.5 post-coïtum and thereafter, including the adult mouse, predominantly in the pericardium, pleural membranes and in the lung.

Human immunodeficiency virus-1 reverse transcriptase immunodominant CD4+ T cell epitopes: a peptide-based multiparametric assessment in the mouse.

We previously identified an immunodominant CD4+ T cell determinant in the carboxy-terminal region of HIV-1 reverse transcriptase (RT528-543). The present study aimed at enumerating all the potential sites of HIV-1 RT recognized by Th cells in the BALB/c (H-2d) mouse model. To achieve this we used a panel of 62 overlapping 15-mer synthetic peptides covering the whole RT sequence to assay the following parameters: (i) immunogenicity in naive BALB/c mice injected either with peptides pools or individual peptides; (ii) antigenicity, as detected by their ability to restimulate in vitro T cells from BALB/c mice primed with native RT; (iii) MHC class II (Ad)-binding capacity as measured by the inhibition of the antigen-specific, Ad-restricted presentation of unfolded apamin (4-Acm) by fixed antigen-presenting cells to Ad/4-Acm-specific, interleukin-2-producing T hybridoma cells; and (iv) the presence of typical or degenerate consensus Ad-binding motifs. The results in this study permitted identification of three novel immunodominant RT mouse CD4+ T cell sites (RT276-290, RT375-389 and RT411-425) located in regions of limited polymorphism among RT from several HIV isolates. Some of these RT segments were found to be in the vicinity of B cell or H-2Kk- or HLA-A2-restricted cytotoxic T lymphocyte epitopes. Finally, the approach used in this study was found to be very efficient for enumerating most T cell recognition sites in a complex protein, a result that would have not been achieved by a single parameter-based analysis.