Études de l'expression des protéines fragile X related 1 (FXR1P) durant le développement des vertébrés

La famille des protéines Fragile X Related (FXR) comprend la protéine FMRP ainsi que les homologues FXR1P et FXR2P. En plus d'une forte homologie de séquence, tous les membres de cette famille de protéines possèdent des domaines caractéristiques aux molécules liant l'ARN ainsi que des signaux d'importation et d'exportation nucléaire. FMRP est l’archétype de cette famille de protéine, puisque son absence cause le retard mental avec X Fragile. Par contre, aucune pathologie n’est associée avec la perte d’expression des homologues FXR1P et FXR2P et ce, même si ces deux protéines ont été mises en évidence dans des processus développementaux chez la souris. En effet, cette famille de protéines semble jouer un rôle primordial durant l’embryogenèse, puisque la délétion de FMR1 et FXR2 provoque des troubles cognitifs, alors que FXR1 semble plutôt jouer un rôle dans la myogenèse et la spermatogenèse chez les mammifères. Cette diversification fonctionnelle de FXR1 semble être attribuable à l’expression complexe de ses différentes isoformes. En effet, chez les mammifères, quatre des six isoformes de FXR1P (70, 74, 78 et 80 kDa) sont exprimées dans tous les tissus à l’exception des muscles striés et cardiaques où elles sont remplacées par deux isoformes dites « muscle spécifique » (82 et 84 kDa). Le nombre élevé d’isoformes de FXR1 rend cette protéine difficile à étudier chez les mammifères. Cette expression de FXR1 est hautement conservée chez tous les vertébrés et peut être décelée chez plusieurs organismes tels le poulet, le poisson zèbre ainsi que chez la grenouille Xenopus laevis. Le xénope s’avère être le modèle exemplaire, puisque l’expression de xFxr1 y est beaucoup plus simple et ce, tout en conservant l’expression tissu spécifique de ces isoformes. En effet, seule une isoforme de 84 kDa est exprimée dans tous les tissus à l’exception des muscles striés et cardiaques où il y a expression d’une isoforme de 88 kDa. Étant donné le rôle dans les cellules musculaires striées, il est impératif de comprendre les implications de l’inactivation de ce gène chez les vertébrés.Fragile X Mental Retardation Protein (FMRP) is part of a mRNA-binding proteins family that includes the Fragile X Related 1 and 2 proteins (FXR1P and FXR2P). These proteins share multiple functional domains typical of mRNA-binding domain (two KH domains and 1 RGG box) as well as a nuclear and a cytoplasmic localization domain. Whereas absence of FMRP is the cause of Fragile X Mental Retardation in human, it is not known whether FXR1P and FXR2P are associated to any pathology and whether these homologous proteins can compensate for the absence of FMRP in the case of the Fragile X syndrome. Knockout mice for FXR proteins are powerful tools that are commonly used in research to shed light on the functions of these proteins and point out their embryonic involvement. However, the Fxr1 knockout mouse didn’t proved to be a good model as the two mentioned above. In mammals, we have shown that FXR1 play a key role in muscle differentiation, since two of the six isoforms are muscle specific and are believed to be essential for the normal development of the cardiac and skeletal muscle. Although essential for embryonic development, it is nearly impossible to study the developmental implication of the differential expression of these tissues specific proteins in mammals due to the large number of FXR1P isoform. Simpler model such as drosophila melanogaster are being used, but this model have only one proteins (dFMRP) which is expressed ubiquitously in this organism and do not represent the tissue specific expression of some of the family member. We choose an intermediate model such as Xenopus laevis, which is an extensively used model for developmental studies, and proceeded with the inactivation of xFxr1. In Xenopus laevis, we found two different xFxr1 proteins isoform; one short isoform (84 KDa) is ubiquitously expressed in every tissues except in muscle, whereas the long isoform (88 KDa) is expressed only in cardiac and skeletal muscle. Specific inactivation of xFxr1 messengers during the early development gave us new insight on the specific functions of these proteins during the embryogenesis and primary myogenesis

The oncometabolite 2-hydroxyglutarate activates the mTOR signalling pathway

The identification of cancer-associated mutations in the tricarboxylic acid (TCA) cycle enzymes isocitrate dehydrogenases 1 and 2 (IDH1/2) highlights the prevailing notion that aberrant metabolic function can contribute to carcinogenesis. IDH1/2 normally catalyse the oxidative decarboxylation of isocitrate into α-ketoglutarate (αKG). In gliomas and acute myeloid leukaemias, IDH1/2 mutations confer gain-of-function leading to production of the oncometabolite R-2-hydroxyglutarate (2HG) from αKG. Here we show that generation of 2HG by mutated IDH1/2 leads to the activation of mTOR by inhibiting KDM4A, an αKG-dependent enzyme of the Jumonji family of lysine demethylases. Furthermore, KDM4A associates with the DEP domain-containing mTOR-interacting protein (DEPTOR), a negative regulator of mTORC1/2. Depletion of KDM4A decreases DEPTOR protein stability. Our results provide an additional molecular mechanism for the oncogenic activity of mutant IDH1/2 by revealing an unprecedented link between TCA cycle defects and positive modulation of mTOR function downstream of the canonical PI3K/AKT/TSC1-2 pathway

An Adaptor Role for Cytoplasmic Sam68 in Modulating Src Activity during Cell Polarization▿

The Src-associated substrate during mitosis with a molecular mass of 68 kDa (Sam68) is predominantly nuclear and is known to associate with proteins containing the Src homology 3 (SH3) and SH2 domains. Although Sam68 is a Src substrate, little is known about the signaling pathway that link them. Src is known to be activated transiently after cell spreading, where it modulates the activity of small Rho GTPases. Herein we report that Sam68-deficient cells exhibit loss of cell polarity and cell migration. Interestingly, Sam68-deficient cells exhibited sustained Src activity after cell attachment, resulting in the constitutive tyrosine phosphorylation and activation of p190RhoGAP and its association with p120rasGAP. Consistently, we observed that Sam68-deficient cells exhibited deregulated RhoA and Rac1 activity. By using total internal reflection fluorescence microscopy, we observed Sam68 near the plasma membrane after cell attachment coinciding with phosphorylation of its C-terminal tyrosines and association with Csk. These findings show that Sam68 localizes near the plasma membrane during cell attachment and serves as an adaptor protein to modulate Src activity for proper signaling to small Rho GTPases

The phosphoinositide 3-kinase (PI3K) pathway and glycogen synthase kinase-3 (GSK-3) positively regulate the activity of metal-responsive transcription factor-1 (MTF-1) in response to zinc ions.

Metal-responsive transcription factor-1 (MTF-1) is a metal-regulatory transcription factor essential for induction of the genes encoding metallothioneins (MTs) in response to transition metal ions. Activation of MTF-1 is dependent on the interaction of zinc with the zinc fingers of the protein. In addition, phosphorylation is essential for MTF-1 transactivation. We previously showed that inhibition of phosphoinositide 3-kinase (PI3K) abrogated Mt expression and metal-induced MTF-1 activation in human hepatocellular carcinoma (HCC) HepG2 and mouse L cells, thus showing that the PI3K signaling pathway positively regulates MTF-1 activity and Mt gene expression. However, it has also been reported that inhibition of PI3K has no significant effects on Mt expression in immortalized epithelial cells and increases Mt expression in HCC cells. To further characterize the role of the PI3K pathway on the activity of MTF-1, transfection experiments were performed in HEK293 and HepG2 cells in presence of glycogen synthase kinase-3 (GSK-3), mTOR-C1 and mTOR-C2 inhibitors as well as of siRNAs targeting Phosphatase and TENsin homolog (PTEN). We showed that inhibition of the mTOR-C2 complex inhibits the activity of MTF-1, in HepG2 and HEK293 cells, while inhibition of the mTOR-C1 complex or of PTEN stimulates MTF-1 activity in HEK293 cells. These results confirm that the PI3K pathway positively regulates MTF-1 activity. Finally, we showed that GSK-3 is required for MTF-1 activation in response to zinc ions.The accepted manuscript in pdf format is listed with the files at the bottom of this page. The presentation of the authors' names and (or) special characters in the title of the manuscript may differ slightly between what is listed on this page and what is listed in the pdf file of the accepted manuscript; that in the pdf file of the accepted manuscript is what was submitted by the author

Cancer-Associated Fibroblasts in a 3D Engineered Tissue Model Induce Tumor-like Matrix Stiffening and EMT Transition

A tumor microenvironment is characterized by its altered mechanical properties. However, most models remain unable to faithfully recreate the mechanical properties of a tumor. Engineered models based on the self-assembly method have the potential to better recapitulate the stroma architecture and composition. Here, we used the self-assembly method based on a bladder tissue model to engineer a tumor-like environment. The tissue-engineered tumor models were reconstituted from stroma-derived healthy primary fibroblasts (HFs) induced into cancer-associated fibroblast cells (iCAFs) along with an urothelium overlay. The iCAFs-derived extracellular matrix (ECM) composition was found to be stiffer, with increased ECM deposition and remodeling. The urothelial cells overlaid on the iCAFs-derived ECM were more contractile, as measured by quantitative polarization microscopy, and displayed increased YAP nuclear translocation. We further showed that the proliferation and expression of epithelial-to-mesenchymal transition (EMT) marker in the urothelial cells correlate with the increased stiffness of the iCAFs-derived ECM. Our data showed an increased expression of EMT markers within the urothelium on the iCAFs-derived ECM. Together, our results demonstrate that our tissue-engineered tumor model can achieve stiffness levels comparable to that of a bladder tumor, while triggering a tumor-like response from the urothelium

SAM68 interaction with U1A modulates U1 snRNP recruitment and regulates mTor pre-mRNA splicing

Src associated in mitosis (SAM68) plays major roles in regulating RNA processing events, such as alternative splicing and mRNA translation, implicated in several developmental processes. It was previously shown that SAM68 regulates the alternative splicing of the mechanistic target of rapamycin (mTor), but the mechanism regulating this process remains elusive. Here, we report that SAM68 interacts with U1 small nuclear ribonucleoprotein (U1 snRNP) to promote splicing at the 5′ splice site in intron 5 of mTor. We also show that this direct interaction is mediated through U1A, a core-component of U1snRNP. SAM68 was found to bind the RRM1 domain of U1A through its C-terminal tyrosine rich region (YY domain). Deletion of the U1A-SAM68 interaction domain or mutation in SAM68-binding sites in intron 5 of mTor abrogates U1A recruitment and 5′ splice site recognition by the U1 snRNP, leading to premature intron 5 termination and polyadenylation. Taken together, our results provide the first mechanistic study by which SAM68 modulates alternative splicing decision, by affecting U1 snRNP recruitment at 5′ splice sites.ISSN:1362-4962ISSN:0301-561

Identification of a Sam68 Ribonucleoprotein Complex Regulated by Epidermal Growth Factor*

Sam68, Src associated in mitosis of 68 kDa, is a known RNA-binding protein and a signaling adaptor protein for tyrosine kinases. However, the proteins associated with Sam68 and the existence of a Sam68 complex, its mass, and regulation are, however, unknown. Herein we identify a large Sam68 complex with a mass >1 MDa in HeLa cells that is composed of ∼40 proteins using an immunoprecipitation followed by a mass spectrometry approach. Many of the proteins identified are RNA-binding proteins and are known components of a previously identified structure termed the spreading initiation center. The large Sam68 complex is a ribonucleoprotein complex, as treatment with RNases caused a shift in the molecular mass of the complex to 200–450 kDa. Moreover, treatment of HeLa cells with phorbol 12-myristate 13-acetate or epidermal growth factor induced the disassociation of Sam68 from the large complex and the appearance of Sam68 within the smaller complex. Actually, in certain cell lines such as breast cancer cell lines MCF-7 and BT-20, Sam68 exists in equilibrium between a large and a small complex. The appearance of the small Sam68 complex in cells correlates with the ability of Sam68 to promote the alternative splicing of CD44 and cell migration. Our findings show that Sam68 exists in equilibrium in transformed cells between two complexes and that extracellular signals, such as epidermal growth factor stimulation, promote alternative splicing by modulating the composition of the Sam68 complex