Littérature scientifique sur le sujet « RalGPS2 »

Increasing evidence has shown competitive endogenous RNAs (ceRNAs) play key roles in numerous cancers. Nevertheless, the ceRNA network that can predict the prognosis of lung adenocarcinoma (LUAD) is still lacking. The aim of the present study was to identify the prognostic value of key ceRNAs in lung tumorigenesis. Differentially expressed (DE) RNAs were identified between LUAD and adjacent normal samples by limma package in R using The Cancer Genome Atlas database (TCGA). Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway function enrichment analysis was performed using the clusterProfiler package in R. Subsequently, the LUAD ceRNA network was established in three steps based on ceRNA hypothesis. Hub RNAs were identified using degree analysis methods based on Cytoscape plugin cytoHubba. Multivariate Cox regression analysis was implemented to calculate the risk score using the candidate ceRNAs and overall survival information. The survival differences between the high-risk and low-risk ceRNA groups were determined by the Kaplan-Meier and log-rank test using survival and survminer package in R. A total of 2,989 mRNAs, 185 lncRNAs, and 153 miRNAs were identified. GO and KEGG pathway function enrichment analysis showed that DE mRNAs were mainly associated with “sister chromatid segregation,” “regulation of angiogenesis,” “cell adhesion molecules (CAMs),” “cell cycle,” and “ECM-receptor interaction.” LUAD-related ceRNA network was constructed, which comprised of 54 nodes and 78 edges. Top ten hub RNAs (hsa-miR-374a-5p, hsa-miR-374b-5p, hsa-miR-340-5p, hsa-miR-377-3p, hsa-miR-21-5p, hsa-miR-326, SNHG1, RALGPS2, and PITX2) were identified according to their degree. Kaplan-Meier survival analyses demonstrated that hsa-miR-21-5p and RALGPS2 had a significant prognostic value. Finally, we found that a high risk of three novel ceRNA interactions (SNHG1-hsa-miR-21-5p-RALGPS2, SNHG1-hsa-miR-326-RALGPS2, and SNHG1-hsa-miR-377-3p-RALGPS2) was positively associated with worse prognosis. Three novel ceRNAs (SNHG1-hsa-miR-21-5p-RALGPS2, SNHG1-hsa-miR-326-RALGPS2, and SNHG1-hsa-miR-377-3p-RALGPS2) might be potential biomarkers for the prognosis and treatment of LUAD.

RalGPS2 is a Ras-independent Guanine Nucleotide Exchange Factor for RalA GTPase that is involved in several cellular processes, including cytoskeletal organization. Previously, we demonstrated that RalGPS2 also plays a role in the formation of tunneling nanotubes (TNTs) in bladder cancer 5637 cells. In particular, TNTs are a novel mechanism of cell–cell communication in the tumor microenvironment, playing a central role in cancer progression and metastasis formation. However, the molecular mechanisms involved in TNTs formation still need to be fully elucidated. Here we demonstrate that mid and high-stage bladder cancer cell lines have functional TNTs, which can transfer mitochondria. Moreover, using confocal fluorescence time-lapse microscopy, we show in 5637 cells that TNTs mediate the trafficking of RalA protein and transmembrane MHC class III protein leukocyte-specific transcript 1 (LST1). Furthermore, we show that RalGPS2 is essential for nanotubes generation, and stress conditions boost its expression both in 5637 and HEK293 cell lines. Finally, we prove that RalGPS2 interacts with Akt and PDK1, in addition to LST1 and RalA, leading to the formation of a complex that promotes nanotubes formation. In conclusion, our findings suggest that in the tumor microenvironment, RalGPS2 orchestrates the assembly of multimolecular complexes that drive the formation of TNTs.

Uterine carcinosarcoma (UCS) is a highly invasive malignant tumor that originated from the uterine epithelium. Many studies suggested that the abnormal changes of alternative splicing (AS) of pre-mRNA are related to the occurrence and metastasis of the tumor. This study investigates the mechanism of alternative splicing events (ASEs) in the tumorigenesis and metastasis of UCS. RNA-seq of UCS samples and alternative splicing event (ASE) data of UCS samples were downloaded from The Cancer Genome Atlas (TCGA) and TCGASpliceSeq databases, several times. Firstly, we performed the Cox regression analysis to identify the overall survival-related alternative splicing events (OSRASEs). Secondly, a multivariate model was applied to approach the prognostic values of the risk score. Afterwards, a coexpressed network between splicing factors (SFs) and OSRASEs was constructed. In order to explore the relationship between the potential prognostic signaling pathways and OSRASEs, we fabricated a network between these pathways and OSRASEs. Finally, validations from multidimension platforms were used to explain the results unambiguously. 1,040 OSRASEs were identified by Cox regression. Then, 6 OSRASEs were incorporated in a multivariable model by Lasso regression. The area under the curve (AUC) of the receiver operator characteristic (ROC) curve was 0.957. The risk score rendered from the multivariate model was corroborated to be an independent prognostic factor ( P < 0.001 ). In the network of SFs and ASEs, junction plakoglobin (JUP) noteworthily regulated RALGPS1-87608-AT ( P < 0.001 , R = 0.455 ). Additionally, RALGPS1-87608-AT ( P = 0.006 ) showed a prominent relationship with distant metastasis. KEGG pathways related to prognosis of UCS were selected by gene set variation analysis (GSVA). The pyrimidine metabolism ( P < 0.001 , R = − 0.470 ) was the key pathway coexpressed with RALGPS1. We considered that aberrant JUP significantly regulated RALGPS1-87608-AT and the pyrimidine metabolism pathway might play a significant part in the metastasis and prognosis of UCS.

Using a yeast two-hybrid system, we identified a novel protein which interacts with ras p21. This protein shares 69% amino acid homology with ral guanine nucleotide dissociation stimulator (ralGDS), a GDP/GTP exchange protein for ral p24. We designated this protein RGL, for ralGDS-like. Using the yeast two-hybrid system, we found that an effector loop mutant of ras p21 was defective in interacting with the ras p21-interacting domain of RGL, suggesting that this domain binds to ras p21 through the effector loop of ras p21. Since ralGDS contained a region highly homologous with the ras p21-interacting domain of RGL, we examined whether ralGDS could interact with ras p21. In the yeast two-hybrid system, ralGDS failed to interact with an effector loop mutant of ras p21. In insect cells, ralGDS made a complex with v-ras p21 but not with a dominant negative mutant of ras p21. ralGDS interacted with the GTP-bound form of ras p21 but not with the GDP-bound form in vitro. ralGDS inhibited both the GTPase-activating activity of the neurofibromatosis gene product (NF1) for ras p21 and the interaction of Raf with ras p21 in vitro. These results demonstrate that ralGDS specifically interacts with the active form of ras p21 and that ralGDS can compete with NF1 and Raf for binding to the effector loop of ras p21. Therefore, ralGDS family members may be effector proteins of ras p21 or may inhibit interactions between ras p21 and its effectors.

Using a yeast two-hybrid system, we identified a novel protein which interacts with ras p21. This protein shares 69% amino acid homology with ral guanine nucleotide dissociation stimulator (ralGDS), a GDP/GTP exchange protein for ral p24. We designated this protein RGL, for ralGDS-like. Using the yeast two-hybrid system, we found that an effector loop mutant of ras p21 was defective in interacting with the ras p21-interacting domain of RGL, suggesting that this domain binds to ras p21 through the effector loop of ras p21. Since ralGDS contained a region highly homologous with the ras p21-interacting domain of RGL, we examined whether ralGDS could interact with ras p21. In the yeast two-hybrid system, ralGDS failed to interact with an effector loop mutant of ras p21. In insect cells, ralGDS made a complex with v-ras p21 but not with a dominant negative mutant of ras p21. ralGDS interacted with the GTP-bound form of ras p21 but not with the GDP-bound form in vitro. ralGDS inhibited both the GTPase-activating activity of the neurofibromatosis gene product (NF1) for ras p21 and the interaction of Raf with ras p21 in vitro. These results demonstrate that ralGDS specifically interacts with the active form of ras p21 and that ralGDS can compete with NF1 and Raf for binding to the effector loop of ras p21. Therefore, ralGDS family members may be effector proteins of ras p21 or may inhibit interactions between ras p21 and its effectors.

Abstract The small GTP-binding protein Ral has been implicated in regulated exocytosis via its interaction with the mammalian exocyst complex. We have previously demonstrated that Ral is involved in exocytosis of Weibel-Palade bodies (WPBs). Little is known about intracellular signaling pathways that promote activation of Ral in response to ligand binding of G protein–coupled receptors. Here we show that RNAi-mediated knockdown of RalGDS, an exchange factor for Ral, results in inhibition of thrombin- and epinephrine-induced exocytosis of WPBs, while overexpression of RalGDS promotes exocytosis of WPBs. A RalGDS variant lacking its exchange domain behaves in a dominant negative manner by blocking release of WPBs. We also provide evidence that RalGDS binds calmodulin (CaM) via an amino-terminal CaM-binding domain. RalGDS association to CaM is required for Ral activation because a cell-permeable peptide comprising this RalGDS CaM-binding domain inhibits Ral activation and WPB exocytosis. Together our findings suggest that RalGDS plays a vital role in the regulation of Ral-dependent WPB exocytosis after stimulation with Ca2+- or cAMP-raising agonists.

RalGPS2 è uno scambiatore appartenente alla famiglia RalGPS, composto da un dominio catalitico Cdc25-like nella regione N-terminale, un motivo PxxP nella regione centrale, e un dominio di omologia alla Pleckstrina (PH) nella regione C-terminale. E’ stato precedentemente dimostrato che RalGPS2 attiva in “vivo” la GTPasi RalA, mentre la regione PH-PxxP si comporta da dominante negativo per l’attività di RalA in cellule NIH3T3 e PC12. Inoltre, se è overespresso RalGPS2 causa cambiamenti morfologici consistenti nelle cellule HEK293, suggerendo che esso possa avere effetti sul citoscheletro. Tutto ciò suggerisce un possibile ruolo di RalGPS2 nella riorganizzazione del citoscheletro anche in linee cellulari tumorali. A tal fine è stata scelta come modello la linea cellulare umana 5637 di cancro alla vescica, in cui la GTPasi RalA è iperattiva. Nel presente lavoro abbiamo dimostrato che RalGPS2 da solo è in grado attivare RalA in “vivo”, mentre la sua deplezione ne abbassa notevolmente i livelli. In più si è dimostrato che la regione PH-PxxP e il dominio PH di RalGPS2 si comportano da dominanti negativi per l’attività di RalA. Inoltre, analisi al confocale hanno rivelato una parziale ma marcata co-localizzazione tra RalA, RalGPS2, il dominio PH e la regione PH-PxxP a livello della membrana plasmatica e in sottili protrusioni di membrana. La presenza di queste protrusioni in cui si localizzava RalA ha suggerito che esse potessero essere nanotubi traforati (TNT). I TNT sono condotti intracellulari per il trasporto di vari componenti cellulari o segnali importanti per la comunicazione cellulare. Siccome i TNT sono stati precedentemente descritti come strutture costituite da actina ma non da tubulina, si è utilizzato questo criterio per caratterizzare tali protrusioni. L’analisi al microscopio confocale ha evidenziato la presenza di protrusioni ricche in actina ma povere in tubulina. Per valutare se effettivamente RalGPS2 e i suoi domini influenzino la formazione dei TNT, si è condotta un’analisi al microscopio confocale in cui si andava a caratterizzare le protrusioni formate dalle cellule. Un’analisi statistica dettagliata ha evidenziato che RalGPS2 supporta la formazione di TNT in cellule 5637. Successivamente si è cercato di analizzare il ruolo degli effettori di RalA nella formazione dei TNT. Un’analisi statistica accurata ha dimostrato che il blocco di Sec5 (subunià del complesso delle esocisti ed effettore di RalA) riduce fortemente la formazione dei TNT. Dunque sia Sec5 che RalGPS2 sembrano giocare un ruolo chiave nella genesi di queste strutture. Per confermare il ruolo di RalGPS2 nella formazione dei TNT e per valutare se esso cooperi assieme a Sec5 in tale processo abbiamo effettuato un saggio di co-immunoprecipiatazione. Tale analisi rivela la presenza di un complesso tra RalA, RalGPS2,LST1 (proteina che induce la formazione dei TNT) e Sec5. Inoltre è stato dimostrato che RalGPS2 supporta la formazione dei TNT maggiormente in condizioni di carenza di nutrienti. I risultati ottenuti ci suggeriscono l’esistenza di due pathway compresenti, ma maggiormente attivati in condizioni diverse. In questa proposta RalGPS2 interagendo con LST1 e RalA determina la formazione di un complesso che in condizioni di stress si attiva e permette l’interazione tra RalA e Sec5. L’interazione RalA-Sec5 determina l’assemblaggio di un complesso multi-proteico che controlla la formazione dei nanotubi. Al contrario in condizioni di stimolo proliferativo, sebbene il complesso RalGPS2-LST1-RalA sia comunque presente e in parte attivo è eclissato dall’attivazione di un altro pathway che ha come protagonisti i GEF della famiglia RalGDS, la GTPasi RalA e Sec5. In queste condizioni infatti i GEF RalGDS sono attivi e interagiscono con RalA attivandola. In questo stato attivo RalA interagisce a sua volta con Sec5 promuovendo l’assemblaggio del complesso delle esocisti e regolando così l’esocitosi.
RalGPS2 is a murine guanine nucleotide exchange factor belonging to RalGPS family; that contains a well conserved CDC25-like domain in the N-terminal region, a PxxP motif in central region and a PH (Pleckstrin Homology) domain in the C-terminus. It has been demonstrated that RalGPS2 can activate RalA in vivo, while the PH-PxxP domain behaves as a dominant negative for RalA activation in NIH3T3 and PC12 cells. Furthermore, when overexpressed, RalGPS2 causes considerable morphological changes in HEK293 cells, suggesting its possible role on cytoskeleton re-organization. These data suggest us a possible role of RalGPS2 and its domains in cytoskeleton re-modelling also in tumour cell lines. For this purpose it has been chosen the human bladder cancer cell line 5637, as a model. In the present work it has been shown that RalGPS2 alone is able to activate RalA in “vivo”, while its depletion significantly lowers RalA levels. Furthermore, it has been demonstrated that PH-PxxP region and PH domain of RalGPS2 behave as dominant negatives for RalA activation. Moreover, confocal analysis reveals a partial, but marked co-localization between RalA, RalGPS2, the PH domain and the PH-PxxP region at the level of plasma membrane end in thin membrane protrusions. The presence of these protrusions in which localize the GTPase RalA suggested us that these structures could be Tunneling Nanotubes (TNTs). TNTs are intercellular conduits and have been shown to enable the transport of various cellular components and signals, they are important for cellular communication between cells. Since nanotubes were initially described to contain actin but not tubulin we used this criterion to characterize the protrusions that we have observed in 5637 cells. Confocal analysis reveals presence of protrusions rich in actin but poor in tubulin. To determinate whether RalGPS2 and its domain induce formation of TNTs, it has been made a confocal analysis in which it has been characterized protrusions formed by cells. Statistical analysis reveals that RalGPS2 supports TNTs formation in 5637 cells. Later, it has been analyzed the role of RalA effectors in TNTs formation. Statistical analysis shown that lack of interaction between RalA and Sec5 (subunit of exocyst complex and RalA effector) strongly reduces nanotubes formation. Therefore, both Sec5 and RalGPS2 seem to play a key role in generation of these structures. To confirm the role of RalGPS2 in TNTs formation and to evaluate whether it cooperates with Sec5 in this process, it has been performed an co-immunoprecipitation assay. This investigation reveals the presence of a complex between RalA,RalGPS2, LST1 (protein which induces TNTs formation) and Sec5. Moreover, it has been demonstrated that RalGPS2 supports TNT formation more in conditions of nutrient deficiency. Results obtained suggest the existence of two coexisting pathways, but more activates under different conditions. In this proposal, interaction between RalGPS2, LST1 and RalA establishes formation of a complex that under stress condition is active and allows the interaction between the RalA and Sec5. RalA-Sec5 interaction determines the assembly of multi-protein complex which controls TNTs formation. On the contrary, in proliferative stimulus conditions, while RalGPS2-LST1-RalA complex is still present and partially activated, it is outclassed by the activation of a distinct pathway in which GEFs of the RalGDS family, the RalA GTPase and Sec5 play a pivotal role. In such conditions, RalGDS GEFs are activated and interact with the RalA GTPase while promoting the GDP-GTP exchange. RalA in its active state also interacts with Sec5, allowing the assembly of the exocyst complex and so regulating the exocytosis.

Noncovalent factors, such as shape complementarity and electrostatic driving forces, almost exclusively cause the affinity and specificity for which two or more biological macromolecules organize into a functioning complex. The human oncoprotein p21Ras (Ras) and a structurally identical but functionally distant analog, Rap1A (Rap), exhibit high selectivity and specificity when binding to downstream effector proteins that cannot be explained through structural analysis alone. Both Ras and Rap bind to Ral guanine nucleotide dissociation stimulator (RalGDS) with affinities that differ tenfold instigating diverse cellular functions; it is hypothesized that this specificity of RalGDS to discriminate between GTPases is largely electrostatic in nature. To investigate this hypothesis, electrostatic fields at the binding interface between mutants of RalGDS bound to Rap or Ras are measured using vibrational Stark effect (VSE) spectroscopy, in which spectral shifts of a probe oscillator’s energy is related directly to that probe’s local electrostatic environment and measured by Fourier transform infrared spectroscopy (FTIR). After calibration, the probe is inserted into a known position in RalGDS where it becomes a highly local, sensitive, and directional reporter of fluctuations of the protein’s electrostatic field caused by structural or chemical perturbations of the protein. The thiocyanate (SCN) vibrational spectroscopic probe was systematically incorporated throughout the binding interface of RalGDS. Changes in the absorption energy of the thiocyanate probe upon binding were directly related to the change of the strength of the local electrostatic field in the immediate vicinity of the probe, thereby creating a comprehensive library of the binding interactions between Ras-RalGDS and Rap-RalGDS. The measured SCN absorption energy on the monomeric protein was compared with solvent-accessible surface area (SASA) calculations with the results highlighting the complex structural and electrostatic nature of protein-water interface. Additional SASA studies of the nine RalGDS mutants that bind to Ras or Rap verified that experimentally measured thiocyanate absorption energies are negatively correlated with exposure to water at the protein-water interface. By changing the solvent composition, we confirmed that the cyanocysteine residues that are more exposed to solvent experienced a large difference in absorption energy. These studies reinforce the hypothesis that differences in the electrostatic environment at the binding interfaces of Ras and Rap are responsible for discriminating binding partners.
text

This examines the interplay of structure and local electrostatic fields in protein-protein and protein-solvent interactions. The partial charges of the protein amino acids and the polarization of the surrounding solvent create a complex system of electrostatic fields at protein-protein and protein-solvent interfaces. An approach incorporating vibrational Stark effect (VSE) spectroscopy, dissociation constant measurements, and molecular dynamics (MD) simulations was used to investigate the electrostatic interactions in these interfaces. Proteins p21Ras (Ras) and Rap1A (Rap) have nearly identical amino acid sequences and structures along the effector-binding region but bind with different affinities to Ral guanine nucleotide dissociation stimulator (RalGDS). A charge reversion mutation at position 31 alters the binding affinity of Ras and Rap with RalGDS from 0.1 [mu]M and 1 [mu]M, to 1 [mu]M and 0.5 [mu]M, respectively. A spectral probe was placed at various locations along the binding interface on the surface of RalGDS as it was docked with Ras and Rap single (position 30 or 31) and double mutants (both positions). By comparing the probes' absorption energies with the respective wild-type (WT) analogs, VSE spectroscopy was able to measure molecular-level electrostatic events across the protein-protein interface. MD simulations provided a basis for deconvoluting the structural and electrostatic changes observed by the probes. The mutation at position 31 was found to be responsible for both structural and electrostatic changes compared to the WT analogs. Furthermore, previous identification of positions N27 and N29 on RalGDS as "hot spots" that help discriminate between structurally similar GTPases was supported. The RalGDS probe-containing variants and three model compounds were placed in aqueous solvents with varying dielectric constants to measure changes in absorption energy. We investigated the ability of the Onsager solvent model to describe the solvent induced changes in absorption energy, while MD simulations were employed to determine the location and solvation of the probes at the protein-solvent interface. The solvent accessible-surface area, a measure of hydration, was determined to correlate well with the change in magnitude of the probe's absorption energy and the displaced solvent by the probe.
text