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Background: Use of ionizing radiation (IR) is a common therapeutic modality for patients with colon carcinoma, although resistance of cancer cells and unintended toxicity reduce clinical outcomes.P...

Noonan syndrome (NS), the most common among the RASopathies, is caused by germline variants in genes encoding components of the RAS-MAPK pathway. Distinct variants, including the recurrent Ser257Leu substitution in RAF1, are associated with severe hypertrophic cardiomyopathy (HCM). Here, we investigated the elusive mechanistic link between NS-associated RAF1S257L and HCM using three-dimensional cardiac bodies and bioartificial cardiac tissues generated from patient-derived induced pluripotent stem cells (iPSCs) harboring the pathogenic RAF1 c.770C>T missense change. We characterize the molecular, structural and functional consequences of aberrant RAF1 –associated signaling on the cardiac models. Ultrastructural assessment of the sarcomere revealed a shortening of the I-bands along the Z disc area in both iPSC-derived RAF1S257L cardiomyocytes, and myocardial tissue biopsies. The disease phenotype was partly reverted by using both MEK inhibition, and a gene-corrected isogenic RAF1L257S cell line. Collectively, our findings uncovered a direct link between a RASopathy gene variant and the abnormal sarcomere structure resulting in a cardiac dysfunction that remarkably recapitulates the human disease. These insights represent a basis to develop future targeted therapeutic approaches.

The RASopathies are a group of genetic syndromes caused by upregulated RAS signaling. Noonan syndrome (NS), the most common entity among the RASopathies, is characterized mainly by short stature, cardiac anomalies and distinctive facial features. Mutations in multiple RAS-MAPK pathway-related genes have been associated with NS and related phenotypes. We describe two unrelated patients presenting with hypertrophic cardiomyopathy (HCM) and dysmorphic features suggestive of NS. One of them died in the neonatal period because of cardiac failure. Targeted sequencing revealed de novo MRAS variants, c.203C > T (p.Thr68Ile) and c.67G > C (p.Gly23Arg) as causative events. MRAS has only recently been related to NS based on the observation of two unrelated affected individuals with de novo variants involving the same codons here found mutated. Gly23 and Thr68 are highly conserved residues, and the corresponding codons are known hotspots for RASopathy-associated mutations in other RAS proteins. Functional analyses documented high level of activation of MRAS mutants due to impaired GTPase activity, which was associated with constitutive plasma membrane targeting, prolonged localization in non-raft microdomains, enhanced binding to PPP1CB and SHOC2 protein, and variably increased MAPK and PI3K-AKT activation. This report provides additional evidence that a narrow spectrum of activating mutations in MRAS represents another rare cause of NS, and that MRAS has to be counted among the RASopathy genes predisposing to HCM. Moreover, our findings further emphasize the relevance of the MRAS-SHOC2-PPP1CB axis in the control of MAPK signaling, and the contribution of both MAPK and PI3K-AKT pathways in MRAS functional upregulation.

Among nanoparticles, magnetic nanoparticles are the most appealing candidate for diagnosis and cancer therapy. The researchers are tempting to improve the particles properties, including the size, shape, coating, and magnetic behavior or heating characteristics. Core shell type of magnetic nanoparticle is an important property that modulates their internalization via normal and cancer cells. In this study, magnetite nanoparticles (MNP) covered by N-(2-aminoethyl)-3-aminopropyltriethoxysilane (aminosilane – APTES) were synthesized by co-precipitation of aqueous solution of ferric chloride and ferrous sulfate iron salts with ammonium hydroxide as a base and functionalized by APTES to increase the viability and affinity of the particles to the cancer cells. The structural and morphological properties of these particles were characterized by transmission electron microscopy (TEM), X-ray diffraction (XRD) and Fourier transformed infrared spectroscopy (FTIR). 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl tetrazolium bromide (MTT assay) was carried out, to check the viability of the cells treatment with MNP and APTES-MNP. To study the cellular uptake in vitro, two prostate cell lines were investigated: PC3 as a cancerous cell line and BPH1 as a benign epithelial cell line (normal cells). Both cell lines were incubated for 24 h with different concentrations of MNP and APTES-MNP (100 and 500 μg/ml and one untreated sample as control). TEM and flow cytometry (FC) analyses were subsequently carried out to monitor the cellular uptake of MNP and APTES-MNP. FC data revealed an increase in cell granularity following the treatment with high concentration of the particles. Data showed that PC3 cancer cells take up more APTES-MNP with respect to control cells than BPH1 benign cells and in contrast BPH1 cell uptake MNP correlated to control cells more efficient than PC3 cells. The results from FC and TEM analyses demonstrate increasing of affinity of particles to cancer cell line (PC3). In this project we investigated the effect of surface functionalization of NP to affinity of the MNP and APTES-MNP on PC3 cells as a malignancy prostate cell and BPH1 benign cells as a normal cells. This approach may help to optimize the efficiency of hyperthermia for prostate cancer through internalization of particles to the cells or attaching to the membrane.

RAF (rapidly accelerated fibrosarcoma) Ser/Thr kinases (ARAF, BRAF, and CRAF) link the RAS (rat sarcoma) protein family with the MAPK (mitogen-activated protein kinase) pathway and control cell growth, differentiation, development, aging, and tumorigenesis. Their activity is specifically modulated by protein–protein interactions, post-translational modifications, and conformational changes in specific spatiotemporal patterns via various upstream regulators, including the kinases, phosphatase, GTPases, and scaffold and modulator proteins. Dephosphorylation of Ser-259 (CRAF numbering) and dissociation of 14-3-3 release the RAF regulatory domains RAS-binding domain and cysteine-rich domain for interaction with RAS-GTP and membrane lipids. This, in turn, results in RAF phosphorylation at Ser-621 and 14-3-3 reassociation, followed by its dimerization and ultimately substrate binding and phosphorylation. This review focuses on structural understanding of how distinct binding partners trigger a cascade of molecular events that induces RAF kinase activation.

International audience; The self-renewal capacity of germline derived stem cells (GSCs) makes them an ideal source for research and use in clinics. Despite the presence of active gene network similarities between embryonic stem cells (ESCs) and GSCs, there are unanswered questions regarding the roles of evolutionary conserved genes in GSCs. To determine the reprogramming potential of germ cell- specific genes, we designed a polycistronic gene cassette expressing Stella, Oct4 and Nanos2 in a lentiviral-based vector. Deep transcriptome analysis showed the activation of a set of pluripotency and germ-cell-specific markers and the downregulation of innate immune system. The global shut down of antiviral genes included MHC class I, interferon response genes and dsRNA 2′-5′-oligoadenylate synthetase are critical pathways that has been affected. Individual expression of each factor highlighted suppressive effect of Nanos2 on genes such as Isg15 and Oasl2. Collectively, to our knowledge this is the first report showing that Nanos2 could be considered as an immunosuppressive factor. Furthermore, our results demonstrate suppression of endogenous retrotransposons that harbor immune response but further analysis require to uncover the correlation between transposon suppression and immune response in germ cell development. © 2018 Elsevier Ltd

Silencing of the fragile X mental retardation 1 (FMR1) gene and consequently lack of synthesis of FMR protein (FMRP) are associated with fragile X syndrome, which is one of the most prevalent inherited intellectual disabilities, with additional roles in increased viral infection, liver disease, and reduced cancer risk. FMRP plays critical roles in chromatin dynamics, RNA binding, mRNA transport, and mRNA translation. However, the underlying molecular mechanisms, including the (sub)cellular FMRP protein networks, remain elusive. Here, we employed affinity pull-down and quantitative LC-MS/MS analyses with FMRP. We identified known and novel candidate FMRP-binding proteins as well as protein complexes. FMRP interacted with 180 proteins, 28 of which interacted with its N terminus. Interaction with the C terminus of FMRP was observed for 102 proteins, and 48 proteins interacted with both termini. This FMRP interactome comprises known FMRP-binding proteins, including the ribosomal proteins FXR1P, NUFIP2, Caprin-1, and numerous novel FMRP candidate interacting proteins that localize to different subcellular compartments, including CARF, LARP1, LEO1, NOG2, G3BP1, NONO, NPM1, SKIP, SND1, SQSTM1, and TRIM28. Our data considerably expand the protein and RNA interaction networks of FMRP, which thereby suggest that, in addition to its known functions, FMRP participates in transcription, RNA metabolism, ribonucleoprotein stress granule formation, translation, DNA damage response, chromatin dynamics, cell cycle regulation, ribosome biogenesis, miRNA biogenesis, and mitochondrial organization. Thus, FMRP seems associated with multiple cellular processes both under normal and cell stress conditions in neuronal as well as non-neuronal cell types, as exemplified by its role in the formation of stress granules.

Background Human pluripotent stem cells (PSCs) open new windows for basic research and regenerative medicine due to their remarkable properties, i.e. their ability to self-renew indefinitely and being pluripotent. There are different, conflicting data related to the role of basic fibroblast growth factor (bFGF) in intracellular signal transduction and the regulation of pluripotency of PSCs. Here, we investigated the effect of bFGF and its downstream pathways in pluripotent vs. differentiated human induced (hi) PSCs. Methods bFGF downstream signaling pathways were investigated in long-term culture of hiPSCs from pluripotent to differentiated state (withdrawing bFGF) using immunoblotting, immunocytochemistry and qPCR. Subcellular distribution of signaling components were investigated by simple fractionation and immunoblotting upon bFGF stimulation. Finally, RAS activity and RAS isoforms were studied using RAS assays both after short- and long-term culture in response to bFGF stimulation. Results Our results revealed that hiPSCs were differentiated into the ectoderm lineage upon withdrawing bFGF as an essential pluripotency mediator. Pluripotency markers OCT4, SOX2 and NANOG were downregulated, following a drastic decrease in MAPK pathway activity levels. Notably, a remarkable increase in phosphorylation levels of p38 and JAK/STAT3 was observed in differentiated hiPSCs, while the PI3K/AKT and JNK pathways remained active during differentiation. Our data further indicate that among the RAS paralogs, NRAS predominantly activates the MAPK pathway in hiPSCs. Conclusion Collectively, the MAPK pathway appears to be the prime signaling pathway downstream of bFGF for maintaining pluripotency in hiPSCs and among the MAPK pathways, the activity of NRAS-RAF-MEK-ERK is decreased during differentiation, whereas p38 is activated and JNK remains constant. Open-Access-Publikationsfonds 2018

Hepatic stellate cells (HSCs) were recently identified as liver-resident mesenchymal stem cells. HSCs are activated after liver injury and involved in pivotal processes, such as liver development, immunoregulation, regeneration, and also fibrogenesis. To date, several studies have reported candidate pathways that regulate the plasticity of HSCs during physiological and pathophysiological processes. Here we analyzed the expression changes and activity of the RAS family GTPases and thereby investigated the signaling networks of quiescent HSCs versus activated HSCs. For the first time, we report that embryonic stem cell-expressed RAS (ERAS) is specifically expressed in quiescent HSCs and down-regulated during HSC activation via promoter DNA methylation. Notably, in quiescent HSCs, the high level of ERAS protein correlates with the activation of AKT, STAT3, mTORC2, and HIPPO signaling pathways and inactivation of FOXO1 and YAP. Our data strongly indicate that in quiescent HSCs, ERAS targets AKT via two distinct pathways driven by PI3Kα/δ and mTORC2, whereas in activated HSCs, RAS signaling shifts to RAF-MEK-ERK. Thus, in contrast to the reported role of ERAS in tumor cells associated with cell proliferation, our findings indicate that ERAS is important to maintain quiescence in HSCs.

The history of the RAS protein family dates back in 1960s, when the highly oncogenic Harvey and Kirsten murine sarcoma viruses (Ha-MSV and Ki-MSV) were discovered by Jennifer Harvey and later Werne...

Successful replication of Human immunodeficiency virus (HIV)-1 depends on the expression of various cellular host factors, such as the interleukin-2 inducible T-cell kinase (ITK), a member of the protein family of TEC-tyrosine kinases. ITK is selectively expressed in T-cells and coordinates signaling pathways downstream of the T-cell receptor and chemokine receptors, including PLC-1 activation, Ca2+-release, transcription factor mobilization, and actin rearrangements. The exact role of ITK during HIV-1 infection is still unknown. We analyzed the function of ITK during HIV-1 replication and showed that attachment, fusion of virions with the cell membrane and entry into Jurkat T-cells was inhibited when ITK was knocked down. In contrast, reverse transcription and provirus expression were not affected by ITK deficiency. Inhibited ITK expression did not affect the CXCR4 receptor on the cell surface, whereas CD4 and LFA-1 integrin levels were slightly enhanced in ITK knockdown cells and heparan sulfate (HS) expression was completely abolished in ITK depleted T-cells. However, neither HS expression nor other attachment factors could explain the impaired HIV-1 binding to ITK-deficient cells, which suggests that a more complex cellular process is influenced by ITK or that not yet discovered molecules contribute to restriction of HIV-1 binding and entry.

Table S1. Primer sequences (5â ˛ to 3â ˛) for qPCR using the SYBR Green system obtained from PrimerBank ( https://pga.mgh.harvard.edu/primerbank/ ). Figure S1. Morphological changes of hiPSCs during 6â day culture of bFGF starvation. Figure S2. Quantitative western blot analysis of cell signaling in hiPSCs with different culture conditions. Figure S3. Subcellular distribution of ERK1/2. Figure S4. Specification and validation of RAS antibodies. (DOCX 2260 kb)

E-RAS is a member of the RAS family specifically expressed in embryonic stem cells, gastric tumors, and hepatic stellate cells. Unlike classical RAS isoforms (H-, N-, and K-RAS4B), E-RAS has, in addition to striking and remarkable sequence deviations, an extended 38-amino acid-long unique N-terminal region with still unknown functions. We investigated the molecular mechanism of E-RAS regulation and function with respect to its sequence and structural features. We found that N-terminal extension of E-RAS is important for E-RAS signaling activity. E-RAS protein most remarkably revealed a different mode of effector interaction as compared with H-RAS, which correlates with deviations in the effector-binding site of E-RAS. Of all these residues, tryptophan 79 (arginine 41 in H-RAS), in the interswitch region, modulates the effector selectivity of RAS proteins from H-RAS to E-RAS features.

[This corrects the article DOI: 10.1371/journal.pgen.1006684.].

The three deleted in liver cancer genes (DLC1–3) encode Rho-specific GTPase-activating proteins (RhoGAPs). Their expression is frequently silenced in a variety of cancers. The RhoGAP activity, which is required for full DLC-dependent tumor suppressor activity, can be inhibited by the Src homology 3 (SH3) domain of a Ras-specific GAP (p120RasGAP). Here, we comprehensively investigated the molecular mechanism underlying cross-talk between two distinct regulators of small GTP-binding proteins using structural and biochemical methods. We demonstrate that only the SH3 domain of p120 selectively inhibits the RhoGAP activity of all three DLC isoforms as compared with a large set of other representative SH3 or RhoGAP proteins. Structural and mutational analyses provide new insights into a putative interaction mode of the p120 SH3 domain with the DLC1 RhoGAP domain that is atypical and does not follow the classical PXXP-directed interaction. Hence, p120 associates with the DLC1 RhoGAP domain by targeting the catalytic arginine finger and thus by competitively and very potently inhibiting RhoGAP activity. The novel findings of this study shed light on the molecular mechanisms underlying the DLC inhibitory effects of p120 and suggest a functional cross-talk between Ras and Rho proteins at the level of regulatory proteins.

RAS effectors specifically interact with the GTP-bound form of RAS in response to extracellular signals and link them to downstream signaling pathways. The molecular nature of effector interaction by RAS is well-studied but yet still incompletely understood in a comprehensive and systematic way. Here, structure-function relationships in the interaction between different RAS proteins and various effectors were investigated in detail by combining our in vitro data with in silico data. Equilibrium dissociation constants were determined for the binding of HRAS, KRAS, NRAS, RRAS1 and RRAS2 to both the RAS binding (RB) domain of CRAF and PI3Kα, and the RAS association (RA) domain of RASSF5, RALGDS and PLCe, respectively, using fluorescence polarization. An interaction matrix, constructed on the basis of available crystal structures, allowed identification of hotspots as critical determinants for RAS-effector interaction. New insights provided by this study are the dissection of the identified hotspots in five distinct regions (R1 to R5) in spite of high sequence variability not only between, but also within, RB/RA domain-containing effectors proteins. Finally, we propose that intermolecular β-sheet interaction in R1 is a central recognition region while R3 may determine specific contacts of RAS versus RRAS isoforms with effectors.

International audience; Juvenile myelomonocytic leukemia (JMML) is a rare and severe myelodysplastic and myeloproliferative neoplasm of early childhood initiated by germline or somatic RAS-activating mutations. Genetic profiling and whole-exome sequencing of a large JMML cohort (118 and 30 cases, respectively) uncovered additional genetic abnormalities in 56 cases (47%). Somatic events were rare (0.38 events/Mb/case) and restricted to sporadic (49/78; 63%) or neurofibromatosis type 1 (NF1)-associated (8/8; 100%) JMML cases. Multiple concomitant genetic hits targeting the RAS pathway were identified in 13 of 78 cases (17%), disproving the concept of mutually exclusive RAS pathway mutations and defining new pathways activated in JMML involving phosphoinositide 3-kinase (PI3K) and the mTORC2 complex through RAC2 mutation. Furthermore, this study highlights PRC2 loss (26/78; 33% of sporadic JMML cases) that switches the methylation/acetylation status of lysine 27 of histone H3 in JMML cases with altered RAS and PRC2 pathways. Finally, the association between JMML outcome and mutational profile suggests a dose-dependent effect for RAS pathway activation, distinguishing very aggressive JMML rapidly progressing to acute myeloid leukemia.

The three deleted in liver cancer genes (DLC1-3) encode Rho-specific GTPase-activating proteins (RhoGAPs). Their expression is frequently silenced in a variety of cancers. The RhoGAP activity, which is required for full DLC-dependent tumor suppressor activity, can be inhibited by the Src homology 3 (SH3) domain of a Ras-specific GAP (p120RasGAP). Here, we comprehensively investigated the molecular mechanism underlying cross-talk between two distinct regulators of small GTP-binding proteins using structural and biochemical methods. We demonstrate that only the SH3 domain of p120 selectively inhibits the RhoGAP activity of all three DLC isoforms as compared with a large set of other representative SH3 or RhoGAP proteins. Structural and mutational analyses provide new insights into a putative interaction mode of the p120 SH3 domain with the DLC1 RhoGAP domain that is atypical and does not follow the classical PXXP-directed interaction. Hence, p120 associates with the DLC1 RhoGAP domain by targeting the catalytic arginine finger and thus by competitively and very potently inhibiting RhoGAP activity. The novel findings of this study shed light on the molecular mechanisms underlying the DLC inhibitory effects of p120 and suggest a functional cross-talk between Ras and Rho proteins at the level of regulatory proteins.

Rho family proteins are involved in an array of cellular processes by modulating cytoskeletal organization, transcription, and cell cycle progression. The signaling functions of Rho family proteins are based on the formation of distinctive protein–protein complexes with their regulators and effectors. A necessary precondition for such differential interactions is an intact molecular switch function, which is a hallmark of most members of the Rho family. Such classical Rho proteins cycle between an inactive GDP-bound state and an active GTP-bound state. They specifically interact via a consensus-binding sites called switch I and II with three structurally and functionally unrelated classes of regulatory proteins, such as guanine nucleotide dissociation inhibitors (GDIs), guanine nucleotide exchange factors (GEFs), and GTPase-activating proteins (GAPs). Extensive studies in the last 25 years have provided invaluable insights into the molecular mechanisms underlying regulation and signal transduction of the Rho family proteins. In this chapter, we will review common features of Rho protein regulations and highlight specific aspects of their structure–function relationships.

Induced pluripotent stem cells are generated by direct reprogramming of somatic cells with the introduction of defined transcription factors or other means. Clinical applications of induced pluripotent stem cells are the latest of stem cell therapy approaches due to overcoming problems associated with insufficient cells from conventional sources and immune rejections. In practice, this is restricted by 4 major barriers including the use of genetic manipulations for delivering the reprogramming factors, low efficiency of this process, slow kinetics of the direct reprogramming, and potential for tumor development. Here, we review the latest achievements in improving reprogramming efficiency by alternative strategies. These alternatives mainly involve the replacement of genetic reprogramming factors with small molecules or other factors.