Fragile X Messenger Ribonucleoprotein Protein and Its Multifunctionality: From Cytosol to Nucleolus and Back.

Silencing of the fragile X messenger ribonucleoprotein 1 (FMR1) gene and a consequent lack of FMR protein (FMRP) synthesis are associated with fragile X syndrome, one of the most common inherited intellectual disabilities. FMRP is a multifunctional protein that is involved in many cellular functions in almost all subcellular compartments under both normal and cellular stress conditions in neuronal and non-neuronal cell types. This is achieved through its trafficking signals, nuclear localization signal (NLS), nuclear export signal (NES), and nucleolar localization signal (NoLS), as well as its RNA and protein binding domains, and it is modulated by various post-translational modifications such as phosphorylation, ubiquitination, sumoylation, and methylation. This review summarizes the recent advances in understanding the interaction networks of FMRP with a special focus on FMRP stress-related functions, including stress granule formation, mitochondrion and endoplasmic reticulum plasticity, ribosome biogenesis, cell cycle control, and DNA damage response.

The Microenvironment of the Pathogenesis of Cardiac Hypertrophy.

Pathological cardiac hypertrophy is a key risk factor for the development of heart failure and predisposes individuals to cardiac arrhythmia and sudden death. While physiological cardiac hypertrophy is adaptive, hypertrophy resulting from conditions comprising hypertension, aortic stenosis, or genetic mutations, such as hypertrophic cardiomyopathy, is maladaptive. Here, we highlight the essential role and reciprocal interactions involving both cardiomyocytes and non-myocardial cells in response to pathological conditions. Prolonged cardiovascular stress causes cardiomyocytes and non-myocardial cells to enter an activated state releasing numerous pro-hypertrophic, pro-fibrotic, and pro-inflammatory mediators such as vasoactive hormones, growth factors, and cytokines, i.e., commencing signaling events that collectively cause cardiac hypertrophy. Fibrotic remodeling is mediated by cardiac fibroblasts as the central players, but also endothelial cells and resident and infiltrating immune cells enhance these processes. Many of these hypertrophic mediators are now being integrated into computational models that provide system-level insights and will help to translate our knowledge into new pharmacological targets. This perspective article summarizes the last decades' advances in cardiac hypertrophy research and discusses the herein-involved complex myocardial microenvironment and signaling components.

Dominant ARF3 variants disrupt Golgi integrity and cause a neurodevelopmental disorder recapitulated in zebrafish.

Vesicle biogenesis, trafficking and signaling via Endoplasmic reticulum-Golgi network support essential developmental processes and their disruption lead to neurodevelopmental disorders and neurodegeneration. We report that de novo missense variants in ARF3, encoding a small GTPase regulating Golgi dynamics, cause a developmental disease in humans impairing nervous system and skeletal formation. Microcephaly-associated ARF3 variants affect residues within the guanine nucleotide binding pocket and variably perturb protein stability and GTP/GDP binding. Functional analysis demonstrates variably disruptive consequences of ARF3 variants on Golgi morphology, vesicles assembly and trafficking. Disease modeling in zebrafish validates further the dominant behavior of the mutants and their differential impact on brain and body plan formation, recapitulating the variable disease expression. In-depth in vivo analyses traces back impaired neural precursors' proliferation and planar cell polarity-dependent cell movements as the earliest detectable effects. Our findings document a key role of ARF3 in Golgi function and demonstrate its pleiotropic impact on development.

The glucocorticoid receptor associates with RAS complexes to inhibit cell proliferation and tumor growth.

Mutations that activate members of the RAS family of GTPases are associated with various cancers and drive tumor growth. The glucocorticoid receptor (GR), a member of the nuclear receptor family, has been proposed to interact with and inhibit the activation of components of the PI3K-AKT and MAPK pathways downstream of RAS. In the absence of activating ligands, we found that GR was present in cytoplasmic KRAS-containing complexes and inhibited the activation of wild-type and oncogenic KRAS in mouse embryonic fibroblasts and human lung cancer A549 cells. The DNA binding domain of GR was involved in the interaction with KRAS, but GR-dependent inhibition of RAS activation did not depend on the nuclear translocation of GR. The addition of ligand released GR-dependent inhibition of RAS, AKT, the MAPK p38, and the MAPKK MEK. CRISPR-Cas9-mediated deletion of GR in A549 cells enhanced tumor growth in xenografts in mice. Patient samples of non-small cell lung carcinomas showed lower expression of NR3C1, the gene encoding GR, compared to adjacent normal tissues and lower NR3C1 expression correlated with a worse disease outcome. These results suggest that glucocorticoids prevent the ability of GR to limit tumor growth by inhibiting RAS activation, which has potential implications for the use of glucocorticoids in patients with cancer.

Physical Interaction between Embryonic Stem Cell-Expressed Ras (ERas) and Arginase-1 in Quiescent Hepatic Stellate Cells.

Embryonic stem cell-expressed Ras (ERas) is an atypical constitutively active member of the Ras family and controls distinct signaling pathways, which are critical, for instance, for the maintenance of quiescent hepatic stellate cells (HSCs). Unlike classical Ras paralogs, ERas has a unique N-terminal extension (Nex) with as yet unknown function. In this study, we employed affinity pull-down and quantitative liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses and identified 76 novel binding proteins for human and rat ERas Nex peptides, localized in different subcellular compartments and involved in various cellular processes. One of the identified Nex-binding proteins is the nonmitochondrial, cytosolic arginase 1 (ARG1), a key enzyme of the urea cycle and involved in the de novo synthesis of polyamines, such as spermidine and spermine. Here, we show, for the first time, a high-affinity interaction between ERas Nex and purified ARG1 as well as their subcellular colocalization. The inhibition of ARG1 activity strikingly accelerates the activation of HSCs ex vivo, suggesting a central role of ARG1 activity in the maintenance of HSC quiescence.

The Pseudo-Natural Product Rhonin Targets RHOGDI.

For the discovery of novel chemical matter generally endowed with bioactivity, strategies may be particularly efficient that combine previous insight about biological relevance, e.g., natural product (NP) structure, with methods that enable efficient coverage of chemical space, such as fragment-based design. We describe the de novo combination of different 5-membered NP-derived N-heteroatom fragments to structurally unprecedented "pseudo-natural products" in an efficient complexity-generating and enantioselective one-pot synthesis sequence. The pseudo-NPs inherit characteristic elements of NP structure but occupy areas of chemical space not covered by NP-derived chemotypes, and may have novel biological targets. Investigation of the pseudo-NPs in unbiased phenotypic assays and target identification led to the discovery of the first small-molecule ligand of the RHO GDP-dissociation inhibitor 1 (RHOGDI1), termed Rhonin. Rhonin inhibits the binding of the RHOGDI1 chaperone to GDP-bound RHO GTPases and alters the subcellular localization of RHO GTPases.

Inhibition of the RacGEF VAV3 by the small molecule IODVA1 impedes RAC signaling and overcomes resistance to tyrosine kinase inhibition in acute lymphoblastic leukemia.

Aberrant RHO guanine nucleotide exchange factor (RhoGEF) activation is chief mechanism driving abnormal activation of their GTPase targets in transformation and tumorigenesis. Consequently, a small-molecule inhibitor of RhoGEF can make an anti-cancer drug. We used cellular, mouse, and humanized models of RAC-dependent BCR-ABL1-driven and Ph-like acute lymphoblastic leukemia to identify VAV3, a tyrosine phosphorylation-dependent RacGEF, as the target of the small molecule IODVA1. We show that through binding to VAV3, IODVA1 inhibits RAC activation and signaling and increases pro-apoptotic activity in BCR-ABL1-transformed cells. Consistent with this mechanism of action, cellular and animal models of BCR-ABL1-induced leukemia in Vav3-null background do not respond to IODVA1. By durably decreasing in vivo RAC signaling, IODVA1 eradicates leukemic propagating activity of TKI-resistant BCR-ABL1(T315I) B-ALL cells after treatment withdrawal. Importantly, IODVA1 suppresses the leukemic burden in the treatment refractory pediatric Ph+ and TKI-resistant Ph+ B-ALL patient-derived xenograft models better than standard-of-care dasatinib or ponatinib and provides a more durable response after treatment withdrawal. Pediatric leukemia samples with diverse genetic lesions show high sensitivity to IODVA1 ex vivo and this sensitivity is VAV3 dependent. IODVA1 thus spearheads a novel class of drugs that inhibits a RacGEF and holds promise as an anti-tumor therapy.

KRAS-related long noncoding RNAs in human cancers.

KRAS is one of the most widely prevalent proto-oncogenes in human cancers. The constitutively active KRAS oncoprotein contributes to both tumor onset and cancer development by promoting cell proliferation and anchorage-independent growth in a MAPK pathway-dependent manner. The expression of microRNAs (miRNAs) and the KRAS oncogene are known to be dysregulated in various cancers, while long noncoding RNAs (lncRNAs) can act as regulators of the miRNAs targeting KRAS oncogene in different cancers and have gradually become a focus of research in recent years. In this review article, we summarize recent advances in the research on lncRNAs that have sponging effects on KRAS-targeting miRNAs as crucial mediators of KRAS expression in different cell types and organs. A deeper understanding of lncRNA function in KRAS-driven cancers is of major fundamental importance and will provide a valuable clinical tool for the diagnosis, prognosis, and eventual treatment of cancers.

Electrostatic Forces Mediate the Specificity of RHO GTPase-GDI Interactions.

Three decades of research have documented the spatiotemporal dynamics of RHO family GTPase membrane extraction regulated by guanine nucleotide dissociation inhibitors (GDIs), but the interplay of the kinetic mechanism and structural specificity of these interactions is as yet unresolved. To address this, we reconstituted the GDI-controlled spatial segregation of geranylgeranylated RHO protein RAC1 in vitro. Various biochemical and biophysical measurements provided unprecedented mechanistic details for GDI function with respect to RHO protein dynamics. We determined that membrane extraction of RHO GTPases by GDI occurs via a 3-step mechanism: (1) GDI non-specifically associates with the switch regions of the RHO GTPases; (2) an electrostatic switch determines the interaction specificity between the C-terminal polybasic region of RHO GTPases and two distinct negatively-charged clusters of GDI1; (3) a non-specific displacement of geranylgeranyl moiety from the membrane sequesters it into a hydrophobic cleft, effectively shielding it from the aqueous milieu. This study substantially extends the model for the mechanism of GDI-regulated RHO GTPase extraction from the membrane, and could have implications for clinical studies and drug development.

RHO GTPase-Related Long Noncoding RNAs in Human Cancers.

RHO GTPases are critical signal transducers that regulate cell adhesion, polarity, and migration through multiple signaling pathways. While all these cellular processes are crucial for the maintenance of normal cell homeostasis, disturbances in RHO GTPase-associated signaling pathways contribute to different human diseases, including many malignancies. Several members of the RHO GTPase family are frequently upregulated in human tumors. Abnormal gene regulation confirms the pivotal role of lncRNAs as critical gene regulators, and thus, they could potentially act as oncogenes or tumor suppressors. lncRNAs most likely act as sponges for miRNAs, which are known to be dysregulated in various cancers. In this regard, the significant role of miRNAs targeting RHO GTPases supports the view that the aberrant expression of lncRNAs may reciprocally change the intensity of RHO GTPase-associated signaling pathways. In this review article, we summarize recent advances in lncRNA research, with a specific focus on their sponge effects on RHO GTPase-targeting miRNAs to crucially mediate gene expression in different cancer cell types and tissues. We will focus in particular on five members of the RHO GTPase family, including RHOA, RHOB, RHOC, RAC1, and CDC42, to illustrate the role of lncRNAs in cancer progression. A deeper understanding of the widespread dysregulation of lncRNAs is of fundamental importance for confirmation of their contribution to RHO GTPase-dependent carcinogenesis.

Optimizing the synthesis and purification of MS2 virus like particles.

Introducing bacteriophage MS2 virus-like particles (VLPs) as gene and drug delivery tools increases the demand for optimizing their production and purification procedure. PEG precipitation method is used efficiently to purify VLPs, while the effects of pH and different electrolytes on the stability, size, and homogeneity of purified MS2 VLPs, and the encapsulated RNA sequences remained to be elucidated. In this regard, a vector, capable of producing VLP with an shRNA packed inside was prepared. The resulting VLPs in different buffers/solutions were assessed for their size, polydispersity index, and ability to protect the enclosed shRNA. We report that among Tris, HEPES, and PBS, with or without NaNO3, and also NaNO3 alone in different pH and ionic concentrations, the 100 mM NaNO3-Tris buffer with pH:8 can be used as a new and optimal MS2 VLP production buffer, capable of inhibiting the VLPs aggregation. These VLPs show a size range of 27-30 nm and suitable homogeneity with minimum 12-month stability at 4 °C. Moreover, the resulting MS2 VLPs were highly efficient and stable for at least 48 h in conditions similar to in vivo. These features of MS2 VLPs produced in the newly introduced buffer make them an appropriate candidate for therapeutic agents' delivery.

The RHO Family GTPases: Mechanisms of Regulation and Signaling.

Much progress has been made toward deciphering RHO GTPase functions, and many studies have convincingly demonstrated that altered signal transduction through RHO GTPases is a recurring theme in the progression of human malignancies. It seems that 20 canonical RHO GTPases are likely regulated by three GDIs, 85 GEFs, and 66 GAPs, and eventually interact with >70 downstream effectors. A recurring theme is the challenge in understanding the molecular determinants of the specificity of these four classes of interacting proteins that, irrespective of their functions, bind to common sites on the surface of RHO GTPases. Identified and structurally verified hotspots as functional determinants specific to RHO GTPase regulation by GDIs, GEFs, and GAPs as well as signaling through effectors are presented, and challenges and future perspectives are discussed.

Spotlight on Accessory Proteins: RTK-RAS-MAPK Modulators as New Therapeutic Targets.

The RTK-RAS-MAPK axis is one of the most extensively studied signaling cascades and is related to the development of both cancers and RASopathies. In the last 30 years, many ideas and approaches have emerged for directly targeting constituent members of this cascade, predominantly in the context of cancer treatment. These approaches are still insufficient due to undesirable drug toxicity, resistance, and low efficacy. Significant advances have been made in understanding the spatiotemporal features of the constituent members of the RTK-RAS-MAPK axis, which are linked and modulated by many accessory proteins. Given that the majority of such modulators are now emerging as attractive therapeutic targets, a very small number of accessory inhibitors have yet to be discovered.

Rigosertib elicits potent anti-tumor responses in colorectal cancer by inhibiting Ras signaling pathway.

BACKGROUND: The therapeutic potency of Rigosertib (RGS) in the treatment of the myelodysplastic syndrome has been investigated previously, but little is known about its mechanisms of action. METHODS: The present study integrates systems and molecular biology approaches to investigate the mechanisms of the anti-tumor effects of RGS, either alone or in combination with 5-FU in cellular and animal models of colorectal cancer (CRC). RESULTS: The effects of RGS were more pronounced in dedifferentiated CRC cell types, compared to cell types that were epithelial-like. RGS inhibited cell proliferation and cell cycle progression in a cell-type specific manner, and that was dependent on the presence of mutations in KRAS, or its down-stream effectors. RGS increased both early and late apoptosis, by regulating the expression of p53, BAX and MDM2 in tumor model. We also found that RGS induced cell senescence in tumor tissues by increasing ROS generation, and impairing oxidant/anti-oxidant balance. RGS also inhibited angiogenesis and metastatic behavior of CRC cells, by regulating the expression of CD31, E-cadherin, and matrix metalloproteinases-2 and 9. CONCLUSION: Our findings support the therapeutic potential of this potent RAS signaling inhibitor either alone or in combination with standard regimens for the management of patients with CRC.

Subcellular Localization and Mitotic Interactome Analyses Identify SIRT4 as a Centrosomally Localized and Microtubule Associated Protein.

The stress-inducible and senescence-associated tumor suppressor SIRT4, a member of the family of mitochondrial sirtuins (SIRT3, SIRT4, and SIRT5), regulates bioenergetics and metabolism via NAD+-dependent enzymatic activities. Next to the known mitochondrial location, we found that a fraction of endogenous or ectopically expressed SIRT4, but not SIRT3, is present in the cytosol and predominantly localizes to centrosomes. Confocal spinning disk microscopy revealed that SIRT4 is found during the cell cycle dynamically at centrosomes with an intensity peak in G2 and early mitosis. Moreover, SIRT4 precipitates with microtubules and interacts with structural (α,ß-tubulin, γ-tubulin, TUBGCP2, TUBGCP3) and regulatory (HDAC6) microtubule components as detected by co-immunoprecipitation and mass spectrometric analyses of the mitotic SIRT4 interactome. Overexpression of SIRT4 resulted in a pronounced decrease of acetylated α-tubulin (K40) associated with altered microtubule dynamics in mitotic cells. SIRT4 or the N-terminally truncated variant SIRT4(ΔN28), which is unable to translocate into mitochondria, delayed mitotic progression and reduced cell proliferation. This study extends the functional roles of SIRT4 beyond mitochondrial metabolism and provides the first evidence that SIRT4 acts as a novel centrosomal/microtubule-associated protein in the regulation of cell cycle progression. Thus, stress-induced SIRT4 may exert its role as tumor suppressor through mitochondrial as well as extramitochondrial functions, the latter associated with its localization at the mitotic spindle apparatus.

Rigosertib potently protects against colitis-associated intestinal fibrosis and inflammation by regulating PI3K/AKT and NF-κB signaling pathways.

AIMS: Rigosertib (RGS) is a PI3K inhibitor that exerts protective effects against tumor progression and cancer-related inflammation. This study was aimed to explore the regulatory effects of RGS on proliferative, pro-fibrotic and inflammatory factors in DSS- induced colitis mice model. MATERIALS AND METHODS: The present study integrates systems and molecular biology approaches to investigate the therapeutic potency of RGS in an experimental model of colitis specifically examining its effects on the PI3K/AKT and NF-κB signaling pathways. KEY FINDINGS: Analysis of time-resolved proteome profiling showed that PI3K-AKT inhibitors regulate expression of many proteins in all stages of inflammation, fibrogenesis and extracellular matrix remodeling. Consistent with our in-silico findings, RGS improved colitis disease activity as assessed by changes in body weight, degree of stool consistency, rectal bleeding and prolapse. RGS also reduced oxidative stress markers and colon histopathological score by decreasing inflammatory responses in colon tissues. Moreover, expression of pro-fibrotic and pro-inflammatory factors including Acta 2, Col 1a1, Col 1a2, IL-1ß, TNF-α, INF-γ, and MCP-1 were suppressed in the mice treated with RGS compared to the control group. The protective effects of RGS were mediated by inactivation of PI3K/AKT and NF-kB signaling pathways. SIGNIFICANCE: This study clearly demonstrates the anti-proliferative, anti-inflammatory and anti-fibrotic effects of RGS in colitis that may have implications for the treatment of colitis and colitis-associated cancer.

Fragile X mental retardation protein protects against tumour necrosis factor-mediated cell death and liver injury.

OBJECTIVE: The Fragile X mental retardation (FMR) syndrome is a frequently inherited intellectual disability caused by decreased or absent expression of the FMR protein (FMRP). Lack of FMRP is associated with neuronal degradation and cognitive dysfunction but its role outside the central nervous system is insufficiently studied. Here, we identify a role of FMRP in liver disease. DESIGN: Mice lacking Fmr1 gene expression were used to study the role of FMRP during tumour necrosis factor (TNF)-induced liver damage in disease model systems. Liver damage and mechanistic studies were performed using real-time PCR, Western Blot, staining of tissue sections and clinical chemistry. RESULTS: Fmr1null mice exhibited increased liver damage during virus-mediated hepatitis following infection with the lymphocytic choriomeningitis virus. Exposure to TNF resulted in severe liver damage due to increased hepatocyte cell death. Consistently, we found increased caspase-8 and caspase-3 activation following TNF stimulation. Furthermore, we demonstrate FMRP to be critically important for regulating key molecules in TNF receptor 1 (TNFR1)-dependent apoptosis and necroptosis including CYLD, c-FLIPS and JNK, which contribute to prolonged RIPK1 expression. Accordingly, the RIPK1 inhibitor Necrostatin-1s could reduce liver cell death and alleviate liver damage in Fmr1null mice following TNF exposure. Consistently, FMRP-deficient mice developed increased pathology during acute cholestasis following bile duct ligation, which coincided with increased hepatic expression of RIPK1, RIPK3 and phosphorylation of MLKL. CONCLUSIONS: We show that FMRP plays a central role in the inhibition of TNF-mediated cell death during infection and liver disease.

From basic researches to new achievements in therapeutic strategies of KRAS-driven cancers.

Among the numerous oncogenes involved in human cancers, KRAS represents the most studied and best characterized cancer-related genes. Several therapeutic strategies targeting oncogenic KRAS (KRAS onc ) signaling pathways have been suggested, including the inhibition of synthetic lethal interactions, direct inhibition of KRAS onc itself, blockade of downstream KRAS onc effectors, prevention of post-translational KRAS onc modifications, inhibition of the induced stem cell-like program, targeting of metabolic peculiarities, stimulation of the immune system, inhibition of inflammation, blockade of upstream signaling pathways, targeted RNA replacement, and oncogene-induced senescence. Despite intensive and continuous efforts, KRAS onc remains an elusive target for cancer therapy. To highlight the progress to date, this review covers a collection of studies on therapeutic strategies for KRAS published from 1995 to date. An overview of the path of progress from earlier to more recent insights highlight novel opportunities for clinical development towards KRASonc-signaling targeted therapeutics.

bFGF-mediated pluripotency maintenance in human induced pluripotent stem cells is associated with NRAS-MAPK signaling.

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.