Integrative multiomics and in silico analysis revealed the role of ARHGEF1 and its screened antagonist in mild and severe COVID-19 patients.

COVID-19 is a sneaking deadly disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The rapid increase in the number of infected patients worldwide enhances the exigency for medicines. However, precise therapeutic drugs are not available for COVID-19; thus, exhaustive research is critically required to unscramble the pathogenic tools and probable therapeutic targets for the development of effective therapy. This study utilizes a chemogenomics strategy, including computational tools for the identification of viral-associated differentially expressed genes (DEGs), and molecular docking of potential chemical compounds available in antiviral, anticancer, and natural product-based libraries against these DEGs. We scrutinized the messenger RNA expression profile of SARS-CoV-2 patients, publicly available on the National Center for Biotechnology Information-Gene Expression Omnibus database, stratified them into different groups based on the severity of infection, superseded by identification of overlapping mild and severe infectious (MSI)-DEGs. The profoundly expressed MSI-DEGs were then subjected to trait-linked weighted co-expression network construction and hub module detection. The hub module MSI-DEGs were then exposed to enrichment (gene ontology + pathway) and protein-protein interaction network analyses where Rho guanine nucleotide exchange factor 1 (ARHGEF1) gene conjectured in all groups and could be a probable target of therapy. Finally, we used the molecular docking and molecular dynamics method to identify inherent hits against the ARHGEF1 gene from antiviral, anticancer, and natural product-based libraries. Although the study has an identified significant association of the ARHGEF1 gene in COVID19; and probable compounds targeting it, using in silico methods, these targets need to be validated by both in vitro and in vivo methods to effectively determine their therapeutic efficacy against the devastating virus.

Apigenin inhibits tumor angiogenesis by hindering microvesicle biogenesis via ARHGEF1.

Extracellular vesicles are essential for intercellular communication and are involved in tumor progression. Inhibiting the direct release of extracellular vesicles seems to be an effective strategy in inhibiting tumor progression, but lacks of investigation. Here, we report a natural flavonoid compound, apigenin, could significantly inhibit the growth of hepatocellular carcinoma by preventing microvesicle secretion. Mechanistically, apigenin primarily targets the guanine nucleotide exchange factor ARHGEF1, inhibiting the activity of small G protein Cdc42, which is essential in regulating the release of microvesicles from tumor cells. In turn, this inhibits tumor angiogenesis related to VEGF90K transported on microvesicles, ultimately impeding tumor progression. Collectively, these findings highlight the therapeutic potential of apigenin and shed light on its anticancer mechanisms through inhibiting microvesicle biogenesis, providing a solid foundation for the refinement and practical application of apigenin.

Regulation of neural stem cell self-renewal, proliferation and differentiation by the RhoA guanine nucleotide exchange factor Arhgef 1.

The role of the Arhgef1 as a RhoA-specific guanine nucleotide exchange factor has been widely investigated in the immune system. Our previous findings reveal that Arhgef 1 is highly expressed in neural stem cells (NSCs) and controls the process of neurite formation. However, the functional role of Arhgef 1 in NSCs remains poorly understood. In order to investigate the role of Arhgef 1 in NSCs, Arhgef 1 expression in NSCs was reduced by using lentivirus-mediated short hairpin RNA interference. Our results indicate that down-regulated expression of Arhgef 1 reduced the self-renewal, proliferation capacity of NSCs and affect cell fate determination. In addition, the comparative transcriptome analysis from RNA-seq data determines the mechanisms of deficits in Arhgef 1 knockdown NSCs. Altogether, our present studies show that Arhgef 1 down-regulation leads to interruption of the cell cycle procession. The importance of Arhgef 1 for regulating self-renewal, proliferation and differentiation in NSCs is reported for the first time.

Structural Design and Analysis of the RHOA-ARHGEF1 Binding Mode: Challenges and Applications for Protein-Protein Interface Prediction.

The interaction between two proteins may involve local movements, such as small side-chains re-positioning or more global allosteric movements, such as domain rearrangement. We studied how one can build a precise and detailed protein-protein interface using existing protein-protein docking methods, and how it can be possible to enhance the initial structures using molecular dynamics simulations and data-driven human inspection. We present how this strategy was applied to the modeling of RHOA-ARHGEF1 interaction using similar complexes of RHOA bound to other members of the Rho guanine nucleotide exchange factor family for comparative assessment. In parallel, a more crude approach based on structural superimposition and molecular replacement was also assessed. Both models were then successfully refined using molecular dynamics simulations leading to protein structures where the major data from scientific literature could be recovered. We expect that the detailed strategy used in this work will prove useful for other protein-protein interface design. The RHOA-ARHGEF1 interface modeled here will be extremely useful for the design of inhibitors targeting this protein-protein interaction (PPI).

Arhgef1 Plays a Vital Role in Platelet Function and Thrombogenesis.

Background Platelets are the cellular mediators of hemostasis and thrombosis, and their function is regulated by a number of molecular mediators, such as small GTP ases. These small GTP ases are themselves regulated by guanine nucleotide exchange factors such as Arhgefs, several of which are found in platelets, including the highly expressed Arhgef1. However, the role of Arhgef1 in platelets has not yet been investigated. Methods and Results We employed mice with genetic deletion of Arhgef1 (ie, Arhgef1-/-) and investigated their platelet phenotype by employing a host of in vivo and in vitro platelet assays. Our results indicate that Arhgef1-/- mice had prolonged carotid artery occlusion and tail bleeding times. Moreover, platelets from these mice exhibited defective aggregation, dense and α granule secretion, α II bß3 integrin activation, clot retraction and spreading, in comparison to their wild-type littermates. Finally, we also found that the mechanism by which Arhgef1 regulates platelets is mediated in part by a defect in the activation of the RhoA-Rho-associated kinase axis, but not Rap1b. Conclusions Our data demonstrate, for the first time, that Arhgef1 plays a critical role in platelet function, in vitro and in vivo.

Neurotoxicity of the pesticide rotenone on neuronal polarization: a mechanistic approach.

Neurons are the most extensive and polarized cells that display a unique single long axon and multiple dendrites, which are compartments exhibiting structural and functional differences. Polarity occurs early in neuronal development and it is maintained by complex subcellular mechanisms throughout cell life. A well-defined and controlled spatio-temporal program of cellular and molecular events strictly regulates the formation of the axon and dendrites from a non-polarized cell. This event is critical for an adequate neuronal wiring and therefore for the normal functioning of the nervous system. Neuronal polarity is very sensitive to the harmful effects of different factors present in the environment. In this regard, rotenone is a crystalline, colorless and odorless isoflavone used as insecticide, piscicide and broad spectrum pesticide commonly used earlier in agriculture. In the present review we will summarize the toxicity mechanism caused by this pesticide in different neuronal cell types, focusing on a particular biological mechanism whereby rotenone could impair neuronal polarization in cultured hippocampal neurons. Recent advances suggest that the inhibition of axonogenesis produced by rotenone could be related with its effect on microtubule dynamics, the actin cytoskeleton and their regulatory pathways, particularly affecting the small RhoGTPase RhoA. Unveiling the mechanism by which rotenone produces neurotoxicity will be instrumental to understand the cellular mechanisms involved in neurodegenerative diseases influenced by this environmental pollutant, which may lead to research focused on the design of new therapeutic strategies.

Inhibition of Cdc42 and Rac1 activities in pheochromocytoma, the adrenal medulla tumor.

Altered Rho GTPase signaling has been linked to many types of cancer. As many small G proteins, Rho GTPases cycle between an active and inactive state thanks to specific regulators that catalyze exchange of GDP into GTP (Rho-GEF) or hydrolysis of GTP into GDP (Rho-GAP). Recent studies have shown that alteration takes place either at the level of Rho proteins themselves (expression levels, point mutations) or at the level of their regulators, mostly RhoGEFs and RhoGAPs. Most reports describe Rho GTPases gain of function that may participate to the tumorigenesis processes. In contrast, we have recently reported that decreased activities of Cdc42 and Rac1 as well as decreased expression of 2 Rho-GEFs, FARP1 and ARHGEF1, correlate with pheochromocytomas, a tumor developing in the medulla of the adrenal gland (Croisé et al., Endocrine Related Cancer, 2016). Here we highlight the major evidence and further study the correlation between Rho GTPases activities and expression levels of ARHGEF1 and FARP1. Finally we also discuss how the decrease of Cdc42 and Rac1 activities may help human pheochromocytomas to develop and comment the possible relationship between FARP1, ARHGEF1 and the 2 Rho GTPases Cdc42 and Rac1 in tumorigenesis.

Arhgef1 is expressed in cortical neural progenitor cells and regulates neurite outgrowth of newly differentiated neurons.

Neurite outgrowth is crucial for the maturation of neurons and the establishment of anatomical connections during development of the nervous system. We report here that Arhgef1, a RhoA guanine nucleotide exchange factor previously found expressed in the early stages of neuronal development to regulate neurite outgrowth, is also highly expressed in cortical neural progenitor cells (NPCs). To better dissect its role in NPCs, we knocked down Arhgef1 expression in these cells and induced differentiated of them into neurons. Notably, silencing of Arhgef1 markedly enhanced neurite outgrowth in neurons derived from NPCs. Furthermore, we showed that Arhgef1 silencing inhibited the activity of RhoA, and pharmacological blockade of RhoA activity promoted neurite outgrowth in NPC-derived neurons. These findings reveal that Arhgef1 controls the process of neurite formation in newborn cortical neurons derived from NPCs.

Arhgef1 negatively regulates neurite outgrowth through activation of RhoA signaling pathways.

Neurite outgrowth is essential for the establishment of functional neuronal connections during brain development. This study identifies that Arhgef1 is predominantly expressed in early neuronal developmental stages and negatively regulates neurite outgrowth. Knockdown of Arhgef1 in either Neuro-2a cells or primary cortical neurons leads to excess growth of neurites, whereas overexpression of Arhgef1 prominently restricts neurite formation. Arhgef1 strongly activates RhoA activity while concomitantly inhibits Rac1 and Cdc42 activities. Pharmacological blockade of RhoA activity restores normal neurite outgrowth in Arhgef1-overexpressed neurons. Importantly, Arhgef1 promotes F-actin polymerization in neurons, probably through inhibiting the activity of the actin-depolymerizing factor cofilin. Collectively, these findings reveal that Arhgef1 functions as a negative regulator of neurite outgrowth through regulating RhoA-cofilin pathway and actin dynamics.