Rho GTPase Activity Research Articles

Adverse clinical outcomes and immunosuppressive microenvironment of RHO-GTPase activation pattern in hepatocellular carcinoma

BackgroundEmerging evidence suggests that Rho GTPases play a crucial role in tumorigenesis and metastasis, but their involvement in the tumor microenvironment (TME) and prognosis of hepatocellular carcinoma (HCC) is not well understood.MethodsWe aim to develop a tumor prognosis prediction system called the Rho GTPases-related gene score (RGPRG score) using Rho GTPase signaling genes and further bioinformatic analyses.ResultsOur work found that HCC patients with a high RGPRG score had significantly worse survival and increased immunosuppressive cell fractions compared to those with a low RGPRG score. Single-cell cohort analysis revealed an immune-active TME in patients with a low RGPRG score, with strengthened communication from T/NK cells to other cells through MIF signaling networks. Targeting these alterations in TME, the patients with high RGPRG score have worse immunotherapeutic outcomes and decreased survival time in the immunotherapy cohort. Moreover, the RGPRG score was found to be correlated with survival in 27 other cancers. In vitro experiments confirmed that knockdown of the key Rho GTPase-signaling biomarker SFN significantly inhibited HCC cell proliferation, invasion, and migration.ConclusionsThis study provides new insight into the TME features and clinical use of Rho GTPase gene pattern at the bulk-seq and single-cell level, which may contribute to guiding personalized treatment and improving clinical outcome in HCC.

P1212 Effects of Enterococcus faecalis-derived Gelatinase E on HCT116 colorectal cancer cells

Abstract Background The leading cause of death in colorectal cancer (CRC) patients is metastasis to crucial organs, such as the lung, brain and liver. The tumour microenvironment (TME) influences metastasis heavily. One important component in the TME of CRC is the microbiota. Microbiota can affect several pathways related to the origin of metastasis and reorganization of the extracellular matrix. One of the possible contributors to early CRC metastasis is Enterococcus faecalis. E. faecalis produces Gelatinase E (GelE), a quorum-sensing protein with collagenolytic properties. The primary goal of our study was to determine the effect of GelE on CRC behaviour, and especially on migration and invasion in HCT-116 human CRC cells. Moreover, we aimed to uncover the underlying pathways by which GelE influences CRC cells. Methods Extracellular GelE was purified from E. faecalis strain V583 from the culture medium. HCT-116 cells were cultured and exposed to different concentrations of GelE. We performed a viability assay using CCK-8 to assess potential toxicity of GelE. To test effects of GelE on collagen structure, we measured stiffness of collagen-I. A transwell assay was used to assess the influence of GelE on invasive capacity of cells through a collagen-I matrix. GelE exposed cells were assessed for shape and motility markers using immunofluorescent staining for β-catenin (cell shape) and loss of E-cadherin (cell motility marker). For mechanistic insight of GelE exposure on HCT116, we performed proteomics analysis on the cells. Results GelE treatment did not affect relative cell viability of HCT116 cells up to a concentration of 100 μg/ml. Exposure to GelE caused a concentration-dependent increase in HCT-116 cell invasion through collagen-I. The immunostainings showed a downregulation of E-cadherin, indicating an induction of cell motility. Immunostaining for β-catenin showed a statistically significant effect on cell shape, becoming more elongated, an additional indication of cell motility. Proteomics showed possible mechanistic pathways that explain the increased motility including activation of the SMAD pathway, RhoGTPAse activation and activation the urokinase-plasminogen system. Reactome database analysis on the proteomics data also revealed an increase in cell-cycle related pathways. Conclusion Our results indicate that E. faecalis-derived GelE drives HCT-116 invasion with an elongated motility. Therefore, drugs inhibiting the activity of GelE could decrease the risk of metastasis in patients with a high E. faecalis V583 abundance in the TME.

Opposite Mechanical Preference of Bone/Nerve Regeneration in 3D-Printed Bioelastomeric Scaffolds/Conduits Consistently Correlated with YAP-Mediated Stem Cell Osteo/Neuro-Genesis.

To systematically unveil how substrate stiffness, a critical factor in directing cell fate through mechanotransduction, correlates with tissue regeneration, novel biodegradable and photo-curable poly(trimethylene carbonate) fumarates (PTMCFs) for fabricating elastomeric 2D substrates and 3D bone scaffolds/nerve conduits, are presented. These substrates and structures with adjustable stiffness serve as a unique platform to evaluate how this mechanical cue affects the fate of human umbilical cord mesenchymal stem cells (hMSCs) and hard/soft tissue regeneration in rat femur bone defect and sciatic nerve transection models; whilst, decoupling from topographical and chemical cues. In addition to a positive relationship between substrate stiffness (tensile modulus: 90-990kPa) and hMSC adhesion, spreading, and proliferation mediated through Yes-associated protein (YAP), opposite mechanical preference is revealed in the osteogenesis and neurogenesis of hMSCs as they are significantly enhanced on the stiff and compliant substrates, respectively. In vivo tissue regeneration demonstrates the same trend: bone regeneration prefers the stiffer scaffolds; while, nerve regeneration prefers the more compliant conduits. Whole-transcriptome analysis further shows that upregulation of Rho GTPase activity and the downstream genes in the compliant group promote nerve repair, providing critical insight into the design strategies of biomaterials for stem cell regulation and hard/soft tissue regeneration through mechanotransduction.

Patterning of the cell cortex by Rho GTPases.

The Rho GTPases - RHOA, RAC1 and CDC42 - are small GTP binding proteins that regulate basic biological processes such as cell locomotion, cell division and morphogenesis by promoting cytoskeleton-based changes in the cell cortex. This regulation results from active (GTP-bound) Rho GTPases stimulating target proteins that, in turn, promote actin assembly and myosin 2-based contraction to organize the cortex. This basic regulatory scheme, well supported by in vitro studies, led to the natural assumption that Rho GTPases function in vivo in an essentially linear matter, with a given process being initiated by GTPase activation and terminated by GTPase inactivation. However, a growing body of evidence based on live cell imaging, modelling and experimental manipulation indicates that Rho GTPase activation and inactivation are often tightly coupled in space and time via signalling circuits and networks based on positive and negative feedback. In this Review, we present and discuss this evidence, and we address one of the fundamental consequences of coupled activation and inactivation: the ability of the Rho GTPases to self-organize, that is, direct their own transition from states of low order to states of high order. We discuss how Rho GTPase self-organization results in the formation of diverse spatiotemporal cortical patterns such as static clusters, oscillatory pulses, travelling wave trains and ring-like waves. Finally, we discuss the advantages of Rho GTPase self-organization and pattern formation for cell function.

Presenilin2 D439A Mutation Induces Dysfunction of Mitochondrial Fusion/Fission Dynamics and Abnormal Regulation of GTPase Activity

Alzheimer’s disease (AD) is an age-related progressive neurodegenerative disease, and approximately 10% of AD cases are early-onset familial AD (EOFAD), which is mainly linked to point mutations in genes encoding presenilins (PS1 and PS2). Mutations in PS2 are extremely rare and have not received enough attention. Recently, studies have found that Rho GTPase activity is closely related to the pathogenesis of AD. In this study, we used transcriptome sequencing in PS2 siRNA-transfected SH-SY5Y cells and found a group of differentially expressed genes (DEGs) related to the regulation of GTPase activity. Among those DEGs, the most significantly downregulated was Rho guanine nucleotide exchange factor 5 (ARHGEF5). GTPase activity in PS2 siRNA-transfected cells was significantly decreased. Then, we found that the expression of ARHGEF5 and the GTPase activity of Mitochondrial Rho GTPase 2 (Miro2) in PS2 D439A mutant SH-SY5Y cells were significantly decreased. We found for the first time that PS2 can bind to Miro2, and the PS2 D439A mutation reduced the binding between PS2 and Miro2, reduced the expression of Miro2, and resulted in an imbalance in mitochondrial fusion/fission dynamics. In conclusion, PS2 gene knockdown may participate in the pathogenesis of AD through the regulation of GTPase activity. The imbalance in mitochondrial dynamics mediated by the PS2 D439A mutation through regulation of the expression and GTPase activity of Miro2 may be a potential pathogenic mechanism of AD.

The RhoGEF Trio is transported by microtubules and affects microtubule stability in migrating neural crest cells

Directed cell migration requires a local fine-tuning of Rho GTPase activity to control protrusion formation, cell-cell contraction, and turnover of cellular adhesions. The Rho guanine nucleotide exchange factor (GEF) TRIO is ideally suited to control RhoGTPase activity because it combines two distinct catalytic domains to control Rac1 and RhoA activity in one molecule. However, at the cellular level, this molecular feature also requires a tight spatiotemporal control of TRIO activity. Here, we analyze the dynamic localization of Trio in Xenopus cranial neural crest (NC) cells, where we have recently shown that Trio is required for protrusion formation and migration. Using live cell imaging, we find that the GEF2 domain, but not the GEF1 domain of Trio, dynamically colocalizes with EB3 at microtubule plus-ends. Microtubule-mediated transport of Trio appears to be relevant for its function in NC migration, as a mutant GEF2 construct lacking the SxIP motif responsible for microtubule plus-end localization was significantly impaired in its ability to rescue the Trio loss-of-function phenotype compared to wild-type GEF2. Furthermore, by analyzing microtubule dynamics in migrating NC cells, we observed that loss of Trio function stabilized microtubules at cell-cell contact sites compared to controls, whereas they were destabilized at the leading edge of NC cells. Our data suggest that Trio is transported by microtubules to distinct subcellular locations where it has different functions in controlling microtubule stability, cell morphology, and cell-cell interaction during directed NC migration.

Rho GTPase activity crosstalk mediated by Arhgef11 and Arhgef12 coordinates cell protrusion-retraction cycles

Rho GTPases play a key role in the spatio-temporal coordination of cytoskeletal dynamics during cell migration. Here, we directly investigate crosstalk between the major Rho GTPases Rho, Rac and Cdc42 by combining rapid activity perturbation with activity measurements in mammalian cells. These studies reveal that Rac stimulates Rho activity. Direct measurement of spatio-temporal activity patterns show that Rac activity is tightly and precisely coupled to local cell protrusions, followed by Rho activation during retraction. Furthermore, we find that the Rho-activating Lbc-type GEFs Arhgef11 and Arhgef12 are enriched at transient cell protrusions and retractions and recruited to the plasma membrane by active Rac. In addition, their depletion reduces activity crosstalk, cell protrusion-retraction dynamics and migration distance and increases migration directionality. Thus, our study shows that Arhgef11 and Arhgef12 facilitate exploratory cell migration by coordinating cell protrusion and retraction by coupling the activity of the associated regulators Rac and Rho.

Molecular mechanism of regulation of RhoA GTPase by phosphorylation of RhoGDI

Rho-specific guanine nucleotide dissociation inhibitors (RhoGDIs) play a crucial role in the regulation of Rho family GTPases. They act as negative regulators that prevent the activation of Rho GTPases by forming complexes with the inactive GDP-bound state of GTPase. Release of Rho GTPase from the RhoGDI-bound complex is necessary for Rho GTPase activation. Biochemical studies provide evidence of a "phosphorylation code," where phosphorylation of some specific residues of RhoGDI selectively releases its GTPase partner (RhoA, Rac1, Cdc42, etc.). This work attempts to understand the molecular mechanism behind this specific phosphorylation-induced reduction in binding affinity. Using several microseconds long atomistic molecular dynamics simulations of the wild-type and phosphorylated states of the RhoA-RhoGDI complex, we propose a molecular-interaction-based mechanistic model for the dissociation of the complex. Phosphorylation induces major structural changes, particularly in the positively charged polybasic region (PBR) of RhoA and the negatively charged N-terminal region of RhoGDI that contribute most to the binding affinity. Molecular mechanics Poisson-Boltzmann surface area binding energy calculations show a significant weakening of interaction on phosphorylation at the RhoA-specific site of RhoGDI. In contrast, phosphorylation at a Rac1-specific site does not affect the overall binding affinity significantly, which confirms the presence of a phosphorylation code. RhoA-specific phosphorylation leads to a reduction in the number of contacts between the PBR of RhoA and the N-terminal region of RhoGDI, which manifests a reduction of the binding affinity. Using hydrogen bond occupancy analysis and energetic perturbation network, we propose a mechanistic model for the allosteric response, i.e., long-range signal propagation from the site of phosphorylation to the PBR and buried geranylgeranyl group in the form of rearrangement and rewiring of hydrogen bonds and salt bridges. Our results highlight the crucial role of specific electrostatic interactions in manifestation of the phosphorylation code.

Salt Behind the Scenes of Systemic Lupus Erythematosus and Rheumatoid Arthritis.

Sodium is vital for human health. High salt intake is a global health problem and is associated with cardiovascular morbidity and mortality. Recent evidence suggests that both innate and adaptive immune systems are affected by sodium. In general, excess salt intake drives immune cells toward a pro-inflammatory phenotype. The incidence of autoimmune diseases, including systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA), is steadily increasing. As excess salt induces a pro-inflammatory state, increased salt intake may have impacts on autoimmune diseases. The relationship between salt intake and autoimmune diseases is most widely studied in patients with SLE or RA. This review aimed to summarize the relationship between salt intake and SLE and RA. Most, but not all, of these studies showed that high salt intake might promote SLE by M1 macrophage shift, increase in Th17/Treg cell ratio, activation of dendritic and follicular helper T cells, and increased secretion of pro-inflammatory cytokines. In RA, apart from driving immune cells toward a pro-inflammatory state, high salt intake also influences cellular signaling pathways, including receptor activator of nuclear factor κB ligand (RANKL), Rho GTPases, and MAPK (mitogen-activated protein kinase). There is now sufficient evidence that excess salt intake may be related to the development and progression of SLE and RA, although there are still knowledge gaps. More studies are warranted to further highlight the relationship between excess salt intake, SLE, and RA. Salt intake may affect cell types and pro-inflammatory cytokines and signaling pathways associated with the development and progression of systemic lupus erythematosus and rheumatoid arthritis. Bcl-6 B-cell lymphoma, 6 Erk extracellular signal-regulated kinases, IFN-γ interferon-gamma, JNK c-Jun N-terminal kinase, IL-4 interleukin 4, IL-6 interleukin 6, MAPK mitogen-activated protein kinase, STAT signal transducer and activator of transcription, Tnf-α tumor necrosis factor, Treg T regulatory cell.

Levamisole Modulation of Podocytes' Actin Cytoskeleton in Nephrotic Syndrome.

Podocytes play a central role in glomerular diseases such as (idiopathic) nephrotic syndrome (iNS). Glucocorticoids are the gold standard therapy for iNS. Nevertheless, frequent relapses are common. In children with iNS, steroid-sparing agents are used to avoid prolonged steroid use and reduce steroid toxicity. Levamisole is one of these steroid-sparing drugs and although clinical effectiveness has been demonstrated, the molecular mechanisms of how levamisole exerts its beneficial effects remains poorly studied. Apart from immunomodulatory capacities, nonimmunological effects of levamisole on podocytes have also been suggested. We aimed to elaborate on the effects of levamisole on human podocytes in iNS. RNA sequencing data from a human podocyte cell line treated with levamisole showed that levamisole modulates the expression of various genes involved in actin cytoskeleton stabilization and remodeling. Functional experiments showed that podocytes exposed to puromycin aminonucleoside (PAN), lipopolysaccharides (LPS), and NS patient plasma resulted in significant actin cytoskeleton derangement, reduced cell motility, and impaired cellular adhesion when compared to controls, effects that could be restored by levamisole. Mechanistic studies revealed that levamisole exerts its beneficial effects on podocytes by signaling through the glucocorticoid receptor and by regulating the activity of Rho GTPases. In summary, our data show that levamisole exerts beneficial effects on podocytes by stabilizing the actin cytoskeleton in a glucocorticoid receptor-dependent manner.

The Muscarinic Acetylcholine M2 Receptor-Induced Nitration of p190A by eNOS Increases RhoA Activity in Cardiac Myocytes.

p190RhoGAP, which exists in two paralogs, p190RhoGAP-A (p190A) and p190RhoGAP-B (p190B), is a GTPase activating protein (GAP) contributing to the regulation of the cellular activity of RhoGTPases. Recent data showed that M2 muscarinic acetylcholine receptor (M2R) stimulation in neonatal rat cardiac myocytes (NRCM) induces the binding of p190RhoGAP to the long isoform of the regulator of G protein signaling 3 (RGS3L). This complex formation alters the substrate preference of p190RhoGAP from RhoA to Rac1. By analyzing carbachol-stimulated GAP activity, we show herein that p190A, but not p190B, alters its substrate preference in NRCM. Based on data that the RhoGAP activity of p190A in endothelial cells is diminished upon nitration by endothelial nitric oxide synthase (eNOS)-derived peroxynitrite, we studied whether carbachol-induced NO/peroxynitrite formation contributes to the carbachol-induced RhoA activation in NRCM. Interestingly, the carbachol-induced RhoA activation in NRCM was suppressed by the eNOS-preferring inhibitor L-NIO as well as the non-selective NOS inhibitor L-NAME. Using L-NIO, we firstly verified the carbachol-induced NO production concurrent with eNOS activation and, secondly, the carbachol-induced nitration of p190A in NRCM. By co-immunoprecipitation, the carbachol-induced complex formation of eNOS, p190A, RGS3L and caveolin-3 was detected. We thus conclude that the NO production by M2R-induced eNOS activation in caveolae in NRCM is required for the nitration of p190A, leading to the binding to RGS3L and the change in substrate preference from RhoA to Rac1. In line with this interpretation, the disruption of caveolae in NRCM by methyl-β-cyclodextrin suppressed carbachol-induced RhoA activation in NRCM to a similar extent as the inhibition of NO production.

Metabolic signature and proteasome activity controls synovial migration of CDC42hiCD14+ cells in rheumatoid arthritis.

Activation of Rho-GTPases in macrophages causes inflammation and severe arthritis in mice. In this study, we explore if Rho-GTPases define the joint destination of pathogenic leukocytes, the mechanism by which they perpetuate rheumatoid arthritis (RA), and how JAK inhibition mitigates these effects. CD14+ cells of 136 RA patients were characterized by RNA sequencing and cytokine measurement to identify biological processes and transcriptional regulators specific for CDC42 hiCD14+ cells, which were summarized in a metabolic signature (MetSig). The effect of hypoxia and IFN-γ signaling on the metabolic signature of CD14+ cells was assessed experimentally. To investigate its connection with joint inflammation, the signature was translated into the single-cell characteristics of CDC42 hi synovial tissue macrophages. The sensitivity of MetSig to the RA disease activity and the treatment effect were assessed experimentally and clinically. CDC42 hiCD14+ cells carried MetSig of genes functional in the oxidative phosphorylation and proteasome-dependent cell remodeling, which correlated with the cytokine-rich migratory phenotype and antigen-presenting capacity of these cells. Integration of CDC42 hiCD14+ and synovial macrophages marked with MetSig revealed the important role of the interferon-rich environment and immunoproteasome expression in the homeostasis of these pathogenic macrophages. The CDC42 hiCD14+ cells were targeted by JAK inhibitors and responded with the downregulation of immunoproteasome and MHC-II molecules, which disintegrated the immunological synapse, reduced cytokine production, and alleviated arthritis. This study shows that the CDC42-related MetSig identifies the antigen-presenting CD14+ cells that migrate to joints to coordinate autoimmunity. The accumulation of CDC42 hiCD14+ cells discloses patients perceptive to the JAKi treatment.

Periodicity, mixed-mode oscillations, and multiple timescales in a phosphoinositide-Rho GTPase network

While rhythmic contractile behavior is commonly observed at the cellular cortex, the primary focus has been on excitable or periodic events described by simple activator-delayed inhibitor mechanisms. We show that Rho GTPase activation in nocodazole-treated mitotic cells exhibits both simple oscillations and complex mixed-mode oscillations. Rho oscillations with a 20- to 30-s period are regulated by phosphatidylinositol (3,4,5)-trisphosphate (PIP3) via an activator-delayed inhibitor mechanism, while a slow reaction with period of minutes is regulated by phosphatidylinositol 4-kinase via an activator-substrate depletion mechanism. Conversion from simple to complex oscillations can be induced by modulating PIP3 metabolism or altering membrane contact site protein E-Syt1. PTEN depletion results in a period-doubling intermediate, which, like mixed-mode oscillations, is an intermediate state toward chaos. In sum, this system operates at the edge of chaos. Small changes in phosphoinositide metabolism can confer cells with the flexibility to rapidly enter ordered states with different periodicities.

Chirality-biased protein expression profile during early stages of bone regeneration.

Introduction: Chirality is a crucial mechanical cue within the extracellular matrix during tissue repair and regeneration. Despite its key roles in cell behavior and regeneration efficacy, our understanding of chirality-biased protein profile in vivo remains unclear. Methods: In this study, we characterized the proteomic profile of proteins extracted from bone defect areas implanted with left-handed and right-handed scaffold matrices during the early healing stage. We identified differentially-expressed proteins between the two groups and detected heterogenic characteristic signatures on day 3 and day 7 time points. Results: Proteomic analysis showed that left-handed chirality could upregulate cell adhesion-related and GTPase-related proteins on day 3 and day 7. Besides, interaction analysis and in vitro verification results indicated that the left-handed chiral scaffold material activated Rho GTPase and Akt1, ultimately leading to M2 polarization of macrophages. Discussion: In summary, our study thus improved understanding of the regenerative processes facilitated by chiral materials by characterizing the protein atlas in the context of bone defect repair and exploring the underlying molecular mechanisms of chirality-mediated polarization differences in macrophages.

TAGAP activates Th17 cell differentiation by promoting RhoA and NLRP3 to accelerate rheumatoid arthritis development.

Rheumatoid arthritis (RA) is a chronic autoimmune disorder that can give rise to joint swelling and inflammation, potentially affecting the entire body, closely linked to the state of T cells. The T-cell activation Rho GTPase activating protein (TAGAP) is associated with many autoimmune diseases including RA and is directly linked to the differentiation of Th17 cells. The present study intends to investigate the influence of TAGAP on the RA progression and its mechanism to empower new treatments for RA. A collagen-induced-arthritis (CIA) rat model was constructed, as well as the extraction of CD4+ T cells. RT-qPCR, H&E staining and safranin O/fast green staining revealed that TAGAP interference reduced TAGAP production in the ankle joint of CIA rats, and joint inflammation and swelling were alleviated, which reveals that TAGAP interference reduces synovial inflammation and cartilage erosion in the rat ankle joint. Expression of inflammatory factors (TNF-α, IL-1β, and IL-17) revealed that TAGAP interference suppressed the inflammatory response. Expression of pro-inflammatory cytokines, matrix-degrading enzymes, and anti-inflammatory cytokines at the mRNA level was detected by RT-qPCR and revealed that TAGAP interference contributed to the remission of RA. Mechanistically, TAGAP interference caused a significant decrease in the levels of RhoA and NLRP3. Assessment of Th17/Treg levels by flow cytometry revealed that TAGAP promotes Th17 cells differentiation and inhibits Treg cells differentiation in vitro and in vivo. In conclusion, TAGAP interference may decrease the differentiation of Th17 cells by suppressing the expression of RhoA and NLRP3 to slow down the RA progression.

Opto-RhoGEFs, an optimized optogenetic toolbox to reversibly control Rho GTPase activity on a global to subcellular scale, enabling precise control over vascular endothelial barrier strength.

The inner layer of blood vessels consists of endothelial cells, which form the physical barrier between blood and tissue. This vascular barrier is tightly regulated and is defined by cell-cell contacts through adherens and tight junctions. To investigate the signaling that regulates vascular barrier strength, we focused on Rho GTPases, regulators of the actin cytoskeleton and known to control junction integrity. To manipulate Rho GTPase signaling in a temporal and spatial manner we applied optogenetics. Guanine-nucleotide exchange factor (GEF) domains from ITSN1, TIAM1, and p63RhoGEF, activating Cdc42, Rac, and Rho, respectively, were integrated into the optogenetic recruitment tool improved light-induced dimer (iLID). This tool allows for Rho GTPase activation at the subcellular level in a reversible and non-invasive manner by recruiting a GEF to a specific area at the plasma membrane, The membrane tag of iLID was optimized and a HaloTag was applied to gain more flexibility for multiplex imaging. The resulting optogenetically recruitable RhoGEFs (Opto-RhoGEFs) were tested in an endothelial cell monolayer and demonstrated precise temporal control of vascular barrier strength by a cell-cell overlap-dependent, VE-cadherin-independent, mechanism. Furthermore, Opto-RhoGEFs enabled precise optogenetic control in endothelial cells over morphological features such as cell size, cell roundness, local extension, and cell contraction. In conclusion, we have optimized and applied the optogenetic iLID GEF recruitment tool, that is Opto-RhoGEFs, to study the role of Rho GTPases in the vascular barrier of the endothelium and found that membrane protrusions at the junction region can rapidly increase barrier integrity independent of VE-cadherin.

Opto-RhoGEFs, an optimized optogenetic toolbox to reversibly control Rho GTPase activity on a global to subcellular scale, enabling precise control over vascular endothelial barrier strength

The inner layer of blood vessels consists of endothelial cells, which form the physical barrier between blood and tissue. This vascular barrier is tightly regulated and is defined by cell-cell contacts through adherens and tight junctions. To investigate the signaling that regulates vascular barrier strength, we focused on Rho GTPases, regulators of the actin cytoskeleton and known to control junction integrity. To manipulate Rho GTPase signaling in a temporal and spatial manner we applied optogenetics. Guanine-nucleotide exchange factor (GEF) domains from ITSN1, TIAM1, and p63RhoGEF, activating Cdc42, Rac, and Rho, respectively, were integrated into the optogenetic recruitment tool improved light-induced dimer (iLID). This tool allows for Rho GTPase activation at the subcellular level in a reversible and non-invasive manner by recruiting a GEF to a specific area at the plasma membrane, The membrane tag of iLID was optimized and a HaloTag was applied to gain more flexibility for multiplex imaging. The resulting optogenetically recruitable RhoGEFs (Opto-RhoGEFs) were tested in an endothelial cell monolayer and demonstrated precise temporal control of vascular barrier strength by a cell-cell overlap-dependent, VE-cadherin-independent, mechanism. Furthermore, Opto-RhoGEFs enabled precise optogenetic control in endothelial cells over morphological features such as cell size, cell roundness, local extension, and cell contraction. In conclusion, we have optimized and applied the optogenetic iLID GEF recruitment tool, that is Opto-RhoGEFs, to study the role of Rho GTPases in the vascular barrier of the endothelium and found that membrane protrusions at the junction region can rapidly increase barrier integrity independent of VE-cadherin.

The Role of ARHGAP1 in Rho GTPase Inactivation during Metastasizing of Breast Cancer Cell Line MCF-7 after Treatment with Doxorubicin.

Breast cancer is the most prevalent cancer type in women worldwide. It proliferates rapidly and can metastasize into farther tissues at any stage due to the gradual invasiveness and motility of the tumor cells. These crucial properties are the outcome of the weakened intercellular adhesion, regulated by small guanosine triphosphatases (GTPases), which hydrolyze to the guanosine diphosphate (GDP)-bound conformation. We investigated the inactivating effect of ARHGAP1 on Rho GTPases involved signaling pathways after treatment with a high dose of doxorubicin. Label-free quantitative proteomic analysis of the proteome isolated from the MCF-7 breast cancer cell line, treated with 1 μM of doxorubicin, identified RAC1, CDC42, and RHOA GTPases that were inactivated by the ARHGAP1 protein. Upregulation of the GTPases involved in the transforming growth factor-beta (TGF-beta) signaling pathway initiated epithelial-mesenchymal transitions. These findings demonstrate a key role of the ARHGAP1 protein in the disruption of the cell adhesion and simultaneously allow for a better understanding of the molecular mechanism of the reduced cell adhesion leading to the subsequent metastasis. The conclusions of this study corroborate the hypothesis that chemotherapy with doxorubicin may increase the risk of metastases in drug-resistant breast cancer cells.

Pleckstrin homology and RhoGEF domain containing G4 (PLEKHG4) leads to the activation of RhoGTPases promoting the malignant phenotypes of thyroid cancer.

Thyroid cancer (TC) is one of the most common endocrine system cancers, and its incidence is elevating. There is an urgent need to develop a deeper understanding of TC pathogenesis and explore new therapeutic target for its treatment. This study aimed to investigate the effects of pleckstrin homology and RhoGEF domain containing G4 (PLEKHG4) on the progression of TC. Herein, 29 pairs of TC and adjacent tissues were used to assess the expression of PLEKHG4. A xenograft model of mouse was established by subcutaneously injected with TC cells. Lung metastasis model was established through left ventricular injection. The results revealed that PLEKHG4 was up-regulated in human TC tissues. PLEKHG4 level was correlated with clinicopathological parameters of TC patients. In vitro assays revealed that PLEKHG4 promoted TC cell proliferation, migration, invasion, and epithelial-mesenchymal transformation. Knockdown of PLEKHG4 led to the opposite effects, and the loss of PLEKHG4 enhanced the apoptosis ability and inhibited the stemness properties of TC cells. These findings were further confirmed by the in vivo growth and lung metastasis of TC tumor. Mechanistically, PLEKHG4 promoted the activation of RhoGTPases RhoA, Cdc42, and Rac1. The inhibitors of these RhoGTPases reversed the PLEKHG4-induced malignant phenotypes. Additionally, ubiquitin-conjugating enzyme E2O (UBE2O), a large E2 ubiquitin-conjugating enzyme acted as an ubiquitin enzyme of PLEKHG4, facilitated its ubiquitination and degradation. In conclusion, PLEKHG4, regulated by UBE2O, promoted the thyroid cancer progression via activating the RhoGTPases pathway. UBE2O/PLEKHG4/RhoGTPases axis is expected to be a novel a therapeutic target for TC treatment.

#3119 DEEP LEARNING IDENTIFIES SUBPHENOTYPES OF DIABETIC KIDNEY DISEASE DRIVEN BY GENETIC VARIATIONS IN THE RHO PATHWAY

Abstract Background and Aims Diabetic kidney disease (DKD) is a leading cause of end-stage kidney disease (ESKD), however therapies targeting causal pathways have been limited by disease heterogeneity. Integrating electronic health record (EHR) data and genomics may uncover hidden subphenotypes in DKD. In this study, we use deep learning to identify a novel genetic variant of ARHGEF18 associated with significantly higher risk of DKD and ESKD (Figure 1A). We further employed quantitative microscopy techniques and biochemical assays to elucidate the mechanistic role of ARHGEF18 and its variant in podocytes. Method DKD patients from the Mount Sinai BioMe Biobank were used in this study. Unsupervised clustering and accounting for population structure in a deep learning framework identified two clusters: Cluster M (mild) and S (severe). We then performed a genome wide association study (GWAS) of patients within each cluster compared with healthy controls. For mechanistic studies of the novel variant, cytoplasmic, focal adhesion, cytoskeletal morphometrics as well as live-cell motility, Rho GTPase activity, and protein degradation experiments were performed using confocal and total internal reflection fluorescence (TIRF) microscopy as well as cell-free biochemical assays using immortalized human podocytes expressing ARHGEF18 wild-type (WT) and mutant transcripts. Results We employed autoencoders and unsupervised clustering of EHR data on 1,372 DKD patients to establish two clusters with differential prevalence of ESKD. There was a greater prevalence of proteinuria in Cluster S compared to Cluster M (Figure 1B). Further exome sequencing study in these patients identified a novel variant in ARHGEF18, a Rho guanine exchange factor highly enriched in podocytes (Figure 1A). Nephroseq database showed an increased ARHGEF18 expression in chronic kidney disease (CKD) kidney biopsy samples compared to healthy controls (Figure 1C). Overexpression of ARHGEF18 mutant transcripts in human immortalized podocytes led to impairments in cell adhesion, focal adhesion architecture, and cell motility. Live TIRF microscopy experiments showed preferential subcellular localization of GEF18 mutant to the periphery of migrating podocytes whereas GEF18 WT localized at the perinuclear/cytoplasmic region (Figure 2A, B). GEF18 mutant cells also displayed an increased RhoA activation (Figure 2C). Upon inhibition of protein synthesis using cycloheximide (CHX), we observed a significantly slower degradation of GEF18 mutant protein over a 12h period indicating increased protein stability (Figure 2D). GEF18 mutant also showed resistance to ubiquitin mediated degradation leading to pathologically increased protein levels. Conclusions We report a novel gain of function variant of ARHGEF18 that drives podocyte dysfunction through impaired protein localization and degradation. Targeting this pathway could help regulate RhoA activation and cytoskeletal rearrangements preventing podocyte effacement in DKD.