Mechanical confinement prevents ectopic platelet release.

Blood platelets are produced by megakaryocytes (MKs), their parent cells, which are in the bone marrow. Once mature, MK pierces through the sinusoid vessel, and the initial protrusion further elongates as proplatelet or buds to release platelets. The mechanisms controlling the decision to initiate proplatelet and platelet formation are unknown. Here, we show that the mechanical properties of the microenvironment prevent proplatelet and platelet release in the marrow stroma while allowing this process in the bloodstream. Loss of marrow confinement following myelosuppression led to inappropriate proplatelet and platelet release into the extravascular space. We further used an inert viscoelastic hydrogel to evaluate the impact of compressive stress. Transcriptional analysis showed that culture in three-dimensional gel induced upregulation of genes related to the Rho-GTPase pathway. We found higher Rho-GTPase activation, myosin light chain phosphorylation and F-actin under mechanical constraints while proplatelet formation was inhibited. The use of latrunculin-A to decrease F-actin promoted microtubule-dependent budding and proplatelet extension inside the gel. Additionally, ex vivo exposure of intact bone marrow to latrunculin-A triggered proplatelet extensions in the interstitial space. In vivo, this confinement-mediated high intracellular tension is responsible for the formation of the peripheral zone, a unique actin-rich structure. Cytoskeleton reorganization induces the disappearance of the peripheral zone upon reaching a liquid milieu to facilitate proplatelet and platelet formation. Hence, our data provide insight into the mechanisms preventing ectopic platelet release in the marrow stroma. Identifying such pathways is especially important for understanding pathologies altering marrow mechanics such as chemotherapy or myelofibrosis.

The metastasis-promoting P1597L mutation in PlexinB1 enhances Ras activity.

BACKGROUND: Metastatic prostate cancer is a leading cause of cancer-related morbidity and mortality in men, yet the underlying molecular mechanisms are poorly understood. Plexins are transmembrane receptors for semaphorins with divergent roles in many forms of cancer. We recently found that a single clinically relevant specific amino acid change (Proline1597Leucine, (P1597L)), found in metastatic deposits of prostate cancer patients, converts PlexinB1 from a metastasis suppressor to a gene that drives prostate cancer metastasis in vivo. However, the mechanism by which PlexinB1(P1597L) promotes metastasis is not known. METHODS: Pull down assays using GST-RalGDS or -GSTRaf1-RBD were used to reveal the effect of mutant or wild-type PlexinB1 expression on Rap and Ras activity respectively. Protein-protein interactions were assessed in GST pulldown assays, Akt/ERK phosphorylation by immunoblotting and protein stability by treatment with cycloheximide. Rho/ROCK activity was monitored by measuring MLC2 phosphorylation and actin stress fiber formation. PlexinB1 function was measured using cell-collapse assays. RESULTS: We show here that the single clinically relevant P1597L amino acid change converts PlexinB1 from a repressor of Ras to a Ras activator. The PlexinB1(P1597L) mutation inhibits the RapGAP activity of PlexinB1, promoting a significant increase in Ras activity. The P1597L mutation also blocks PlexinB1-mediated reduction in Rho/ROCK activity, restraining the decrease in MLC2 phosphorylation and actin stress fiber formation induced by overexpression of wild-type PlexinB1. PlexinB1(P1597L) has little effect on the interaction of PlexinB1 with small GTPases or receptor tyrosine kinases and does not inhibit PlexinB1-stimulated Akt or ERK phosphorylation. These results indicate that the mutation affects Rho signalling via the Rap/Ras pathway. The PlexinB1(P1597L) mutation inhibits morphological cell collapse induced by wild-type PlexinB1 expression, suggesting that the mutation induces a loss of an inhibitory tumour suppressor function. CONCLUSION: These results suggest that the clinically relevant P1597L mutation in PlexinB1 may transform PlexinB1 from a suppressor to a driver of metastasis in mouse models of prostate cancer by reducing the RapGAP activity of PlexinB1, leading to Ras activation. These findings highlight the PlexinB1-Rap-Ras pathway for therapeutic intervention in prostate cancer.

Neutrophil-specific interactome of ARHGAP25 reveals novel partners and regulatory insights.

ARHGAP25, a crucial molecule in immunological processes, serves as a Rac-specific GTPase-activating protein. Its role in cell migration and phagocyte functions, affecting the outcome of complex immunological diseases such as rheumatoid arthritis, renders it a promising target for drug research. Despite its importance, our knowledge of its intracellular interactions is still limited. This study employed proteomic analysis of glutathione S-transferase (GST)-tag pulldowns and co-immunoprecipitation from neutrophilic granulocyte cell lysate, revealing 76 candidates for potential physical interactions that complement ARHGAP25's known profile. Notably, four small GTPases (RAC2, RHOG, ARF4, and RAB27A) exhibited high affinity for ARHGAP25. The ARHGAP25-RAC2 and ARHGAP25-RHOG interactions appeared to be affected by the activation state of the small GTPases, suggesting a GTP-GDP cycle-dependent interaction. In silico dimer prediction pinpointed ARHGAP25's GAP domain as a credible binding interface, suggesting its suitability for GTP hydrolysis. Additionally, a list of Fc receptor-related kinases, phosphatases, and three of the 14-3-3 members were identified as potential partners, with in silico predictions highlighting eight binding sites, presenting novel insight on a potential regulatory mechanism for ARHGAP25.

Inhibiting endothelial Rhoj blocks profibrotic vascular intussusception and angiocrine factors to sustain lung regeneration.

Lung regeneration after fibrosis requires formation of functional new vasculature, which is essential for gas exchange and cellular cross-talk with other lung cells. It remains unknown how the lung vasculature can be regenerated without fibrosis. Here, we tested the role of N6-methyladenosine (m6A) modification of forkhead box protein O1 (Foxo1) mRNA in lung regeneration after pneumonectomy (PNX) in mice, a model for lung regrowth after surgical resection. Endothelial cell (EC)-specific knockout of methyltransferase-like 3 (Mettl3) and Foxo1 caused nonproductive intussusceptive angiogenesis (IA), which impaired regeneration and enhanced fibrosis. This nonproductive IA was characterized by enhanced endothelial proliferation and increased vascular splitting with increased numbers of pillar ECs. Endothelial-selective knockout of Mettl3 in mice stimulated nonproductive IA and up-regulation of profibrotic factors after PNX, promoting regeneration to fibrotic transition. EC-specific mutation of m6A modification sites in the Foxo1 gene in mice revealed that endothelial Mettl3 modified A504 and A2035 sites in the Foxo1 mRNA to maintain pro-regenerative endothelial glycolysis, ensuring productive IA and lung regeneration without fibrosis. Suppression of Mettl3-Foxo1 signaling stimulated a subset of hyperglycolytic and hyperproliferative 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (Pfkfb3)+, Ras homolog family member J (Rhoj)+, and platelet-derived growth factor subunit B (Pdgfb)+ ECs in both human and mouse lungs with fibrosis. Inhibiting this Pfkfb3+Rhoj+Pdgfb+ EC subset normalized IA, alleviated fibrosis, and restored regeneration in bleomycin (BLM)-injured mouse lungs. We found that m6A modification of Foxo1 in the mouse vasculature promoted lung regeneration over fibrosis after PNX and BLM injury.

miR-135b-5p promotes gastric carcinogenesis by targeting CLIP4-mediated JAK2/STAT3 signal pathway.

BACKGROUND: Gastric cancer (GC) is a common cancer worldwide; however, its molecular and pathogenic mechanisms remain unclear. MicroRNAs (miRNAs), which target key genes in GC, are associated with tumor promotion or suppression. Therefore, identifying new miRNA mechanisms could improve the novel diagnostic and therapeutic strategies for patients with GC. METHODS: To explore the biological functions of miR-135b-5p in GC, bioinformatic analysis and in vitro functional assays, including colony formation, wound healing, Transwell, and EdU assays, were used to assess the proliferative, invasive, and migratory capacities of GC cells. Target genes were predicted using RNA-seq and online databases. Dual-luciferase reporter assay, fluorescence in situ hybridization and western blotting were used to confirm the regulatory relationship between miR-135b-5p and CLIP4. The role of CLIP4 in tumor progression was assessed using clinical samples and both in vitro and in vivo assays. The tumor-suppressive mechanism of CLIP4 in GC was elucidated using rescue assays. RESULTS: Our study identified that miR-135b-5p as one of the top three over-expressed miRNAs in GC tissues, with RT-qPCR confirming its upregulation. Functional analysis showed that upregulated miR-135b-5p promoted malignant phenotypes in GC cells. Mechanistic research indicated that miR-135b-5p acts as a cancer promoter by targeting CLIP4. Moreover, our study suggested that CLIP4 exerts its tumor-suppressive function by inhibiting the JAK2/STAT3 signaling pathway. CONCLUSION: This study reveals a novel mechanism by which miR-135b-5p exerts its tumor-promoting functions by targeting CLIP4. The tumor-suppressive function of CLIP4 by inactivating the JAK2/STAT3 pathway is also elucidated. Regulatory mechanism of CLIP4 by miR-135b-5p provides a promising novel therapeutic strategy for GC patients.

Exercise Attenuate Diaphragm Atrophy in COPD Mice via Inhibiting the RhoA/ROCK Signaling.

Background: Exercise is an indispensable component of pulmonary rehabilitation with strong anti-inflammatory effects. However, the mechanisms by which exercise prevents diaphragmatic atrophy in COPD (chronic obstructive pulmonary disease) remain unclear. Methods: Forty male C57BL/6 mice were assigned to the control (n=16) and smoke (n=24) groups. Mice in the smoke group were exposed to the cigarette smoke (CS) for six months. They were then divided into model and exercise training groups for 2 months. Histological changes were observed in lung and diaphragms. Subsequently, agonist U46639 and antagonist Y27632 of RhoA/ROCK were subjected to mechanical stretching in LPS-treated C2C12 myoblasts. The expression levels of Atrogin-1, MuRF-1, MyoD, Myf5, IL-1ß, TNF-α, and RhoA/ROCK were determined by Western blotting. Results: Diaphragmatic atrophy and increased RhoA/ROCK expression were observed in COPD mice. Exercise training attenuated diaphragmatic atrophy, decreased the expression of MuRF-1, and increased MyoD expression in COPD diaphragms. Exercise also affects the upregulation of RhoA/ROCK and inflammation-related proteins. In in vitro experiments with C2C12 myoblasts, LPS remarkably increased the level of inflammation and protein degradation, whereas Y27632 or combined with mechanical stretching prevented this phenomenon considerably. Conclusion: RhoA/ROCK plays an important role in the prevention of diaphragmatic atrophy in COPD.

Fluoride-induced hypertension by regulating RhoA/ROCK pathway and phenotypic transformation of vascular smooth muscle cells: In vitro and in vivo evidence.

Fluoride exposure has been implicated as a potential risk factor for hypertension, but the underlying mechanisms remain unclear. This study investigated the role of the RhoA/ROCK signaling pathway in fluoride-induced hypertension. Male Wistar rats were divided into different groups and exposed to varying concentrations of sodium fluoride (NaF) or sodium chloride (NaCl) via drinking water. The rats' blood pressure was measured, and their aortic tissue was utilized for high-throughput sequencing analysis. Additionally, rat and A7r5 cell models were established using NaF and/or Fasudil. The study evaluated the effects of fluoride exposure on blood pressure, pathological changes in the aorta, as well as the protein/mRNA expression levels of phenotypic transformation indicators (a-SMA, calp, OPN) in vascular smooth muscle cells (VSMCs), along with the RhoA/ROCK signaling pathway (RhoA, ROCK1, ROCK2, MLC/p-MLC). The results demonstrated that fluoride exposure in rats led to increased blood pressure. High-throughput sequencing analysis revealed differential gene expression associated with vascular smooth muscle contraction, with the RhoA/ROCK signaling pathway emerging as a key regulator. Pathological changes in the rat aorta, such as elastic membrane rupture and collagen fiber deposition, were observed following NaF exposure. However, fasudil, a ROCK inhibitor, mitigated these pathological changes. Both in vitro and in vivo models confirmed the activation of the RhoA/ROCK signaling pathway and the phenotypic transformation of VSMCs from a contractile to a synthetic state upon fluoride exposure. Fasudil effectively inhibited the activities of ROCK1 and ROCK2 and attenuated the phenotypic transformation of VSMCs. In conclusion, fluoride has the potential to induce hypertension through the activation of the RhoA/ROCK signaling pathway and phenotypic changes in vascular smooth muscle cells. These results provide new insights into the mechanism of fluoride-induced hypertension.

Investigating Ras homolog gene family member C (RhoC) and Ki67 expression following external beam radiation therapy show increased RhoC expression in relapsing prostate cancer xenografts.

Ras homolog gene family member C (RhoC) is a GTPase involved in cell migration, implicated in epithelial-mesenchymal transition and treatment resistance and metastasis of cancer. For example, RhoC has been shown to be involved in resistance to radiation in cervical carcinoma. Here, the effect of X-ray irradiation on RhoC expression in prostate cancer (PCa) xenografts was investigated in both xenografts in regression and relapse. Male BALB/cAnNRj-Foxn1nu/nu mice were inoculated with 4-6 million LNCaP-FGC cells and established xenografts were irradiated with X-rays (200 kV, 1 Gymin-1), 5, 10 or 15 Gy using a Gulmay Medical X-ray system. Expression of RhoC and Ki67, a known proliferation marker, was investigated in xenografts, given 15 Gy, 7 days (midst response as measured by size) or 3 weeks (relapse) post irradiation. Staining was quantified using the Halo software (v2.3.2089.34) with the Indica Labs - cytonuclear v1.6 algorithm. RhoC and Ki67 staining was divided into weak, medium, and strong staining and the percentage of cells stained, single and dual staining, was quantified. The HALO software was further used to classify the tissue in each section so that analysis of RhoC and Ki67 expression in cancer cells, stroma and necrotic areas could be done separately. The results showed that RhoC expression in cancer and stroma cells was significantly higher in relapsed xenografts than in those in regression. This was not seen for Ki67 staining, where the percentage of stained cells were the same in regressing and relapsing tumors. RhoC could be a useful biomarker to confirm relapse following external beam radiation therapy.

Gnao1 is a molecular switch that regulates the Rho signaling pathway in differentiating neurons.

GNAO1 encodes G protein subunit alpha O1 (Gαo). Pathogenic variations in GNAO1 cause developmental delay, intractable seizures, and progressive involuntary movements from early infancy. Because the functional role of GNAO1 in the developing brain remains unclear, therapeutic strategies are still unestablished for patients presenting with GNAO1-associated encephalopathy. We herein report that siRNA-mediated depletion of Gnao1 perturbs the expression of transcripts associated with Rho GTPase signaling in Neuro2a cells. Consistently, siRNA treatment hampered neurite outgrowth and extension. Growth cone formation was markedly disrupted in monolayer neurons differentiated from iPSCs from a patient with a pathogenic variant of Gαo (p.G203R). This variant disabled neuro-spherical assembly, acquisition of the organized structure, and polarized signals of phospho-MLC2 in cortical organoids from the patient's iPSCs. We confirmed that the Rho kinase inhibitor Y27632 restored these morphological phenotypes. Thus, Gαo determines the self-organizing process of the developing brain by regulating the Rho-associated pathway. These data suggest that Rho GTPase pathway might be an alternative target of therapy for patients with GNAO1-associated encephalopathy.

Differential expression of genes in the RhoA/ROCK pathway in the hippocampus and cortex following intermittent hypoxia and high-intensity interval training.

Structural neuroplasticity such as neurite extension and dendritic spine dynamics is enhanced by brain-derived neurotrophic factor (BDNF) and impaired by types of inhibitory molecules that induce growth cone collapse and actin depolymerization, for example, myelin-associated inhibitors, chondroitin sulfate proteoglycans, and negative guidance molecules. These inhibitory molecules can activate RhoA/rho-associated coiled-coil containing protein kinase (ROCK) signaling (known to restrict structural plasticity). Intermittent hypoxia (IH) and high-intensity interval training (HIIT) are known to upregulate BDNF that is associated with improvements in learning and memory and greater functional recovery following neural insults. We investigated whether the RhoA/ROCK signaling pathway is also modulated by IH and HIIT in the hippocampus, cortex, and lumbar spinal cord of male Wistar rats. The gene expression of 25 RhoA/ROCK signaling pathway components was determined following IH, HIIT, or IH combined with HIIT (30 min/day, 5 days/wk, 6 wk). IH included 10 3-min bouts that alternated between hypoxia (15% O2) and normoxia. HIIT included 10 3-min bouts alternating between treadmill speeds of 50 cm·s-1 and 15 cm·s-1. In the hippocampus, IH and HIIT significantly downregulated Acan and NgR2 mRNA that are involved in the inhibition of neuroplasticity. However, IH and IH + HIIT significantly upregulated Lingo-1 and NgR3 in the cortex. This is the first time IH and HIIT have been linked to the modulation of plasticity-inhibiting pathways. These results provide a fundamental step toward elucidating the interplay between the neurotrophic and inhibitory mechanisms involved in experience-driven neural plasticity that will aid in optimizing physiological interventions for the treatment of cognitive decline or neurorehabilitation.NEW & NOTEWORTHY Intermittent hypoxia (IH) and high-intensity interval training (HIIT) enhance neuroplasticity and upregulate neurotrophic factors in the central nervous system (CNS). We provide evidence that IH and IH + HIIT also have the capacity to regulate genes involved in the RhoA/ROCK signaling pathway that is known to restrict structural plasticity in the CNS. This provides a new mechanistic insight into how these interventions may enhance hippocampal-related plasticity and facilitate learning, memory, and neuroregeneration.

Astrocytes control quiescent NSC reactivation via GPCR signaling-mediated F-actin remodeling.

The transitioning of neural stem cells (NSCs) between quiescent and proliferative states is fundamental for brain development and homeostasis. Defects in NSC reactivation are associated with neurodevelopmental disorders. Drosophila quiescent NSCs extend an actin-rich primary protrusion toward the neuropil. However, the function of the actin cytoskeleton during NSC reactivation is unknown. Here, we reveal the fine filamentous actin (F-actin) structures in the protrusions of quiescent NSCs by expansion and super-resolution microscopy. We show that F-actin polymerization promotes the nuclear translocation of myocardin-related transcription factor, a microcephaly-associated transcription factor, for NSC reactivation and brain development. F-actin polymerization is regulated by a signaling cascade composed of G protein-coupled receptor Smog, G protein αq subunit, Rho1 guanosine triphosphatase, and Diaphanous (Dia)/Formin during NSC reactivation. Further, astrocytes secrete a Smog ligand folded gastrulation to regulate Gαq-Rho1-Dia-mediated NSC reactivation. Together, we establish that the Smog-Gαq-Rho1 signaling axis derived from astrocytes, an NSC niche, regulates Dia-mediated F-actin dynamics in NSC reactivation.

Understanding P-Rex regulation: structural breakthroughs and emerging perspectives.

Rho GTPases are a family of highly conserved G proteins that regulate numerous cellular processes, including cytoskeleton organisation, migration, and proliferation. The 20 canonical Rho GTPases are regulated by ∼85 guanine nucleotide exchange factors (GEFs), with the largest family being the 71 Diffuse B-cell Lymphoma (Dbl) GEFs. Dbl GEFs promote GTPase activity through the highly conserved Dbl homology domain. The specificity of GEF activity, and consequently GTPase activity, lies in the regulation and structures of the GEFs themselves. Dbl GEFs contain various accessory domains that regulate GEF activity by controlling subcellular localisation, protein interactions, and often autoinhibition. This review focuses on the two phosphatidylinositol (3,4,5)-trisphosphate (PI(3,4,5)P3)-dependent Rac exchangers (P-Rex), particularly the structural basis of P-Rex1 autoinhibition and synergistic activation. First, we discuss structures that highlight the conservation of P-Rex catalytic and phosphoinositide binding activities. We then explore recent breakthroughs in uncovering the structural basis for P-Rex1 autoinhibition and detail the proposed minimal two-step model of how PI(3,4,5)P3 and Gßγ synergistically activate P-Rex1 at the membrane. Additionally, we discuss the further layers of P-Rex regulation provided by phosphorylation and P-Rex2-PTEN coinhibitory complex formation, although these mechanisms remain incompletely understood. Finally, we leverage the available data to infer how cancer-associated mutations in P-Rex2 destabilise autoinhibition and evade PTEN coinhibitory complex formation, leading to increased P-Rex2 GEF activity and driving cancer progression and metastasis.

Determining Isoprenoid-Facilitated Monomeric GTPase Turnover in Primary Human Trabecular Meshwork Cultures.

Members of the Rho family of small monomeric GTPases regulate a plethora of critical cellular functions including gene expression, cell cycle progression, and the dynamic modeling of the actin cytoskeleton. Diversity among Rho family members is derived, in part, from variations in their subcellular distribution. Localization of newly synthesized (naïve) Rho proteins to target subcellular compartments is largely governed by lipid modifications, including posttranslational prenylation. Here, using well-established and widely available contemporary methodologies, detailed protocols by which to semiquantitatively evaluate the functional consequence of posttranslational prenylation in human trabecular meshwork cells are described. We propose the novel concept that posttranslational prenylation itself is a key regulator of mammalian Rho GTPase protein expression and turnover.

Neutrophil-secreted S100A8/A9 participates in fatty liver injury and fibrosis by promoting myofibroblast migration.

Fatty liver, which is induced by abnormal lipid metabolism, is one of the most common causes of chronic liver disease globally and causes liver fibrosis. During this process, bone marrow-derived mesenchymal stromal cells (BMSCs) and hepatic stellate cells (HSCs) migrate toward the injured liver and participate in fibrogenesis by transdifferentiating into myofibroblasts. S100A8/A9 is a powerful inducer of cell migration and is involved in liver injury. But there are few reports about the effects of S100A8/A9 on BMSC/HSC migration. In the current study, we found that S100A8/A9 expression was increased during fatty liver injury/fibrogenesis. Moreover, S100A8/A9 expression had a positive correlation with fibrosis marker gene expressions in the injured liver. S100A8/A9 was mainly produced by neutrophils in the fibrotic liver. In vitro, neutrophil-secreted S100A8/A9 promoted BMSC/HSC migration via remodeling of microfilaments. Using specific siRNA and inhibitor, we proved that S100A8/A9-induced BMSC/HSC migration is dependent on TLR4/Rho GTPases signaling. Moreover, S100A8/A9 knock-down alleviated liver injury and fibrogenesis in vivo, while injection of S100A9 neutralizing antibody performed similar roles. We proved that S100A8/A9 was involved in liver injury and fibrogenesis via inducing BMSC/HSC migration. Our research reveals a new mechanism underlying BMSC/HSC migration in liver fibrosis and suggests S100A8/A9 as a potential therapeutic target of liver fibrosis. KEY MESSAGES: S100A8/A9 is secreted by neutrophils and increased in fatty liver injury. Neutrophil-secreted S100A8/A9 is a mediator of BMSC/HSC migration in vitro. S100A8/A9-induced BMSC/HSC migration is dependent on TLR4/Rho GTPases signaling. S100A8/A9 blockade alleviates liver injury and fibrogenesis in vivo.

Hierarchical determinants in cytotoxic necrotizing factor (CNF) toxins driving Rho G-protein deamidation versus transglutamination.

The cytotoxic necrotizing factor (CNF) family of AB-type bacterial protein toxins catalyze two types of modification on their Rho GTPase substrates: deamidation and transglutamination. It has been established that E. coli CNF1 and its close homolog proteins catalyze primarily deamidation and Bordetella dermonecrotic toxin (DNT) catalyzes primarily transglutamination. The rapidly expanding microbial genome sequencing data have revealed that there are at least 13 full-length variants of CNF1 homologs. CNFx from E. coli strain GN02091 is the most distant from all other members of the CNF family with 50%-55% sequence identity at the protein level and 0.45-0.52 nucleotide substitutions per site at the DNA level. CNFx modifies RhoA, Rac1, and Cdc42, and like CNF1, activates downstream SRE-dependent mitogenic signaling pathways in human HEK293T cells, but at a 1,000-fold higher EC50 value. Unlike other previously characterized CNF toxins, CNFx modifies Rho proteins primarily through transglutamination, as evidenced by gel-shift assay and confirmed by MALDI mass spectral analysis, when coexpressed with Rho-protein substrates in E. coli BL21 cells or through direct treatment of HEK293T cells. A comparison of CNF1 and CNFx sequences identified two critical active-site residues corresponding to positions 832 and 862 in CNF1. Reciprocal site-specific mutations at these residues in each toxin revealed hierarchical rules that define the preference for deamidase versus a transglutaminase activity in CNFs. An additional unique Cys residue at the C-terminus of CNFx was also discovered to be critical for retarding cargo delivery.IMPORTANCECytotoxic necrotizing factor (CNF) toxins not only play important virulence roles in pathogenic E. coli and other bacterial pathogens, but CNF-like genes have also been found in an expanding number of genomes from clinical isolates. Harnessing the power of evolutionary relationships among the CNF toxins enabled the deciphering of the hierarchical active-site determinants that define whether they modify their Rho GTPase substrates through deamidation or transglutamination. With our finding that a distant CNF variant (CNFx) unlike other known CNFs predominantly transglutaminates its Rho GTPase substrates, the paradigm of "CNFs deamidate and DNTs transglutaminate" could finally be attributed to two critical amino acid residues within the active site other than the previously identified catalytic Cys-His dyad residues. The significance of our approach and research findings is that they can be applied to deciphering enzyme reaction determinants and substrate specificities for other bacterial proteins in the development of precision therapeutic strategies.

Low-intensity pulsed ultrasound combined with ST36 modulate gastric smooth muscle contractile marker expression via RhoA/Rock and MALAT1/miR-449a/DLL1 signaling in diabetic rats.

BACKGROUND: Low-intensity pulsed ultrasound (LIPUS) combined with acupoint can promote gastric motility of diabetic rats. The switch of gastric smooth muscle cell (GSMCs) phenotype was related to the diabetes-induced gastric dysfunction, but the mechanism is not clearly elucidated. This study was aimed at exploring the underlying mechanism of LIPUS stimulation application in diabetic gastroparesis rats. METHODS: In this study, Sprague-Dawley male rats were divided into three groups: control group (CON), diabetic gastroparesis group (DGP), and LIPUS-treated group (LIPUS). LIPUS irradiation was performed bilaterally at ST36 for 20 min per day for 4 weeks. The gastric emptying rate was measured by ultrasound examination. Contraction ability of GSMCs was assessed by muscle strip experiment. The expression of related proteins or mRNAs including α-SMA, SM22α, MHC, RhoA, Rock2, p-MYPT1, MYPT1, p-MLC, MLC, MALAT1, miR-449a, and DLL1 was detected by different methods such as western blotting, RT-qPCR, immunohistochemistry, and immunofluorescence staining, as appropriate. KEY RESULTS: (a) LIPUS stimulation at ST36 could improve the gastric motility dysfunction of diabetic rats. (b) LIPUS increased RhoA, Rock2, p-MYPT1, and p-MLC expression level. (c) MALAT1 and DLL1 contents were decreased, but the level of miR-449a was increased in the LIPUS group. CONCLUSIONS & INFERENCES: LIPUS may affect the contractile marker expression of gastric smooth muscle through the RhoA/Rock and MALAT1/miR-449a/DLL1 pathway to ameliorate DGP.

Inhibition of the Rho/MRTF pathway improves the response of BRAF-resistant melanoma to PD1/PDL1 blockade.

Metastatic cutaneous melanoma is a fatal skin cancer. Resistance to targeted and immune therapies limits the benefits of current treatments. Identifying and adding anti-resistance agents to current treatment protocols can potentially improve clinical responses. Myocardin-related transcription factor (MRTF) is a transcriptional coactivator whose activity is indirectly regulated by actin and the Rho family of GTPases. We previously demonstrated that development of BRAF inhibitor (BRAFi) resistance frequently activates the Rho/MRTF pathway in human and mouse BRAFV600E melanomas. In clinical trials, pretreatment with BRAFi reduces the benefit of immune therapies. We aimed to test the efficacy of concurrent treatment with our MRTF pathway inhibitor CCG-257081 and anti-PD1 in vivo and to examine its effects on the melanoma immune microenvironment. Because MRTF pathway activation upregulates the expression of immune checkpoint inhibitor genes/proteins, we asked whether CCG-257081 can improve the response to immune checkpoint blockade. CCG-257081 reduced the expression of PDL1 in BRAFi-resistant melanoma cells and decreased surface PDL1 levels on both BRAFi-sensitive and -resistant melanoma cells. Using our recently described murine vemurafenib-resistant melanoma model, we found that CCG-257081, in combination with anti-PD1 immune therapy, reduced tumor growth and increased survival. Moreover, anti-PD1/CCG-257081 co-treatment increased infiltration of CD8+ T cells and B cells into the tumor microenvironment and reduced tumor-associated macrophages. Here, we propose CCG-257081 as an anti-resistance and immune therapy-enhancing anti-melanoma agent.

RhoG facilitates a conformational transition in the guanine nucleotide exchange factor complex DOCK5/ELMO1 to an open state.

The dedicator of cytokinesis (DOCK)/engulfment and cell motility (ELMO) complex serves as a guanine nucleotide exchange factor (GEF) for the GTPase Rac. RhoG, another GTPase, activates the ELMO-DOCK-Rac pathway during engulfment and migration. Recent cryo-EM structures of the DOCK2/ELMO1 and DOCK2/ELMO1/Rac1 complexes have identified closed and open conformations that are key to understanding the autoinhibition mechanism. Nevertheless, the structural details of RhoG-mediated activation of the DOCK/ELMO complex remain elusive. Herein, we present cryo-EM structures of DOCK5/ELMO1 alone and in complex with RhoG and Rac1. The DOCK5/ELMO1 structure exhibits a closed conformation similar to that of DOCK2/ELMO1, suggesting a shared regulatory mechanism of the autoinhibitory state across DOCK-A/B subfamilies (DOCK1-5). Conversely, the RhoG/DOCK5/ELMO1/Rac1 complex adopts an open conformation that differs from that of the DOCK2/ELMO1/Rac1 complex, with RhoG binding to both ELMO1 and DOCK5. The alignment of the DOCK5 phosphatidylinositol (3,4,5)-trisphosphate binding site with the RhoG C-terminal lipidation site suggests simultaneous binding of RhoG and DOCK5/ELMO1 to the plasma membrane. Structural comparison of the apo and RhoG-bound states revealed that RhoG facilitates a closed-to-open state conformational change of DOCK5/ELMO1. Biochemical and surface plasmon resonance (SPR) assays confirm that RhoG enhances the Rac GEF activity of DOCK5/ELMO1 and increases its binding affinity for Rac1. Further analysis of structural variability underscored the conformational flexibility of the DOCK5/ELMO1/Rac1 complex core, potentially facilitating the proximity of the DOCK5 GEF domain to the plasma membrane. These findings elucidate the structural mechanism underlying the RhoG-induced allosteric activation and membrane binding of the DOCK/ELMO complex.

Structured RhoGEF recruitment drives myosin II organization on large exocytic vesicles.

The Rho family of GTPases plays a crucial role in cellular mechanics by regulating actomyosin contractility through the parallel induction of actin and myosin assembly and function. Using exocytosis of large vesicles in the Drosophila larval salivary gland as a model, we followed the spatiotemporal regulation of Rho1, which in turn creates distinct organization patterns of actin and myosin. After vesicle fusion, low levels of activated Rho1 reach the vesicle membrane and drive actin nucleation in an uneven, spread-out pattern. Subsequently, the Rho1 activator RhoGEF2 distributes as an irregular meshwork on the vesicle membrane, activating Rho1 in a corresponding punctate pattern and driving local myosin II recruitment, resulting in vesicle constriction. Vesicle membrane buckling and subsequent crumpling occur at local sites of high myosin II concentrations. These findings indicate that distinct thresholds for activated Rho1 create a biphasic mode of actomyosin assembly, inducing anisotropic membrane crumpling during exocrine secretion.

Formin Binding Protein 1 (FNBP1) regulates non-canonical Wnt signaling and vertebrate gastrulation.

The Formin protein Daam1 is required for Wnt-induced cytoskeletal changes during gastrulation, though how it accomplishes this remains unresolved. Here we report the characterization of Formin Binding Protein 1 (FNBP1) as a binding partner of Daam1. The interaction of Daam1 with FNBP1 and its domains required for this interaction were delineated. Immunofluorescence studies showed FNBP1 co-localizes with Daam1, and is an integral component of the actin cytoskeletal complex that is responsive to Wnt stimulation. Specifically, FNBP1 can induce intracellular tubule-like structures and localize to focal adhesions suggesting a role for FNBP1 in cell migration. Functional FNBP1 studies in Xenopus embryos uncover a critical role for FNBP1 in regulating vertebrate gastrulation. Additionally, suboptimal doses of Daam1 and FNBP1 synergize to produce severe gastrulation defects, indicating FNBP1 and Daam1 may function within the same signaling pathway. These results together show FNBP1 is an integral component of Daam1-regulated non-canonical Wnt signaling required for vertebrate gastrulation.