GTPase Activity Research Articles

ASAP1 and ARF1 regulate myogenic differentiation in rhabdomyosarcoma by modulating TAZ activity.

Despite aggressive, multimodal therapies, the prognosis of patients with refractory or recurrent rhabdomyosarcoma (RMS) has not improved in four decades. Because RMS resembles skeletal muscle precursor cells, differentiation-inducing therapy has been proposed for patients with advanced disease. In RAS-mutant PAX fusion-negative RMS (FN-FMS) preclinical models, MEK1/2 inhibition (MEKi) induces differentiation, slows tumor growth, and extends survival. However, the response is short-lived. A better understanding of the molecular mechanisms regulating FN-RMS differentiation could improve differentiation therapy. Here, we identified a role in FN-RMS differentiation for ASAP1, an ARF GTPase-activating protein (ARF GAP) with both pro-invasive and tumor suppressor functions. We found that ASAP1 knockdown inhibited differentiation in FN-RMS cells. Interestingly, knockdown of the GTPases ARF1 or ARF5, targets of ASAP1 GAP activity, also blocked differentiation of FN-RMS. We discovered that loss of ARF pathway components blocked myogenic transcription factor expression. Therefore, we examined the effects on transcriptional regulators. MEKi led to the phosphorylation and inactivation of WWTR1 (TAZ), a homolog of the pro-proliferative transcriptional co-activator YAP1 regulated by the Hippo pathway. However, loss of ASAP1 or ARF1 blocked this inactivation, which inhibits MEKi-induced differentiation. Finally, MEKi-induced differentiation was rescued by dual knockdown of ASAP1 and WWTR1. This study shows that ASAP1 and ARF1 are necessary for myogenic differentiation, providing a deeper understanding of differentiation in FN-RMS and illuminating an opportunity to advance differentiation therapy. Implications: ASAP1 and ARF1 regulate MEKi-induced differentiation of FN-RMS cells by modulating WWTR1 (TAZ) activity, supporting YAP1/TAZ inhibition as a FN-RMS differentiation therapy strategy.

Transcriptional Profiling of Tofacitinib Treatment in Juvenile Idiopathic Arthritis: Implications for Treatment Response Prediction.

To assess changes in gene expression following tofacitinib treatment and investigate transcription patterns as potential predictors of treatment response in patients with active juvenile idiopathic arthritis (JIA). Whole blood samples were collected from JIA patients at baseline and after 18 weeks of open-label tofacitinib treatment (clinical trial NCT02592434). Patients who achieved a JIA-American College of Rheumatology (ACR) response of 70 or above at week 18 were classified as treatment-responders (TR) while those with at most a JIA-ACR30 response were classified as poor-responders (PR). Differential gene expression and gene ontology (GO) over-representation analyses were performed to compare RNA expression between week 18 and baseline samples, as well as between PR and TR samples at baseline. Samples from 67 patients at baseline and 60 at week 18 were analyzed. Following 18 weeks of tofacitinib treatment across all JIA subjects, 883 genes showed significant differential expression (week 18 - baseline). The most strongly downregulated genes were over-represented within IL-7, type I, and type II interferon pathways, while upregulated genes were enriched in ontologies related to neuronal cell processes and cell signaling. Comparing PR and TR at baseline, 663 genes showed differential expression. Upregulated genes were over-represented within ontologies including activation of MAPK activity (p=9.40x10-5), myeloid cell development (p=8.13 x10-5), activation of GTPase activity (p=0.00015), and organelle transport along microtubules (p=0.00021). Tofacitinib treatment in JIA downregulated genes in interferon and IL-7 signaling pathways regardless of effectiveness. Furthermore, baseline upregulation of MAPK signaling may predict poor response to tofacitinib treatment in JIA.

Identification of key genes in diabetic nephropathy based on lipid metabolism.

Diabetic nephropathy (DN) is a common systemic microvascular complication of diabetes with a high incidence rate. Notably, the disturbance of lipid metabolism is associated with DN progression. The present study aimed to identify lipid metabolism-related hub genes associated with DN for improved diagnosis of DN. The gene expression profile data of DN and healthy samples (GSE142153) were obtained from the Gene Expression Omnibus database, and the lipid metabolism-related genes were obtained from the Molecular Signatures Database. Differentially expressed genes (DEGs) between DN and healthy samples were analyzed. The weighted gene co-expression network analysis (WGCNA) was performed to examine the relationship between genes and clinical traits to identify the key module genes associated with DN. Next, the Venn Diagram R package was used to identify the lipid metabolism-related genes associated with DN and their protein-protein interaction (PPI) network was constructed. Subsequently, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were performed. The hub genes were identified using machine-learning algorithms. The Gene Set Enrichment Analysis (GSEA) was used to analyze the functions of the hub genes. The present study also investigated the immune infiltration discrepancies between DN and healthy samples, and assessed the correlation between the immune cells and hub genes. Finally, the expression levels of key genes were verified by reverse transcription-quantitative (RT-q)PCR. The present study determined 1,445 DEGs in DN samples. In addition, 694 DN-related genes in MEyellow and MEturquoise modules were identified by WGCNA. Next, the Venn Diagram R package was used to identify 17 lipid metabolism-related genes and to construct a PPI network. GO analysis revealed that these 17 genes were markedly associated with 'phospholipid biosynthetic process' and 'cholesterol biosynthetic process', while the KEGG analysis showed that they were enriched in 'glycerophospholipid metabolism' and 'fatty acid degradation'. In addition, SAMD8 and CYP51A1 were identified through the intersections of two machine-learning algorithms. The results of GSEA revealed that the 'mitochondrial matrix' and 'GTPase activity' were the markedly enriched GO terms in both SAMD8 and CYP51A1. Their KEGG pathways were mainly concentrated in the 'pathways of neurodegeneration-multiple diseases'. Immune infiltration analysis showed that nine types of immune cells had different expression levels in DN (diseased) and healthy samples. Notably, SAMD8 and CYP51A1 were both markedly associated with activated B cells and effector memory CD8 T cells. Finally, RT-qPCR confirmed the high expression of SAMD8 and CYP51A1 in DN. In conclusion, lipid metabolism-related genes SAMD8 and CYP51A1 may play key roles in DN. The present study provides fundamental information on lipid metabolism that may aid the diagnosis and treatment of DN.

Pharmacologic restoration of GTP hydrolysis by mutant RAS.

Approximately 3.4 million patients worldwide are diagnosed each year with cancers that harbor pathogenic mutations in one of three RAS proto-oncogenes (KRAS, NRAS and HRAS)1,2. These mutations impair the GTPase activity of RAS, leading to activation of downstream signaling and proliferation3-6. Long-standing efforts to restore the hydrolase activity of RAS mutants have been unsuccessful, extinguishing any consideration towards a viable therapeutic strategy7. Here we show that tri-complex inhibitors, that is, molecular glues with the ability to recruit cyclophilin A (CYPA) to the active state of RAS have a dual mechanism of action: not only do they prevent activated RAS from binding to its effectors, but, unexpectedly, they also stimulate GTP hydrolysis. Drug-bound CYPA complexes modulate residues in the switch II motif of RAS to coordinate the nucleophilic attack on the ɣ phosphate of GTP, in a mutation-specific manner. RAS mutants most sensitive to stimulation of GTPase activity were more susceptible to treatment compared to mutants whose hydrolysis could not be enhanced, suggesting that pharmacologic stimulation of hydrolysis potentiates the therapeutic effects of tri-complex inhibitors for specific RAS mutants. This study lays the foundation for developing a new class of therapeutics that inhibit cancer growth by stimulating mutant GTPase activity.

CK and LRRK2 Involvement in Neurodegenerative Diseases

Neurodegenerative diseases (NDDs) are currently the most widespread neuronal pathologies in the world. Among these, the most widespread are Alzheimer’s disease (AD), dementia, Parkinson’s disease (PD), amyotrophic lateral sclerosis (ALS), and Huntington’s disease (HD)—all characterized by a progressive loss of neurons in specific regions of the brain leading to varied clinical symptoms. At the basis of neurodegenerative diseases, an emerging role is played by genetic mutations in the leucine-rich repeat kinase 2 (LRRK2) gene that cause increased LRRK2 activity with consequent alteration of neuronal autophagy pathways. LRRK2 kinase activity requires GTPase activity which functions independently of kinase activity and is required for neurotoxicity and to potentiate neuronal death. Important in the neurodegeneration process is the upregulation of casein kinase (CK), which causes the alteration of the AMPK pathway by enhancing the phosphorylation of α-synuclein and huntingtin proteins, known to be involved in PD and HD, and increasing the accumulation of the amyloid-β protein (Aβ) for AD. Recent research has identified CK of the kinases upstream of LRRK2 as a regulator of the stability of the LRRK2 protein. Based on this evidence, this review aims to understand the direct involvement of individual kinases in NDDs and how their crosstalk may impact the pathogenesis and early onset of neurodegenerative diseases.

CAMP/PKA-mediated in vitro angiogenesis: A novel interplay between PKA, RhoA/ROCK, and VEGFR2 signalling

Abstract Background and Aims Others and we have previously demonstrated that cAMP/PKA signalling stabilise endothelial cell (EC) barrier function via modulating the activities of Rho GTPases, mainly RhoA and Rac1. In the present study we analysed whether cAMP/PKA also modulates EC angiogenesis capacity and whether Rho GTPases are involved in this function. Methods The study was carried out on cultured human umbilical vein ECs (HUVECs). RhoA and Rac1 activities were genetically manipulated by lentiviral-mediated over expression of dominant negative (DN) and constitutive active (CA) forms of RhoA and Rac1. Pharmacologically, specific activation and inhibition of PKA was achieved by using cAMP analogue N6-Benzoyl-cAMP (5 mM) and PKI (1 µM), respectively. Pharmacologically, Rac1 was inhibited using NSC 23766 (10 µM) and RhoA was activated using Rho activator I (Cytoskeleton Inc.). In vitro angiogenesis was analysed by 3-D spheroid assay and matrigel tube formation assay. Results Pharmacologically, specific activation of PKA induced EC migration (Wound healing assay), tube formation, and 3-D spheroids and potentiated the VEGF-induced in vitro angiogenesis. These pro-angiogenesis activities were abrogated by highly specific cell-permeable peptide inhibitor (PKI) of PKA. Activation of cAMP/PKA-signalling resulted in an increased VEGFR2 and Akt phosphorylation, increased Rac1 activity, and a strong inhibition of RhoA/Rock pathway. Pharmacological activation of RhoA but not inhibition of Rac1 abrogated PKA-induced VEGFR2 phosphorylation, tube formation and 3-D spheroids. Lentiviral mediated over-expression of constitutive active form of RhoA but not Rac1 abrogated PKA-induced VEGFR2 and Akt phosphorylation and angiogenesis. Moreover, pharmacological inhibition of Akt also abrogated PKA-mediated VEGFR2 phosphorylation and angiogenesis. Likewise, over expression of either dominant negative RhoA or pharmacological inhibitors of Rho kinase (ROCK) promoted basal and potentiated PKA-mediated VEGFR2 and Akt phosphorylation and angiogenesis. Conclusion We describe for the first time a novel interplay between PKA, RhoA/ROCK, Akt, and VEGFR2 signalling and angiogenesis. Inhibition of RhoA/ROCK plays an important role in mediating PKA-induced angiogenesis and activation of RhoA/ROCK signalling may offer an important target to intervene therapeutic angiogenesis.

RAB4A is a master regulator of cancer cell stemness upstream of NUMB–NOTCH signaling

Cancer stem cells (CSCs) are a group of specially programmed tumor cells that possess the characteristics of perpetual cell renewal, increased invasiveness, and often, drug resistance. Hence, eliminating CSCs is a major challenge for cancer treatment. Understanding the cellular programs that maintain CSCs, and identifying the critical regulators for such programs, are major undertakings in both basic and translational cancer research. Recently, we have reported that RAB4A is a major regulator of epithelial-to-mesenchymal transition (EMT) and it does so mainly through regulating the activation of RAC1 GTPase. In the current study, we have delineated a new signaling circuitry through which RAB4A transmits its control of cancer stemness. Using in vitro and in vivo studies, we show that RAB4A, as the upstream regulator, relays signal stepwise to NUMB, NOTCH1, RAC1, and then SOX2 to control the self-renewal property of multiple cancer cells of diverse tissue origins. Knockdown of NUMB, or overexpression of NICD (the active fragment NOTCH1) or SOX2, rescued the in vitro sphere-forming and in vivo tumor-forming abilities that were lost upon RAB4A knockdown. Furthermore, we discovered that the chain of control is mostly through transcriptional regulation at every step of the pathway. The discovery of the novel signaling axis of RAB4A–NUMB–NOTCH–SOX2 opens the path for further expansion of the signaling chain and for the identification of new regulators and interacting proteins important for CSC functions, which can be explored to develop new and effective therapies.

Abl kinases regulate FGF signaling independent of Crk phosphorylation to prevent Peters anomaly.

Peters anomaly, the most common cause of congenital corneal opacity, stems from corneal-lenticular adhesion. Despite numerous identified mutations, a cohesive molecular framework of the disease's etiology remains elusive. Here, we identified Abl kinases as pivotal regulators of FGF signaling, as genetic ablation of Abl kinases restores lens induction even in the absence of FGF signaling. Intriguingly, both Abl kinase deficiency and increased FGF-Ras activity result in Peters anomaly independent of ERK signaling, which can be rescued by allelic deletion of Abl substrate, Crk. However, contrary to the prevailing belief that Abl kinases regulate Crk proteins by direct phosphorylation, mutations at Abl kinase phosphorylation sites on Crk and CrkL did not yield any observable effects. Instead, our findings reveal that Abl kinases phosphorylate Ptpn12, which in turn inhibits p130Cas phosphorylation and Crk recruitment, crucial for Rho GTPases activation and cytoskeletal dynamics. Consequently, Abl kinase deficiency reduces actomyosin contractility within the lens vesicle and genetically interacts with RhoA inhibition. Conversely, Rac1 deletion mitigates Peters anomaly in models with aberrant FGF, Abl kinase and RhoA signaling. Our results demonstrate that Abl kinases regulate FGF signaling to balance RhoA and Rac1 activity via the Ptpn12-p130Cas pathway, suggesting that targeting tension-mediated lens vesicle separation could be a therapeutic strategy for Peters anomaly.

A homozygous variant in ARHGAP39 is associated with lethal cerebellar vermis hypoplasia in a consanguineous Saudi family

Cerebellar vermis hypoplasia refers to a varying degree of incomplete development of the cerebellum and vermis. A Saudi family with four affected individuals with cerebellar vermis hypoplasia, facial dysmorphology, visual impairment, skeletal, and cardiac abnormalities was ascertained in this study. Three out of four patients could not survive longer and had died in early infancy. Genetic analysis of the youngest affected was performed by genome-wide homozygosity mapping coupled with whole exome sequencing (WES), followed by Sanger validation. Genome-wide genotyping analysis mapped the phenotype to chromosome 8q24.3. Using an autosomal recessive model, considering deleterious variants with minor allele frequency of less than 0.001 in WES data, a homozygous missense variant (NM_025251.2; ARHGAP39; c.1301G > T; p.Cys434Phe) was selected as a potential candidate for the phenotype. The variant (c.1301G > T) in the ARHGAP39 is in the region of homozygosity on chromosome 8q24.3. ARHGAP39 is a Rho GTPase-activating protein 39 and has been known to regulate apoptosis, cell migration, neurogenesis, and cerebral and hippocampal dendritic spine morphology. Mice homozygous for arhgap39 knockouts have shown premature embryonic lethality. Our findings present the first ever human phenotype associated with ARHGAP39 alteration.

Comprehensive proteomic analysis of buffalo milk extracellular vesicles

Extracellular vesicles are secretory vesicles involved in cell-to-cell communication via their encapsulated cargo of proteins, lipids, and nucleic acids. Bovine milk provides a rich source of extracellular vesicles (mEVs) that have been studied as therapeutics and drug delivery systems. Therefore, insight into the mEV cargo, such as its proteome, may help in understanding the molecular mechanism underlying the potential health benefits attributed to the mEVs. Hence, mEVs were isolated from healthy buffalo milk after screening the milk somatic cell count. The total proteins of mEVs were analyzed using LC-MS, and 331 proteins were found commonly present among three buffalo milk samples. These proteins were primarily derived from extracellular regions and lysosomes. The major biological roles associated with the proteins were immune response, metabolism, and cell cycle regulation. The molecular functions of the proteins were transporter activity, catalytic activity, and GTPase activity. Further, comparative analysis with the previously available bovine mEVs proteome data showed 114 proteins to be newly identified in the buffalo mEVs. The biological pathways associated with these proteins may play a major role in muscle development. These findings shed a light on the potential health benefits of buffalo mEVs as therapeutics as well as drug delivery vehicles.

EPHB4-RASA1 Inhibition of PIEZO1 Ras Activation Drives Lymphatic Valvulogenesis.

EPHB4 (ephrin receptor B4) and the RASA1 (p120 Ras GTPase-activating protein) are necessary for the development of lymphatic vessel (LV) valves. However, precisely how EPHB4 and RASA1 regulate LV valve development is unknown. In this study, we examine the mechanisms by which EPHB4 and RASA1 regulate the development of LV valves. We used LV-specific inducible EPHB4-deficient mice and EPHB4 knockin mice that express a form of EPHB4 that is unable to bind RASA1 yet retains protein tyrosine kinase activity (EPHB4 2YP) to study the role of EPHB4 and RASA1 in LV valve development in the embryo and LV valve maintenance in adults. We also used human dermal lymphatic endothelial cells in vitro to study the role of EPHB4 and RASA1 as regulators of LV valve specification induced by oscillatory shear stress, considered the trigger for LV valve specification in vivo. LV valve specification, continued valve development postspecification, and LV valve maintenance were blocked upon induced loss of EPHB4 in LV. LV specification and maintenance were also impaired in EPHB4 2YP mice. Defects in LV development were reversed by inhibition of the Ras-MAPK (mitogen-activated protein kinase) signaling pathway. In human dermal lymphatic endothelial cells, loss of expression of EPHB4 or its ephrin b2 ligand, loss of expression of RASA1, and inhibition of physical interaction between EPHB4 and RASA1 resulted in dysregulated oscillatory shear stress-induced Ras-MAPK activation and impaired expression of LV specification markers that could be rescued by Ras-MAPK pathway inhibition. The same results were observed when human dermal lymphatic endothelial cells were stimulated with the Yoda1 agonist of the PIEZO1 oscillatory shear stress sensor. Although Yoda1 increased the number of LV valves when administered to wild-type embryos, it did not increase LV valve number when administered to EPHB4 2YP embryos. EPHB4 is necessary for LV valve specification, continued valve development postspecification, and valve maintenance. LV valve specification requires physical interaction between EPHB4 and RASA1 to limit activation of the Ras-MAPK pathway in lymphatic endothelial cells. Specifically, EPHB4-RASA1 physical interaction is necessary to dampen Ras-MAPK activation induced through the PIEZO1 oscillatory shear stress sensor. These findings reveal the mechanism by which EPHB4 and RASA1 regulate the development of LV valves.

Nonphototrophic hypocotyl 3 domain proteins: traffic directors, hitchhikers, or both?

The nonphototrophic hypocotyl 3 (NPH3) domain is plant specific and of unknown function. It is nearly always attached to an N-terminal BTB domain and a largely unstructured C-terminal region. Recent reports revealed NPH3-domain GTPase activity and connection to intracellular trafficking, condensate formation, membrane attachment of the C-terminal region for some NPH3-domain proteins and, at the physiological level, drought-related function for at least one NPH3-domain protein. We integrate these new ideas of NPH3-domain protein function into two, nonexclusive, working models: the 'traffic director' model, whereby NPH3-domain proteins regulate intracellular trafficking and, the 'hitchhiker' model whereby NPH3-domain proteins ride the trafficking system to find ubiquitination targets. Determining which model best applies to uncharacterized NPH3-domain proteins will contribute to understanding intracellular trafficking and environmental responses.

Sirtuin 1 regulates the phenotype and functions of dendritic cells through Ido1 pathway in obesity

Sirtuin 1 (SIRT1) is a class III histone deacetylase (HDAC3) that plays a crucial role in regulating the activation and differentiation of dendritic cells (DCs) as well as controlling the polarization and activation of T cells. Obesity, a chronic inflammatory condition, is characterized by the activation of immune cells in various tissues. We hypothesized that SIRT1 might influence the phenotype and functions of DCs through the Ido1 pathway, ultimately leading to the polarization towards pro-inflammatory T cells in obesity. In our study, we observed that SIRT1 activity was reduced in bone marrow-derived DCs (BMDCs) from obese animals. These BMDCs exhibited elevated oxidative phosphorylation (OXPHOS) and increased extracellular acidification rates (ECAR), along with enhanced expression of class II MHC, CD86, and CD40, and elevated secretion of IL-12p40, while the production of TGF-β was reduced. The kynurenine pathway activity was decreased in BMDCs from obese animals, particularly under SIRT1 inhibition. SIRT1 positively regulated the expression of Ido1 in DCs in a PPARγ-dependent manner. To support these findings, ATAC-seq analysis revealed that BMDCs from obese mice had differentially regulated open chromatin regions compared to those from lean mice, with reduced chromatin accessibility at the Sirt1 genomic locus in BMDCs from obese WT mice. Gene Ontology (GO) enrichment analysis indicated that BMDCs from obese animals had disrupted metabolic pathways, including those related to GTPase activity and insulin response. Differential expression analysis showed reduced levels of Pparg and Sirt1 in BMDCs from obese mice, which was challenged and confirmed using BMDCs from mice with conditional knockout of Sirt1 in dendritic cells (SIRT1∆). This study highlights that SIRT1 controls the metabolism and functions of DCs through modulation of the kynurenine pathway, with significant implications for obesity-related inflammation.

A RhoGAP controls apical actin polymerization by inhibiting formin in Arabidopsis pollen tubes

Formin is an important player in promoting apical actin polymerization in pollen tubes, but the mechanism regulating its activity remains unknown. We here identify REN1, a Rho GTPase-activating protein, as a negative regulator of formins in Arabidopsis pollen tubes. Specifically, we found that depletion of REN1 promotes apical actin polymerization and increases the amount of filamentous actin in pollen tubes. Interestingly, the effect of REN1 loss of function phenocopies the effect of formin gain of function, as it causes the formation of supernumerary membrane-derived actin bundles, which leads to tube swelling and membrane deformation. Importantly, inhibition of formins suppresses the phenotypic defects in ren1 mutant pollen tubes. We further demonstrate that REN1 physically interacts with the Arabidopsis formin protein AtFH5, predominantly with the C terminus, and inhibits the ability of AtFH5 to nucleate and assemble actin invitro. Depletion of AtFH5 partially suppresses the phenotype in ren1 mutant pollen tubes, demonstrating that REN1 regulates apical actin polymerization at least partially through inhibiting AtFH5. We thus uncover a novel mechanism regulating formins and actin polymerization in plants.

Axon guidance genes are regulated by TDP-43 and RGNEF through long-intron removal.

Rho guanine nucleotide exchange factor (RGNEF) is a guanine nucleotide exchange factor (GEF) mainly involved in regulating the activity of Rho-family GTPases. It is a bi-functional protein, acting both as a guanine exchange factor and as an RNA-binding protein. RGNEF is known to act as a destabilizing factor of neurofilament light chain RNA (NEFL) and it could potentially contribute to their sequestration in nuclear cytoplasmic inclusions. Most importantly, RGNEF inclusions in the spinal motor neurons of ALS patients have been shown to co-localize with inclusions of TDP-43, the major well-known RNA-binding protein aggregating in the brain and spinal cord of human patients. Therefore, it can be hypothesized that loss-of-function of both proteins following aggregation may contribute to motor neuron death/survival in ALS patients. To further characterize their relationship, we have compared the transcriptomic profiles of neuronal cells depleted of TDP-43 and RGNEF and show that these two factors predominantly act in an antagonistic manner when regulating the expression of axon guidance genes. From a mechanistic point of view, our experiments show that the effect of these genes on the processivity of long introns can explain their mode of action. Taken together, our results show that loss-of-function of factors co-aggregating with TDP-43 can potentially affect the expression of commonly regulated neuronal genes in a very significant manner, potentially acting as disease modifiers. This finding further highlights that neurodegenerative processes at the RNA level are the result of combinatorial interactions between different RNA-binding factors that can be co-aggregated in neuronal cells. A deeper understanding of these complex scenarios may lead to a better understanding of pathogenic mechanisms occurring in patients, where more than one specific protein may be aggregating in their neurons.

RGS Proteins in Sympathetic Nervous System Regulation: Focus on Adrenal RGS4.

The sympathetic nervous system (SNS) consists largely of two different types of components: neurons that release the neurotransmitter norepinephrine (NE, noradrenaline) to modulate homeostasis of the innevrvated effector organ or tissue and adrenal chromaffin cells, which synthesize and secrete the hormone epinephrine (Epi, adrenaline) and some NE into the blood circulation to act at distant organs and tissues that are not directly innervated by the SNS. Like almost every physiological process in the human body, G protein-coupled receptors (GPCRs) tightly modulate both NE release from sympathetic neuronal terminals and catecholamine (CA) secretion from the adrenal medulla. Regulator of G protein Signaling (RGS) proteins, acting as guanosine triphosphatase (GTPase)-activating proteins (GAPs) for the Gα subunits of heterotrimeric guanine nucleotide-binding proteins (G proteins), play a central role in silencing G protein signaling from a plethora of GPCRs. Certain RGS proteins and, in particular, RGS4, have been implicated in regulation of SNS activity and of adrenal chromaffin cell CA secretion. More specifically, recent studies have implicated RGS4 in regulation of NE release from cardiac sympathetic neurons by means of terminating free fatty acid receptor (FFAR)-3 calcium signaling and in regulation of NE and Epi secretion from the adrenal medulla by means of terminating cholinergic calcium signaling in adrenal chromaffin cells. Thus, in this review, we provide an overview of the current literature on the involvement of RGS proteins, with a particular focus on RGS4, in these two processes, i.e., NE release from sympathetic nerve terminals & CA secretion from adrenal chromaffin cells. We also highlight the therapeutic potential of RGS4 pharmacological manipulation for diseases characterized by sympathetic dysfunction or SNS hyperactivity, such as heart failure and hypertension.

The p. S178L mutation in Tbc1d24 disrupts endosome-mediated synaptic vesicle trafficking of cochlear hair cells and leads to hearing impairment in mice.

The ribbon synapses of cochlear inner hair cells (IHCs) employ efficient vesicle resupply to enable fast and sustained release rates. However, the molecular mechanisms of these physiological activities remain unelucidated. Previous studies showed that the RAB-specific GTPase-activating protein TBC1D24 controls the endosomal trafficking of the synaptic vesicles (SVs) in Drosophila and mammalian neurons, and mutations in TBC1D24 may lead to non-syndromic hearing loss or hearing loss associated with the DOORS syndrome in humans. In this study, we generated a knock-in mouse model for the p. S178L mutation in TBC1D24, which leads to autosomal dominant non-syndromic hearing loss (DFNA65). The p.S178L mutant mice show mild hearing loss and progressively declined wave I amplitude of the auditory brainstem responses. Despite the normal gross and cellular morphology of the cochlea, transmission electron microscopy reveals accumulation of endosome-like vacuoles and a lower-than-normal number of SVs directly associated with the ribbons in the IHCs. Consistently, patch clamp of the IHCs shows reduced exocytosis under prolonged stimulus. ARF6, a TBC1D24-interacting protein also involved in endosomal membrane trafficking, was underexpressed in the cochleae of the mutant mouse and has weakened in vitro interaction with the p.S178L mutant TBC1D24. Our results suggest an important role of TBC1D24 in maintaining endosomal-mediated vesicle recycling and sustained exocytosis of hair cell ribbon synapses.

Structural basis of antimicrobial membrane coat assembly by human GBP1.

Guanylate-binding proteins (GBPs) are interferon-inducible guanosine triphosphate hydrolases (GTPases) mediating host defense against intracellular pathogens. Their antimicrobial activity hinges on their ability to self-associate and coat pathogen-associated compartments or cytosolic bacteria. Coat formation depends on GTPase activity but how nucleotide binding and hydrolysis prime coat formation remains unclear. Here, we report the cryo-electron microscopy structure of the full-length human GBP1 dimer in its guanine nucleotide-bound state and describe the molecular ultrastructure of the GBP1 coat on liposomes and bacterial lipopolysaccharide membranes. Conformational changes of the middle and GTPase effector domains expose the isoprenylated C terminus for membrane association. The α-helical middle domains form a parallel, crossover arrangement essential for coat formation and position the extended effector domain for intercalation into the lipopolysaccharide layer of gram-negative membranes. Nucleotide binding and hydrolysis create oligomeric scaffolds with contractile abilities that promote membrane extrusion and fragmentation. Our data offer a structural and mechanistic framework for understanding GBP1 effector functions in intracellular immunity.

Effect of Pathogenic Mutations on the Formation of High-Order Dynamin 2 Assemblies in Living Cells.

Mutations in dynamin 2 (DNM2) have been associated with two distinct movement disorders: Charcot-Marie-Tooth neuropathies (CMT) and centronuclear myopathy (CNM). Most of these mutations are clustered in the pleckstrin homology domain (PHD), which engages in intramolecular interactions that limit dynamin self-assembly and GTPase activation. CNM mutations interfere with these intramolecular interactions and suppress the formation of the autoinhibited state. CMT mutations are located primarily on the opposite surface of the PHD, which is specialized for phosphoinositide binding. It has been speculated that the distinct locations and interactions of residues mutated in CMT and CNM explain why each set of mutations causes either one disease or the other, despite their close proximity within the PHD sequence. We previously reported that at least one CMT-causing mutant, lacking residues 555DEE557 (ΔDEE), displays the same inability to undergo autoinhibition as observed in CNM-linked mutants. Here, we show that both the DNM2ΔDEE and CNM-linked DNM2A618T mutants form larger and more stable structures on the plasma membrane than that of wild-type DNM2 (DNM2WT). However, DNM2A618T forms cytoplasmic inclusions at concentrations lower than those of either DNM2WT or DNM2ΔDEE, suggesting that CNM-linked mutations confer more severe gain-of-function properties than the ΔDEE mutation.

Characterization and Physiological Differences of Two Primary Cultures of Human Normal and Hypertrophic Scar Dermal Fibroblasts: A Pilot Study.

Background/Objectives: Dermal fibroblasts (DFs) are key participants in skin hypertrophic scarring, and their properties are being studied to identify the molecular and cellular mechanisms underlying the pathogenesis of skin scarring. Methods: In the present work, we performed a comparative analysis of DFs isolated from normal skin (normal dermal fibroblasts, NDFs), and hypertrophic scar skin (hypertrophic scar fibroblasts, HTSFs). The fibroblasts were karyotyped and phenotyped, and experiments on growth rate, wound healing, and single-cell motility were conducted. Results: Comparative analysis revealed a minor karyotype difference between cells. However, HTSFs are characterized by higher proliferation level and motility compared to NDFs. These significant differences may be associated with quantitative and qualitative differences in the cell secretome. A proteomic comparison of NDF and HTSF found that differences were associated with metabolic proteins reflecting physiological differences between the two cells lines. Numerous unique proteins were found only in the vesicular phase of vHTSFs. Some proteins involved in cell proliferation (protein-glutamine gamma-glutamyltransferase K) and cell motility (catenin delta-1), which regulate gene transcription and the activity of Rho family GTPases and downstream cytoskeletal dynamics, were identified. A number of proteins which potentially play a role in fibrosis and inflammation (mucin-5B, CD97, adhesion G protein-coupled receptor E2, antileukoproteinase, protein S100-A8 and S100-A9, protein caspase recruitment domain-containing protein 14) were detected in vHTSFs. Conclusions: A comparative analysis of primary cell cultures revealed their various properties, especially in the cell secretome. These proteins may be considered promising target molecules for developing treatment or prevention strategies for pathological skin scarring.