GTPase Gene Research Articles

The Rho GTPase gene family plays a crucial role in key cellular functions. RAC3, one of the three genes in this family, along with RAC1 and RAC2, is highly expressed in the brain. It is specifically involved in neuronal differentiation, maturation, and migration. Therefore, dysregulation of RAC3 can lead to neurodevelopmental abnormalities. Here we report two siblings, born to a nonconsanguineous couple, with global developmental delay, intellectual disability, and facial dysmorphism. Whole exome sequencing revealed a pathogenic heterozygous variant, c.184G>A (p.Glu62Lys), in exon 3 of the RAC3 gene [NM_005052.3], which is associated with neurodevelopmental disorder (NDD) with structural brain anomalies and dysmorphic facies. RAC3-associated disorder should be considered as a differential diagnosis in children with NDDs and characteristic facial dysmorphism, including arched eyebrows, hypertelorism, and prominent eyes, along with central nervous system abnormalities. In our cases, we observed novel MRI brain findings that had not been previously reported, thereby expanding the spectrum of brain anomalies associated with this condition.

Membrane trafficking is a crucial function of all cells and is regulated at multiple levels from vesicle formation, packaging, and localization to fusion, exocytosis, and endocytosis. Rab GTPase proteins are core regulators of eukaryotic membrane trafficking, but developmental roles of specific Rab GTPases are less well characterized, potentially because of their essentiality for basic cellular function. Caenorhabditis elegans gonad development entails the coordination of cell growth, proliferation, and migration-processes in which membrane trafficking is known to be required. Here, we take an organ-focused approach to Rab GTPase function in vivo to assess the roles of Rab genes in reproductive system development. We performed a whole-body RNAi screen of the entire Rab family in C. elegans to uncover Rabs essential for gonad development. Notable gonad defects resulted from RNAi knockdown of rab-1, the key regulator of ER-Golgi trafficking. We then examined the effects of tissue-specific RNAi knockdown of rab-1 in somatic reproductive system and germline cells. We interrogated the dual functions of the distal tip cell as both a leader cell of gonad organogenesis and the germline stem cell niche. We find that rab-1 functions cell-autonomously and non-cell-autonomously to regulate both somatic gonad and germline development. Gonad migration, elongation, and gamete differentiation-but surprisingly not germline stem niche function-are highly sensitive to rab-1 RNAi.

ROP small GTPases function as signaling hubs that mediate various physiological processes, including plant defense. Their specific roles in strawberry resistance against gray mold remain uncharacterized. In this study, we identified 53 ROP genes across the genomes of six Rosaceae species. Based on sequence homology, they were classified into three distinct phylogenetic clades. Detailed analysis of FveROP proteins revealed the presence of highly conserved catalytic G-domains, which are essential for their GTPase activity. By conducting transient overexpression experiments in strawberry fruits challenged with the gray mold pathogen Botrytis cinerea, we investigated the impact of the FveROP4 gene on disease resistance. The overexpression of both wild-type and constitutively active forms of FveROP4 enhanced resistance against B. cinerea infection. Subsequent analysis revealed that overexpression of FveROP4 and FveCAROP4 genes led to increased accumulation of reactive oxygen species. Moreover, FveROP4 was localized on the plasma membrane, where it interacted directly with FveRBOHF1, corroborating the results obtained through yeast two-hybrid and luciferase complementation imaging assays. The study findings may provide valuable insights for investigating the mechanisms of ROP signaling in regulating the immune response in strawberries and could significantly contribute to strawberry breeding programs aimed at developing new cultivars with enhanced fruit shelf life.

Membrane trafficking is a crucial function of all cells and is regulated at multiple levels from vesicle formation, packaging, and localization to fusion, exocytosis, and endocytosis. Rab GTPase proteins are core regulators of eukaryotic membrane trafficking, but developmental roles of specific Rab GTPases are less well characterized, potentially because of their essentiality for basic cellular function. C. elegans gonad development entails the coordination of cell growth, proliferation, and migration—processes in which membrane trafficking is known to be required. Here we take an organ-focused approach to Rab GTPase function in vivo to assess the roles of Rab genes in reproductive system development. We performed a whole-body RNAi screen of the entire Rab family in C. elegans to uncover Rabs essential for gonad development. Notable gonad defects resulted from RNAi knockdown of rab-1, the key regulator of ER-Golgi trafficking. We then examined the effects of tissue-specific RNAi knockdown of rab-1 in somatic reproductive system and germline cells. We interrogated the dual functions of the distal tip cell (DTC) as both a leader cell of gonad organogenesis and the germline stem cell niche. We find that rab-1 functions cell-autonomously and non-cell-autonomously to regulate both somatic gonad and germline development. Gonad migration, elongation, and gamete differentiation—but surprisingly not germline stem niche function—are highly sensitive to rab-1 RNAi.

The discovery and simulation analysis of a novel mutation c.40 G < T (V14F) in the NRAS gene in patients with colorectal cancer in Saudi Arabia

Autosomal dominant optic atrophy (DOA) is a progressive form of blindness caused by degeneration of retinal ganglion cells and their axons, mainly caused by mutations in the OPA1 mitochondrial dynamin like GTPase (OPA1) gene. OPA1 encodes a dynamin-like GTPase present in the mitochondrial inner membrane. When associated with OPA1 mutations, DOA can present not only ocular symptoms but also multi-organ symptoms (DOA plus). DOA plus often results from point mutations in the GTPase domain, which are assumed to have dominant-negative effects. However, the presence of mutations in the GTPase domain does not always result in DOA plus. Therefore, an experimental system to distinguish between DOA and DOA plus is needed. In this study, we found that loss-of-function mutations of the dOPA1 gene in Drosophila can imitate the pathology of optic nerve degeneration observed in DOA. We successfully rescued this degeneration by expressing the human OPA1 (hOPA1) gene, indicating that hOPA1 is functionally interchangeable with dOPA1 in the fly system. However, mutations previously identified did not ameliorate the dOPA1 deficiency phenotype. By expressing both WT and DOA plus mutant hOPA1 forms in the optic nerve of dOPA1 mutants, we observed that DOA plus mutations suppressed the rescue, facilitating the distinction between loss-of-function and dominant-negative mutations in hOPA1. This fly model aids in distinguishing DOA from DOA plus and guides initial hOPA1 mutation treatment strategies.

Autosomal dominant optic atrophy (DOA) is a progressive form of blindness caused by degeneration of retinal ganglion cells and their axons, mainly caused by mutations in the OPA1 mitochondrial dynamin like GTPase (OPA1) gene. OPA1 encodes a dynamin-like GTPase present in the mitochondrial inner membrane. When associated with OPA1 mutations, DOA can present not only ocular symptoms but also multi-organ symptoms (DOA plus). DOA plus often results from point mutations in the GTPase domain, which are assumed to have dominant-negative effects. However, the presence of mutations in the GTPase domain does not always result in DOA plus. Therefore, an experimental system to distinguish between DOA and DOA plus is needed. In this study, we found that loss-of-function mutations of the dOPA1 gene in Drosophila can imitate the pathology of optic nerve degeneration observed in DOA. We successfully rescued this degeneration by expressing the human OPA1 (hOPA1) gene, indicating that hOPA1 is functionally interchangeable with dOPA1 in the fly system. However, mutations previously identified did not ameliorate the dOPA1 deficiency phenotype. By expressing both WT and DOA plus mutant hOPA1 forms in the optic nerve of dOPA1 mutants, we observed that DOA plus mutations suppressed the rescue, facilitating the distinction between loss-of-function and dominant-negative mutations in hOPA1. This fly model aids in distinguishing DOA from DOA plus and guides initial hOPA1 mutation treatment strategies.

A key mechanism employed by plants to adapt to salinity stress involves maintaining ion homeostasis via the actions of ion transporters. While the function of cation transporters in maintaining ion homeostasis in plants has been extensively studied, little is known about the roles of their anion counterparts in this process. Here, we describe a mechanism of salt adaptation in plants. We characterized the chloride channel (CLC) gene AtCLCf, whose expression is regulated by WRKY transcription factor under salt stress in Arabidopsis thaliana. Loss-of-function atclcf seedlings show increased sensitivity to salt, whereas AtCLCf overexpression confers enhanced resistance to salt stress. Salt stress induces the translocation of GFP-AtCLCf fusion protein to the plasma membrane (PM). Blocking AtCLCf translocation using the exocytosis inhibitor brefeldin-A or mutating the small GTPase gene AtRABA1b/BEX5 (RAS GENES FROM RAT BRAINA1b homolog) increases salt sensitivity in plants. Electrophysiology and liposome-based assays confirm the Cl−/H+ antiport function of AtCLCf. Therefore, we have uncovered a mechanism of plant adaptation to salt stress involving the NaCl-induced translocation of AtCLCf to the PM, thus facilitating Cl− removal at the roots, and increasing the plant’s salinity tolerance.

SummaryBackgroundParkinson's disease is a progressive neurodegenerative disorder with multifactorial causes, among which genetic risk factors play a part. The RAB GTPases are regulators and substrates of LRRK2, and variants in the LRRK2 gene are important risk factors for Parkinson's disease. We aimed to explore genetic variability in RAB GTPases within cases of familial Parkinson's disease.MethodsWe did whole-exome sequencing in probands from families in Canada and Tunisia with Parkinson's disease without a genetic cause, who were recruited from the Centre for Applied Neurogenetics (Vancouver, BC, Canada), an international consortium that includes people with Parkinson's disease from 36 sites in 24 countries. 61 RAB GTPases were genetically screened, and candidate variants were genotyped in relatives of the probands to assess disease segregation by linkage analysis. Genotyping was also done to assess variant frequencies in individuals with idiopathic Parkinson's disease and controls, matched for age and sex, who were also from the Centre for Applied Neurogenetics but unrelated to the probands or each other. All participants were aged 18 years or older. The sequencing and genotyping findings were validated by case–control association analyses using bioinformatic data obtained from publicly available clinicogenomic databases (AMP-PD, GP2, and 100 000 Genomes Project) and a private German clinical diagnostic database (University of Tübingen). Clinical and pathological findings were summarised and haplotypes were determined. In-vitro studies were done to investigate protein interactions and enzyme activities.FindingsBetween June 1, 2010, and May 31, 2017, 130 probands from Canada and Tunisia (47 [36%] female and 83 [64%] male; mean age 72·7 years [SD 11·7; range 38–96]; 109 White European ancestry, 18 north African, two east Asian, and one Hispanic] underwent whole-exome sequencing. 15 variants in RAB GTPase genes were identified, of which the RAB32 variant c.213C>G (Ser71Arg) cosegregated with autosomal dominant Parkinson's disease in three families (nine affected individuals; non-parametric linkage Z score=1·95; p=0·03). 2604 unrelated individuals with Parkinson's disease and 344 matched controls were additionally genotyped, and five more people originating from five countries (Canada, Italy, Poland, Turkey, and Tunisia) were identified with the RAB32 variant. From the database searches, in which 6043 individuals with Parkinson's disease and 62 549 controls were included, another eight individuals were identified with the RAB32 variant from four countries (Canada, Germany, UK, and USA). Overall, the association of RAB32 c.213C>G (Ser71Arg) with Parkinson's disease was significant (odds ratio [OR] 13·17, 95% CI 2·15–87·23; p=0·0055; I2=99·96%). In the people who had the variant, Parkinson's disease presented at age 54·6 years (SD 12·75, range 31–81, n=16), and two-thirds had a family history of parkinsonism. RAB32 Ser71Arg heterozygotes shared a common haplotype, although penetrance was incomplete. Findings in one individual at autopsy showed sparse neurofibrillary tangle pathology in the midbrain and thalamus, without Lewy body pathology. In functional studies, RAB32 Arg71 activated LRRK2 kinase to a level greater than RAB32 Ser71.InterpretationRAB32 Ser71Arg is a novel genetic risk factor for Parkinson's disease, with reduced penetrance. The variant was found in individuals with Parkinson's disease from multiple ethnic groups, with the same haplotype. In-vitro assays show that RAB32 Arg71 activates LRRK2 kinase, which indicates that genetically distinct causes of familial parkinsonism share the same mechanism. The discovery of RAB32 Ser71Arg also suggests several genetically inherited causes of Parkinson's disease originated to control intracellular immunity. This shared aetiology should be considered in future translational research, while the global epidemiology of RAB32 Ser71Arg needs to be assessed to inform genetic counselling.FundingNational Institutes of Health, the Canada Excellence Research Chairs program, Aligning Science Across Parkinson's, the Michael J Fox Foundation for Parkinson's Research, and the UK Medical Research Council.

Salinity in plants generates an osmotic and ionic imbalance inside cells that compromises the viability of the plant. Rab GTPases, the largest family within the small GTPase superfamily, play pivotal roles as regulators of vesicular trafficking in plants, including the economically important and globally cultivated tomato (Solanum lycopersicum). Despite their significance, the specific involvement of these small GTPases in tomato vesicular trafficking and their role under saline stress remains poorly understood. In this work, we identified and classified 54 genes encoding Rab GTPases in cultivated tomato, elucidating their genomic distribution and structural characteristics. We conducted an analysis of duplication events within the S. lycopersicum genome, as well as an examination of gene structure and conserved motifs. In addition, we investigated the transcriptional profiles for these Rab GTPases in various tissues of cultivated and wild tomato species using microarray-based analysis. The results showed predominantly low expression in most of the genes in both leaves and vegetative meristem, contrasting with notably high expression levels observed in seedling roots. Also, a greater increase in gene expression in shoots from salt-tolerant wild tomato species was observed under normal conditions when comparing Solanum habrochaites, Solanum pennellii, and Solanum pimpinellifolium with S. lycopersicum. Furthermore, an expression analysis of Rab GTPases from Solanum chilense in leaves and roots under salt stress treatment were also carried out for their characterization. These findings revealed that specific Rab GTPases from the endocytic pathway and the trans-Golgi network (TGN) showed higher induction in plants exposed to saline stress conditions. Likewise, disparities in gene expression were observed both among members of the same Rab GTPase subfamily and between different subfamilies. Overall, this work emphasizes the high degree of conservation of Rab GTPases, their high functional diversification in higher plants, and the essential role in mediating salt stress tolerance and suggests their potential for further exploration of vesicular trafficking mechanisms in response to abiotic stress conditions.

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.

The small GTPases of the Rho family are known to regulate various biological processes in filamentous fungi. In this study, we investigated the impact of deleting Rho proteins on the growth and cellulase production of Trichoderma reesei. Our findings revealed that deletion of cdc42 led to the most severe growth defect and impaired cellulase production. Conversely, overexpression of cdc42 resulted in a hyperbranched phenotype, significantly enhancing cellulase production. Furthermore, the cdc42-overexpressing (OCdc42) strain showed an increased expression of multiple cellulase genes and Rho GTPase genes. Analysis of the secretome in the OCdc42 strain unveiled an increased abundance and diversity of extracellular proteins compared to the parent strain. These discoveries provide valuable insights into the functionality of Rho GTPases in T. reesei and offer potential targets for engineering fungi to improve plant biomass deconstruction in biorefineries.

Recent studies have shown that several members of the G-protein-coupled receptors (GPCR) superfamily play crucial roles in the maintenance of ion-water homeostasis of the sperm and Sertoli cells, development of the germ cells, formation of the blood barrier, and maturation of sperm. The GPCR, guanyl-nucleotide exchange factor, membrane traffic protein, and small GTPase genes were analyzed by microarray and bioinformatics (3513 sperm and Sertoli cell genes). In the microarray analyses of three human cases with different nonobstructive azoospermia sperm, the expression of GOLGA8IP, OR2AT4, PHKA1, A2M, OR56A1, SEMA3G, LRRC17, APP, ARHGAP33, RABGEF1, NPY2R, GHRHR, LTB4R2, GRIK5, OR6K6, NAPG, OR6C65, VPS35, FPR3, and ARL4A was upregulated, while expression of MARS, SIRPG, OGFR, GPR150, LRRK1, and NGEF was downregulated. There was an increase in GBP3, GBP3, TNF, TGFB3, and CLTC expression in the Sertoli cells of three human cases with NOA, whereas expression of PAQR4, RRAGD, RAC2, SERPINB8, IRPB1, MRGPRF, RASA2, SIRPG, RGS2, RAP2A, RAB2B, ARL17, SERINC4, XIAP, DENND4C, ANKRA2, CSTA, STX18, and SNAP23 were downregulated. A combined analysis of Enrich Shiny Gene Ontology (GO), STRING, and Cytoscape was used to predict proteins' molecular interactions and then to recognize master pathways. Functional enrichment analysis showed that the biological process (BP), regulation of protein metabolic process, regulation of small GTPase-mediated signal transduction were significantly expressed in up-/downregulated differentially expressed genes (DEGs) in sperm. In molecular function (MF) experiments of DEGs that were up-/downregulated, it was found that GPCR activity, guanyl ribonucleotide binding, GTPase activity and nucleoside-triphosphatase activity were overexpressed. An analysis of GO enrichment findings of Sertoli cells showed BP and MF to be common DEGs. When these gene mutations have been validated, they can be used to create new GPCR antagonists or agonists that are receptor-selective.

Multiple regulators were involved in glutelin synthesis and subunit accumulation in response to temperature and nitrogen during rice grain-filling stage

Salmonella enterica serovar Enteritidis is a globally significant zoonotic foodborne pathogen. Chicken liver is a vital organ that has been recently implicated in several reported human salmonellosis outbreaks in the U.S. One promising strategy for reducing Salmonella in chickens could be through supplementation with natural antimicrobial additives. Ethanolic extracted cranberry pomace (CPOH) is an excellent source of bioactive polyphenolic compounds with antioxidant and antimicrobial activities. However, the protective effect of CPOH against S. Enteritidis-induced chicken hepatic cell damage remains unclear. In this study, we used a chicken hepatoma cell (LMH) infection model to investigate the protective effects and potential mechanisms of CPOH. CPOH increased the viability of S. Enteritidis-infected LMH cells. Furthermore, CPOH reduced the adhesion and invasion of S. Enteritidis to LMH cells. CPOH downregulated the expression of Rho GTPase genes that are essential for Salmonella's entry into LMH cells. Additionally, the expression of antioxidant regulatory genes, such as Nrf2, HO-1, Txn, and Gclc, was increased. Our data show that CPOH effectively protected LMH cells from cell damage through the inhibition of S. Enteritidis adhesion and invasion, as well as the induction of the expression of master antioxidant genes. These findings offer opportunities to develop sustainable, safe, and economic strategies to reduce the colonization and pathogenesis of Salmonella.

Clostridioides difficile (C. difficile) toxins A (TcdA) and B (TcdB) cause antibiotic-associated colitis in part by disrupting epithelial barrier function. Accurate in vitro models are necessary to detect early toxicity kinetics, investigate disease etiology, and develop preclinical models for new therapies. Properties of cancer cell lines and organoids inherently limit these efforts. We developed adult stem cell-derived monolayers of differentiated human colonic epithelium (hCE) with barrier function, investigated the impact of toxins on apical/basal aspects of monolayers, and evaluated whether a leaky epithelial barrier enhances toxicity. Single-cell RNA-sequencing (scRNAseq) mapped C. difficile-relevant genes to human lineages. Transcriptomics compared hCE to Caco-2, informed timing of in vitro stem cell differentiation, and revealed transcriptional responses to TcdA. Transepithelial electrical resistance (TEER) and fluorescent permeability assays measured cytotoxicity. Contribution of TcdB toxicity was evaluated in a diclofenac-induced leaky gut model. scRNAseq demonstrated broad and variable toxin receptor expression. Absorptive colonocytes in vivo displayed increased toxin receptor, Rho GTPase, and cell junction gene expression. Advanced TcdA toxicity generally decreased cytokine/chemokine and increased tight junction and death receptor genes. Differentiated Caco-2 cells remained immature whereas hCE monolayers were similar to mature colonocytes in vivo. Basal exposure of TcdA/B caused greater toxicity and apoptosis than apical exposure. Apical exposure to toxins was enhanced by diclofenac. Apical/basal toxicities are uncoupled with more rapid onset and increased magnitude postbasal toxin exposure. Leaky junctions enhance toxicity of apical TcdB exposure. hCE monolayers represent a physiologically relevant and sensitive system to evaluate the impact of microbial toxins on gut epithelium.NEW & NOTEWORTHY Novel human colonocyte monolayer cultures, benchmarked by transcriptomics for physiological relevance, detect early cytopathic impacts of Clostridioides difficile toxins TcdA and TcdB. A fluorescent ZO-1 reporter in primary human colonocytes is used to track epithelial barrier disruption in response to TcdA. Basal TcdA/B exposure generally caused more rapid onset and cytotoxicity than apical exposure. Transcriptomics demonstrate changes in tight junction, chemokine, and cytokine receptor gene expression post-TcdA exposure. Diclofenac-induced leaky epithelium enhanced apical exposure toxicity.

The Ras superfamily of small guanosine triphosphatases (GTPases) are a large group of small GTP-binding proteins, which play crucial roles in basic cellular processes in all eukaryotes. In this study, by analyzing the gene structure, temporal and spatial expression patterns, a total of 108 Ras superfamily genes were identified in the genome of the Pacific white shrimp Litopenaeus vannamei. We found these genes included not only the classical Ras GTPase superfamily members, but also some unconventional and novel Ras GTPase proteins, which have unknown functions and unique expression patterns. All Ras superfamily genes of L. vannamei were highly conserved within the core G domain and closely related in phylogeny, but they might have two different evolutionary origins. In addition, different Ras GTPase genes exhibited distinct expression patterns in different tissues, development/molting stages and WSSV infection samples of L. vannamei, suggesting that they may have a high functional specialization, and play important roles in regulating the biological processes of cell differentiation, growth and development, immune response, etc. This study provides important clues for the structure, classification, evolution and function of Ras superfamily in shrimp.

MoRts1, a regulatory subunit of PP2A, is required for fungal development and pathogenicity of Magnaporthe oryzae

Effects of chloroquine and hydroxychloroquine on the sensitivity of pancreatic cancer cells to targeted therapies.

Plants are frequently subjected to abiotic and biotic stress which causes major impediments in their growth and development. It is emerging that small guanosine triphosphatases (small GTPases), also known as monomeric GTP-binding proteins, assist plants in managing environmental stress. Small GTPases function as tightly regulated molecular switches that get activated with the aid of guanosine triphosphate (GTP) and deactivated by the subsequent hydrolysis of GTP to guanosine diphosphate (GDP). All small GTPases except Rat sarcoma (Ras) are found in plants, including Ras-like in brain (Rab), Rho of plant (Rop), ADP-ribosylation factor (Arf) and Ras-like nuclear (Ran). The members of small GTPases in plants interact with several downstream effectors to counteract the negative effects of environmental stress and disease-causing pathogens. In this review, we describe processes of stress alleviation by developing pathways involving several small GTPases and their associated proteins which are important for neutralizing fungal infections, stomatal regulation, and activation of abiotic stress-tolerant genes in plants. Previous reviews on small GTPases in plants were primarily focused on Rab GTPases, abiotic stress, and membrane trafficking, whereas this review seeks to improve our understanding of the role of all small GTPases in plants as well as their interactome in regulating mechanisms to combat abiotic and biotic stress. This review brings to the attention of scientists recent research on small GTPases so that they can employ genome editing tools to precisely engineer economically important plants through the overexpression/knock-out/knock-in of stress-related small GTPase genes.