GTPase Activity Research Articles

Epigenetic modulation rescues neurodevelopmental deficits in Syngap1+/- mice.

SYNGAP1 is a Ras GTPase-activating protein that plays a crucial role during brain development and in synaptic plasticity. Sporadic heterozygous mutations in SYNGAP1 affect social and emotional behaviour observed in intellectual disability (ID) and autism spectrum disorder (ASD). Although neurophysiological deficits have been extensively studied, the epigenetic landscape of SYNGAP1 mutation-mediated intellectual disability is unexplored. Here, we have found that the p300/CBP specific acetylation marks of histones are significantly repressed in the hippocampus of adolescent Syngap1+/- mice. Additionally, we observed decreased dendritic branching of newly born DCX+ neurons in these mice, suggesting altered adult hippocampal neurogenesis. To establish the causal relationship of Syngap1+/- phenotype and the altered histone acetylation signature we have treated 2-4 months old Syngap1+/- mice with glucose-derived carbon nanosphere (CSP) conjugated potent small molecule activator (TTK21) of p300/CBP lysine acetyltransferase (CSP-TTK21). The enhancement of the p300/CBP specific acetylation marks of histones by CSP-TTK21 restored synaptic functions, increased dendritic branching of DCX+ neurons, enables the capability to reorganise cortical circuits in response to change in the sensory stimuli, and improves behavioural measures in Syngap1+/- mice that are very closely comparable to wild type littermates. Further, hippocampal RNA-Seq analysis of these mice revealed that the expression of many critical genes such as Adcy1, Ntrk3, Egr1, and Foxj1 which are key regulators of synaptic plasticity and neurogenesis and are well associated with ID/ASD reversed upon CSP-TTK21 treatment. This study could be the first demonstration of the reversal of autistic behaviour and neural wiring upon the modulation of altered epigenetic modification(s).

G3BP1 Regulates the Cell Cycle by Promoting IFNβ Production to Promote PCV2 Replication and Promotes Nuclear Transfer of Viral Proteins by Direct Binding

Porcine circovirus type 2 (PCV2) is a significant pathogen responsible for porcine circovirus-associated diseases (PCVAD), and it is widely prevalent in pig farms, leading to huge economic losses for the pig industry. Currently, the ability of PCV2 to enhance its own replication by using the antiviral inflammatory factors IFNα, IFNβ, and IL-2 and its complex immune escape mechanism remain unclear, which has attracted wide attention. Research has indicated that GTPase-activating protein (SH3 domain)-binding protein 1 (G3BP1) is involved in the innate immune response to a variety of viruses, primarily by regulating and composing stress granules (SGs) to inhibit viral replication. Our initial studies identified elevated G3BP1 expression during PCV2 infection, paradoxically promoting PCV2 replication. In light of this phenomenon, this study aims to elucidate how PCV2 regulates G3BP1 to enhance its replication. Our findings demonstrate that G3BP1 overexpression further activates PCV2-induced expression of RIG-I, MDA5, cGAS and STING, thereby promoting IFNβ production and affecting cell cycle arrest in the S phase, facilitating PCV2 replication. Moreover, interactions were observed between PCV2 Cap protein and G3BP1’s RGG domain, and between PCV2 Rep protein and G3BP1’s NTF2 and RRM domains, potentially promoting viral protein nuclear transfer. In summary, PCV2 enhances its replication by modulating G3BP1 to induce IFNβ production and directly binds viral proteins to promote viral protein nuclear transfer. This research provides a foundation for further investigation into the immune evasion mechanisms of PCV2.

TBC1D20 coordinates vesicle transport and actin remodeling to regulate ciliogenesis.

TBC1D20 deficiency causes Warburg Micro Syndrome in humans, characterized by multiple eye abnormalities, severe intellectual disability, and abnormal sexual development, but the molecular mechanisms remain unknown. Here, we identify TBC1D20 as a novel Rab11 GTPase-activating protein that coordinates vesicle transport and actin remodeling to regulate ciliogenesis. Depletion of TBC1D20 promotes Rab11 vesicle accumulation and actin deconstruction around the centrosome, facilitating the initiation of ciliogenesis even in cycling cells. Further investigations reveal enhanced Rab11-MICAL1 interaction upon TBC1D20 loss, activating the monooxygenase domain of MICAL1 and inducing F-actin depolymerization around the centrosome. This actin network reorganization facilitates vesicle trafficking and docking, ultimately promoting ciliogenesis. In summary, our work uncovers a coordinated ciliogenesis initiation mechanism via Rab11 activation. These findings underscore a pivotal role for TBC1D20 in early ciliogenesis, advancing our understanding of its spatiotemporal regulation and offering insights into the disease pathogenesis associated with TBC1D20 mutations.

Differential GTP-dependent in-vitro polymerization of recombinant Physcomitrella FtsZ proteins

Bacterial cell division and plant chloroplast division require selfassembling Filamentous temperature-sensitive Z (FtsZ) proteins. FtsZ proteins are GTPases sharing structural and biochemical similarities with eukaryotic tubulin. In the moss Physcomitrella, the morphology of the FtsZ polymer networks varies between the different FtsZ isoforms. The underlying mechanism and foundation of the distinct networks is unknown. Here, we investigated the interaction of Physcomitrella FtsZ2-1 with FtsZ1 isoforms via co-immunoprecipitation and mass spectrometry, and found protein-protein interaction in vivo. We tagged FtsZ1-2 and FtsZ2-1 with different fluorophores and expressed both in E. coli, which led to the formation of defined structures within the cells and to an influence on bacterial cell division and morphology. Furthermore, we have optimized the purification protocols for FtsZ1-2 and FtsZ2-1 expressed in E. coli and characterized their GTPase activity and polymerization in vitro. Both FtsZ isoforms showed GTPase activity. Stoichiometric mixing of both proteins led to a significantly increased GTPase activity, indicating a synergistic interaction between them. In light scattering assays, we observed GTP-dependent assembly of FtsZ1-2 and of FtsZ2-1 in a protein concentration dependent manner. Stoichiometric mixing of both proteins resulted in significantly faster polymerization, again indicating a synergistic interaction between them. Under the same conditions used for GTPase and light scattering assays both FtsZ isoforms formed filaments in a GTP-dependent manner as visualized by transmission electron microscopy (TEM). Taken together, our results reveal that Physcomitrella FtsZ1-2 and FtsZ2-1 are functionally different, can synergistically interact in vivo and in vitro, and differ in their properties from FtsZ proteins from bacteria, archaea and vascular plants.

Dynamine 3 as a Diagnostic and Prognostic Biomarker in Pancreatic Cancer: Implications for Early Detection and Targeted Therapy

ABSTRACT Background Dynamins are defined as a group of molecules with GTPase activity that play a role in the formation of endocytic vesicles and Golgi apparatus. Among them, DNM3 has gained recognition in oncology for its tumor suppressor role. Based on this, the aim of this study is to investigate the effects of the DNM3 gene in patients diagnosed with pancreatic cancer using bioinformatics databases. Materials and Methods For differential gene expression analysis, TCGA TARGET GTEx study on the UCSC Xena, GSE196009, GSE211398, GSE151580 datasets on The Gene Expression Omnibus (GEO) were utilized; for the analysis of changes in gene expression according to stages GEPIA2 were utilized; for the analysis of changes in gene expression according to clinical and pathological characteristics, UALCAN was employed; for Overall Survival (OS) analysis, Kaplan-Meier Plotter was used; for gene alteration analysis, cBioPortal was utilized; for immune cell infiltration analysis, Tumor Immune Estimation Resource (TIMER) and TIMER2.0 were employed; for protein-protein interaction and gene enrichment analyses, STRING was used; for enrichment analyses Enrichr was used; for Gene Set Correlation Enrichment Analysis Gscore was used on GSE15471; for essentiality of DNM3 gene in pancratic cancer cell lines DepMap was used; and for the detection of miRNAs, miRDB was utilized; ENCORI was used for gene-miRNA correlation and miRNA prognosis analyses. Results In the pancreatic adenocarcinoma (PAAD) cohort, DNM3 gene expression was higher in tumor samples, and there was no significant difference in expression among cancer stages. High levels of DNM3 gene expression were associated with longer OS in PAAD. A weak positive correlation was observed between DNM3 gene expression and B-Cell and CD + T Cell infiltrations, while a moderate positive correlation was found with CD8+ T Cell, Macrophage, Neutrophil, and Dendritic Cell infiltrations in TIMER. NK cell by QUANTISEQ, CD 4+ T Cell by TIMER, T cell regulatory (Tregs) by CIBERSORT-ABS infiltrations were positively associated with DNM3 gene expression and decreased risk in prognosis. Common lymphoid progenitor by XCELL and MDSC by TIDE infiltrations were negatively associated with DNM3 gene expression and increased risk of prognosis. Macrophage M1 by QUANTISEQ was positively associated with DNM3 gene expression and increased risk in prognosis. DNM3 gene appears to be associated with various pathways related to inflammation and the immune system. Amplification of the DNM3 gene was detected in 5 out of 175 patients. Enrichment was observed in pathways such as bacterial invasion of epithelial cells, endocytosis, endocrine and other factor-regulated calcium reabsorption, synaptic vesicle cycle, and phospholipase D signaling pathway. According to Gscore, DNM3 gene was associated with Fc epsilon RI signaling pathway, HALLMARK MTORC1 SIGNALING, HALLMARK EPITHELIAL MESENCHYMAL TRANSITION gene sets. According to ENCORI, DNM3 gene was negatively correlated with hsa-miR-203a-3p and increased expression of this miRNA was associated with adverse prognosis in PAAD. Conclusions The DNM3 gene may play a tumor suppressor role in pancreatic cancer, similar to its role in other malignancies. The contribution of immune cells may also be significant in this effect. However, in vitro studies are needed to elucidate the mechanisms triggered in pancreatic cancer.

Synergistic activity of Pitstop-2 and 1,6-hexanediol in aggressive human lung cancer cells

Metastatic cancer cells undergo metabolic reprogramming, which involves changes in the metabolic fluxes, including endocytosis, nucleocytoplasmic transport, and mitochondrial metabolism, to satisfy their massive demands for energy, cell division, and proliferation compared to normal cells. We have previously demonstrated the ability of two different types of compounds to interfere with linchpins of metabolic reprogramming, Pitstop-2 and 1,6-hexanediol (1,6-HD). 1,6-HD disrupts glycolysis enzymes and mitochondrial function, enhancing reactive oxygen species production and reducing cellular ATP levels, while Pitstop-2 impedes clathrin-mediated endocytosis and small GTPases activity. Besides, both compounds interfere with the integrity of nuclear pore complexes, the gatekeepers for all nucleocytoplasmic transport. Herein, we investigate the possible synergistic effects of both compounds on lowly, highly metastatic, and erlotinib-resistant non-small cell lung cancer. We observe a synergistic cytotoxic effect on erlotinib-resistant cells. Moreover, motility assays show that the compounds combination significantly impedes the motility of all cells. Drug safety and tolerability assessments were validated using the in vivo model organism Caenorhabditis elegans, where fairly high doses showed negligible impact on survival, development, or behavioral parameters. Our findings propose that the 1,6-HD and Pitstop-2 combination may usher in the design of potent strategies for treating advanced lung cancer.

Molecular Mechanisms of Alzheimer's Disease Induced by Amyloid-β and Tau Phosphorylation Along with RhoA Activity: Perspective of RhoA/Rho-Associated Protein Kinase Inhibitors for Neuronal Therapy.

Amyloid-β peptide (Aβ) is a critical cause of Alzheimer's disease (AD). It is generated from amyloid precursor protein (APP) through cleavages by β-secretase and γ-secretase. γ-Secretase, which includes presenilin, is regulated by several stimuli. Tau protein has also been identified as a significant factor in AD. In particular, Tau phosphorylation is crucial for neuronal impairment, as phosphorylated Tau detaches from microtubules, leading to the formation of neurofibrillary tangles and the destabilization of the microtubule structure. This instability in microtubules damages axons and dendrites, resulting in neuronal impairment. Notably, Aβ is linked to Tau phosphorylation. Another crucial factor in AD is neuroinflammation, primarily occurring in the microglia. Microglia possess several receptors that bind with Aβ, triggering the expression and release of an inflammatory factor, although their main physiological function is to phagocytose debris and pathogens in the brain. NF-κB activation plays a major role in neuroinflammation. Additionally, the production of reactive oxygen species (ROS) in the microglia contributes to this neuroinflammation. In microglia, superoxide is produced through NADPH oxidase, specifically NOX2. Rho GTPases play an essential role in regulating various cellular processes, including cytoskeletal rearrangement, morphology changes, migration, and transcription. The typical function of Rho GTPases involves regulating actin filament formation. Neurons, with their complex processes and synapse connections, rely on cytoskeletal dynamics for structural support. Other brain cells, such as astrocytes, microglia, and oligodendrocytes, also depend on specific cytoskeletal structures to maintain their unique cellular architectures. Thus, the aberrant regulation of Rho GTPases activity can disrupt actin filaments, leading to altered cell morphology, including changes in neuronal processes and synapses, and potentially contributing to brain diseases such as AD.

Targeting CFTR restoring aggrephagy to suppress HSC activation and alleviate liver fibrosis.

Multiple studies have shown that hepatic fibrosis, a progressive condition that represents the endpoint of various chronic liver diseases, is primarily marked by the extensive activation of hepatic stellate cells (HSCs). However, the exact impact of cystic fibrosis transmembrane conductance regulator (CFTR) on HSCs during the development of hepatic fibrosis remains unclear. In our study, we measured CFTR levels in tissue samples and in HSCs activated by TGF-β stimulation. We established mouse models of liver fibrosis using carbon tetrachloride (CCl4) and bile duct ligation (BDL). In vitro, we investigated the specific mechanisms of CFTR action in HSCs by exploring aggrephagy. We employed co-immunoprecipitation (co-IP) experiments to identify potential downstream targets of CFTR. Finally, through rescue experiments, we examined the impact of GTPase-activating protein - binding protein 1 (G3BP1) on CFTR-mediated activation of hepatic stellate cells. In activated HSCs induced by TGF-β, the reduction of CFTR, various liver fibrosis models, and fibrotic tissue samples were identified. In vitro functional experiments confirmed that CFTR promoted the expression of fibrosis-related markers and aggrephagy in HSCs. Mechanistically, we found that CFTR directly interacts with G3BP1, thereby further promoting the TGF-β/Smad2/3 pathway. The inhibition of G3BP1 caused by CFTR knockdown reduced extracellular matrix deposition, contributing to alleviating liver fibrosis. We emphasize that CFTR activates aggrephagy and promotes HSC activation and hepatic fibrosis by targeting G3BP1, participating in the TGF-β/Smad2/3 signaling pathway. Overall, CFTR has been identified as a potential therapeutic target for liver fibrosis.

A TSC2 recurrent variant c.5126C>T in a Han-Chinese family with tuberous sclerosis complex.

To identify the disease-causing variant in a family with tuberous sclerosis complex (TSC). This study including a Han-Chinese pedigree recruited from the Third Xiangya Hospital, Central South University, Changsha, Hunan, China was conducted between February, 2019 and January, 2023. Detailed clinical examinations were performed on the proband and other family members of a Han-Chinese family with TSC. Whole exome sequencing of the proband and Sanger sequencing of all family members were performed, followed by variant pathogenicity prediction and conservation analysis. SWISS-MODEL and PyMOL software were used for protein modelling and creating the three-dimensional structure model illustration of the critical GTPase-activating protein (GAP) domain. The variant was classified following the American College of Medical Genetics and Genomics (ACMG) standards and guidelines. The female proband exhibited typical features of TSC, including hypomelanotic macules, angiofibromas, shagreen patches, seizures, brain lesions, cognitive impairment, renal abnormalities, and cardiovascular abnormalities. A recurrent c.5126C>T variant in the TSC complex subunit 2 gene (TSC2) was identified as the genetic cause of TSC in this family, classified as "pathogenic" according to ACMG standards and guidelines. The c.5126C>T variant leads to an amino acid change from proline to leucine at position 1709 (p.P1709L) in the functional GAP domain of tuberin protein, which may impair tumor growth inhibition of the hamartin-tuberin complex. This study reported a Han-Chinese TSC patient with a recurrent variant TSC2 c.5126C>T (p.P1709L). These findings broaden the phenotypic spectrum of TSC caused by this variant and may contribute to improving TSC genetic diagnoses as well as understanding of its mechanisms.

IQGAP3 activates Hedgehog signaling to confer stemness and metastasis via up-regulating GLI1 in lung cancer

Lung cancer ranks as the most prevalent malignant neoplasm worldwide, contributing significantly to cancer-related mortality. Stemness is a well-recognized factor underlying radiotherapy resistance, recurrence and metastasis in non-small-cell lung cancer (NSCLC) patients. Our prior investigations have established the role of IQ motif containing GTPase-activating protein 3 (IQGAP3) in mediating radiotherapy resistance in lung cancer, but its impact on lung cancer stemness remains unexplored. Our bioinformatics analysis results revealed a significant correlation between IQGAP3 and lung cancer stemness. Moreover, we found that IQGAP3 depletion in lung cancer cells resulted in reduced migration, invasion and sphere-forming capabilities. Through RNA sequencing, we identified GLI1 as a pivotal downstream effector of IQGAP3. The knockdown of IQGAP3 led to the downregulation of GLI1 mRNA and protein levels, which impeded the activation of the Hedgehog-GLI1 signaling pathway. Further, our results also indicated that GLI1 is the primary effector mediating IQGAP3’s biological functions in lung cancer. These findings elucidate the role of IQGAP3 in promoting lung cancer stemness and metastasis through the Hedgehog pathway, facilitated by GLI1, highlighting the potential of IQGAP3 as a promising therapeutic target for lung cancer treatment.

Novel endocytosis inhibitors block entry of HIV-1 Tat into neural cells.

Many pathogens including viruses enter cells by endocytosis. We identified and evaluated novel endocytosis inhibitors capable of blocking the entry of the HIV-1 Transactivation of Transcription protein (Tat) protein into neuronal cells and investigated their potential protective properties against Tat-induced neurotoxicity. In this study, the compounds Les-6631 and Les-6633 were synthesized and assessed. The effects of these compounds on the internalization of dextran and the cell-penetrating peptide (CPP) Tat-Cy5 complex in nerve cells were examined. In addition, the ability of these compounds to protect against oxidative stress and DNA damage induced by the full-length Tat protein was investigated. Les-6631 and Les-6633 were found to inhibit endocytosis better than the classical endocytosis inhibitor chlorpromazine, thereby effectively preventing the entry of the Tat protein into nerve cells. Moreover, compounds demonstrated the capacity to reduce oxidative stress and protect DNA from Tat-induced damage. In a neuro-AIDS model, both compounds proved effective in preventing neurotoxicity associated with HIV-1 infection, indicating its potential for therapeutic applications. Les-6631 and Les-6633 thus can protect cells from the harmful effects of pathogens. Their use in a neuro-AIDS model suggests a potential application in protective therapies for the nervous system in patients with HIV.NEW & NOTEWORTHY This study identifies novel rhodadyn-based inhibitors, Les-6631 and Les-6633, which selectively block dynamin's GTPase activity while sparing clathrin-mediated pathways. They effectively inhibit cellular uptake, protect neural cells from HIV-1 Tat-induced oxidative stress, and reduce mitochondrial and DNA damage. Their selective dynamin inhibition and antioxidant properties highlight their therapeutic potential for neurodegeneration and viral infections, offering cell protection without disrupting essential endocytic functions.

Magnolol as an Antibacterial Agent Against Drug-resistant Bacteria Targeting Filamentous Temperature-sensitive Mutant Z.

The emergence of multiple drug-resistant bacteria poses critical health threats worldwide. It is urgently needed to develop potent and safe antibacterial agents with novel bactericidal mechanisms to treat these infections. In this study, magnolol was identified as a potential bacterial cell division inhibitor by a cell-based screening approach. This compound showed good antibacterial activity against a number of Gram-positive pathogens (minimum inhibitory concentration 8-16 µg/mL) including methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus. Further results obtained from biochemical experiments demonstrated that magnolol could markedly disrupt GTPase activity and filamentous temperature-sensitive mutant Z (FtsZ) polymerization, consistent with the impediment to cell division in the bacteria tested. The in vivo antibacterial activity of magnolol was evaluated with a Galleria mellonella larvae model. The results showed that magnolol significantly increased the survival rate of larvae infected with methicillin-resistant S. aureus. The interaction pattern of magnolol with FtsZ was investigated through molecular docking. The finding may offer meaningful insights into the mechanism of action of the compound. The results point to magnolol as a promising antimicrobial compound that inhibits cell division by affecting FtsZ polymerization and has the potential to be developed into an effective antimicrobial drug by further structure modification.

Disruption of Molecular Interactions between the G3BP1 Stress Granule Host Protein and the Nucleocapsid (NTD-N) Protein Impedes SARS-CoV-2 Virus Replication.

The Ras GTPase-activating protein SH3-domain-binding protein 1 (G3BP1) serves as a formidable barrier to viral replication by generating stress granules (SGs) in response to viral infections. Interestingly, viruses, including SARS-CoV-2, have evolved defensive mechanisms to hijack SG proteins like G3BP1 for the dissipation of SGs that lead to the evasion of the host's immune responses. Previous research has demonstrated that the interaction between the NTF2-like domain of G3BP1 (G3BP1NTF-2) and the intrinsically disordered N-terminal domain (NTD-N1-25) of the N-protein plays a crucial role in regulating viral replication and pathogenicity. Interestingly, the current study identified an additional upstream stretch of residues (128KDGIIWVATEG138) (N128-138) within the N-terminal domain of the N-protein (NTD-N41-174) that also forms molecular contacts with the G3BP1 protein, as revealed through in silico analysis, site-directed mutagenesis, and biochemical analysis. Remarkably, WIN-62577, and fluspirilene, the small molecules targeting the conserved peptide-binding pocket in G3BP1NTF-2, not only disrupted the protein-protein interactions (PPIs) between NTD-N41-174 and G3BP1NTF-2 but also exhibited significant antiviral efficacy against SARS-CoV-2 replication with EC50 values of ∼1.8 and ∼1.3 μM, respectively. The findings of this study, validated by biophysical thermodynamics and biochemical investigations, advance the potential of developing therapeutics targeting the SG host protein against SARS-CoV-2, which may also serve as a broad-spectrum antiviral target.

The PH domain in the ArfGAP ASAP1 drives catalytic activation through an unprecedented allosteric mechanism.

ASAP1 is a multidomain Arf GTPase-activating protein (ArfGAP) that catalyzes GTP hydrolysis on the small GTPase Arf1 and is implicated in cancer progression. The PH domain of ASAP1 enhances its activity greater than 7 orders of magnitude but the underlying mechanisms remain poorly understood. Here, we combined Nuclear Magnetic Resonance (NMR), Molecular Dynamic (MD) simulations and mathematical modeling of functional data to build a comprehensive structural-mechanistic model of the complex of Arf1 and the ASAP1 PH domain on a membrane surface. Our results support a new conceptual model in which the PH domain contributes to efficient catalysis not only by membrane recruitment but by acting as a critical component of the catalytic interface, binding Arf·GTP and allosterically driving it towards the catalytic transition state. We discuss the biological implications of these results and how they may apply more broadly to poorly understood membrane-dependent regulatory mechanisms controlling catalysis of the ArfGAP superfamily as well as other peripheral membrane enzymes.

RAB37 suppresses the EMT, migration and invasion of gastric cancer cells by mediating autophagic degradation of β-catenin.

Gastric cancer, characterized by its high morbidity and mortality rates, exhibits low levels of RAB37. The role and molecular mechanisms of RAB37, a small GTPase, in the pathogenesis of gastric cancer are still unclear. We assessed RAB37 expression in gastric cancer cells using quantitative Polymerase Chain Reaction (qPCR), Western blot, and immunohistochemical staining (IHC), and analyzed EMT marker proteins and autophagy changes via Western blot, immunofluorescence (IF), and transmission electron microscopy (TEM). Co-immunoprecipitation (co-IP) was used to identify protein-protein interactions. We studied the migration and invasion of gastric cancer cells using wound healing and transwell assays in vitro and a mouse pulmonary metastasis model in vivo. Overexpression of RAB37 suppressed EMT, invasion, and migration while enhancing autophagy in gastric cancer cells, which was dependent on its GTPase activity. However, all these effects could be reversed by the autophagy inhibitor chloroquine. Regarding the molecular mechanism, RAB37 strengthened the interaction between p62 and β-catenin, which consequently enhanced the p62-mediated autophagic degradation of β-catenin. Furthermore, RAB37 curbed the pulmonary metastasis of both general and cisplatin-resistant gastric cancer cells. The low level of RAB37 reduces interaction between p62 and β-catenin and then the autophagic degradation of β-catenin, thereby promoting the EMT, invasion, and migration in gastric cancer cells. The low expression of RAB37 in gastric cancer suggests a potential therapeutic target, especially for cisplatin-resistant gastric cancer.

APKC/Par3/Par6 polarity complexes regulate podocyte motility and crescent formation in the progression of ANCA-associated vasculitis.

Podocyte bridging may be a key initial event occurring early in crescent formation. This study aims to probe the underlying mechanism of atypical protein kinase C (aPKC)/protease-activated receptor 3(Par3)/Par6 polarity complexes on podocyte motility and crescent formation during the progression of antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV). The effects of anti-TNF-α monoclonal antibody (mAb) on the crescent formation, localization and expression of aPKC/Par3/Par6 polarity complexes, and activities of small GTPases (Rho/Rac1/Cdc42) were explored in an AAV mouse model. Podocytes were stimulated in vitro by TNF-α and myeloperoxidase (MPO)-ANCA positive serum collected from patients with microscopic polyangiitis (MPA). Then, podocyte motility, aPKC/Par3/Par6 polarity protein expression and small GTPases activity were measured. The impacts of heat shock protein 70 (HSP70), a type of molecular chaperone, on the phosphorylation of aPKC was evaluated. Anti-TNF-α mAb inhibited crescent formation and restored the localization of aPKC/Par3/Par6 polarity complexes in the glomerulus of the AAV mouse model. Both MPO-ANCA-positive serum and TNF-α stimulation significantly induced podocyte motility by inhibiting of aPKC phosphorylation and detachment of aPKC/Par3/Par6 polarity complexes. Overexpression of HSP70 increased p-aPKC level and inhibited podocyte motility stimulated by either MPO-ANCA-positive serum or TNF-α. The podocyte polarity preserved by aPKC/Par3/Par6 polarity complexes, especially the phosphorylation status of aPKC, may play an important role in the crescent formation of AAV. The inhibition of TNF-α prevents the crescent formation in AAV via, at least partly, inhibiting podocyte polarity loss and motility.

Ionic control of small GTPase HRas using calmodulin.

HRas is a small GTPase that plays physiologically important roles in various intracellular signal transduction processes, such as cell growth and proliferation. The structure and action mechanisms of HRas have been well-characterized, leading to widespread its use as a molecular switch in bionanomachines. calmodulin, a calcium ion-binding protein, acts as an ion-binding molecular switch and activates the target enzymes. We previously demonstrated that the fusion protein of HRas (M13-HRas) with the calmodulin target peptide M13 at the N-terminus of HRas exhibits reversible regulation of GTPase activity and the interaction between M13-HRas and the downstream signaling factor Raf by calcium ions with calmodulin. In this study, we prepared two new HRas fusion proteins with the M13 peptide at the C-terminus (HRas-M13) and both termini (M13-HRas-M13) of HRas and analyzed the calcium-dependent regulation of HRas function. M13-HRas-M13 more efficiently controlled GTPase, interaction with Raf, and the HRas regulator GEF by calcium ions with calmodulin.

Identified VCAM1 as prognostic gene in gastric cancer by co-expression network analysis

The diffuse gastric cancer (DGC) is a malignant tumor distinct from intestinal gastric cancer (IGC). This study aims to identify genetic variances and potential diagnostic and therapeutic approaches for diverse types of gastric cancer utilizing an extensive dataset. Data from RNA sequencing and clinical pathological details were acquired from The Cancer Genome Atlas (TCGA) database and the Gene Expression Omnibus (GEO) dataset. Co‐expression gene modules were constructed via Weighted Gene Co‐Expression Network Analysis (WGCNA), followed by deciphering gene functions and protein–protein interaction networks within significantly associated modules. In total, analysis was conducted on 56,753 genes from 247 individuals with gastric cancer. Particularly, 621 genes from the green module exhibited strong associations with the Lauren type of gastric cancer. The prominent genes in the green module showed enrichment in processes such as signal transduction, immune response, and the positive regulation of GTPase activity. Noteworthy among these, VCAM1 was identified as the central gene linked to patients’ prognosis. Moreover, 72 gastric cancer specimens were collected from The First Affiliated Hospital of University of Science and Technology of China. Immunohistochemical analysis demonstrated a significantly higher expression of VCAM1 in DGC compared to IGC (p = 0.019). Furthermore, it was confirmed that VCAM1 expression serves as a prognostic indicator for patients with DGC (p = 0.002), a correlation not observed in IGC (p = 0.760). In conclusion, this study identifies VCAM1 as a promising diagnostic and prognostic factor, suggesting novel avenues for diagnostic and therapeutic approaches in gastric cancer.

Exploiting 19F NMR in a Multiplexed Assay for Small GTPase Activity.

Small GTPases (smG) are a 150-member family of proteins, comprising five subfamilies: Ras, Rho, Arf, Rab, and Ran-GTPases. These proteins function as molecular switches, toggling between two distinct nucleotide-bound states. Using traditional multidimensional heteronuclear NMR, even for single smGs, numerous experiments, high protein concentrations, expensive isotope labeling, and long analysis times are necessary. 19F NMR of fluorinated proteins or ligands can overcome these drawbacks. Using indole position-specific 19F labeling of the proteins, the activities of several smGs were measured in a multiplexed fashion. We investigated 4-, 5-, 6-, and 7-fluoro tryptophan containing smGs to study nucleotide binding. Distinct resonances for GDP- or GTP-bound states of three different 19F-labeled smGs, RhoA, K-Ras, and Rac1, were observed, and the kinetics of exchange and hydrolysis were measured. This multiplexed system will permit screening of nucleotide-specific ligands of smGs under true physiological conditions.

Sdd3 regulates the biofilm formation of Candida albicans via the Rho1-PKC-MAPK pathway.

Candida albicans, the most frequently isolated fungal pathogen in humans, forms biofilms that enhance resistance to antifungal drugs and host immunity, leading to frequent treatment failure. Understanding the molecular mechanisms governing biofilm formation is crucial for developing anti-biofilm therapies. In this study, we conducted a genetic screen to identify novel genes that regulate biofilm formation in C. albicans. One identified gene is ORF19.6693, a homolog of the Saccharomyces cerevisiae SDD3 gene. The sdd3∆/∆ mutant exhibited severe defects in biofilm formation and significantly reduced chitin content in the cell wall. Overexpression of the constitutively active version of the Rho1 GTPase Rho1G18V, an upstream activator of the protein kinase C (PKC)-mitogen-activated protein kinase (MAPK) cell-wall integrity pathway, rescued these defects. Affinity purification, mass spectrometry, and co-immunoprecipitation revealed Sdd3's physical interaction with Bem2, the GTPase-activating protein of Rho1. Deletion of SDD3 significantly reduced the amount of the active GTP-bound form of Rho1, thereby diminishing PKC-MAPK signaling and downregulating chitin synthase genes CHS2 and CHS8. Taken together, our studies identify a new biofilm regulator, Sdd3, in C. albicans that modulates Rho1 activity through its inhibitory interaction with Bem2, thereby regulating the PKC-MAPK pathway to control chitin biosynthesis, which is critical for biofilm formation. As an upstream component of the pathway and lacking a homolog in mammals, Sdd3 has the potential to serve as an antifungal target for biofilm infections.IMPORTANCEThe human fungal pathogen Candida albicans is categorized as a critical priority pathogen on the World Health Organization's Fungal Priority Pathogens List. A key virulence attribute of this pathogen is its ability to form biofilms on the surfaces of indwelling medical devices. Fungal cells in biofilms are highly resistant to antifungal drugs and host immunity, leading to treatment failure. This study conducted a genetic screen to discover novel genes that regulate biofilm formation. We found that deletion of the SDD3 gene caused severe biofilm defects. Sdd3 negatively regulates the Rho1 GTPase, an upstream activator of the protein kinase C-mitogen-activated protein kinase pathway, through direct interaction with Bem2, the GTPase-activating protein of Rho1, resulting in a significant decrease in chitin content in the fungal cell wall. This chitin synthesis defect leads to biofilm formation failure. Given its essential role in biofilm formation, Sdd3 could serve as an antifungal target for biofilm infections.