Search Results (12)

HBV infection is challenging to cure due to the persistence of viral covalently closed circular viral DNA (cccDNA). The dedicator of cytokinesis 11 (DOCK11) is recognized as a guanine nucleotide exchange factor (GEF) for CDC42 that has been reported to be required for HBV persistence. DOCK11 is expressed in both the cytoplasm and nucleus of human hepatocytes and is functionally associated with retrograde trafficking proteins Arf-GAP with GTPase domain, ankyrin repeat, and pleckstrin homology domain-containing protein 2 (AGAP2), and ADP-ribosylation factor 1 (ARF1), together with the HBV capsid, in the trans-Golgi network (TGN). This opens an alternative retrograde trafficking route for HBV from early endosomes (EEs) to the TGN and then to the endoplasmic reticulum (ER), thereby avoiding lysosomal degradation. DOCK11 also facilitates the association of cccDNA with H3K4me3 and RNA Pol II for activating cccDNA transcription. In addition, DOCK11 plays a crucial role in the host DNA repair system, being essential for cccDNA synthesis. This function can be inhibited by 10M-D42AN, a novel DOCK11-binding peptide, leading to the suppression of HBV replication both in vitro and in vivo. Treatment with a combination of 10M-D42AN and entecavir may represent a promising therapeutic strategy for patients with chronic hepatitis B (CHB). Consequently, DOCK11 may be seen as a potential candidate molecule in the development of molecularly targeted drugs against CHB. Full article

Mangrove-associated fungi are rich sources of novel and bioactive compounds. A total of 102 fungal strains were isolated from the medicinal mangrove Acanthus ilicifolius collected from the South China Sea. Eighty-four independent culturable isolates were identified using a combination of morphological characteristics and internal transcribed spacer (ITS) sequence analyses, of which thirty-seven strains were selected for phylogenetic analysis. The identified fungi belonged to 22 genera within seven taxonomic orders of one phyla, of which four genera Verticillium, Neocosmospora, Valsa, and Pyrenochaeta were first isolated from mangroves. The cytotoxic activity of organic extracts from 55 identified fungi was evaluated against human lung cancer cell lines (A-549), human cervical carcinoma cell lines (HeLa), human hepatoma cells (HepG2), and human acute lymphoblastic leukemia cell lines (Jurkat). The crude extracts of 31 fungi (56.4%) displayed strong cytotoxicity at the concentration of 50 μg/mL. Furthermore, the fungus Penicillium sp. (HS-N-27) still showed strong cytotoxic activity at the concentration of 25 µg/mL. Integrating cytotoxic activity-guided strategy and fingerprint analysis, a well-known natural Golgi-disruptor and Arf-GEFs inhibitor, brefeldin A, was isolated from the target active strain HS-N-27. It displayed potential activity against A549, HeLa and HepG2 cell lines with the IC₅₀ values of 101.2, 171.9 and 239.1 nM, respectively. Therefore, combining activity-guided strategy with fingerprint analysis as a discovery tool will be implemented as a systematic strategy for quick discovery of active compounds. Full article

Brefeldin A (1), a potent cytotoxic natural macrolactone, was produced by the marine fungus Penicillium sp. (HS-N-29) from the medicinal mangrove Acanthus ilicifolius. Series of its ester derivatives 2–16 were designed and semi-synthesized, and their structures were characterized by spectroscopic methods. Their cytotoxic activities were evaluated against human chronic myelogenous leukemia K562 cell line in vitro, and the preliminary structure–activity relationships revealed that the hydroxy group played an important role. Moreover, the monoester derivatives exhibited stronger cytotoxic activity than the diester derivatives. Among them, brefeldin A 7-O-2-chloro-4,5-difluorobenzoate (7) exhibited the strongest inhibitory effect on the proliferation of K562 cells with an IC₅₀ value of 0.84 µM. Further evaluations indicated that 7 induced cell cycle arrest, stimulated cell apoptosis, inhibited phosphorylation of BCR-ABL, and thereby inactivated its downstream AKT signaling pathway. The expression of downstream signaling molecules in the AKT pathway, including mTOR and p70S6K, was also attenuated after 7-treatment in a dose-dependent manner. Furthermore, molecular modeling of 7 docked into 1 binding site of an ARF1–GDP-GEF complex represented well-tolerance. Taken together, 7 had the potential to be served as an effective antileukemia agent or lead compound for further exploration. Full article

Contrary to the opinion recently offered by Dimou et al., our previously published biochemical, subcellular and genetic data supported our contention that AN11127 corresponds to the A. nidulans gene encoding Sec12, which is the guanine nucleotide exchange factor (GEF) specific for SAR1. We add here additional bioinformatics evidence that fully disprove the otherwise negative evidence reported by Dimou et al., highlighting the dangers associated with the lax interpretation of genomic data. On the positive side, we establish guidelines for the identification of this key secretory gene in other species of Ascomycota and Basidiomycota, including species of medical and applied interest. Full article

IQSEC2 is a guanine nucleotide exchange factor (GEF) for ADP-ribosylation factor 6 (Arf6), of which protein is exclusively localized to the postsynaptic density of the excitatory synapse. Human genome studies have revealed that the IQSEC2 gene is associated with X-linked neurodevelopmental disorders, such as intellectual disability (ID), epilepsy, and autism. In this study, we examined the behavior and synapse function in IQSEC2 knockout (KO) mice that we generated using CRIPSR/Cas9-mediated genome editing to solve the relevance between IQSEC2 deficiency and the pathophysiology of neurodevelopmental disorders. IQSEC2 KO mice exhibited autistic behaviors, such as overgrooming and social deficits. We identified that up-regulation of c-Fos expression in the medial prefrontal cortex (mPFC) induced by social stimulation was significantly attenuated in IQSEC2 KO mice. Whole cell electrophysiological recording identified that synaptic transmissions mediated by α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR), N-methyl-D-aspartate receptor (NMDAR), and γ-aminobutyric acid receptor (GABAR) were significantly decreased in pyramidal neurons in layer 5 of the mPFC in IQSEC2 KO mice. Reexpression of IQSEC2 isoform 1 in the mPFC of IQSEC2 KO mice using adeno-associated virus (AAV) rescued both synaptic and social deficits, suggesting that impaired synaptic function in the mPFC is responsible for social deficits in IQSEC2 KO mice. Full article

Several small guanosine triphosphatases (GTPases) from the Ras protein superfamily regulate glucose-stimulated insulin secretion in the pancreatic islet β-cell. The Rho family GTPases Cdc42 and Rac1 are primarily involved in relaying key signals in several cellular functions, including vesicle trafficking, plasma membrane homeostasis, and cytoskeletal dynamics. They orchestrate specific changes at each spatiotemporal region within the β-cell by coordinating with signal transducers, guanine nucleotide exchange factors (GEFs), GTPase-activating factors (GAPs), and their effectors. The Arf family of small GTPases is involved in vesicular trafficking (exocytosis and endocytosis) and actin cytoskeletal dynamics. Rab-GTPases regulate pre-exocytotic and late endocytic membrane trafficking events in β-cells. Several additional functions for small GTPases include regulating transcription factor activity and mitochondrial dynamics. Importantly, defects in several of these GTPases have been found associated with type 2 diabetes (T2D) etiology. The purpose of this review is to systematically denote the identities and molecular mechanistic steps in the glucose-stimulated insulin secretion pathway that leads to the normal release of insulin. We will also note newly identified defects in these GTPases and their corresponding regulatory factors (e.g., GDP dissociation inhibitors (GDIs), GEFs, and GAPs) in the pancreatic β-cells, which contribute to the dysregulation of metabolism and the development of T2D. Full article

Vesicle trafficking plays an important role in delivering a diverse range of cargoes between different membranous systems in eukaryotes. It is well documented that the brefeldin A (BFA)-inhibited guanine nucleotide exchange factor (GEF), named BIG, regulates vesicle budding at the trans-Golgi network (TGN) and recycling endosomes through activating the ADP-ribosylation factor (ARFs). Among the five BIGs in Arabidopsis, BIG5 is characterized to mediate ARF-dependent trafficking at the plasma membrane or endosomes while the members from BIG1 to BIG4 (BIG1-BIG4) at the TGN in the secretory pathway. However, evidence is increasing to suggest that BIG5 can function redundantly with BIG1-BIG4 to regulate vesicular trafficking in response to various intra- and extra-cellular stimuli. In this study, our genetic analysis showed that BIG5 played an overlapping role at least with BIG3 in cell proliferation. To elucidate molecular mechanisms underlying the BIG5- and BIG3-regulated biological processes, we examined the effect of BIGs on expression patterns of the two transmembrane proteins, PINFORMED 2 (PIN2) epically localized in root epidermal cells and the regulator of G protein signaling 1 (RGS1) localized in the plasma membrane. Our data showed that the PIN2 polar distribution was slightly reduced in big3 big5 in the absence of BFA, and it was significantly reduced by the treatment of 0.1 µM BFA in big3 big5. Further analysis revealed that BFA bodies derived from the plasma membrane were only observed in wild type (WT), big3 and big5 cells, but not in the big3 big5 cells. These results indicate that BIG5 and BIG3 are functionally redundant in the endosome recycling pathway from the plasma membrane to TGN. On the other hand, the single BIG3 or BIG5 mutation had no effect on the plasma membrane expression of RGS1, whereas the double mutations in BIG3 and BIG5 led to a significant amount of RGS1 retained in the vesicle, indicating that BIG3 and BIG5 act redundantly in mediating protein trafficking. Furthermore, transmission electron microscopy assays showed that Golgi ultrastructure in big3 big5 cells was abnormal and similar to that in BFA-treated WT cells. Taken together, our data provide several new lines of evidence supporting that BIGs play a redundant role in vesicular trafficking and probably also in maintaining the Golgi structural integrity in Arabidopsis. Full article

Monomeric GTPases, which belong to the Ras superfamily, are small proteins involved in many biological processes. They are fine-tuned regulated by guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs). Several families have been identified in organisms from different kingdoms. Overall, the most studied families are Ras, Rho, Rab, Ran, Arf, and Miro. Recently, a new family named Big Ras GTPases was reported. As a general rule, the proteins of all families have five characteristic motifs (G1–G5), and some specific features for each family have been described. Here, we present an exhaustive analysis of these small GTPase families in fungi, using 56 different genomes belonging to different phyla. For this purpose, we used distinct approaches such as phylogenetics and sequences analysis. The main functions described for monomeric GTPases in fungi include morphogenesis, secondary metabolism, vesicle trafficking, and virulence, which are discussed here. Their participation during fungus–plant interactions is reviewed as well. Full article

Golgi trafficking depends on the small GTPase Arf1 which, upon activation, drives the assembly of different coats onto budding vesicles. Two related types of guanine nucleotide exchange factors (GEFs) activate Arf1 at different Golgi sites. In yeast, Gea1 in the cis-Golgi and Gea2 in the medial-Golgi activate Arf1 to form COPI-­coated vesicles for retrograde cargo sorting, whereas Sec7 generates clathrin/adaptor­-coated vesicles at the trans-Golgi network (TGN) for forward cargo transport. A central question is how the same activated Arf1 protein manages to assemble different coats depending on the donor Golgi compartment. A previous study has postulated that the interaction between Gea1 and COPI would channel Arf1 activation for COPI vesicle budding. Here, we found that the p24 complex, a major COPI vesicle cargo, promotes the binding of Gea1 with COPI by increasing the COPI association to the membrane independently of Arf1 activation. Furthermore, the p24 complex also facilitates the interaction of Arf1 with its COPI effector. Therefore, our study supports a mechanism by which the p24 complex contributes to program Arf1 activation by Gea1 for selective COPI coat assembly at the cis-Golgi compartment. Full article

The cuticle is the outermost layer of plant aerial tissue that interacts with the environment and protects plants against water loss and various biotic and abiotic stresses. ADP ribosylation factor guanine nucleotide exchange factor proteins (ARF-GEFs) are key components of the vesicle trafficking system. Our study discovers that AtMIN7, an Arabidopsis ARF-GEF, is critical for cuticle formation and related leaf surface defense against the bacterial pathogen Pseudomonas syringae pathovar tomato (Pto). Our transmission electron microscopy and scanning electron microscopy studies indicate that the atmin7 mutant leaves have a thinner cuticular layer, defective stomata structure, and impaired cuticle ledge of stomata compared to the leaves of wild type plants. GC–MS analysis further revealed that the amount of cutin monomers was significantly reduced in atmin7 mutant plants. Furthermore, the exogenous application of either of three plant hormones—salicylic acid, jasmonic acid, or abscisic acid—enhanced the cuticle formation in atmin7 mutant leaves and the related defense responses to the bacterial Pto infection. Thus, transport of cutin-related components by AtMIN7 may contribute to its impact on cuticle formation and related defense function. Full article

The small GTPase ADP-ribosylation factor 6 (Arf6) anchors at the plasma membrane to orchestrate key functions, such as membrane trafficking and regulating cortical actin cytoskeleton rearrangement. A number of studies have identified key players that interact with Arf6 to regulate actin dynamics in diverse cell processes, yet it is still unknown whether Arf6 can directly signal to the wave regulatory complex to mediate actin assembly. By reconstituting actin dynamics on supported lipid bilayers, we found that Arf6 in co-ordination with Rac1(Ras-related C3 botulinum toxin substrate 1) can directly trigger actin polymerization by recruiting wave regulatory complex components. Interestingly, we demonstrated that Arf6 triggers actin assembly at the membrane directly without recruiting the Arf guanine nucleotide exchange factor (GEF) ARNO (ARF nucleotide-binding site opener), which is able to activate Arf1 to enable WRC-dependent actin assembly. Furthermore, using labelled E. coli, we demonstrated that actin assembly by Arf6 also contributes towards efficient phagocytosis in THP-1 macrophages. Taken together, this study reveals a mechanism for Arf6-driven actin polymerization. Full article

ADP-ribosylation factor-guanine nucleotide exchange factors (ARF-GEFs) act as key regulators of vesicle trafficking in all eukaryotes. In Arabidopsis, there are eight ARF-GEFs, including three members of the GBF1 subfamily and five members of the BIG subfamily. These ARF-GEFs have different subcellular localizations and regulate different trafficking pathways. Until now, the roles of these BIG-subfamily ARF-GEFs have not been fully revealed. Here, analysis of the BIGs expression patterns showed that BIG3 and BIG5 have similar expression patterns. big5-1 displayed a dwarf growth and big3-1 big5-1 double mutant showed more severe defects, indicating functional redundancy between BIG3 and BIG5. Moreover, both big5-1 and big3-1 big5-1 exhibited a reduced sensitivity to Brassinosteroid (BR) treatment. Brefeldin A (BFA)-induced BR receptor Brassinosteroid insensitive 1 (BRI1) aggregation was reduced in big5-1 mutant, indicating that the action of BIG5 is required for BRI1 recycling. Furthermore, BR-induced dephosphorylation of transcription factor BZR1 was decreased in big3-1 big5-1 double mutants. The introduction of the gain-of-function of BZR1 mutant BZR1-1D in big3-1 big5-1 mutants can partially rescue the big3-1 big5-1 growth defects. Our findings revealed that BIG5 functions redundantly with BIG3 in plant growth and gravitropism, and BIG5 participates in BR signal transduction pathway through regulating BRI1 trafficking. Full article