MAPK3-MYB36-ARF1 module regulates the tanshinone formation in Salvia miltiorrhiza.

Salvia miltiorrhiza, known as Danshen, is a traditional Chinese medicinal plant with significant cardiovascular benefits, attributed to its secondary metabolites, particularly tanshinones. Despite their medicinal value, tanshinones occur in low natural abundance, necessitating research to increase their content. This study explores the role of the ARF transcription factor (SmARF1) in tanshinone accumulation in Danshen. Overexpressing SmARF1 in hairy roots significantly increased tanshinone levels. EMSA and Dual-LUC assays revealed that SmMYB36, a transcription factor interacting with SmMAPK3, binds to and regulates the SmARF1 promoter. SmMYB36 alone inhibited the expression of SmARF1 gene, while its interaction with SmMAPK3 enhanced SmARF1 promoter activity. This MAPK3-MYB36-ARF1 module elucidates a complex regulatory mechanism for tanshinone biosynthesis, offering insights for targeted enhancement of tanshinone content through advanced biotechnological approaches.

Variations associated with neurodevelopmental disorders affect ARF1 function and cortical development.

ADP-ribosylation factors (ARFs) are a family of small GTPases that regulate vesicle trafficking and actin dynamics in cells. Recent genetic analyses have revealed associations between variations in ARF genes and neurodevelopmental disorders, although their pathophysiological significance remains unclear. In this study, we conducted biochemical, cell biological, and in vivo analyses of ARF1 variants linked to neurodevelopmental disorders. The mant-GDP dissociation assay revealed that ARF1-p.R19C, -p.F51L, -p.R99C, and -p.R99H exhibit higher GDP/GTP exchange activity compared to ARF1 wild type (WT). The GTPase-activating protein (GAP) increased the GTPase activity of WT, p.R19C, p.Y35H, p.F51L, p.P131L, and p.P131R, but not of p.Y35D, p.T48I, p.R99C, and p.R99H. The transient expression of p.R99C, p.R99H, and p.K127E in mammalian cells resulted in the disruption of the Golgi apparatus. In utero electroporation-mediated gene transfer into the cortical neurons of embryonic mice demonstrated that p.R99C, p.R99H, and p.K127E cause a migration defect. Expression of these variants resulted in the expansion of the Golgi apparatus in migrating cortical neurons. These findings suggest that the ARF1 variants linked to neurodevelopmental disorders, specifically p.R99C, p.R99H, and p.K127E, disrupt the structure of the Golgi apparatus, thereby leading to a developmental defect of cortical neurons.

THUMPD3-AS1 inhibits ovarian cancer cell apoptosis through the miR-320d/ARF1 axis.

Ovarian cancer is one of the most common gynecologic malignancies that has a poor prognosis. THUMPD3-AS1 is an oncogenic long noncoding RNA (lncRNA) in several cancers. Moreover, miR-320d is downregulated and inhibited proliferation in ovarian cancer cells, whereas ARF1 was upregulated and promoted the malignant progression in epithelial ovarian cancer. Nevertheless, the role of THUMPD3-AS1 in ovarian cancer and the underlying mechanism has yet to be elucidated. Human normal ovarian epithelial cells (IOSE80) and ovarian cancer cell lines (CAVO3, A2780, SKOV3, OVCAR3, and HEY) were adopted for in vitro experiments. The functional roles of THUMPD3-AS1 in cell viability and apoptosis were determined using CCK-8, flow cytometry, and TUNEL assays. Western blot was performed to assess the protein levels of ARF1, Bax, Bcl-2, and caspase 3, whereas RT-qPCR was applied to measure ARF1 mRNA, THUMPD3-AS1, and miR-320d levels. The targeting relationship between miR-320d and THUMPD3-AS1 or ARF1 was validated with dual luciferase assay. THUMPD3-AS1 and ARF1 were highly expressed in ovarian cancer cells, whereas miR-320d level was lowly expressed. THUMPD3-AS1 knockdown was able to repress cell viability and accelerate apoptosis of OVCAR3 and SKOV3 cells. Also, THUMPD3-AS1 acted as a sponge of miR-320d, preventing the degradation of ARF1. MiR-320d downregulation reversed the tumor suppressive function induced by THUMPD3-AS1 depletion. Additionally, miR-320d overexpression inhibited ovarian cancer cell viability and accelerated apoptosis, which was overturned by overexpression of ARF1. THUMPD3-AS1 inhibited ovarian cancer cell apoptosis by modulation of miR-320d/ARF1 axis. The discoveries might provide a prospective target for ovarian cancer treatment.

ADP-ribosylation factor 1 (ARF1) protein interacts with elicitor PvNLP7 from Plasmopara viticola to mediate PvNLP7-triggered immunity.

Revealing the effector-host molecular interactions is crucial for understanding the host immunity against Plasmopara viticola and devising innovative disease management strategies. As a pathogenic oomycete causing grapevine downy mildew, Plasmopara viticola employs various effectors to manipulate the defense systems of host plants. One of these P. viticola derived effectors is necrosis- and ethylene-inducing peptide 1 (Nep1) -like protein (PvNLP7), which has been known to elicit cell death and immune responses in plants. However, the underlying molecular mechanisms remain obscure, prompting the focus of this study. Through yeast two-hybrid screening, we have identified the Vitis rotundifolia ADP-ribosylation factor (VrARF1) as a host interactor of PvNLP7. This interaction is corroborated through bimolecular fluorescence complementation (BiFC) and co-immunoprecipitation (Co-IP) assays. Heterologous expression of VrARF1 in Nicotiana benthamiana verifies its accumulation in both the cytoplasm and nucleus, and induction of cell death. Moreover, the VrARF1 gene is strongly induced during early P. viticola infection and upon PvNLP7 transient expression. Overexpression of the VrARF1 gene in grapevine and N. benthamiana enhances resistance to P. viticola and Phytophthora capsici, respectively, via induction of defense related genes PR1 and PR2. Conversely, virus-induced gene silencing (VIGS) of NbARF1 in N. benthamiana, homologous to VrARF1, markedly attenuates PvNLP7-triggered cell death and reduces the expression of four PTI marker genes (PTI5, Acre31, WRKY7 and Cyp71D20) and two defense related genes (PR1 and PR2), rendering plants transiently transformed with PvNLP7 more susceptible to oomycete P. capsici. These findings highlight the role of ARF1 in mediating PvNLP7-induced immunity and indicate its potential as a target for engineering disease-resistant transgenic plants against oomycete pathogens.

dAsap regulates cellular protrusions via an Arf6-dependent actin regulatory pathway in S2R+ cells.

Membrane protrusions are fundamental to cellular functions like migration, adhesion, and communication and depend upon dynamic reorganization of the cytoskeleton. GAP-dependent GTP hydrolysis of Arf proteins regulates actin-dependent membrane remodeling. Here, we show that dAsap regulates membrane protrusions in S2R+ cells by a mechanism that critically relies on its ArfGAP domain and relocalization of actin regulators, SCAR, and Ena. While our data reinforce the preference of dAsap for Arf1 GTP hydrolysis in vitro, we demonstrate that induction of membrane protrusions in S2R+ cells depends on Arf6 inactivation. This study furthers our understanding of how dAsap-dependent GTP hydrolysis maintains a balance between active and inactive states of Arf6 to regulate cell shape.

Arf1 GTPase Regulates Golgi-Dependent G2/M Transition and Spindle Organization in Oocyte Meiosis.

ADP-ribosylation factor 1 (Arf1) is a small GTPase belonging to the Arf family. As a molecular switch, Arf1 is found to regulate retrograde and intra-Golgi transport, plasma membrane signaling, and organelle function during mitosis. This study aimed to explore the noncanonical roles of Arf1 in cell cycle regulation and cytoskeleton dynamics in meiosis with a mouse oocyte model. Arf1 accumulated in microtubules during oocyte meiosis, and the depletion of Arf1 led to the failure of polar body extrusion. Unlike mitosis, it finds that Arf1 affected Myt1 activity for cyclin B1/CDK1-based G2/M transition, which disturbed oocyte meiotic resumption. Besides, Arf1 modulated GM130 for the dynamic changes in the Golgi apparatus and Rab35-based vesicle transport during meiosis. Moreover, Arf1 is associated with Ran GTPase for TPX2 expression, further regulating the Aurora A-polo-like kinase 1 pathway for meiotic spindle assembly and microtubule stability in oocytes. Further, exogenous Arf1 mRNA supplementation can significantly rescue these defects. In conclusion, results reported the noncanonical functions of Arf1 in G2/M transition and meiotic spindle organization in mouse oocytes.

YUCCA2 (YUC2)-Mediated 3-Indoleacetic Acid (IAA) Biosynthesis Regulates Chloroplast RNA Editing by Relieving the Auxin Response Factor 1 (ARF1)-Dependent Inhibition of Editing Factors in Arabidopsis thaliana.

Although recent research progress on the abundant C-to-U RNA editing events in plant chloroplasts and mitochondria has uncovered many recognition factors and their molecular mechanisms, the intrinsic regulation of RNA editing within plants remains largely unknown. This study aimed to establish a regulatory relationship in Arabidopsis between the plant hormone auxin and chloroplast RNA editing. We first analyzed auxin response elements (AuxREs) present within promoters of chloroplast editing factors reported to date. We found that each has more than one AuxRE, suggesting a potential regulatory role of auxin in their expression. Further investigation unveiled that the depletion of auxin synthesis gene YUC2 reduces the expression of several editing factors. However, in yuc2 mutants, only the expression of CRR4, DYW1, ISE2, and ECD1 editing factors and the editing efficiency of their corresponding editing sites, ndhD-2 and rps14-149, were simultaneously suppressed. In addition, exogenous IAA and the overexpression of YUC2 enhanced the expression of these editing factors and the editing efficiency at the ndhD-2 and rps14-149 sites. These results suggested a direct effect of auxin upon the editing of the ndhD-2 and rps14-149 sites through the modulation of the expression of the editing factors. We further demonstrated that ARF1, a downstream transcription factor in the auxin-signaling pathway, could directly bind to and inactivate the promoters of CRR4, DYW1, and ISE2 in a dual-luciferase reporter system, thereby inhibiting their expression. Moreover, the overexpression of ARF1 in Arabidopsis significantly reduced the expression of the three editing factors and the editing efficiency at the ndhD-2 and rps14-149 sites. These data suggest that YUC2-mediated auxin biosynthesis governs the RNA-editing process through the ARF1-dependent signal transduction pathway.

Extracellular Glutamine Promotes Intestinal Porcine Epithelial Cell Proliferation via Arf1-mTORC1 Pathway Independently of Rag GTPases.

Glutamine (Gln) is the major energy source of intestinal porcine epithelial cells (IPEC-J2 cells) and plays a critical role in the nutritional physiological function of the intestine. However, the underlying mechanism requires further investigation. Here, the Gln-sensing pathway in IPEC-J2 cells was investigated. The results showed that Gln increased the cell proliferation. Subsequently, an analysis of the phosphorylated proteome revealed that Gln markedly upregulated ribosomal protein S6 (RPS6) phosphorylation at serine 235/236, suggesting that Gln activated the mTORC1 pathway. mTOR inhibition revealed that Gln promotes cell proliferation through the mTORC1 pathway. Similarly, blocking ADP-ribosylation factor 1 (Arf1) activity indicated that Gln-induced mTORC1 activation promoted cell proliferation in an Arf1-dependent manner. Additionally, the RagA/B pathway did not participate in Gln-induced mTORC1 activation. Collectively, these findings suggest that Gln-induced mTORC1 activation promotes IPEC-J2 cell proliferation via Arf1, not Rag GTPases. These results broaden our understanding of functional-cell-sensing amino acids, particularly Gln, that are regulated by mTORC1.

Golgi-dependent reactivation and regeneration of Drosophila quiescent neural stem cells.

The ability of stem cells to switch between quiescent and proliferative states is crucial for maintaining tissue homeostasis and regeneration. In Drosophila, quiescent neural stem cells (qNSCs) extend a primary protrusion, a hallmark of qNSCs. Here, we have found that qNSC protrusions can be regenerated upon injury. This regeneration process relies on the Golgi apparatus that acts as the major acentrosomal microtubule-organizing center in qNSCs. A Golgi-resident GTPase Arf1 and its guanine nucleotide exchange factor Sec71 promote NSC reactivation and regeneration via the regulation of microtubule growth. Arf1 physically associates with its new effector mini spindles (Msps)/XMAP215, a microtubule polymerase. Finally, Arf1 functions upstream of Msps to target the cell adhesion molecule E-cadherin to NSC-neuropil contact sites during NSC reactivation. Our findings have established Drosophila qNSCs as a regeneration model and identified Arf1/Sec71-Msps pathway in the regulation of microtubule growth and NSC reactivation.

ARF1 maintains intestinal homeostasis by modulating gut microbiota and stem cell function.

AIMS: The small GTPase protein ARF1 has been shown to be involved in the lipolysis pathway and to selectively kill stem cells in Drosophila melanogaster. However, the role of ARF1 in mammalian intestinal homeostasis remains elusive. This study aimed to explore the role of ARF1 in intestinal epithelial cells (IECs) and reveal the possible mechanism. MATERIALS AND METHODS: IEC-specific ARF1 deletion mouse model was used to evaluate the role of ARF1 in intestine. Immunohistochemistry and immunofluorescence analyses were performed to detect specific cell type markers, and intestinal organoids were cultured to assess intestinal stem cell (ISC) proliferation and differentiation. Fluorescence in situ hybridization, 16S rRNA-seq analysis, and antibiotic treatments were conducted to elucidate the role of gut microbes in ARF1-mediated intestinal function and the underlying mechanism. Colitis was induced in control and ARF1-deficient mice by dextran sulfate sodium (DSS). RNA-seq was performed to elucidate the transcriptomic changes after ARF1 deletion. KEY FINDINGS: ARF1 was essential for ISC proliferation and differentiation. Loss of ARF1 increased susceptibility to DSS-induced colitis and gut microbial dysbiosis. Gut microbiota depletion by antibiotics could rescue the intestinal abnormalities to a certain extent. Furthermore, RNA-seq analysis revealed alterations in multiple metabolic pathways. SIGNIFICANCE: This work is the first to elucidate the essential role of ARF1 in regulating gut homeostasis, and provides novel insights into the pathogenesis of intestinal diseases and potential therapeutic targets.

Pooled analysis of frontal lobe transcriptomic data identifies key mitophagy gene changes in Alzheimer's disease brain.

Background: The growing prevalence of Alzheimer's disease (AD) is becoming a global health challenge without effective treatments. Defective mitochondrial function and mitophagy have recently been suggested as etiological factors in AD, in association with abnormalities in components of the autophagic machinery like lysosomes and phagosomes. Several large transcriptomic studies have been performed on different brain regions from AD and healthy patients, and their data represent a vast source of important information that can be utilized to understand this condition. However, large integration analyses of these publicly available data, such as AD RNA-Seq data, are still missing. In addition, large-scale focused analysis on mitophagy, which seems to be relevant for the aetiology of the disease, has not yet been performed. Methods: In this study, publicly available raw RNA-Seq data generated from healthy control and sporadic AD post-mortem human samples of the brain frontal lobe were collected and integrated. Sex-specific differential expression analysis was performed on the combined data set after batch effect correction. From the resulting set of differentially expressed genes, candidate mitophagy-related genes were identified based on their known functional roles in mitophagy, the lysosome, or the phagosome, followed by Protein-Protein Interaction (PPI) and microRNA-mRNA network analysis. The expression changes of candidate genes were further validated in human skin fibroblast and induced pluripotent stem cells (iPSCs)-derived cortical neurons from AD patients and matching healthy controls. Results: From a large dataset (AD: 589; control: 246) based on three different datasets (i.e., ROSMAP, MSBB, & GSE110731), we identified 299 candidate mitophagy-related differentially expressed genes (DEG) in sporadic AD patients (male: 195, female: 188). Among these, the AAA ATPase VCP, the GTPase ARF1, the autophagic vesicle forming protein GABARAPL1 and the cytoskeleton protein actin beta ACTB were selected based on network degrees and existing literature. Changes in their expression were further validated in AD-relevant human in vitro models, which confirmed their down-regulation in AD conditions. Conclusion: Through the joint analysis of multiple publicly available data sets, we identify four differentially expressed key mitophagy-related genes potentially relevant for the pathogenesis of sporadic AD. Changes in expression of these four genes were validated using two AD-relevant human in vitro models, primary human fibroblasts and iPSC-derived neurons. Our results provide foundation for further investigation of these genes as potential biomarkers or disease-modifying pharmacological targets.

The crescent-like Golgi ribbon is shaped by the Ajuba/PRMT5/Aurora-A complex-modified HURP.

BACKGROUND: Golgi apparatus (GA) is assembled as a crescent-like ribbon in mammalian cells under immunofluorescence microscope without knowing the shaping mechanisms. It is estimated that roughly 1/5 of the genes encoding kinases or phosphatases in human genome participate in the assembly of Golgi ribbon, reflecting protein modifications play major roles in building Golgi ribbon. METHODS: To explore how Golgi ribbon is shaped as a crescent-like structure under the guidance of protein modifications, we identified a protein complex containing the scaffold proteins Ajuba, two known GA regulators including the protein kinase Aurora-A and the protein arginine methyltransferase PRMT5, and the common substrate of Aurora-A and PRMT5, HURP. Mutual modifications and activation of PRMT5 and Aurora-A in the complex leads to methylation and in turn phosphorylation of HURP, thereby producing HURP p725. The HURP p725 localizes to GA vicinity and its distribution pattern looks like GA morphology. Correlation study of the HURP p725 statuses and GA structure, site-directed mutagenesis and knockdown-rescue experiments were employed to identify the modified HURP as a key regulator assembling GA as a crescent ribbon. RESULTS: The cells containing no or extended distribution of HURP p725 have dispersed GA membranes or longer GA. Knockdown of HURP fragmentized GA and HURP wild type could, while its phosphorylation deficiency mutant 725A could not, restore crescent Golgi ribbon in HURP depleted cells, collectively indicating a crescent GA-constructing activity of HURP p725. HURP p725 is transported, by GA membrane-associated ARF1, Dynein and its cargo adaptor Golgin-160, to cell center where HURP p725 forms crescent fibers, binds and stabilizes Golgi assembly factors (GAFs) including TRIP11, GRASP65 and GM130, thereby dictating the formation of crescent Golgi ribbon at nuclear periphery. CONCLUSIONS: The Ajuba/PRMT5/Aurora-A complex integrates the signals of protein methylation and phosphorylation to HURP, and the HURP p725 organizes GA by stabilizing and recruiting GAFs to its crescent-like structure, therefore shaping GA as a crescent ribbon. Therefore, the HURP p725 fiber serves a template to construct GA according to its shape. Video Abstract.

Kinetics of Arf1 inactivation regulates Golgi organisation and function in non-adherent fibroblasts.

Arf1 belongs to the Arf family of small GTPases that localise at the Golgi and plasma membrane. Active Arf1 plays a crucial role in regulating Golgi organisation and function. In mouse fibroblasts, loss of adhesion triggers a consistent drop (∼50%) in Arf1 activation that causes the Golgi to disorganise but not fragment. In suspended cells, the trans-Golgi (GalTase) disperses more prominently than cis-Golgi (Man II), accompanied by increased active Arf1 (detected using GFP-ABD: ARHGAP10 Arf1 binding domain) associated with the cis-Golgi compartment. Re-adhesion restores Arf1 activation at the trans-Golgi as it reorganises. Arf1 activation at the Golgi is regulated by Arf1 Guanine nucleotide exchange factors (GEFs), GBF1, and BIG1/2. In non-adherent fibroblasts, the cis-medial Golgi provides a unique setting to test and understand the role GEF-mediated Arf1 activation has in regulating Golgi organisation. Labelled with Man II-GFP, non-adherent fibroblasts treated with increasing concentrations of Brefeldin-A (BFA) (which inhibits BIG1/2 and GBF1) or Golgicide A (GCA) (which inhibits GBF1 only) comparably decrease active Arf1 levels. They, however, cause a concentration-dependent increase in cis-medial Golgi fragmentation and fusion with the endoplasmic reticulum (ER). Using selected BFA and GCA concentrations, we find a change in the kinetics of Arf1 inactivation could mediate this by regulating cis-medial Golgi localisation of GBF1. On loss of adhesion, a ∼50% drop in Arf1 activation over 120 min causes the Golgi to disorganise. The kinetics of this drop, when altered by BFA or GCA treatment causes a similar decline in Arf1 activation but over 10 min. This causes the Golgi to now fragment which affects cell surface glycosylation and re-adherent cell spreading. Using non-adherent fibroblasts this study reveals the kinetics of Arf1 inactivation, with active Arf1 levels, to be vital for Golgi organisation and function.

Profiling of phytohormone-specific microRNAs and characterization of the miR160-ARF1 module involved in glandular trichome development and artemisinin biosynthesis in Artemisia annua.

MicroRNAs (miRNAs) play crucial roles in plant development and secondary metabolism through different modes of sequence-specific interaction with their targets. Artemisinin biosynthesis is extensively regulated by phytohormones. However, the function of phytohormone-responsive miRNAs in artemisinin biosynthesis remains enigmatic. Thus, we combined the analysis of transcriptomics, small RNAs, and the degradome to generate a comprehensive resource for identifying key miRNA-target circuits involved in the phytohormone-induced process of artemisinin biosynthesis in Artemisia annua. In total, 151 conserved and 52 novel miRNAs and their 4132 targets were determined. Based on the differential expression analysis, miR160 was selected as a potential miRNA involved in artemisinin synthesis. Overexpressing MIR160 significantly impaired glandular trichome formation and suppressed artemisinin biosynthesis in A. annua, while repressing its expression resulted in the opposite effect, indicating that miR160 negatively regulates glandular trichome development and artemisinin biosynthesis. RNA ligase-mediated 5' RACE and transient transformation assays showed that miR160 mediates the RNA cleavage of Auxin Response Factor 1 (ARF1) in A. annua. Furthermore, ARF1 was shown to increase artemisinin synthesis by activating AaDBR2 expression. Taken together, our results reveal the intrinsic link between the miR160-ARF1 module and artemisinin biosynthesis, and may expedite the innovation of metabolic engineering approaches for high and stable production of artemisinin in the future.

Targeting ARF1-IQGAP1 interaction to suppress colorectal cancer metastasis and vemurafenib resistance.

INTRODUCTION: Acquired resistance to BRAF inhibitor vemurafenib is frequently observed in metastatic colorectal cancer (CRC), and it is a thorny issue that results in treatment failure. As adaptive responses for vemurafenib treatment, a series of cellular bypasses are response for the adaptive feedback reactivation of ERK signaling, which warrant further investigation. OBJECTIVES: We identified ARF1 (ADP-ribosylation factor 1) as a novel regulator of both vemurafenib resistance and cancer metastasis, its molecular mechanism and potential inhibitor were investigated in this study. METHODS: DIA-based quantitative proteomics and RNA-seq were performed to systematic analyze the profiling of vemurafenib-resistant RKO cells (RKO-VR) and highly invasive RKO cells (RKO-I8), respectively. Co­immunoprecipitation assay was performed to detect the interaction of ARF1 and IQGAP1 (IQ-domain GTPase activating protein 1). An ELISA-based drug screen system on FDA-approved drug library was established to screen the compounds against the interaction of ARF1-IQGAP1.The biological functions of ARF1 and LY2835219 were determined by transwell, western blotting, Annexin V-FITC/PI staining and in vivo experimental metastasis assays. RESULTS: We found that ARF1 strongly interacted with IQGAP1 to activate ERK signaling in VR and I8 CRC cells. Deletion of IQGAP1 or inactivation of ARF1 (ARF-T48S) restored the invasive ability induced by ARF1. As ARF1-IQGAP1 interaction is essential for ERK activation, we screened LY2835219 as novel inhibitor of ARF1-IQGAP1 interaction, which inactivated ERK signaling and suppressed CRC metastasis and vemurafenib-resistance in vitro and in vivo with no observed side effect. Furthermore, LY2835219 in combined treatment with vemurafenib exerted significantly inhibitory effect on ARF1-mediated cancer metastasis than used independently. CONCLUSION: This study uncovers that ARF1-IQGAP1 interaction-mediated ERK signaling reactivation is critical for vemurafenib resistance and cancer metastasis, and that LY2835219 is a promising therapeutic agent for CRC both as a single agent and in combination with vemurafenib.

Molecular mechanisms of PI4K regulation and their involvement in viral replication.

Lipid phosphoinositides are master signaling molecules in eukaryotic cells and key markers of organelle identity. Because of these important roles, the kinases and phosphatases that generate phosphoinositides must be tightly regulated. Viruses can manipulate this regulation, with the Type III phosphatidylinositol 4-kinases (PI4KA and PI4KB) being hijacked by many RNA viruses to mediate their intracellular replication through the formation of phosphatidylinositol 4-phosphate (PI4P)-enriched replication organelles (ROs). Different viruses have evolved unique approaches toward activating PI4K enzymes to form ROs, through both direct binding of PI4Ks and modulation of PI4K accessory proteins. This review will focus on PI4KA and PI4KB and discuss their roles in signaling, functions in membrane trafficking and manipulation by viruses. Our focus will be the molecular basis for how PI4KA and PI4KB are activated by both protein-binding partners and post-translational modifications, with an emphasis on understanding the different molecular mechanisms viruses have evolved to usurp PI4Ks. We will also discuss the chemical tools available to study the role of PI4Ks in viral infection.

The olfactory receptor OR51E2 activates ERK1/2 through the Golgi-localized Gßγ-PI3Kγ-ARF1 pathway in prostate cancer cells.

The olfactory receptor OR51E2 is ectopically expressed in prostate tissues and regulates prostate cancer progression, but its function and regulation in oncogenic mitogen-activate protein kinase (MAPK) activation are poorly defined. Here we demonstrate that ß-ionone, an OR51E2 agonist, dose-dependently activates extracellular signal-regulated kinases 1 and 2 (ERK1/2) in prostate cancer cells, with an EC50 value of approximate 20 µM and an efficiency comparable to other receptor agonists. We also find that CRISPR-Cas9-mediated knockout of Golgi-translocating Gγ9 subunit, phosphoinositide 3-kinase γ (PI3Kγ) and the small GTPase ADP-ribosylation factor 1 (ARF1), as well as pharmacological inhibition of Gßγ, PI3Kγ and Golgi-localized ARF1, each abolishes ERK1/2 activation by ß-ionone. We further show that ß-ionone significantly promotes ARF1 translocation to the Golgi and activates ARF1 that can be inhibited by Gγ9 and PI3Kγ depletion. Collectively, our data demonstrate that OR51E2 activates ERK1/2 through the Gßγ-PI3Kγ-ARF1 pathway that occurs spatially at the Golgi, and also provide important insights into MAPK hyper-activation in prostate cancer.

Blocking the cytohesin-2/ARF1 axis by SecinH3 ameliorates osteoclast-induced bone loss via attenuating JNK-mediated IRE1 endoribonuclease activity.

cytohesin-2 is a guanine nucleotide exchange factor to activate ARF1 and ARF6, which are involved in various biological processes, including signal transduction, cell differentiation, cell structure organization, and survival. Nevertheless, there is a lack of evidence revealing the role of cytohesin-2 in osteoclast differentiation and in the development of osteoporosis. In this study, we find cytohesin-2 and ARF1 positively regulate osteoclast differentiation and function. Blocking the cytohesin-2 /ARF1 axis with SecinH3 or by genetic silencing of cytohesin-2 inhibits osteoclast formation and function in vitro. In vivo treatment with SecinH3 ameliorates ovariectomy-induced osteoporosis. Mechanistically, RNA-sequencing combined with molecular biological methodologies reveal that the regulatory function of cythohesin-2/ARF1 axis in osteoclast differentiation is mainly dependent on activating the JNK pathway. Further, in addition to the common viewpoint that JNK is activated by IRE1 via its kinase activity, we found that JNK can act upstream and regulate the endoribonuclease activity of IRE1 to promote XBP1 splicing. Both SecinH3 and silencing of cytohesin-2 inhibit JNK activation and IRE1 endoribonuclease activity, leading to the suppression of osteoclast differentiation. Taken together, our findings add new insights into the regulation between JNK and IRE1, and reveal that inhibiting the cytohesin-2/ARF1/JNK/IRE1 axis might represent a potential new strategy for the treatment of post-menopause osteoporosis.

Structural basis for activation of Arf1 at the Golgi complex.

The Golgi complex is the central sorting station of the eukaryotic secretory pathway. Traffic through the Golgi requires activation of Arf guanosine triphosphatases that orchestrate cargo sorting and vesicle formation by recruiting an array of effector proteins. Arf activation and Golgi membrane association is controlled by large guanine nucleotide exchange factors (GEFs) possessing multiple conserved regulatory domains. Here we present cryoelectron microscopy (cryoEM) structures of full-length Gea2, the yeast paralog of the human Arf-GEF GBF1, that reveal the organization of these regulatory domains and explain how Gea2 binds to the Golgi membrane surface. We find that the GEF domain adopts two different conformations compatible with different stages of the Arf activation reaction. The structure of a Gea2-Arf1 activation intermediate suggests that the movement of the GEF domain primes Arf1 for membrane insertion upon guanosine triphosphate binding. We propose that conformational switching of Gea2 during the nucleotide exchange reaction promotes membrane insertion of Arf1.

Hepatocellular carcinoma-associated antigen 59 and ADP-ribosylation factor 1 with poly (lactic-co-glycolic acid): A promising candidate as nanovaccine against haemonchosis.

Haemonchus contortus (H. contortus) ADP-ribosylation factor 1 (Hc-ARF1) and Hepatocellular carcinoma-associated antigen 59 (Hc-HCA59) are recognized to largely regulate the immune responses of host cells. However, studies about the protective efficacy of the two molecules are poorly unknown. In this research, combinations of recombinant Hc-HCA59 (rHc-HCA59) and Hc-ARF1 (rHc-ARF1) proteins were amalgamated with poly (lactic-co-glycolic acid) (PLGA) nanoparticles adjuvant in order to investigate their protection potential against H. contortus in goats. The results demonstrated that the levels of IgG, IgA, IgE, and IL-4 were noticeably enhanced in the rHc-HCA59 and rHc-ARF1 (rHc-HCA59+rHc-ARF1) group before H. contortus third-stage larvae (L3) challenge. After the L3 challenge, the levels of IL-17, IL-9, and TGF-ß were considerably upregulated in the rHc-HCA59+rHc-ARF1 group. In the meantime, the abomasal worm burdens and the fecal eggs were reduced by 63.2% and 69.4% respectively in the rHc-HCA59+rHc-ARF1 group. According to the studies, PLGA nanoparticles immobilized with rHc-HCA59 and rHc-ARF1 proteins conferred partial protection and were expected to be a potential candidate for developing nano vaccines to combat goat haemonchosis.