Point mutations in Arf1 reveal cooperative effects of the N-terminal extension and myristate for GTPase-activating protein catalytic activity.

The ADP-ribosylation factors (Arfs) constitute a family of small GTPases within the Ras superfamily, with a distinguishing structural feature of a hypervariable N-terminal extension of the G domain modified with myristate. Arf proteins, including Arf1, have roles in membrane trafficking and cytoskeletal dynamics. While screening for Arf1:small molecule co-crystals, we serendipitously solved the crystal structure of the non-myristoylated engineered mutation [L8K]Arf1 in complex with a GDP analogue. Like wild-type (WT) non-myristoylated Arf1•GDP, we observed that [L8K]Arf1 exhibited an N-terminal helix that occludes the hydrophobic cavity that is occupied by the myristoyl group in the GDP-bound state of the native protein. However, the helices were offset from one another due to the L8K mutation, with a significant change in position of the hinge region connecting the N-terminus to the G domain. Hypothesizing that the observed effects on behavior of the N-terminus affects interaction with regulatory proteins, we mutated two hydrophobic residues to examine the role of the N-terminal extension for interaction with guanine nucleotide exchange factors (GEFs) and GTPase Activating Proteins (GAPs. Different than previous studies, all mutations were examined in the context of myristoylated Arf. Mutations had little or no effect on spontaneous or GEF-catalyzed guanine nucleotide exchange but did affect interaction with GAPs. [F13A]myrArf1 was less than 1/2500, 1/1500, and 1/200 efficient as substrate for the GAPs ASAP1, ARAP1 and AGAP1; however, [L8A/F13A]myrArf1 was similar to WT myrArf1. Using molecular dynamics simulations, the effect of the mutations on forming alpha helices adjacent to a membrane surface was examined, yet no differences were detected. The results indicate that lipid modifications of GTPases and consequent anchoring to a membrane influences protein function beyond simple membrane localization. Hypothetical mechanisms are discussed.

Electrostatic Impact of Brefeldin A on Thiocyanate Probes Surrounding the Interface of Arf1-BFA-ARNO4M, a Protein-Drug-Protein Complex.

Protein-protein interactions regulate many cellular processes, making them ideal drug candidates. Design of such drugs, however, is hindered by a lack of understanding of the factors that contribute to the interaction specificity. Specific protein-protein complexes possess both structural and electrostatic complementarity, and while structural complementarity of protein complexes has been extensively investigated, fundamental understanding of the complicated networks of electrostatic interactions at these interfaces is lacking, thus hindering the rational design of orthosterically binding small molecules. To better understand the electrostatic interactions at protein interfaces and how a small molecule could contribute to and fit within that environment, we used a model protein-drug-protein system, Arf1-BFA-ARNO4M, to investigate how small molecule brefeldin A (BFA) perturbs the Arf1-ARNO4M interface. By using nitrile probe labeled Arf1 sites and measuring vibrational Stark effects as well as temperature dependent infrared shifts, we measured changes in the electric field and hydrogen bonding at this interface upon BFA binding. At all five probe locations of Arf1, we found that the vibrational shifts resulting from BFA binding corroborate trends found in Poisson-Boltzmann calculations of surface potentials of Arf1-ARNO4M and Arf1-BFA-ARNO4M, where BFA contributes negative electrostatic potential to the protein interface. The data also corroborate previous hypotheses about the mechanism of interfacial binding and confirm that alternating patches of hydrophobic and polar interactions lead to BFA binding specificity. These findings demonstrate the impact of BFA on this protein-protein interface and have implications for the design of other interfacial drug candidates.

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.

Immunity-linked genes are stimulated by a membrane stress pathway linked to Golgi function and the ARF-1 GTPase.

Infection response and other immunity-linked genes (ILGs) were first named in Caenorhabditis elegans-based expression after pathogen challenge, but many are also up-regulated when lipid metabolism is perturbed. Why pathogen attack and metabolic changes both increase ILGs is unclear. We find that ILGs are activated when phosphatidylcholine (PC) levels change in membranes of secretory organelles in C. elegans. RNAi targeting of the ADP-ribosylation factor arf-1, which disrupts the Golgi and secretory function, also activates ILGs. Low PC limits ARF-1 function, suggesting a mechanism for ILG activation via lipid metabolism, as part of a membrane stress response acting outside the ER. RNAi of selected ILGs uncovered defects in the secretion of two GFP reporters and the accumulation of a pathogen-responsive complement C1r/C1s, Uegf, Bmp1 (CUB) domain fusion protein. Our data argue that up-regulation of some ILGs is a coordinated response to changes in trafficking and may act to counteract stress on secretory function.

ARF1 prevents aberrant type I interferon induction by regulating STING activation and recycling.

Type I interferon (IFN) signalling is tightly controlled. Upon recognition of DNA by cyclic GMP-AMP synthase (cGAS), stimulator of interferon genes (STING) translocates along the endoplasmic reticulum (ER)-Golgi axis to induce IFN signalling. Termination is achieved through autophagic degradation or recycling of STING by retrograde Golgi-to-ER transport. Here, we identify the GTPase ADP-ribosylation factor 1 (ARF1) as a crucial negative regulator of cGAS-STING signalling. Heterozygous ARF1 missense mutations cause a previously unrecognized type I interferonopathy associated with enhanced IFN-stimulated gene expression. Disease-associated, GTPase-defective ARF1 increases cGAS-STING dependent type I IFN signalling in cell lines and primary patient cells. Mechanistically, mutated ARF1 perturbs mitochondrial morphology, causing cGAS activation by aberrant mitochondrial DNA release, and leads to accumulation of active STING at the Golgi/ERGIC due to defective retrograde transport. Our data show an unexpected dual role of ARF1 in maintaining cGAS-STING homeostasis, through promotion of mitochondrial integrity and STING recycling.

Myr-Arf1 conformational flexibility at the membrane surface sheds light on the interactions with ArfGAP ASAP1.

ADP-ribosylation factor 1 (Arf1) interacts with multiple cellular partners and membranes to regulate intracellular traffic, organelle structure and actin dynamics. Defining the dynamic conformational landscape of Arf1 in its active form, when bound to the membrane, is of high functional relevance and key to understanding how Arf1 can alter diverse cellular processes. Through concerted application of nuclear magnetic resonance (NMR), neutron reflectometry (NR) and molecular dynamics (MD) simulations, we show that, while Arf1 is anchored to the membrane through its N-terminal myristoylated amphipathic helix, the G domain explores a large conformational space, existing in a dynamic equilibrium between membrane-associated and membrane-distal conformations. These configurational dynamics expose different interfaces for interaction with effectors. Interaction with the Pleckstrin homology domain of ASAP1, an Arf-GTPase activating protein (ArfGAP), restricts motions of the G domain to lock it in what seems to be a conformation exposing functionally relevant regions.

Arf1 Ablation in Colorectal Cancer Cells Activates a Super Signal Complex in DC to Enhance Anti-Tumor Immunity.

The anti-tumor immune response relies on interactions among tumor cells and immune cells. However, the molecular mechanisms by which tumor cells regulate DCs as well as DCs regulate T cells remain enigmatic. Here, the authors identify a super signaling complex in DCs that mediates the Arf1-ablation-induced anti-tumor immunity. They find that the Arf1-ablated tumor cells release OxLDL, HMGB1, and genomic DNA, which together bound to a coreceptor complex of CD36/TLR2/TLR6 on DC surface. The complex then is internalized into the Rab7-marked endosome in DCs, and further joined by components of the NF-κB, NLRP3 inflammasome and cGAS-STING triple pathways to form a super signal complex for producing different cytokines, which together promote CD8+ T cell tumor infiltration, cross-priming and stemness. Blockage of the HMGB1-gDNA complex or reducing expression in each member of the coreceptors or the cGAS/STING pathway prevents production of the cytokines. Moreover, depletion of the type I IFNs and IL-1ß cytokines abrogate tumor regression in mice bearing the Arf1-ablated tumor cells. These findings reveal a new molecular mechanism by which dying tumor cells releasing several factors to activate the triple pathways in DC for producing multiple cytokines to simultaneously promote DC activation, T cell infiltration, cross-priming and stemness.

The Arf-GAP Age2 localizes to the late-Golgi via a conserved amphipathic helix.

Arf GTPases are central regulators of the Golgi complex, which serves as the nexus of membrane-trafficking pathways in eukaryotic cells. Arf proteins recruit dozens of effectors to modify membranes, sort cargos, and create and tether transport vesicles, and are therefore essential for orchestrating Golgi trafficking. The regulation of Arf activity is controlled by the action of Arf-GEFs which activate via nucleotide exchange, and Arf-GAPs which inactivate via nucleotide hydrolysis. The localization dynamics of Arf GTPases and their Arf-GAPs during Golgi maturation have not been reported. Here we use the budding yeast model to examine the temporal localization of the Golgi Arf-GAPs. We also determine the mechanisms used by the Arf-GAP Age2 to localize to the Golgi. We find that the catalytic activity of Age2 and a conserved sequence in the unstructured C-terminal domain of Age2 are both required for Golgi localization. This sequence is predicted to form an amphipathic helix and mediates direct binding of Age2 to membranes in vitro. We also report the development of a probe for sensing active Arf1 in living cells and use this probe to characterize the temporal dynamics of Arf1 during Golgi maturation.

The development of resistance to an inhibitor of a cellular protein reveals a critical interaction between the enterovirus protein 2C and a small GTPase Arf1.

The cellular protein GBF1, an activator of Arf GTPases (ArfGEF: Arf guanine nucleotide exchange factor), is recruited to the replication organelles of enteroviruses through interaction with the viral protein 3A, and its ArfGEF activity is required for viral replication, however how GBF1-dependent Arf activation supports the infection remains enigmatic. Here, we investigated the development of resistance of poliovirus, a prototype enterovirus, to increasing concentrations of brefeldin A (BFA), an inhibitor of GBF1. High level of resistance required a gradual accumulation of multiple mutations in the viral protein 2C. The 2C mutations conferred BFA resistance even in the context of a 3A mutant previously shown to be defective in the recruitment of GBF1 to replication organelles, and in cells depleted of GBF1, suggesting a GBF1-independent replication mechanism. Still, activated Arfs accumulated on the replication organelles of this mutant even in the presence of BFA, its replication was inhibited by a pan-ArfGEF inhibitor LM11, and the BFA-resistant phenotype was compromised in Arf1-knockout cells. Importantly, the mutations strongly increased the interaction of 2C with the activated form of Arf1. Analysis of other enteroviruses revealed a particularly strong interaction of 2C of human rhinovirus 1A with activated Arf1. Accordingly, the replication of this virus was significantly less sensitive to BFA than that of poliovirus. Thus, our data demonstrate that enterovirus 2Cs may behave like Arf1 effector proteins and that GBF1 but not Arf activation can be dispensable for enterovirus replication. These findings have important implications for the development of host-targeted anti-viral therapeutics.

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.

C9orf72 Toxic Species Affect ArfGAP-1 Function.

Compelling evidence indicates that defects in nucleocytoplasmic transport contribute to the pathogenesis of amyotrophic lateral sclerosis (ALS). In particular, hexanucleotide (G4C2) repeat expansions in C9orf72, the most common cause of genetic ALS, have a widespread impact on the transport machinery that regulates the nucleocytoplasmic distribution of proteins and RNAs. We previously reported that the expression of G4C2 hexanucleotide repeats in cultured human and mouse cells caused a marked accumulation of poly(A) mRNAs in the cell nuclei. To further characterize the process, we set out to systematically identify the specific mRNAs that are altered in their nucleocytoplasmic distribution in the presence of C9orf72-ALS RNA repeats. Interestingly, pathway analysis showed that the mRNAs involved in membrane trafficking are particularly enriched among the identified mRNAs. Most importantly, functional studies in cultured cells and Drosophila indicated that C9orf72 toxic species affect the membrane trafficking route regulated by ADP-Ribosylation Factor 1 GTPase Activating Protein (ArfGAP-1), which exerts its GTPase-activating function on the small GTPase ADP-ribosylation factor 1 to dissociate coat proteins from Golgi-derived vesicles. We demonstrate that the function of ArfGAP-1 is specifically affected by expanded C9orf72 RNA repeats, as well as by C9orf72-related dipeptide repeat proteins (C9-DPRs), indicating the retrograde Golgi-to-ER vesicle-mediated transport as a target of C9orf72 toxicity.

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.

Arf1-PI4KIIIß positive vesicles regulate PI(3)P signaling to facilitate lysosomal tubule fission.

Formation and fission of tubules from autolysosomes, endolysosomes, or phagolysosomes are required for lysosome reformation. However, the mechanisms governing these processes in these different lysosomal organelles are poorly understood. Thus, the role of phosphatidylinositol-4-phosphate (PI(4)P) is unclear as it was shown to promote the formation of tubules from phagolysosomes but was proposed to inhibit tubule formation on autolysosomes because the loss of PI4KIIIß causes extensive lysosomal tubulation. Using super-resolution live-cell imaging, we show that Arf1-PI4KIIIß positive vesicles are recruited to tubule fission sites from autolysosomes, endolysosomes, and phagolysosomes. Moreover, we show that PI(4)P is required to form autolysosomal tubules and that increased lysosomal tubulation caused by loss of PI4KIIIß represents impaired tubule fission. At the site of fission, we propose that Arf1-PI4KIIIß positive vesicles mediate a PI(3)P signal on lysosomes in a process requiring the lipid transfer protein SEC14L2. Our findings indicate that Arf1-PI4KIIIß positive vesicles and their regulation of PI(3)P are critical components of the lysosomal tubule fission machinery.

STED imaging of endogenously tagged ARF GTPases reveals their distinct nanoscale localizations.

ADP-ribosylation factor (ARF) GTPases are major regulators of cellular membrane homeostasis. High sequence similarity and multiple, possibly redundant functions of the five human ARFs make investigating their function a challenging task. To shed light on the roles of the different Golgi-localized ARF members in membrane trafficking, we generated CRISPR-Cas9 knockins (KIs) of type I (ARF1 and ARF3) and type II ARFs (ARF4 and ARF5) and mapped their nanoscale localization with stimulated emission depletion (STED) super-resolution microscopy. We find ARF1, ARF4, and ARF5 on segregated nanodomains on the cis-Golgi and ER-Golgi intermediate compartments (ERGIC), revealing distinct roles in COPI recruitment on early secretory membranes. Interestingly, ARF4 and ARF5 define Golgi-tethered ERGIC elements decorated by COPI and devoid of ARF1. Differential localization of ARF1 and ARF4 on peripheral ERGICs suggests the presence of functionally different classes of intermediate compartments that could regulate bi-directional transport between the ER and the Golgi. Furthermore, ARF1 and ARF3 localize to segregated nanodomains on the trans-Golgi network (TGN) and are found on TGN-derived post-Golgi tubules, strengthening the idea of distinct roles in post-Golgi sorting. This work provides the first map of the nanoscale organization of human ARF GTPases on cellular membranes and sets the stage to dissect their numerous cellular roles.

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.

The small molecule inhibitor NAV-2729 has a complex target profile including multiple ADP-ribosylation factor regulatory proteins.

The ADP-ribosylation factor (Arf) GTPases and their regulatory proteins are implicated in cancer progression. NAV-2729 was previously identified as a specific inhibitor of Arf6 that reduced progression of uveal melanoma in an orthotopic xenograft. Here, our goal was to assess the inhibitory effects of NAV-2729 on the proliferation of additional cell types. We found NAV-2729 inhibited proliferation of multiple cell lines, but Arf6 expression did not correlate with NAV-2729 sensitivity, and knockdown of Arf6 affected neither cell viability nor sensitivity to NAV-2729. Furthermore, binding to native Arf6 was not detected; however, we determined that NAV-2729 inhibited both Arf exchange factors and Arf GTPase-activating proteins. ASAP1, a GTPase-activating protein linked to cancer progression, was further investigated. We demonstrated that NAV-2729 bound to the PH domain of ASAP1 and changed ASAP1 cellular distribution. However, ASAP1 knockdown did not fully recapitulate the cytoskeletal effects of NAV-2729 nor affect cell proliferation. Finally, our screens identified 48 other possible targets of NAV-2729. These results illustrate the complexities of defining targets of small molecules and identify NAV-2729 as a model PH domain-binding inhibitor.

An interaction between ß'-COP and the ArfGAP, Glo3, maintains post-Golgi cargo recycling.

The essential COPI coat mediates retrieval of transmembrane proteins at the Golgi and endosomes following recruitment by the small GTPase, Arf1. ArfGAP proteins regulate COPI coats, but molecular details for COPI recognition by ArfGAPs remain elusive. Biochemical and biophysical data reveal how ß'-COP propeller domains directly engage the yeast ArfGAP, Glo3, with a low micromolar binding affinity. Calorimetry data demonstrate that both ß'-COP propeller domains are required to bind Glo3. An acidic patch on ß'-COP (D437/D450) interacts with Glo3 lysine residues located within the BoCCS (binding of coatomer, cargo, and SNAREs) region. Targeted point mutations in either Glo3 BoCCS or ß'-COP abrogate the interaction in vitro, and loss of the ß'-COP/Glo3 interaction drives Ste2 missorting to the vacuole and aberrant Golgi morphology in budding yeast. These data suggest that cells require the ß'-COP/Glo3 interaction for cargo recycling via endosomes and the TGN, where ß'-COP serves as a molecular platform to coordinate binding to multiple proteins, including Glo3, Arf1, and the COPI F-subcomplex.

A neurodevelopmental disorder associated with an activating de novo missense variant in ARF1.

ADP-ribosylation factor 1 (ARF1) is a small GTPase that regulates membrane traffic at the Golgi apparatus and endosomes through recruitment of several coat proteins and lipid-modifying enzymes. Here, we report a pediatric patient with an ARF1-related disorder because of a monoallelic de novo missense variant (c.296 G > A; p.R99H) in the ARF1 gene, associated with developmental delay, hypotonia, intellectual disability and motor stereotypies. Neuroimaging revealed a hypoplastic corpus callosum and subcortical white matter abnormalities. Notably, this patient did not exhibit periventricular heterotopias previously observed in other patients with ARF1 variants (including p.R99H). Functional analysis of the R99H-ARF1 variant protein revealed that it was expressed at normal levels and properly localized to the Golgi apparatus; however, the expression of this variant caused swelling of the Golgi apparatus, increased the recruitment of coat proteins such as coat protein complex I, adaptor protein complex 1 and GGA3 and altered the morphology of recycling endosomes. In addition, we observed that the expression of R99H-ARF1 prevented dispersal of the Golgi apparatus by the ARF1-inhibitor brefeldin A. Finally, protein interaction analyses showed that R99H-ARF1 bound more tightly to the ARF1-effector GGA3 relative to wild-type ARF1. These properties were similar to those of the well-characterized constitutively active Q71L-ARF1 mutant, indicating that the pathogenetic mechanism of the R99H-ARF1 variant involves constitutive activation with resultant Golgi and endosomal alterations. The absence of periventricular nodular heterotopias in this R99H-ARF1 subject also indicates that this finding may not be a consistent phenotypic expression of all ARF1-related disorders.

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.

Arf1 facilitates mast cell proliferation via the mTORC1 pathway.

Mast cells are one of major players in allergic responses. Mast cell activation via the high affinity IgE receptor (FcεRI) causes degranulation and release of de novo synthesized proinflammatory cytokines in a process that involves vesicle trafficking. Considering that the GTPase ADP-ribosylation factor 1 (Arf1) orchestrates and maintains membrane traffic and organelle structure, it seems likely that Arf1 contributes to mast cell activation. Actually, it has been reported that pharmaceutical blockade of the Arf1 pathway suppresses cytokine secretion and mast cell degranulation. However, physiological roles of Arf1 in mast cells remain elusive. Here, by using a genetic approach, we demonstrate that Arf1 is required for optimal mTORC1 activation upon IL-3 and facilitates mast cell proliferation. On the other hand, contrary to our expectation, Arf1-deficiency had little impact on FcεRI-induced degranulation nor cytokine secretion. Our findings reveal an unexpected role of Arf1 in mast cell expansion and its potential as a therapeutic target in the mast cell proliferative disorders.