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The small GTPase ARL4C participates in the regulation of cell migration, cytoskeletal rearrangements, and vesicular trafficking in epithelial cells. The ARL4C signaling cascade starts by the recruitment of the ARF-GEF cytohesins to the plasma membrane, which, in turn, bind and activate the small GTPase ARF6. However, the role of ARL4C-cytohesin-ARF6 signaling during hippocampal development remains elusive. Here, we report that the E3 ubiquitin ligase Cullin 5/RBX2 (CRL5) controls the stability of ARL4C and its signaling effectors to regulate hippocampal morphogenesis. Both RBX2 knockout and Cullin 5 knockdown cause hippocampal pyramidal neuron mislocalization and development of multiple apical dendrites. We used quantitative mass spectrometry to show that ARL4C, Cytohesin-1/3, and ARF6 accumulate in the RBX2 mutant telencephalon. Furthermore, we show that depletion of ARL4C rescues the phenotypes caused by Cullin 5 knockdown, whereas depletion of CYTH1 or ARF6 exacerbates overmigration. Finally, we show that ARL4C, CYTH1, and ARF6 are necessary for the dendritic outgrowth of pyramidal neurons to the superficial strata of the hippocampus. Overall, we identified CRL5 as a key regulator of hippocampal development and uncovered ARL4C, CYTH1, and ARF6 as CRL5-regulated signaling effectors that control pyramidal neuron migration and dendritogenesis.

The unique membrane composition of cilia is maintained by a diffusion barrier at the transition zone that is breached when the BBSome escorts signaling receptors out of cilia. Understanding how the BBSome removes proteins from cilia has been hampered by a lack of structural information. Here, we present a nearly complete Cα model of BBSome purified from cow retina. The model is based on a single-particle cryo-electron microscopy density map at 4.9-Å resolution that was interpreted with the help of comprehensive Rosetta-based structural modeling constrained by crosslinking mass spectrometry data. We find that BBSome subunits have a very high degree of interconnectivity, explaining the obligate nature of the complex. Furthermore, like other coat adaptors, the BBSome exists in an autoinhibited state in solution and must thus undergo a conformational change upon recruitment to membranes by the small GTPase ARL6/BBS3. Our model provides the first detailed view of the machinery enabling ciliary exit.

The peptidoglycan fragments γ-d-glutamyl- meso-diaminopimelic acid (iE-DAP) and muramyl-dipeptide (MDP) are microbial-specific metabolites that activate intracellular pattern recognition receptors and stimulate immune signaling pathways. While extensive structure-activity studies have demonstrated that these bacterial cell wall metabolites trigger NOD1- and NOD2-dependent signaling, their direct binding to these innate immune receptors or other proteins in mammalian cells has not been established. To characterize these fundamental microbial metabolite-host interactions, we synthesized a series of peptidoglycan metabolite photoaffinity reporters and evaluated their cross-linking to NOD1 and NOD2 in mammalian cells. We show that active iE-DAP and MDP photoaffinity reporters selectively cross-linked NOD1 and NOD2, respectively, and not their inactive mutants. We also discovered MDP reporter cross-linking to Arf GTPases, which interacted most prominently with GTP-bound Arf6 and coimmunoprecipitated with NOD2 upon MDP stimulation. Notably, MDP binding to NOD2 and Arf6 was abrogated with loss-of-function NOD2 mutants associated with Crohn's disease. Our studies demonstrate peptidoglycan metabolite photoaffinity reporters can capture their cognate immune receptors in cells and reveal unpredicted ligand-induced interactions with other cellular cofactors. These photoaffinity reporters should afford useful tools to discover and characterize other peptidoglycan metabolite-interacting proteins.

Mutations in the gene Centrosomal Protein 290 kDa (CEP290) result in multiple ciliopathies ranging from the neonatal lethal disorder Meckel-Gruber Syndrome to multi-systemic disorders such as Joubert Syndrome and Bardet-Biedl Syndrome to nonsyndromic diseases like Leber Congenital Amaurosis (LCA) and retinitis pigmentosa. Results from model organisms and human genetics studies, have suggest that mutations in genes encoding protein components of the transition zone (TZ) and other cilia-associated proteins can function as genetic modifiers and be a source for CEP290 pleiotropy. We investigated the zebrafish cep290fh297/fh297 mutant, which encodes a nonsense mutation (p.Q1217*). This mutant is viable as adults, exhibits scoliosis, and undergoes a slow, progressive cone degeneration. The cep290fh297/fh297 mutants showed partial mislocalization of the transmembrane protein rhodopsin but not of the prenylated proteins rhodopsin kinase (GRK1) or the rod transducin subunit GNB1. Surprisingly, photoreceptor degeneration did not trigger proliferation of Müller glia, but proliferation of rod progenitors in the outer nuclear layer was significantly increased. To determine if heterozygous mutations in other cilia genes could exacerbate retinal degeneration, we bred cep290fh297/fh297 mutants to arl13b, ahi1, and cc2d2a mutant zebrafish lines. While cep290fh297/fh297 mutants lacking a single allele of these genes did not exhibit accelerated photoreceptor degeneration, loss of one alleles of arl13b or ahi1 reduced visual performance in optokinetic response assays at 5 days post fertilization. Our results indicate that the cep290fh297/fh297 mutant is a useful model to study the role of genetic modifiers on photoreceptor degeneration in zebrafish and to explore how progressive photoreceptor degeneration influences regeneration in adult zebrafish.

Endogenous sphingolipids (ceramide) and related synthetic molecules (FTY720, SH-BC-893) reduce nutrient access by decreasing cell surface expression of a subset of nutrient transporter proteins. Here, we report that these sphingolipids disrupt endocytic recycling by inactivating the small GTPase ARF6. Consistent with reported roles for ARF6 in maintaining the tubular recycling endosome, MICAL-L1-positive tubules were lost from sphingolipid-treated cells. We propose that ARF6 inactivation may occur downstream of PP2A activation since: (1) sphingolipids that fail to activate PP2A did not reduce ARF6-GTP levels; (2) a structurally unrelated PP2A activator disrupted tubular recycling endosome morphology and transporter localization; and (3) overexpression of a phosphomimetic mutant of the ARF6 GEF GRP1 prevented nutrient transporter loss. ARF6 inhibition alone was not toxic; however, the ARF6 inhibitors SecinH3 and NAV2729 dramatically enhanced the killing of cancer cells by SH-BC-893 without increasing toxicity to peripheral blood mononuclear cells, suggesting that ARF6 inactivation contributes to the anti-neoplastic actions of sphingolipids. Taken together, these studies provide mechanistic insight into how ceramide and sphingolipid-like molecules limit nutrient access and suppress tumor cell growth and survival.

A diffusion barrier at the transition zone enables the compartmentalization of signaling molecules by cilia. The BBSome and the small guanosine triphosphatase Arl6, which triggers BBSome coat polymerization, are required for the exit of activated signaling receptors from cilia, but how diffusion barriers are crossed when membrane proteins exit cilia remains to be determined. In this study, we found that activation of the ciliary G protein-coupled receptors (GPCRs) Smoothened and SSTR3 drove the Arl6-dependent assembly of large, highly processive, and cargo-laden retrograde BBSome trains. Single-molecule imaging revealed that the assembly of BBSome trains enables the lateral transport of ciliary GPCRs across the transition zone. However, the removal of activated GPCRs from cilia was inefficient because a second periciliary diffusion barrier was infrequently crossed. We conclude that exit from cilia is a two-step process in which BBSome/Arl6 trains first move activated GPCRs through the transition zone before a periciliary barrier can be crossed.

Lipidated small GTPases and their regulators need to bind to membranes to propagate actions in the cell, but an integrated understanding of how the lipid bilayer exerts its effect has remained elusive. Here we focused on ADP ribosylation factor (Arf) GTPases, which orchestrate a variety of regulatory functions in lipid and membrane trafficking, and their activation by the guanine-nucleotide exchange factor (GEF) Brag2, which controls integrin endocytosis and cell adhesion and is impaired in cancer and developmental diseases. Biochemical and structural data are available that showed the exceptional efficiency of Arf activation by Brag2 on membranes. We determined the high-resolution crystal structure of unbound Brag2 containing the GEF (Sec7) and membrane-binding (pleckstrin homology) domains, revealing that it has a constitutively active conformation. We used this structure to analyze the interaction of uncomplexed Brag2 and of the myristoylated Arf1/Brag2 complex with a phosphatidylinositol bisphosphate (PIP2) -containing lipid bilayer, using coarse-grained molecular dynamics. These simulations revealed that the system forms a close-packed, oriented interaction with the membrane, in which multiple PIP2 lipids bind the canonical lipid-binding site and unique peripheral sites of the PH domain, the Arf GTPase and, unexpectedly, the Sec7 domain. We cross-validated these predictions by reconstituting the binding and kinetics of Arf and Brag2 in artificial membranes. Our coarse-grained structural model thus suggests that the high efficiency of Brag2 requires interaction with multiple lipids and a well-defined orientation on the membrane, resulting in a local PIP2 enrichment, which has the potential to signal toward the Arf pathway.

ObjectiveCoagulation initiation by tissue factor (TF) is regulated by cellular inhibitors, cell surface availability of procoagulant phosphatidylserine, and thiol-disulfide exchange. How these mechanisms contribute to keeping TF in a noncoagulant state and to generating prothrombotic TF remain incompletely understood.Approach and resultsHere, we study the activation of TF in primary macrophages by a combination of pharmacological, genetic, and biochemical approaches. We demonstrate that primed macrophages effectively control TF cell surface activity by receptor internalization. After cell injury, ATP signals through the purinergic receptor P2rx7 induce release of TF+ microvesicles. TF cell surface availability for release onto microvesicles is regulated by the GTPase arf6 associated with integrin α4β1. Furthermore, microvesicles proteome analysis identifies activation of Gαi2 as a participating factor in the release of microvesicles with prothrombotic activity in flowing blood. ATP not only prevents TF and phosphatidylserine internalization but also induces TF conversion to a conformation with high affinity for its ligand, coagulation factor VII. Although inhibition of dynamin-dependent internalization also exposes outer membrane procoagulant phosphatidylserine, the resulting TF+ microvesicles distinctly lack protein disulfide isomerase and high affinity TF and fail to produce fibrin strands typical for microvesicles generated by thrombo-inflammatory P2rx7 activation.ConclusionsThese data show that procoagulant phospholipid exposure is not sufficient and that TF affinity maturation is required to generate prothrombotic microvesicles from a variety of cell types. These findings are significant for understanding TF-initiated thrombosis and should be considered in designing functional microvesicles-based diagnostic approaches.

Virulence of the human fungal pathogen Candida albicans depends on the switch from budding to filamentous growth, which requires sustained membrane traffic and polarized growth. In many organisms, small GTPases of the Arf (ADP-ribosylation factor) family regulate membrane/protein trafficking, yet little is known about their role in fungal filamentous growth. To investigate these GTPases in C. albicans, we generated loss of function mutants in all 3 Arf proteins, Arf1-Arf3, and 2 Arf-like proteins, Arl1 and Arl3. Our results indicate that of these proteins, Arf2 is required for viability and sensitivity to antifungal drugs. Repressible ARF2 expression results in defects in filamentous growth, cell wall integrity and virulence, likely due to alteration of the Golgi. Arl1 is also required for invasive filamentous growth and, although arl1/arl1 cells can initiate hyphal growth, hyphae are substantially shorter than that of the wild-type, due to the inability of this mutant to maintain hyphal growth at a single site. We show that this defect does not result from an alteration of phospholipid distribution and is unlikely to result from the sole Golgin Imh1 mislocalization, as Imh1 is not required for invasive filamentous growth. Rather, our results suggest that the arl1/arl1 hyphal growth defect results from increased secretion in this mutant. Strikingly, the arl1/arl1 mutant is drastically reduced in virulence during oropharyngeal candidiasis. Together, our results highlight the importance of Arl1 and Arf2 as key regulators of hyphal growth and virulence in C. albicans and identify a unique function of Arl1 in secretion.

Breast cancer (BC) is the most common type of cancer found in women. ADP-ribosylation factors (ARFs) are a group of small proteins that bind to GTP and are involved in controlling different cellular functions. The function and evolution of multiple ARFs in BC have remained to be fully elucidated, despite existing studies on this protein family in Homo sapiens and other species. In the present study, a systematic analysis of ARF expression levels in BC tissues compared to normal breast tissues was performed using data from The Cancer Genome Atlas database. The analysis revealed significantly higher expression of ARFs in BC tissues. In addition, the prognostic significance of ARF1 and ARF3-6 expression levels was assessed in patients with BC. Of note, elevated ARF1 expression was associated with reduced rates of distant metastasis-free survival (DMFS), overall survival (OS) and recurrence-free survival (RFS) in affected individuals. Similarly, patients with high expression levels of ARF3 had lower post-progression survival (PPS) rates. In addition, patients with higher ARF4 expression had worse PPS and patients with high ARF5 expression exhibited lower DMFS. Patients with high ARF6 expression had worse DMFS, OS, RFS and predictive power score values. Furthermore, the expression of ARF was found to be strongly linked to the infiltration of various immune cell types, namely dendritic cells, macrophages, neutrophils, CD8+ T cells and B cells. These significant associations offer a solid foundation for the potential utilization of new therapeutic targets and predictive markers for the treatment of BC. [ABSTRACT FROM AUTHOR]

BackgroundRetinitis pigmentosa is a phenotype with diverse genetic causes. Due to this genetic heterogeneity, genome-wide identification and analysis of protein-altering DNA variants by exome sequencing is a powerful tool for novel variant and disease gene discovery. In this study, exome sequencing analysis was used to search for potentially causal DNA variants in a two-generation pedigree with apparent dominant retinitis pigmentosa.MethodsVariant identification and analysis of three affected members (mother and two affected offspring) was performed via exome sequencing. Parental samples of the index case were used to establish inheritance. Follow-up testing of 94 additional retinitis pigmentosa pedigrees was performed via retrospective analysis or Sanger sequencing.Results and conclusionsA total of 136 high quality coding variants in 123 genes were identified which are consistent with autosomal dominant disease. Of these, one of the strongest genetic and functional candidates is a c.269A>G (p.Tyr90Cys) variant in ARL3. Follow-up testing established that this variant occurred de novo in the index case. No additional putative causal variants in ARL3 were identified in the follow-up cohort, suggesting that if ARL3 variants can cause adRP it is an extremely rare phenomenon.

The retinal features of Bardet-Biedl syndrome (BBS) are insufficiently characterized in Arab populations. This retrospective study investigated the retinal features and genotypes of BBS in Saudi patients managed at a single tertiary eye care center. Data analysis of the identified 46 individuals from 31 families included visual acuity (VA), systemic manifestations, multimodal retinal imaging, electroretinography (ERG), family pedigrees, and genotypes. Patients were classified to have cone-rod, rod-cone, or generalized photoreceptor dystrophy based on the pattern of macular involvement on the retinal imaging. Results showed that nyctalopia and subnormal VA were the most common symptoms with 76% having VA ≤ 20/200 at the last visit (age: 5-35). Systemic features included obesity 91%, polydactyly 56.5%, and severe cognitive impairment 33%. The predominant retinal phenotype was cone-rod dystrophy 75%, 10% had rod-cone dystrophy and 15% had generalized photoreceptor dystrophy. ERGs were undetectable in 95% of patients. Among the 31 probands, 61% had biallelic variants in BBSome complex genes, 32% in chaperonin complex genes, and 6% had biallelic variants in ARL6 ; including six previously unreported variants. Interfamilial and intrafamilial variabilities were noted, without a clear genotype-phenotype correlation. Most BBS patients had advanced retinopathy and were legally blind by early adulthood, indicating a narrow therapeutic window for rescue strategies.

R-Ras small GTPase enhances cell spreading and motility via RalBP1/RLIP76, an R-Ras effector that links GTP-R-Ras to activation of Arf6 and Rac1 GTPases. Here, we report that RLIP76 performs these functions by binding cytohesin-2/ARNO, an Arf GTPase guanine exchange factor, and connecting it to R-Ras at recycling endosomes. RLIP76 formed a complex with R-Ras and ARNO by binding ARNO via its N-terminus (residues 1-180) and R-Ras via residues 180-192. This complex was present in Rab11-positive recycling endosomes and the presence of ARNO in recycling endosomes required RLIP76, and was not supported by RLIP76(Δ1-180) or RLIP76(Δ180-192). Spreading and migration required RLIP76(1-180), and RLIP76(Δ1-180) blocked ARNO recruitment to recycling endosomes, and spreading. Arf6 activation with an ArfGAP inhibitor overcame the spreading defects in RLIP76-depleted cells or cells expressing RLIP76(Δ1-180). Similarly, RLIP76(Δ1-180) or RLIP76(Δ180-192) suppressed Arf6 activation. Together these results demonstrate that RLIP76 acts as a scaffold at recycling endosomes by binding activated R-Ras, recruiting ARNO to activate Arf6, thereby contributing to cell spreading and migration.

Microtubule dynamics and polarity stem from the polymerization of αβ-tubulin heterodimers. Five conserved tubulin cofactors/chaperones and the Arl2 GTPase regulate α- and β-tubulin assembly into heterodimers and maintain the soluble tubulin pool in the cytoplasm, but their physical mechanisms are unknown. Here, we reconstitute a core tubulin chaperone consisting of tubulin cofactors TBCD, TBCE, and Arl2, and reveal a cage-like structure for regulating αβ-tubulin. Biochemical assays and electron microscopy structures of multiple intermediates show the sequential binding of αβ-tubulin dimer followed by tubulin cofactor TBCC onto this chaperone, forming a ternary complex in which Arl2 GTP hydrolysis is activated to alter αβ-tubulin conformation. A GTP-state locked Arl2 mutant inhibits ternary complex dissociation in vitro and causes severe defects in microtubule dynamics in vivo. Our studies suggest a revised paradigm for tubulin cofactors and Arl2 functions as a catalytic chaperone that regulates soluble αβ-tubulin assembly and maintenance to support microtubule dynamics.

A long-held tenet of heterotrimeric G protein signal transduction is that it is triggered by G protein-coupled receptors (GPCRs) at the PM. Here, we demonstrate that Gi is activated in the Golgi by GIV/Girdin, a non-receptor guanine-nucleotide exchange factor (GEF). GIV-dependent activation of Gi at the Golgi maintains the finiteness of the cyclical activation of ADP-ribosylation factor 1 (Arf1), a fundamental step in vesicle traffic in all eukaryotes. Several interactions with other major components of Golgi trafficking-e.g., active Arf1, its regulator, ArfGAP2/3, and the adaptor protein β-COP-enable GIV to coordinately regulate Arf1 signaling. When the GIV-Gαi pathway is selectively inhibited, levels of GTP-bound Arf1 are elevated and protein transport along the secretory pathway is delayed. These findings define a paradigm in non-canonical G protein signaling at the Golgi, which places GIV-GEF at the crossroads between signals gated by the trimeric G proteins and the Arf family of monomeric GTPases.

The BBSome is a coat-like ciliary trafficking complex composed of proteins mutated in Bardet-Biedl syndrome (BBS). A critical step in BBSome-mediated sorting is recruitment of the BBSome to membranes by the GTP-bound Arf-like GTPase ARL6. We have determined crystal structures of Chlamydomonas reinhardtii ARL6-GDP, ARL6-GTP and the ARL6-GTP-BBS1 complex. The structures demonstrate how ARL6-GTP binds the BBS1 β-propeller at blades 1 and 7 and explain why GTP- but not GDP-bound ARL6 can recruit the BBSome to membranes. Single point mutations in the ARL6-GTP-BBS1 interface abolish the interaction of ARL6 with the BBSome and prevent the import of BBSomes into cilia. Furthermore, we show that BBS1 with the M390R mutation, responsible for 30% of all reported BBS disease cases, fails to interact with ARL6-GTP, thus providing a molecular rationale for patient pathologies.

The sorting of signaling receptors into and out of cilia relies on the BBSome, a complex of Bardet-Biedl syndrome (BBS) proteins, and on the intraflagellar transport (IFT) machinery. GTP loading onto the Arf-like GTPase ARL6/BBS3 drives assembly of a membrane-apposed BBSome coat that promotes cargo entry into cilia, yet how and where ARL6 is activated remains elusive. Here, we show that the Rab-like GTPase IFT27/RABL4, a known component of IFT complex B, promotes the exit of BBSome and associated cargoes from cilia. Unbiased proteomics and biochemical reconstitution assays show that, upon disengagement from the rest of IFT-B, IFT27 directly interacts with the nucleotide-free form of ARL6. Furthermore, IFT27 prevents aggregation of nucleotide-free ARL6 in solution. Thus, we propose that IFT27 separates from IFT-B inside cilia to promote ARL6 activation, BBSome coat assembly, and subsequent ciliary exit, mirroring the process by which BBSome mediates cargo entry into cilia.

Key message: PvArf regulate proline biosynthesis by physically interacting with PvP5CS1 to improve salt tolerance in switchgrass. The genetic factors that contribute to stress resiliency are yet to be determined. Here, we identified three ADP-ribosylation factors, PvArf1, PvArf-B1C, and PvArf-related, which contribute to salinity tolerance in transgenic switchgrass (Panicum virgatum L.). Switchgrass overexpressing each of these genes produced approximately twofold more biomass than wild type (WT) under normal growth conditions. Transgenic plants accumulated modestly higher levels of proline under normal conditions, but this level was significantly increased under salt stress providing better protection to transgenic plants compared to WT. We found that PvArf genes induce proline biosynthesis genes under salt stress to positively regulate proline accumulation, and further demonstrated that PvArf physically interact with PvP5CS1. Moreover, the transcript levels of two key ROS-scavenging enzyme genes were significantly increased in the transgenic plants compared to WT, leading to reduced H2O2 accumulation under salt stress conditions. PvArf genes also protect cells against stress-induced changes in Na+ and K+ ion concentrations. Our findings uncover that ADP-ribosylation factors are key determinants of biomass yield in switchgrass, and play pivotal roles in salinity tolerance by regulating genes involved in proline biosynthesis. [ABSTRACT FROM AUTHOR]

Background: A novel risk scoring system, predicated on DNA damage response (DDR), was developed to enhance prognostic predictions and potentially inform the creation of more effective therapeutic protocols for sepsis., Methods: To thoroughly delineate the expression profiles of DDR markers within the context of sepsis, an analytical approach utilizing single-cell RNA-sequencing (scRNA-seq) was implemented. Our study utilized single-cell analysis techniques alongside weighted gene co-expression network analysis (WGCNA) to pinpoint the genes that exhibit the most substantial associations with DNA damage response (DDR). Through Cox proportional hazards LASSO regression, we distinguished DDR-associated genes and established a risk model, enabling the stratification of patients into high- and low-risk groups. Subsequently, we carried out an analysis to determine our model's predictive accuracy regarding patient survival. Moreover, we examined the distinct biological characteristics, various signal transduction routes, and immune system responses in sepsis patients, considering different risk categories and outcomes related to survival. Lastly, we conducted experimental validation of the identified genes through in vivo and in vitro assays, employing RT-PCR, ELISA, and flow cytometry., Results: Both single-cell RNA sequencing (scRNA-seq) and bulk transcriptomic analyses have demonstrated a strong correlation between DNA damage response (DDR) levels and sepsis prognosis. Specific cell subtypes, including monocytes, megakaryocytes, CD4+ T cells, and neutrophils, have shown elevated DDR activity. Cells with increased DDR scores exhibited more robust and numerous interactions with other cell populations. The weighted gene co-expression network analysis (WGCNA) and single-cell analyses revealed 71 DDR-associated genes. We developed a four-gene risk scoring system using ARL4C, CD247, RPL7, and RPL31, identified through univariate COX, LASSO COX regression, and log-rank (Mantel-Cox) tests. Nomograms, calibration plots, and decision curve analyses (DCA) regarding these specific genes have provided significant clinical benefits for individuals diagnosed with sepsis. The study suggested that individuals categorized as lower-risk demonstrated enhanced infiltration of immune cells, upregulated expression of immune regulators, and a more prolific presence of immune-associated functionalities and pathways. RT-qPCR analyses on a sepsis rat model revealed differential gene expression predominantly in the four targeted genes. Furthermore, ARL4C knockdown in sepsis model in vivo and vitro caused increased inflammatory response and a worse prognosis., Conclusion: The delineated DDR expression landscape offers insights into sepsis pathogenesis, whilst our riskScore model, based on a robust four-gene signature, could underpin personalized sepsis treatment strategies., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Lin, Zeng, Yang, Xu, Jin and Wei.)

Ursolic acid (UA), a natural pentacyclic triterpenoid, is known to exhibit various biological activities and anticancer effects. However, the underlying anticancer mechanism is not fully understood to date. The present study aimed to investigate the antimetastatic effect of UA through ADP‑ribosylation factor like GTPase 4C (ARL4C) in colon cancer. A lung metastasis model of colon cancer in nude mice was established through tail vein injection. A Cell Counting Kit‑8 assay was used to investigate the proliferation of colon cancer cells. Transwell assays were used to detect cell migration and invasion. The expression levels of proteins including ARL4C, matrix metallopeptidase 2 (MMP2), phosphorylated (p)‑AKT and p‑mTOR were measured using western blot analysis. Immunohistochemistry was used to determine the protein expression level in tissues. ARL4C ubiquitination levels were analysed using immunoprecipitation and western blotting. The results indicated that UA inhibits the metastasis of colon cancer in vivo and in vitro . The expression of ARL4C in human colon cancer tissue was significantly higher than that in adjacent tissues and its high expression level was associated with lymph node metastases and tumour stage. UA treatment significantly decreased ARL4C and MMP2 protein levels and inhibited the AKT/mTOR signalling pathway. Overexpression of ARL4C reversed the inhibitory effect of UA on the invasion and migration of HCT‑116 and SW480 cells, as well as the expression and secretion of MMP2 protein. In addition, UA and an AKT signalling pathway inhibitor (LY294002) induced the ubiquitination of the ARL4C protein, which was reversed by a proteasome inhibitor (MG‑132). Collectively, it was revealed in the present study that UA served as a novel solution to relieve colon cancer metastasis by inducing the ubiquitination‑mediated degradation of ARL4C by modulating the AKT signalling pathway. Thus, UA may be a promising treatment option to prolong the survival of patients with colon cancer metastasis.

Atherosclerosis is one of the leading causes of death worldwide. miR-26 is a potential biomarker of atherosclerosis. Standardized diagnostic tests for miR-26 (MIR26-DX) have been developed, but the fastest progress has been in predicting the efficacy of IFN-α therapy for hepatocellular carcinoma (HCC, phase 3). MiR-26 slows atherosclerosis development by suppressing ACC1/2, ACLY, ACSL3/4, ALDH3A2, ALPL, BMP2, CD36, COL1A1, CPT1A, CTGF, DGAT2, EHHADH, FAS, FBP1, GATA4, GSK3β, G6PC, Gys2, HMGA1, HMGB1, LDLR, LIPC, IL-1β, IL-6, JAG2, KCNJ2, MALT1, β-MHC, NF-κB, PCK1, PLCβ1, PYGL, RUNX2, SCD1, SMAD1/4/5/7, SREBF1, TAB3, TAK1, TCF7L2, and TNF-α expression. Many agents targeting these genes, such as the ACC1/2 inhibitors GS-0976, PF-05221304, and MK-4074; the DGAT2 inhibitors IONIS-DGAT2Rx, PF-06427878, PF-0685571, and PF-07202954; the COL1A1 inhibitor HT-100; the stimulants 68 Ga-CBP8 and RCT-01; the CPT1A inhibitors etomoxir, perhexiline, and teglicar; the FBP1 inhibitors CS-917 and MB07803; and the SMAD7 inhibitor mongersen, have been investigated in clinical trials. Interestingly, miR-26 better reduced intima-media thickness (IMT) than PCSK9 or CT-1 knockout. Many PCSK9 inhibitors, including alirocumab, evolocumab, inclisiran, AZD8233, Civi-007, MK-0616, and LIB003, have been investigated in clinical trials. Recombinant CT-1 was also investigated in clinical trials. Therefore, miR-26 is a promising target for agent development. miR-26 promotes foam cell formation by reducing ABCA1 and ARL4C expression. Multiple materials can be used to deliver miR-26, but it is unclear which material is most suitable for mass production and clinical applications. This review focuses on the potential use of miR-26 in treating atherosclerosis to support the development of agents targeting it., (© 2024. The Author(s).)

ADP-ribosylation factors (Arfs) are members of the Ras GTPase superfamily. The function of Arfs is dependent on GTPase-activating proteins (GAPs) and guanine nucleotide exchange factors (GEFs), which allow Arfs to cycle between the GDP-bound and GTP-bound forms. Arf GAPs have been shown to be present in integrin adhesion complexes, which include focal adhesions. Integrin adhesion complexes are composed of integrins, scaffolding proteins and signaling proteins and regulate cell proliferation, survival, differentiation and migration. Understanding the role of Arf GAPs in the regulation of integrin adhesion complexes is relevant to understanding normal physiology and cancer. In this review, we will discuss the contribution of the Arf GAP family members to the regulation of integrin adhesion complexes, examining the diverse mechanisms by which they control integrin adhesion complex formation, maturation and dissolution. GIT1 and ARAP2 serve as GAPs for Arf6, regulating Rac1 and other effectors by mechanisms still being defined. In contrast, GIT2 regulates Rac1 independent of Arf6. AGAP2 binds to and regulates focal adhesion kinase (FAK). ARAP2 and ACAP1, both Arf6 GAPs, regulate membrane trafficking of integrins through different endocytic pathways, exerting opposite effects on focal adhesions. ASAP1 not only regulates actin cytoskeleton remodeling through its interaction with nonmuscle myosin 2A, but is also important in integrin recycling. These examples illustrate the diversity and versatility of Arf GAPs as regulators of integrin adhesion complex structure and function. [ABSTRACT FROM AUTHOR]

Arf GTPase-activating proteins (Arf GAPs) were first identified as regulators of the small GTP-binding proteins ADP-ribosylation factors (Arfs). The Arf GAPs are a large family of proteins in metazoans, outnumbering the Arfs that they regulate. The members of the Arf GAP family have complex domain structures and some have been implicated in particular cellular functions, such as cell migration, or with particular pathologies, such as tumor invasion and metastasis. The specific effects of Arfs sometimes depend on the Arf GAP involved in their regulation. These observations have led to speculation that the Arf GAPs themselves may affect cellular activities in capacities beyond the regulation of Arfs. Recently, 2 Arf GAPs, ASAP1 and AGAP1, have been found to bind directly to and influence the activity of myosins and kinesins, motor proteins associated with filamentous actin and microtubules, respectively. The Arf GAP-motor protein interaction is critical for cellular behaviors involving the actin cytoskeleton and microtubules, such as cell migration and other cell movements. Arfs, then, may function with molecular motors through Arf GAPs to regulate microtubule and actin remodeling. [ABSTRACT FROM AUTHOR]

A switch in the response of commissural axons to the repellent Slit is crucial for ensuring that they cross the ventral midline only once. However, the underlyingmechanisms remain to be elucidated. We have found that both endocytosis and recycling of Robo1 receptor are crucial for modulating Slit sensitivity in vertebrate commissural axons. Robo1 endocytosis and its recycling back to the cell surface maintained the stability of axonal Robo1 during Slit stimulation. We identified Arf6 guanosine triphosphatase and its activators, cytohesins, as previously unknowncomponents inSlit-Robo1 signalling in vertebrate commissural neurons. Slit-Robo1 signalling activated Arf6. The Arf6-deficient mice exhibited marked defects in commissural axon midline crossing. Our data showed that a Robo1 endocytosis-triggered and Arf6-mediated positive-feedback strengthens the Slit response in commissural axons upon their midline crossing. Furthermore, the cytohesin-Arf6 pathways modulated this self-enhancement of the Slit response before and after midline crossing, resulting in a switch that reinforced robust regulation of axon midline crossing. Our study provides insights into endocytic trafficking-mediated mechanisms for spatiotemporally controlled axonal responses and uncovers new players in the midline switch in Slit responsiveness of commissural axons. [ABSTRACT FROM AUTHOR]

Abiotic stress poses constant challenges for plant survival and is a serious problem for global agricultural productivity. On a molecular level, stress conditions result in elevation of reactive oxygen species (ROS) production causing oxidative stress associated with oxidation of proteins and nucleic acids as well as impairment of membrane functions. Adaptation of root growth to ROS accumulation is facilitated through modification of auxin and cytokinin hormone homeostasis. Here, we report that in Arabidopsis root meristem, ROS-induced changes of auxin levels correspond to decreased abundance of PIN auxin efflux carriers at the plasma membrane (PM). Specifically, increase in H2O2 levels affects PIN2 endocytic recycling. We show that the PIN2 intracellular trafficking during adaptation to oxidative stress requires the function of the ADP-ribosylation factor (ARF)-guanine-nucleotide exchange factor (GEF) BEN1, an actin-associated regulator of the trafficking from the PM to early endosomes and, presumably, indirectly, trafficking to the vacuoles. We propose that H2O2 levels affect the actin dynamics thus modulating ARF-GEF-dependent trafficking of PIN2. This mechanism provides a way how root growth acclimates to stress and adapts to a changing environment. [ABSTRACT FROM AUTHOR]

The Arf family of small GTP‐binding and ‐hydrolyzing proteins are some of the most important intracellular regulators of membrane dynamics. In this study, we identified the Golgi‐localized Arf family G protein Arf1 in Candida albicans and confirmed its conserved function in regulating the secretory pathway. Interestingly, deletion of ARF1 resulted in intracellular reactive oxygen species (ROS) accumulation, and induced formation of the endoplasmic reticulum (ER)–mitochondria encounter structure (ERMES). Moreover, N‐acetylcysteine‐mediated ROS scavenging in the arf1Δ/Δ strain attenuated ERMES formation, indicating that intracellular ROS accumulation resulting from ARF1 deletion facilitated ERMES formation. In addition, Arf1 regulated many key physiological processes in C. albicans, including cell cycle progression, morphogenesis and virulence. This study uncovers a role for Arf family G proteins in regulating ERMES formation and sheds new light on the crucial contribution of ROS to membrane dynamics. [ABSTRACT FROM AUTHOR]

The inositol polyphosphate 5′-phosphatase E (INPP5E) localizes to cilia. We showed that the carrier protein phosphodiesterase 6 delta subunit (PDE6δ) mediates the sorting of farnesylated INPP5E into cilia due to high affinity binding and release by the ADP-ribosylation factor (Arf)-like protein Arl3·GTP. However, the dynamics of INPP5E transport into and inside the ciliary compartment are not fully understood. Here, we investigate the movement of INPP5E using live cell fluorescence microscopy and fluorescence recovery after photobleaching (FRAP) analysis. We show that PDE6δ and the dynein transport system are essential for ciliary sorting and entry of INPP5E. However, its innerciliary transport is regulated solely by the intraflagellar transport (IFT) system, independent from PDE6δ activity and INPP5E farnesylation. By contrast, movement of Arl3 into and within cilia occurs freely by diffusion and IFT-independently. The farnesylation defective INPP5E CaaX box mutant loses the exclusive ciliary localization. The accumulation of this mutant at centrioles after photobleaching suggests an affinity trap mechanism for ciliary entry, that in case of the wild type is overcome by the interaction with PDE6δ. Collectively, we postulate a three-step mechanism regulating ciliary localization of INPP5E, consisting of farnesylation- and PDE6δ-mediated targeting, INPP5E-PDE6δ complex diffusion into the cilium with transfer to the IFT system, and retention inside cilia. [ABSTRACT FROM AUTHOR]

Arf6 (ADP-ribosylation factor 6) is known to play important roles in membrane dynamics through the regulation of actin filament reorganization for multiple cellular processes such as cytokinesis, phagocytosis, cell migration and tumor cell invasion. However, the functions of Arf6 in mammalian oocyte meiosis have not been clarified. In present study we showed that Arf6 expressed in mouse oocytes and was mainly distributed around the spindle during meiosis. Depletion of Arf6 by morpholino microinjection caused oocytes failing to extrude first polar body. Further analysis indicated that Arf6 knock down caused the aberrant actin distribution, which further induced the failure of meiotic spindle movement. And the loss of oocyte polarity also confirmed this. The regulation of Arf6 on actin filaments in mouse oocytes might be due to its effects on the phosphorylation level of cofilin and the expression of Arp2/3 complex. Moreover, we found that the decrease of Arf6 caused the disruption of spindle formation, indicating the multiple roles of Arf6 on cytoskeleton dynamics in meiosis. In summary, our results indicated that Arf6 was involved in mouse oocyte meiosis through its functional roles in actin-mediated spindle movement and spindle organization. [ABSTRACT FROM AUTHOR]

ADP-ribosylation factors (ARF) GTPases are activated by guanine nucleotide exchange factors (GEFs) to support cellular homeostasis. Key to understanding spatio-temporal regulation of ARF signaling is the mechanism of GEF recruitment to membranes. Small GEFs are recruited through phosphoinositide (PIP) binding by a pleckstrin homology (PH) domain downstream from the catalytic Sec7 domain (Sec7d). The large GEFs lack PH domains, and their recruitment mechanisms are poorly understood. We probed Golgi recruitment of GBF1, a GEF catalyzing ARF activation required for Golgi homeostasis. We show that the homology downstream of Sec7d-1 (HDS1) regulates Golgi recruitment of GBF1. We document that GBF1 binds phosphoinositides, preferentially PI3P, PI4P and PI(4,5)P2, and that lipid binding requires the HDS1 domain. Mutations within HDS1 that reduce GBF1 binding to specific PIPs in vitro inhibit GBF1 targeting to Golgi membranes in cells. Our data imply that HDS1 and PH domains are functionally analogous in that each uses lipid-based membrane information to regulate GEF recruitment. Lipid-based recruitment of GBF1 extends the paradigm of lipid regulation to small and large GEFs and suggests that lipid-based mechanisms evolved early during GEF diversification. [ABSTRACT FROM AUTHOR]

ADP-ribosylation factor 6 (Arf6), a member of the ADP-ribosylation factor family, is overexpressed in different types of cancer cell and promotes invasion, metastasis and drug resistance. However, the potential functions of Arf6 in gastric cancer (GC), and the molecular mechanism underlying these functions, remain to be fully elucidated. In the present study, the results demonstrated that in vitro knockdown of Arf6 decreased proliferation, colony formation, migration and invasion in SGC-7901 cells. Arf6 knockdown also markedly decreased the activity of the extracellular signal-regulated kinase 1/2 (ERK1/2) signaling pathway. Furthermore, knockdown of Arf6 was associated with elevated chemosensitivity of SGC-7901 cells to 5-fluorouracil through inactivation of the ERK1/2 signaling pathway. Taken together, these results suggest that Arf6 is involved in regulating proliferation, migration, invasion and drug resistance in GC, and may be a potential therapeutic target for the treatment of GC. [ABSTRACT FROM AUTHOR]

The ADP-ribosylation factor (Arf) small G proteins act as molecular switches to coordinate multiple downstream pathways that regulate membrane dynamics. Their activation is spatially and temporally controlled by the guanine nucleotide exchange factors (GEFs). Members of the evolutionarily conserved GBF/Gea family of Arf GEFs are well known for their roles in formation of coat protein complex I (COPI) vesicles, essential for maintaining the structure and function of the Golgi apparatus. However, studies over the past 10 years have found new functions for these GEFs, along with their substrate Arf1, in lipid droplet metabolism, clathrin-independent endocytosis, signalling at the plasma membrane, mitochondrial dynamics and transport along microtubules. Here, we describe these different functions, focussing in particular on the emerging theme of GFB1 and Arf1 regulation of organelle movement on microtubules. [ABSTRACT FROM AUTHOR]