Adenosine Diphosphate Ribosylation Factor Research Articles

Vibrio MARTX toxin processing and degradation of cellular Rab GTPases by the cytotoxic effector Makes Caterpillars Floppy

Vibrio vulnificus causes life-threatening wound and gastrointestinal infections, mediated primarily by the production of a Multifunctional-Autoprocessing Repeats-In-Toxin (MARTX) toxin. The most commonly present MARTX effector domain, the Makes Caterpillars Floppy-like (MCF) toxin, is a cysteine protease stimulated by host adenosine diphosphate (ADP) ribosylation factors (ARFs) to autoprocess. Here, we show processed MCF then binds and cleaves host Ras-related proteins in brain (Rab) guanosine triphosphatases within their C-terminal tails resulting in Rab degradation. We demonstrate MCF binds Rabs at the same interface occupied by ARFs. Moreover, we show MCF preferentially binds to ARF1 prior to autoprocessing and is active to cleave Rabs only subsequent to autoprocessing. We then use structure prediction algorithms to demonstrate that structural composition, rather than sequence, determines Rab target specificity. We further determine a crystal structure of aMCF as a swapped dimer, revealing an alternative conformation we suggest represents the open, activated state of MCF with reorganized active site residues. The cleavage of Rabs results in Rab1B dispersal within cells and loss of Rab1B density in the intestinal tissue of infected mice. Collectively, our work describes an extracellular bacterial mechanism whereby MCF is activated by ARFs and subsequently induces the degradation of another small host guanosine triphosphatase (GTPase), Rabs, to drive organelle damage, cell death, and promote pathogenesis of these rapidly fatal infections.

Adenosine diphosphate ribosylation factor 6 inhibition protects burn sepsis induced lung injury through preserving vascular integrity and suppressing ASC inflammasome transmission

BackgroundSevere burns are devastating injuries with significant immune dysfunction and result in substantial mortality and morbidity due to sepsis induced organ failure. Acute lung injury is the most common type of organ injury in sepsis, however, the mechanisms of which are poorly understood and effective therapeutic measures are limited. This study is aimed to investigate the effect of a small Guanosine triphosphatase (GTPase), Adenosine diphosphate ribosylation factor 6 (ARF6), on burn sepsis induced lung injury, and discuss the possible mechanisms. MethodsBurn sepsis was established in male C57BL/6 mice. Mice were anesthetised by intramuscular injection of ketamine and xylazine hydrochloride, then 30% TBSA full thickness burn followed by sub-eschar injection of lipopolysaccharide. Animals were treated with intraperitoneal injection of a small molecule inhibitor of ARF6: NAV-2729, or vehicle, right after the burn and sepsis stimuli were inflicted. Lung tissues were harvested for histopathological observation and the acute lung injury scores were calculated. Organ permeability, Vascular Endothelial Cadherin (VE-cadherin) expression, inflammatory cytokine levels and myeloperoxidase activity in lung tissues were detected. Rat pulmonary microvascular endothelial cells (PMVECs) were stimulated by burn sepsis serum with or without 10 μM NAV-2729. The ARF6 activation, VE-cadherin expression, inflammasome activity, adapter protein apoptosis speck-like protein containing a caspase recruiting domain (ASC) specks and cytokines secretion were determined. Student’s t test was used for comparison between two groups. Multiple comparisons among groups were performed by using analysis of variance, with Tukey’s test for the post hoc test. ResultsNAV-2729 treatment attenuated burn sepsis induced lung injury and promoted survival of burn septic mice by preserving VE-cadherin expression in endothelial cell adherent junction and limited vascular hyperpermeability in lung tissues. Moreover, inflammatory cytokine expression and inflammatory injury in lung tissues were alleviated. Mechanistically, NAV-2729 enhanced vascular integrity by inhibiting ARF6 activation and restoring VE-cadherin expression in PMVECs. In addition, NAV-2729 inhibited ARF6-dependent phagocytosis of ASC specks, thus preventing inflammation propagation mediated by cell-to-cell transmission of ASC specks. ConclusionsARF6 inhibition preserved vascular integrity by restoring expression of VE-cadherin and suppressed the spread of inflammation by affecting phagocytosis of ASC specks, thus protected against sepsis induced lung injury and improve survival of burn septic animals. The findings of this study implied potential therapeutics by which ARF6 inhibition can protect lung function from septic induced lung injury and improve outcomes in burn sepsis.

Selection and Validation of Reference Genes in Virus-Infected Sweet Potato Plants.

Quantitative real-time PCR (qRT-PCR) in sweet potatoes requires accurate data normalization; however, there are insufficient studies on appropriate reference genes for gene expression analysis. We examined variations in the expression of eight candidate reference genes in the leaf and root tissues of sweet potatoes (eight nonvirus-infected or eight virus-infected samples). Parallel analyses with geNorm, NormFinder, and Best-Keeper show that different viral infections and origin tissues affect the expression levels of these genes. Based on the results of the evaluation of the three software, the adenosine diphosphate-ribosylation factor is suitable for nonvirus or virus-infected sweet potato leaves. Cyclophilin and ubiquitin extension proteins are suitable for nonvirus-infected sweet potato leaves. Phospholipase D1 alpha is suitable for virus-infected sweet potato leaves. Actin is suitable for roots of nonvirus-infected sweet potatoes. Glyceraldehyde-3-phosphate dehydrogenase is suitable for virus-infected sweet potato roots. The research provides appropriate reference genes for further analysis in leaf and root samples of viruses in sweet potatoes.

Arf6 regulates endometriotic epithelial-mesenchymal transition and mitochondrial distribution

Objective: To investigate the role of adenosine diphosphate ribosylation factor 6 (Arf6) in the pathogenesis of endometriosis. Methods: Endometrial tissues were sampled from women who were hospitalized in the Affiliated Hospital of Medical School of Ningbo University and Ningbo Women and Children's Hospital from November 2020 to May 2021 with endometriosis (n=44, endometriosis group) and without endometriosis (n=17, control group). The expression of Arf6 protein in the endometrial tissues was detected by western blot. Endometrial epithelial cells from both groups were primary cultured and the distribution of intracellular mitochondria was detected by immunofluorescence. The expression of Arf6 protein was down-regulated by small interference RNA (siRNA), the distribution of mitochondria in cells with decreased Arf6 protein expression was observed, and the expression of mitochondria-related proteins development and differentiation enhancing factor 1 (DDEF1, also called AMAP1), reactive oxygen species 1 (ROS1) and epithelial-mesenchymal transition (EMT)-related proteins E-cadherin, vimentin were detected. Transwell assay was used to detect the changes in the migration ability of the cells. Results: Compared with the control group, ectopic endometrial tissue of endometriosis group showed high expression of Arf6 protein (0.174±0.019 vs 0.423±0.033; t=29.630, P<0.01); and in ectopic endometrial epithelial cells, mitochondria were distributed near the edge of the cell membrane. While Arf6 expression was down-regulated by siRNA, the distribution of mitochondria in ectopic cells returned to natural, close to the control level. In addition, the expression levels of AMAP1 and ROS1 in ectopic cells after Arf6 protein knockdown were significantly decreased. Transwell assay results indicated that knockdown of Arf6 could reduce the migration ability of ectopic epithelial cells [migration cell count: (34.3±7.5) cells]; and immunofluorescence verified low expression of E-cadherin but high expression of vimentin in ectopic epithelial cells, whereas knockdown of Arf6 protein E-cadherin expression increased but vimentin expression decreased. Conclusions: High expression of Arf6 protein in ectopic endometrial epithelial cells leads to the distribution of mitochondria tending to membrane marginalization, while inducing EMT, which are involved in the mechanism of endoheterosis pathogenesis.

IQSEC3 Deletion Impairs Fear Memory Through Upregulation of Ribosomal S6K1 Signaling in the Hippocampus

BackgroundIQSEC3, a gephyrin-binding GABAergic (gamma-aminobutyric acidergic) synapse-specific guanine nucleotide exchange factor, was recently reported to regulate activity-dependent GABAergic synapse maturation, but the underlying signaling mechanisms remain incompletely understood. MethodsWe generated mice with conditional knockout (cKO) of Iqsec3 to examine whether altered synaptic inhibition influences hippocampus-dependent fear memory formation. In addition, electrophysiological recordings, immunohistochemistry, and behavioral assays were used to address our question. ResultsWe found that Iqsec3-cKO induces a specific reduction in GABAergic synapse density, GABAergic synaptic transmission, and maintenance of long-term potentiation in the hippocampal CA1 region. In addition, Iqsec3-cKO mice exhibited impaired fear memory formation. Strikingly, Iqsec3-cKO caused abnormally enhanced activation of ribosomal P70-S6K1–mediated signaling in the hippocampus but not in the cortex. Furthermore, inhibiting upregulated S6K1 signaling by expressing dominant-negative S6K1 in the hippocampal CA1 of Iqsec3-cKO mice completely rescued impaired fear learning and inhibitory synapse density but not deficits in long-term potentiation maintenance. Finally, upregulated S6K1 signaling was rescued by IQSEC3 wild-type, but not by an ARF-GEF (adenosine diphosphate ribosylation factor–guanine nucleotide exchange factor) inactive IQSEC3 mutant. ConclusionsOur results suggest that IQSEC3-mediated balanced synaptic inhibition in hippocampal CA1 is critical for the proper formation of hippocampus-dependent fear memory.

The Coordinated KNR6-AGAP-ARF1 Complex Modulates Vegetative and Reproductive Traits by Participating in Vesicle Trafficking in Maize.

The KERNEL NUMBER PER ROW6 (KNR6)-mediated phosphorylation of an adenosine diphosphate ribosylation factor (Arf) GTPase-activating protein (AGAP) forms a key regulatory module for the numbers of spikelets and kernels in the ear inflorescences of maize (Zea mays L.). However, the action mechanism of the KNR6–AGAP module remains poorly understood. Here, we characterized the AGAP-recruited complex and its roles in maize cellular physiology and agronomically important traits. AGAP and its two interacting Arf GTPase1 (ARF1) members preferentially localized to the Golgi apparatus. The loss-of-function AGAP mutant produced by CRISPR/Cas9 resulted in defective Golgi apparatus with thin and compact cisternae, together with delayed internalization and repressed vesicle agglomeration, leading to defective inflorescences and roots, and dwarfed plants with small leaves. The weak agap mutant was phenotypically similar to knr6, showing short ears with fewer kernels. AGAP interacted with KNR6, and a double mutant produced shorter inflorescence meristems and mature ears than the single agap and knr6 mutants. We hypothesized that the coordinated KNR6–AGAP–ARF1 complex modulates vegetative and reproductive traits by participating in vesicle trafficking in maize. Our findings provide a novel mechanistic insight into the regulation of inflorescence development, and ear length and kernel number, in maize.

De novo ARF3 variants cause neurodevelopmental disorder with brain abnormality.

An optimal Golgi transport system is important for mammalian cells. The adenosine diphosphate (ADP) ribosylation factors (ARF) are key proteins for regulating cargo sorting at the Golgi network. In this family, ARF3 mainly works at the trans-Golgi network (TGN), and no ARF3-related phenotypes have yet been described in humans. We here report the clinical and genetic evaluations of two unrelated children with de novo pathogenic variants in the ARF3 gene: c.200A > T (p.Asp67Val) and c.296G > T (p.Arg99Leu). Although the affected individuals presented commonly with developmental delay, epilepsy and brain abnormalities, there were differences in severity, clinical course and brain lesions. In vitro subcellular localization assays revealed that the p.Arg99Leu mutant localized to Golgi apparatus, similar to the wild-type, whereas the p.Asp67Val mutant tended to show a disperse cytosolic pattern together with abnormally dispersed Golgi localization, similar to that observed in a known dominant negative variant (p.Thr31Asn). Pull-down assays revealed that the p.Asp67Val had a loss-of-function effect and the p.Arg99Leu variant had increased binding of the adaptor protein, Golgi-localized, γ-adaptin ear-containing, ARF-binding protein 1 (GGA1), supporting the gain of function. Furthermore, in vivo studies revealed that p.Asp67Val transfection led to lethality in flies. In contrast, flies expressing p.Arg99Leu had abnormal rough eye, as observed in the gain-of-function variant p.Gln71Leu. These data indicate that two ARF3 variants, the possibly loss-of-function p.Asp67Val and the gain-of-function p.Arg99Leu, both impair the Golgi transport system. Therefore, it may not be unreasonable that they showed different clinical features like diffuse brain atrophy (p.Asp67Val) and cerebellar hypoplasia (p.Arg99Leu).

TNF-α/NF-κB signaling epigenetically represses PSD4 transcription to promote alcohol-related hepatocellular carcinoma progression.

BackgroundChronic alcohol consumption is more frequently associated with advanced, aggressive hepatocellular carcinoma (HCC) tumors. Alcohol adversely impacts ER/Golgi membrane trafficking and Golgi protein N‐glycosylation in hepatocytes; these effects have been attributed (in part) to dysregulated adenosine diphosphate‐ribosylation factor (ARF) GTPase signaling. Here, we investigated the role of the ARF GTPase guanine exchange factor PSD4 in HCC progression.MethodsR‐based bioinformatics analysis was performed on publicly available array data. Modulating gene expression was accomplished via lentiviral vectors. Gene expression was analyzed using quantitative real‐time PCR and immunoblotting. PSD4 promoter methylation was assessed using quantitative methylation‐specific PCR. Phospho‐p65(S276)/DNMT1 binding to the PSD4 promoter was analyzed via chromatin immunoprecipitation. We constructed ethanol/DEN‐induced and DEN only‐induced transgenic murine models of HCC.ResultsWe identified PSD4 as a hypermethylated, suppressed gene in alcohol‐related HCC tumors; however, PSD4 was not dysregulated in all‐cause HCC tumors. Certain HCC cell lines also displayed varying degrees of PSD4 downregulation. PSD4 overexpression or knockdown decreased and increased cell migration and invasiveness, respectively. Mechanistically, PSD4 transcription was repressed by TNF‐α‐induced phospho‐p65(S276)’s recruitment of DNA methyltransferase 1 (DNMT1), resulting in PSD4 promoter methylation. PSD4 inhibited pro‐EMT CDC42 activity, resulting in downregulation of E‐cadherin and upregulation of N‐cadherin and vimentin. Hepatocyte‐specific PSD4 overexpression reduced ethanol/DEN‐induced HCC tumor progression and EMT marker expression in vivo.ConclusionsPSD4 is a hypermethylated, suppressed gene in alcohol‐related HCC tumors that negatively modulated pro‐EMT CDC42 activity. Furthermore, we present a novel phospho‐NF‐κB p65(S276)/DNMT1‐mediated promoter methylation mechanism by which TNF‐α/NF‐κB signaling represses PSD4 transcription in HCC cells.

Expression of ASAP1 and FAK in gastric cancer and its clinicopathological significance.

The present study aimed to analyze the expression levels of adenosine diphosphate ribosylation factor guanylate kinase 1 (ASAP1) and focal adhesion kinase (FAK) in gastric cancer (GC) tissues in order to explore their association with clinicopathological features and prognosis. A total of 32 patients with GC were enrolled in the present study. All patients had complete clinical follow-up data and paraffin-embedded normal gastric mucosal tissues. The expression levels of ASAP1 and FAK in these tissues were measured by immunohistochemistry. The associations of ASAP1 and FAK expression with clinicopathological factors and the survival of patients with GC were subsequently analyzed. The expression levels of ASAP1 (59.4%) and FAK (68.8%) in GC tissues were significantly higher than those in normal gastric mucosal tissues (28.1 and 40.6%, P<0.05). The expression levels of ASAP1 and FAK were associated with depth of invasion, lymph node metastasis and pathological stage (P<0.05). ASAP1 expression was positively associated with FAK expression (P<0.001). In addition, ASAP1 and FAK expression levels were negatively associated with disease-free survival time and overall survival time (P<0.05). The 5-year overall survival rate was significantly higher in patients with negative ASAP1 or FAK expression compared with that in patients with positive ASAP1 or FAK expression (P<0.05). In conclusion, ASAP1 and FAK were highly expressed in human GC tissues and may serve a synergistic role in promoting tumorigenesis, progression, invasion and metastasis in patients with GC. ASAP1 and FAK expression in GC were associated with patient's survival. Therefore, ASAP1 and FAK may represent novel molecular markers for the pathophysiology and prognosis of GC.

Regulation of mTORC1 by Small GTPases in Response to Nutrients

Mechanistic target of rapamycin complex 1 (mTORC1) is a highly evolutionarily conserved serine/threonine kinase that regulates cell growth and metabolism in response to multiple environmental cues, such as nutrients, hormones, energy, and stress. Deregulation of mTORC1 can lead to diseases such as diabetes, obesity, and cancer. A series of small GTPases, including Rag, Ras homolog enriched in brain (Rheb), adenosine diphosphate ribosylation factor 1 (Arf1), Ras-related protein Ral-A, Ras homolog (Rho), and Rab, are involved in regulating mTORC1 in response to nutrients, and mTORC1 is differentially regulated via these small GTPases according to specific conditions. Leucine and arginine sensing are considered to be well-confirmed amino acid-sensing signals, activating mTORC1 via a Rag GTPase-dependent mechanism as well as the Ragulator complex and vacuolar H+-adenosine triphosphatase (v-ATPase). Glutamine promotes mTORC1 activation via Arf1 independently of the Rag GTPase. In this review, we summarize current knowledge regarding the regulation of mTORC1 activity by small GTPases in response to nutrients, focusing on the function of small GTPases in mTORC1 activation and how small GTPases are regulated by nutrients.

Expression of ArfGAP3 in Vaginal Anterior Wall of Patients With Pelvic Floor Organ Prolapse in Pelvic Organ Prolapse and Non-Pelvic Organ Prolapse Patients.

Purpose of InvestigationThe purpose of this study was to study the expression of adenosine diphosphate ribosylation factor GTPase-activating protein 3 (ArfGAP3) in the anterior vaginal wall of patients with pelvic organ prolapse (POP).Materials and MethodsFrom July 2016 to July 2018, the anterior vaginal wall of 31 POP patients (pelvic organ prolapse quantification [POP-Q] II-III [n = 10] and POP-Q IV [n = 21]) with pelvic floor dysfunction-related symptoms who underwent vaginal hysterectomy were enrolled in POP group in the Department of Gynecology of Wuhan University People’s Hospital. The anterior vaginal wall of 28 non-POP patients who underwent vaginal hysterectomy was selected as control group. The expression of 3 groups was determined by immunohistochemical staining, Western blotting, and quantitative real-time fluorescence polymerase chain reaction.ResultsThe expression levels of ArfGAP3 of POP-Q II-III and POP-Q IV groups were lower than the control group (P < 0.05), and there were significant differences between POP-Q II-III and POP-Q IV groups (P < 0.05).ConclusionsThe expression of ArfGAP3 in the anterior vaginal wall of POP patients decreased, which was related to the pathogenesis and clinical grading of POP.

The Role of ARF Family Proteins and Their Regulators and Effectors in Cancer Progression: A Therapeutic Perspective.

The Adenosine diphosphate-Ribosylation Factor (ARF) family belongs to the RAS superfamily of small GTPases and is involved in a wide variety of physiological processes, such as cell proliferation, motility and differentiation by regulating membrane traffic and associating with the cytoskeleton. Like other members of the RAS superfamily, ARF family proteins are activated by Guanine nucleotide Exchange Factors (GEFs) and inactivated by GTPase-Activating Proteins (GAPs). When active, they bind effectors, which mediate downstream functions. Several studies have reported that cancer cells are able to subvert membrane traffic regulators to enhance migration and invasion. Indeed, members of the ARF family, including ARF-Like (ARL) proteins have been implicated in tumorigenesis and progression of several types of cancer. Here, we review the role of ARF family members, their GEFs/GAPs and effectors in tumorigenesis and cancer progression, highlighting the ones that can have a pro-oncogenic behavior or function as tumor suppressors. Moreover, we propose possible mechanisms and approaches to target these proteins, toward the development of novel therapeutic strategies to impair tumor progression.

Abstract 4367: The small GTPase ARF6 is necessary for melanomagenesis

Abstract ARF6 is a member of the adenosine diphosphate (ADP)-ribosylation factor (ARF) family of small GTPases and is part of the RAS superfamily. We have shown that pharmacologic inhibition of ARF6 can inhibit proliferation of uveal melanoma and invasion/metastasis of cutaneous melanoma. Most cutaneous melanomas are defined by mutually-exclusive driver mutations in BRAF, NRAS, or NF1. These subtypes share similar mechanisms of oncogenesis because they align along a continuum of RAS signaling. Mutations in the PI3K pathway occur in a fraction of all melanoma subtypes, indicating a selective advantage for PI3K/AKT activation beyond BRAF or RAS activation. We and others have reported ARF6-dependent ERK activation occurring upon either extracellular ligand stimulation or mutant oncoprotein signaling. Recently, ARF6 has also been shown to be upstream of PI3K signaling. Based on these findings, we hypothesized that ARF6 is critical for both the RAF and PI3K arms of the RAS pathway and that loss of ARF6 will impede tumor growth driven by these oncogenes. We tested this hypothesis using human melanoma cell lines and with an inducible, genetically engineered mouse model (GEMM) of melanoma. Our data reveal that pharmacologic inhibition of ARF6 can reduce proliferation of BRAF, NRAS and NF1-mutated melanoma cells. Furthermore, loss of Arf6 causes a reduction in mean tumor size and improves survival in BRAFV600E/CDKN2ANULL/PTENNULL/AKTE17K and BRAFV600E/CDKN2ANULL/PTENNULL mice. These results indicate that ARF6 is necessary for melanomagenesis and for oncogenic BRAF and/or PI3K signaling. Our data suggest that ARF6 may be an actionable node in the RAS pathway and that therapeutic targeting of ARF6 has the potential to improve current treatment methods for progressive disease. Citation Format: Lehi Acosta, Aaron Rogers, Jingfu Peng, Alan Mueller, Zongzhong Tong, Donghan Shin, Jae Hyuk Yoo, Dean Y. Li, Shannon J. Odelberg, Sheri L. Holmen, Allie H. Grossmann. The small GTPase ARF6 is necessary for melanomagenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4367.

Plasma membrane proteome analysis identifies a role of barley membrane steroid binding protein in root architecture response to salinity.

Although the physiological consequences of plant growth under saline conditions have been well described, understanding the core mechanisms conferring plant salt adaptation has only started. We target the root plasma membrane proteomes of two barley varieties, cvs. Steptoe and Morex, with contrasting salinity tolerance. In total, 588 plasma membrane proteins were identified by mass spectrometry, of which 182 were either cultivar or salinity stress responsive. Three candidate proteins with increased abundance in the tolerant cv. Morex were involved either in sterol binding (a GTPase-activating protein for the adenosine diphosphate ribosylation factor [ZIGA2], and a membrane steroid binding protein [MSBP]) or in phospholipid synthesis (phosphoethanolamine methyltransferase [PEAMT]). Overexpression of barley MSBP conferred salinity tolerance to yeast cells, whereas the knock-out of the heterologous AtMSBP1 increased salt sensitivity in Arabidopsis. Atmsbp1 plants showed a reduced number of lateral roots under salinity, and root-tip-specific expression of barley MSBP in Atmsbp1 complemented this phenotype. In barley, an increased abundance of MSBP correlates with reduced root length and lateral root formation as well as increased levels of auxin under salinity being stronger in the tolerant cv. Morex. Hence, we concluded the involvement of MSBP in phytohormone-directed adaptation of root architecture in response to salinity.

RASSF4 controls SOCE and ER-PM junctions through regulation of PI(4,5)P2.

RAS association domain family 4 (RASSF4) is involved in tumorigenesis and regulation of the Hippo pathway. In this study, we identify new functional roles of RASSF4. First, we discovered that RASSF4 regulates store-operated Ca2+ entry (SOCE), a fundamental Ca2+ signaling mechanism, by affecting the translocation of the endoplasmic reticulum (ER) Ca2+ sensor stromal interaction molecule 1 (STIM1) to ER-plasma membrane (PM) junctions. It was further revealed that RASSF4 regulates the formation of ER-PM junctions and the ER-PM tethering function of extended synaptotagmins E-Syt2 and E-Syt3. Moreover, steady-state PM phosphatidylinositol 4,5-bisphosphate (PI[4,5]P2) levels, important for localization of STIM1 and E-Syts at ER-PM junctions, were reduced in RASSF4-knockdown cells. Furthermore, we demonstrated that RASSF4 interacts with and regulates the activity of adenosine diphosphate ribosylation factor 6 (ARF6), a small G protein and upstream regulator of type I phosphatidylinositol phosphate kinases (PIP5Ks) and PM PI(4,5)P2 levels. Overall, our study suggests that RASSF4 controls SOCE and ER-PM junctions through ARF6-dependent regulation of PM PI(4,5)P2 levels, pivotal for a variety of physiological processes.

ADP-Ribosylation Factor 1 Regulates Proliferation, Migration, and Fusion in Early Stage of Osteoclast Differentiation.

Small G-protein adenosine diphosphate (ADP)-ribosylation factors (ARFs) regulate a variety of cellular functions, including actin cytoskeleton remodeling, plasma membrane reorganization, and vesicular transport. Here, we propose the functional roles of ARF1 in multiple stages of osteoclast differentiation. ARF1 was upregulated during receptor activator of nuclear factor kappa-B ligand (RANKL)-induced osteoclast differentiation and transiently activated in an initial stage of their differentiation. Differentiation of ARF1-deficient osteoclast precursors into mature osteoclasts temporarily increased in pre-maturation stage of osteoclasts followed by reduced formation of mature osteoclasts, indicating that ARF1 regulates the osteoclastogenic process. ARF1 deficiency resulted in reduced osteoclast precursor proliferation and migration as well as increasing cell-cell fusion. In addition, ARF1 silencing downregulated c-Jun N-terminal kinase (JNK), Akt, osteopontin, and macrophage colony-stimulating factor (M-CSF)-receptor c-Fms as well as upregulating several fusion-related genes including CD44, CD47, E-cadherin, and meltrin-α. Collectively, we showed that ARF1 stimulated proliferation and migration of osteoclast precursors while suppressing their fusion, suggesting that ARF1 may be a plausible inter-player that mediates the transition to osteoclast fusion at multiple steps during osteoclast differentiation

Genome-wide association studies in biliary atresia.

Biliary atresia (BA) is a model complex disease resulting from interactions between multiple susceptibility loci and environmental factors. This perception is based on a heterogeneous phenotype extending beyond an absent extrahepatic bile duct to include gut and cardiovascular anomalies, and the association of BA with viral infections. Refractory jaundice and progression to cirrhosis shortly after birth can be fatal without surgical correction, and further suggests a pathogenesis during liver and bile duct development. Conclusive proof for a developmental origin would require documentation of disease progression in the perinatal or fetal liver, an impossible task for obvious reasons. We review three different sets of genome-wide association studies (GWAS) from three different cohorts of BA patients by three different groups of investigators, which address this knowledge gap. Knockdown of each susceptibility gene identified by GWAS in zebrafish embryos impairs excretion of bile from the liver, duplicating the characteristic diagnostic finding seen in affected children. This finding is associated with impaired intrahepatic biliary network formation in zebrafish morphants. Although distinct, these susceptibility genes share several functions including roles in mechanisms for organogenesis (glypican 1 or GPC1, and adenosine diphosphate ribosylation factor 6, or ARF6) or a greater expression in fetal liver than in adult liver (adducin 3 or ADD3). Together, these studies emphasize the importance of the human evidence, and present opportunities to map novel pathways which explain the phenotypic heterogeneity of BA.

An ACAP1 coat complex acting in endocytic recycling.

A key function of coat proteins is the sorting of protein cargoes into intracellular transport pathways. For many years, however, it has been unclear whether this role of coat proteins would apply to pathways of endocytic recycling. This issue has been clarified in recent years through the identification of multiple coat complexes acting in the recycling pathways. Leading this charge have been studies on a coat complex defined by ACAP1 (adenosine diphosphate ribosylation factor GTPase-activating proteins with Coiled-coil, Ankryin repeat and PH domains 1), which acts in the sorting of cargoes at the recycling endosome for their return to the plasma membrane. This chapter describes the methods used to characterize this role of ACAP1.

Proteomic profiling reveals autoimmune targets in sarcoidosis.

There is a need to further characterize the antibody repertoire in relation to sarcoidosis and potentially related autoantigens. We investigated bronchoalveolar lavage (BAL) and serum samples from patients with sarcoidosis and healthy and diseased control subjects to discover sarcoidosis-associated autoantigens. Antigen microarrays built on 3,072 protein fragments were used to screen for IgG reactivity in 73 BAL samples from subjects with sarcoidosis, subjects with asthma, and healthy subjects. A set of 131 targets were selected for subsequent verification on suspension bead arrays using 272 additional BAL samples and 141 paired sera. Reactivity to four antigens was furthermore analyzed in 22 unprocessed BAL samples from patients with fibrosis and 269 plasma samples from patients diagnosed with myositis. Reactivity toward zinc finger protein 688 and mitochondrial ribosomal protein L43 were discovered with higher frequencies in patients with sarcoidosis, for mitochondrial ribosomal protein L43 especially in patients with non-Löfgren syndrome. Increased reactivity toward nuclear receptor coactivator 2 was also observed in patients with non-Löfgren syndrome as compared with patients with Löfgren syndrome. The antigen representing adenosine diphosphate-ribosylation factor GTPase activating protein 1 revealed high reactivity frequency in all sample groups but with significantly higher level of IgG reactivities in patients with sarcoidosis. Autoantigen reactivity was present in most BAL and serum samples analyzed, and the results revealed high interindividual heterogeneity, with most of the reactivities observed in single individuals only. Four proteins are here proposed as sarcoidosis-associated autoimmune targets and of interest for further validation in independent cohorts.

Overexpression of an adenosine diphosphate-ribosylation factor gene from the halophytic grass Spartina alterniflora confers salinity and drought tolerance in transgenic Arabidopsis

Adenosine diphosphate-ribosylation factors (ARFs) are small guanine nucleotide-binding proteins that play an important role in intracellular protein trafficking necessary for undertaking multiple physiological functions in plant growth and developmental processes. However, little is known about the mechanism of ARF functioning at the molecular level, as well as its involvement in abiotic stress tolerance. In this study, we demonstrated the direct involvement of an ARF gene SaARF from a grass halophyte Spartina alterniflora in abiotic stress adaptation for the first time. SaARF, which encodes a protein with predicted molecular mass of 21 kDa, revealed highest identity with ARF of Oryza sativa. The SaARF gene is transcriptionally regulated by salt, drought, cold, and ABA in the leaves and roots of S. alterniflora. Arabidopsis plants overexpressing SaARF showed improved seed germination and survival of seedlings under salinity stress. Similarly, SaARF transgenic Arabidopsis plants were more tolerant to drought stress, compared to wild-type plants, by maintaining chlorophyll synthesis, increasing osmolyte synthesis, and stabilizing membrane integrity. Oxidative damage due to moisture stress in transgenic Arabidopsis was also reduced possibly by activating antioxidant genes, AtSOD1 and AtCAT. Our results suggest that enhanced drought and salinity tolerance conferred by the SaARF gene may be due to its role in mediating multiple abiotic stress tolerance mechanisms.