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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.

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., Competing Interests: The authors declare that they have no competing interests., (Copyright: © 2024 Yang et al.)

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).)

The ability to remodel and move cellular membranes, and the cargoes regulated by these membranes, allows for specialised functions to occur in distinct regions of the cell in a process known as cellular polarisation. The ability to collectively co-ordinate such polarisation between cells allows for the genesis of multicellularity, such as the formation of organs. During tumourigenesis, the rules for such tissue polarisation become dysregulated, allowing for collective polarity rearrangements that can drive metastasis. In this review, we focus on how membrane trafficking underpins collective cell invasion and metastasis in cancer. We examine this through the lens of the ADP-ribosylation factor (ARF) subfamily of small GTPases, focusing on how the ARF regulatory network - ARF activators, inactivators, effectors, and modifications - controls ARF GTPase function., (© 2023 The Author(s).)

Carcinogenesis is a multistep process wherein cells accumulate multiple genetic alterations and progress to a more malignant phenotype. It has been proposed that sequential accumulation of gene abnormalities in specific genes drives the transition from non-tumorous epithelia through a preneoplastic lesion/benign tumor to cancer. Histologically, oral squamous cell carcinoma (OSCC) progresses in multiple ordered steps that begin with mucosal epithelial cell hyperplasia, which is followed by dysplasia, carcinoma in situ and invasive carcinoma. It is therefore hypothesized that genetic alteration-mediated multistep carcinogenesis would be involved in the development of OSCC; however, the detailed molecular mechanisms are unknown. We clarified the comprehensive gene expression patterns and carried out an enrichment analysis using DNA microarray data from a pathological specimen of OSCC (including a non-tumor region, carcinoma in situ lesion and invasive carcinoma lesion). The expression of numerous genes and signal activation were altered in the development of OSCC. Among these, the p63 expression was increased and the MEK/ERK-MAPK pathway was activated in carcinoma in situ lesion and in invasive carcinoma lesion. Immunohistochemical analyses revealed that p63 was initially upregulated in carcinoma in situ and ERK was sequentially activated in invasive carcinoma lesions in OSCC specimens. ADP-ribosylation factor (ARF)-like 4c (ARL4C), the expression of which is reportedly induced by p63 and/or the MEK/ERK-MAPK pathway in OSCC cells, has been shown to promote tumorigenesis. Immunohistochemically, in OSCC specimens, ARL4C was more frequently detected in tumor lesions, especially in invasive carcinoma lesions, than in carcinoma in situ lesions. Additionally, ARL4C and phosphorylated ERK were frequently merged in invasive carcinoma lesions. Loss-of-function experiments using inhibitors and siRNAs revealed that p63 and MEK/ERK-MAPK cooperatively induce the expression of ARL4C and cell growth in OSCC cells. These results suggest that the stepwise activation of p63 and MEK/ERK-MAPK contributes to OSCC tumor cell growth through regulation of ARL4C expression., Competing Interests: Declaration of Competing Interest None of the authors have any relevant financial relationship(s) with a commercial interest., (Copyright © 2023 Elsevier GmbH. All rights reserved.)

Patrick T. Caswell discusses work from Bryant and colleagues (2023. J. Cell Biol.https://doi.org/10.1083/jcb.202206115) which shows how ARF3 impacts metastasis in prostate cancer by regulating a switch between modes of collective invasion., (© 2023 Caswell.)

Background: Accumulating evidence has revealed that CD8+ T cell exhaustion (Tex) results in worse immunotherapy outcomes. However, the molecular functions and mechanisms of action of Tex in chemoresistance needed to be elucidated., Methods: The populations of tumor-infiltrating CD8+ T cells (TILCD8Ts) in chemoresistant and chemosensitive groups of the GSE25066 dataset were calculated using CIBERSORT. Differentially expressed genes (DEGs) between TILCD8Ts and other immune cells were explored by integrating 16 immune cell datasets downloaded from the gene expression omnibus (GEO) database. Gene ontology (GO)/Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment, univariate and multivariate Cox regression, and least absolute shrinkage and selection operator (LASSO) regression of TILCD8T-specific upregulated genes were used to construct a chemoresistant TILCD8T signature (cr-TILCD8TSig). Clinical prognostic data, genomic alterations, chemotherapy response, and immunotherapy response were compared between the different cr-TILCD8TSig subgroups in the GSE25066 and the cancer genome atlas breast cancer (TCGA-BRCA) cohorts., Results: A cr-TILCD8TSig with exhausted features was identified, consisting of seven genes ( TCF7, RARRES3, ARL4C, ITK, CDH3, GZMB , and KLRD1 ), which were identified from 104 TILCD8Ts-specific DEGs. Our results showed that compared to the cr-TILCD8TSig-low subgroup, the -high subgroup had a poorer distant relapse-free survival (DRFS) in the GSE25066 cohort and worse progression-free survival (PFS) in the TCGA-BRCA cohort. Univariate and multivariate Cox regression analyses also demonstrated that cr-TILCD8TSig was an independent prognostic factor in the two independent cohorts. Furthermore, cr-TILCD8TSig-low patients benefited more from chemotherapy and immunotherapy than cr-TILCD8TSig-high patients. Besides, we found cell transmembrane signal transduction and the ECM may provide the molecular basis for resistance to antitumor agents in the cr-TILCD8Sig-high subgroup. For genomic alterations, we revealed that mutations in PIK3CA, DMD, and APOB were more common in the cr-TILCD8Sig-high subgroup than in the cr-TILCD8Sig-low subgroup. A nomogram was finally constructed with good discrimination and calibration., Conclusions: cr-TILCD8TSig is a useful tool to independently predict prognosis, chemotherapy response, and immunotherapy outcomes in patients with breast cancer., Competing Interests: Author MS was employed by Guangdong ACXEL Micro & Nano Tech Co., Ltd. The remaining 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. The handling editor YZ declared a shared parent affiliation with the authors DQC and DC at the time of the review., (Copyright © 2023 Cai, Cai, Zou, Chen, Jian, Shi, Lin and Chen.)

Non-small cell lung cancer (NSCLC) is the main histological subtype of lung cancer with a high incidence and mortality. Circular RNAs (circRNAs) exert vital functions in various cancers by acting as a sponge of miRNAs to abolish their inhibitory effect on target genes. This study aims to explore the biological function of circRNA NEDD4 binding protein 2 like 2 (circ-N4BP2L2) in NSCLC. We found that circ-N4BP2L2 was upregulated in NSCLC tissues and cells by using RT-qPCR. A549 cells were transfected with pcDNA-circN4BP2L2 or sh-circN4BP2L2 to obtain circN4BP2L2-overexpressed or -silenced cells, and then cell proliferation, invasion and apoptosis were determined. The results showed that knockdown of circ-N4BP2L2 repressed cell proliferation, invasion as well as mitochondrial function, and promoted cell apoptosis; while overexpression of circ-N4BP2L2 resulted in the opposite results. Mechanistically, the targeting correlations between miR-135a-5p and circ-N4BP2L2 or ADP-ribosylation factorlike 5B (ARL5B) were confirmed by using dual luciferase reporter, RNA pull-down and RNA immunoprecipitation assays. In addition, we found that circ-N4BP2L2 could promote the expression of ARL5B by serving as a sponge of miR-135a-5p. Moreover, rescue assays revealed that silencing miR-135a-5p or overexpressing ARL5B was able to abate the effects of circ-N4BP2L2 knockdown on malignant phenotypes and mitochondrial function of A549 cells. Finally, tumorigenicity assay demonstrated that circ-N4BP2L2 facilitated NSCLC tumor growth in vivo. Taken together, circ-N4BP2L2 enhanced NSCLC progression via the miR-135a-5p/ARL5B axis, which may provide a novel therapeutic target of NSCLC., (© 2023 Wiley Periodicals LLC.)

Purpose: Bardet-Biedl Syndrome (BBS) is an autosomal recessive systemic disorder characterized by retinitis pigmentosa, polydactyly, obesity, intellectual disability, renal impairments, and hypogonadism. The purpose of this study was to determine the ocular characteristics of a boy with BBS caused by a novel homozygous variant in the ARL6 (alternative named BBS3) gene who had been originally diagnosed with retinitis punctata albescens., Methods: This was an observational case study. The patient underwent ophthalmological examinations, electroretinography, and genetic analyses using whole-exome sequencing., Results: A 7-year-old boy was examined in our hospital with complaints of a progressive reduction of his visual acuity and night blindness in both eyes. There was no family history of eye diseases and no consanguineous marriage. Fundus examinations showed numerous white spots in the deep retina and retinal pigment epithelium. Fundus autofluorescence showed hypofluorescence consistent with these spots. Both the scotopic and photopic components of the full-field electroretinographies were non-detectable. Based on these clinical findings, this boy was suspected to have retinitis punctata albescens. Subsequent genetic testing using whole-exome sequencing revealed a novel homozygous variants in the ARL6/BBS3 gene (NM_001278293.3:c.528G>A, (p.Trp176Ter)). A systemic examination by the pediatric department revealed that this boy had a history of a surgical excision of polydactyly on his left foot when he was born, and that he was mildly obese. There were no prominent intellectual or gonadal dysfunctions, no craniofacial or dental abnormalities, no congenital heart disease, and no hearing impairment. He was then clinically and genetically diagnosed with BBS., Conclusion and Importance: In children with night blindness and progressive visual dysfunction, it is important for ophthalmologists to consult clinical geneticists and pediatricians to rule out the possibility of systemic diseases such as BBS., Competing Interests: The authors have no conflicts of interest to disclose., (Copyright © 2022 the Author(s). Published by Wolters Kluwer Health, Inc.)

When intracellular, pathogenic Salmonella reside in a membrane compartment composed of interconnected vacuoles and tubules, the formation of which depends on the translocation of bacterial effectors into the host cell. Cytoskeletons and their molecular motors are prime targets for these effectors. In this study, we show that the microtubule molecular motor KIF1Bβ (a splice variant of KIF1B), a member of the kinesin-3 family, is a key element for the establishment of the Salmonella replication niche as its absence is detrimental to the stability of bacterial vacuoles and the formation of associated tubules. Kinesin-3 interacts with the Salmonella effector SifA but also with SKIP (also known as PLEKHM2), a host protein complexed to SifA. The interaction with SifA is essential for the recruitment of kinesin-3 on Salmonella vacuoles whereas that with SKIP is incidental. In the non-infectious context, however, the interaction with SKIP is essential for the recruitment and activity of kinesin-3 only on a fraction of the lysosomes. Finally, our results show that, in infected cells, the presence of SifA establishes a kinesin-1 and kinesin-3 recruitment pathway that is analogous to and functions independently of that mediated by the Arl8a and Arl8b GTPases. This article has an associated First Person interview with the first author of the paper., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2022. Published by The Company of Biologists Ltd.)

Protein expression profiling in the serum is used to identify novel biomarkers and investigate the signaling pathways in various diseases. The aim of the present study was to evaluate serum biomarkers associated with coronary artery stenosis resulting from atherosclerosis. The study included 4 groups of subjects: group A and B with and without coronary lesions, respectively, were selected from a previously reported cohort study on coronary atherosclerosis, control group C comprised of asymptomatic subjects and group D was used for independent validation of the microarray data by ELISA. Labeled serum proteins were profiled by an Explorer antibody array, which included 656 specific antibodies in two replicates (FullMoon Biosystems, USA). Cadherin-P, interleukin-5, glutathione S-transferase Mu, and neuronal nitric oxide synthase were sex-independently increased in Group A compared with those in group B. The microarray data on cadherin-P were externally validated in an independent group D using ELISA. Fibroblast growth factor-1, FGF-2, collagen II, granulocyte-macrophage colony-stimulating factor, IL-1 alpha, angiopoietin-2, granulocyte colony-stimulating factor, lymphocyte cell-specific protein tyrosine kinase, and IkappaB kinase b were increase in men in group A compared with group B. Cyclin-dependent kinase 1, DNA fragmentation factor subunit alpha DFF45/ICAD, adenovirus type 2 E1A, calponin, ADP-ribosylation factor-6, muscle-specific actin, thyroid hormone receptor alpha, and alpha-methylacyl-CoA racemase were specifically increased in women in Group A compared with group B. Alterations in the levels of specific proteins may point to the signaling pathways contributing to coronary atherosclerosis, and these proteins will be useful biomarkers for the progression of cardiovascular diseases., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Nadezhda Gumanova reports was provided by National Research Center for Preventive Medicine., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Circular RNAs (circRNAs) act as essential regulators in breast cancer (BC) progression. In this paper, we aimed to investigate the functions of circARL8B in BC. The levels of circARL8B, ADP Ribosylation Factor Like GTPase 8B (ARL8B), miR-653-5p and high-mobility group AT-hook 2 (HMGA2) mRNA were examined by qRT-PCR. The stability of circARL8B was determined by RNase R assay and Actinomycin D assay. Cell viability and metastasis were evaluated by Cell Counting Kit-8 (CCK-8) assay and transwell assay, respectively. The levels of cellular phospholipids and triglycerides were measured using relevant kits. Protein levels were measured by western blot analysis. The association between miR-653-5p and circARL8B or HMGA2 was verified by dual-luciferase reporter assay. A murine xenograft model was established to explore the function of circARL8B in vivo. CircARL8B was increased in BC tissues and cells. CircARL8B silencing inhibited cell viability, migration, invasion and fatty acid metabolism in BC cells in vitro and blocked tumor growth in vivo. MiR-653-5p was identified as the target of circARL8B and miR-653-5p was negatively modulated by circARL8B. The suppressive role of circARL8B silencing in BC cell progression was abolished by miR-653-5p downregulation. Moreover, HMGA2 was the target gene of miR-653-5p. HMGA2 overexpression abrogated the effect of miR-653-5p on BC cell development. In addition, circARL8B knockdown might block PGE2/PI3K/AKT/GSK-3β/Wnt/β-catenin pathway. Silencing of circARL8B inhibited cell viability, migration, invasion and fatty acid metabolism via miR-653-5p/HMGA2 axis in BC., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

ADP-ribosylation modification considered as a model to study histone post-translational modification in chromatin modification. Despite it was reported in many plants, the study of ARFs gene family in longan was still unclear. In this study, 14 longan ARFs genes were identified using the longan genome (the third-generation genome) and further divided into two major groups, including the DlARF in the I-II group and the ARF-like (DlARL) in the III-V group, according to their structure and evolutionary characteristics. Whole-genome duplication (WGD) and segmental duplication events played a major role in the expansion of the DlARFs gene family, the synteny and phylogenetic analyses provided a deeper insight into the evolutionary characteristics of the DlARFs. Protein-protein interactions suggested that some DlARFs proteins may interact to participate in biological processes. Promoter analysis showed more stress response elements in DlARF5, DlGB1, DlARL1, DlARL2, and DlARL8a, suggesting that they may participate in abiotic stress. Expression profiles of DlARFs by quantitative real-time PCR (qRT-PCR) showed that they were abundant accumulation during early somatic embryogenesis (SE). Expression pattern analysis of RNA-seq and qRT-PCR revealed that some ARFs members regulated early SE, and respond to exogenous hormones and abiotic stress such as abscisic acid (ABA), gibberellin A3 (GA3), salicylic acid (SA), methyl jasmonate (MeJA), cold, and heat. Our study provides new insights for further research on the potential function of DlARFs, which may be useful for the improvement of longan., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Long-range movement of organelles within the cytoplasm relies on coupling to microtubule motors, a process that is often mediated by adaptor proteins. In many cases, this coupling involves organelle- or adaptor-induced activation of the microtubule motors by conformational reversal of an autoinhibited state. Herein, we show that a similar regulatory mechanism operates for an adaptor protein named SKIP (also known as PLEKHM2). SKIP binds to the small guanosine triphosphatase (GTPase) ARL8 on the lysosomal membrane to couple lysosomes to the anterograde microtubule motor kinesin-1. Structure-function analyses of SKIP reveal that the C-terminal region comprising three pleckstrin homology (PH) domains interacts with the N-terminal region comprising ARL8- and kinesin-1-binding sites. This interaction inhibits coupling of lysosomes to kinesin-1 and, consequently, lysosome movement toward the cell periphery. We also find that ARL8 does not just recruit SKIP to the lysosomal membrane but also relieves SKIP autoinhibition, promoting kinesin-1-driven, anterograde lysosome transport. Finally, our analyses show that the largely disordered middle region of SKIP mediates self-association and that this self-association enhances the interaction of SKIP with kinesin-1. These findings indicate that SKIP is not just a passive connector of lysosome-bound ARL8 to kinesin-1 but is itself subject to intra- and inter-molecular interactions that regulate its function. We anticipate that similar organelle- or GTPase-induced conformational changes could regulate the activity of other kinesin adaptors., Competing Interests: Declaration of Interests The authors declare no competing interests., (Published by Elsevier Inc.)

Ciliogenesis is a coordinated process initiated by the recruitment and fusion of pre-ciliary vesicles at the distal appendages of the mother centriole through mechanisms that remain unclear. Here, we report that EFA6A (also known as PSD), an exchange factor for the small G protein Arf6, is involved in early stage of ciliogenesis by promoting the fusion of distal appendage vesicles forming the ciliary vesicle. EFA6A is present in the vicinity of the mother centriole before primary cilium assembly and prior to the arrival of Arl13B-containing vesicles. During ciliogenesis, EFA6A initially accumulates at the mother centriole and later colocalizes with Arl13B along the ciliary membrane. EFA6A depletion leads to the inhibition of ciliogenesis, the absence of centrosomal Rab8-positive structures and the accumulation of Arl13B-positive vesicles around the distal appendages. Our results uncover a novel fusion machinery, comprising EFA6A, Arf6 and Arl13B, that controls the coordinated fusion of ciliary vesicles docked at the distal appendages of the mother centriole., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2021. Published by The Company of Biologists Ltd.)

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 H 2 O 2 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.

Background: Accurate prediction of the prognosis of RCC using a single biomarker is challenging due to the genetic heterogeneity of the disease. However, it is essential to develop an accurate system to allow better patient selection for optimal treatment strategies. ARL4C, ECT2, SOD2, and STEAP3 are novel molecular biomarkers identified in earlier studies as survival-related genes by comprehensive analyses of 43 primary RCC tissues and RCC cell lines., Methods: To develop a prognostic model based on these multiple biomarkers, the expression of four biomarkers ARL4C, ECT2, SOD2, and STEAP3 in primary RCC tissue were semi-quantitatively investigated by immunohistochemical analysis in an independent cohort of 97 patients who underwent nephrectomy, and the clinical significance of these biomarkers were analyzed by survival analysis using Kaplan-Meier curves. The prognostic model was constructed by calculation of the contribution score to prognosis of each biomarker on Cox regression analysis, and its prognostic performance was validated., Results: Patients whose tumors had high expression of the individual biomarkers had shorter cancer-specific survival (CSS) from the time of primary nephrectomy. The prognostic model based on four biomarkers segregated the patients into a high- and low-risk scored group according to defined cut-off value. This approach was more robust in predicting CSS compared to each single biomarker alone in the total of 97 patients with RCC. Especially in the 36 metastatic RCC patients, our prognostic model could more accurately predict early events within 2 years of diagnosis of metastasis. In addition, high risk-scored patients with particular strong SOD2 expression had a much worse prognosis in 25 patients with metastatic RCC who were treated with molecular targeting agents., Conclusions: Our findings indicate that a prognostic model based on four novel biomarkers provides valuable data for prediction of clinical prognosis and useful information for considering the follow-up conditions and therapeutic strategies for patients with primary and metastatic RCC.

In this issue of Structure, Das et al. (2019) probe the structure and dynamics of dimeric cytohesins (guanine nucleotide exchange factors for ADP-ribosylation factors), in solution and on membranes. Unleashing an arsenal of methods, they demonstrate that a pleckstrin-homology domain-mediated membrane recruitment promotes conformational changes that prime cytohesins for allosteric activation., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Flaviviruses, including dengue virus and Zika virus, extensively remodel the cellular endomembrane network to generate replication organelles that promote viral genome replication and virus production. However, it remains unclear how these membranes and associated cellular proteins act during the virus cycle. Here, we show that atlastins (ATLs), a subset of ER resident proteins involved in neurodegenerative diseases, have dichotomous effects on flaviviruses-with ATL2 depletion leading to replication organelle defects, and ATL3 depletion to changes in virus production pathways. We characterized non-conserved functional domains in ATL paralogues and show that the ATL interactome is profoundly reprogrammed following dengue virus infection. Screen analysis confirmed non-redundant ATL functions and identified a specific role for ATL3, and its interactor ARF4, in vesicle trafficking and virion maturation. Our data identify ATLs as central hubs targeted by flaviviruses to establish their replication organelle and to achieve efficient virion maturation and secretion.

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.

The mechanism of GTPase specificity remains incompletely understood. In this issue of Structure, Lin et al. (2019) present crystal structures of Rab35 in complex with two effectors, ACAP2 and RUSC2, respectively, unraveling the molecular basis for the exquisite mutual specificity between Rab35 and these effectors., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

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.

Keratinocytes maintain epidermal integrity through cellular differentiation. This process enhances intraorganelle digestion in keratinocytes to sustain nutritional and calcium-ionic stresses observed in upper skin layers. However, the molecular mechanisms governing keratinocyte differentiation and concomitant increase in lysosomal function is poorly understood. Here, by using primary neonatal human epidermal keratinocytes, we identified the molecular link between signaling pathways and cellular differentiation/lysosome biogenesis. Incubation of keratinocytes with CaCl 2 induces differentiation with increased cell size and early differentiation markers. Further, differentiated keratinocytes display enhanced lysosome biogenesis generated through ATF6-dependent ER stress signaling, but independent of mTOR-MiT/TFE pathway. In contrast, chemical inhibition of mTORC1 accelerates calcium-induced keratinocyte differentiation, suggesting that activation of autophagy promotes the differentiation process. Moreover, differentiation of keratinocytes results in lysosome dispersion and Golgi fragmentation, and the peripheral lysosomes showed colocalization with Golgi-tethering proteins, suggesting that these organelles possibly derived from Golgi. In line, inhibition of Golgi function, but not the depletion of Golgi-tethers or altered lysosomal acidity, abolishes keratinocyte differentiation and lysosome biogenesis. Thus, ER stress regulates lysosome biogenesis and keratinocyte differentiation to maintain epidermal homeostasis.

Epigenome-wide DNA methylation has not been studied in men perinatally exposed to PCBs and dioxins. Therefore, we examined whether perinatal exposure to polychlorinated biphenyls (PCBs) and polychlorinated dibenzofurans (PCDFs) induces sustained methylation changes lasting to early adulthood. We used the Illumina HumanMethylation450 BeadChip to assess DNA methylation in whole blood among Yucheng second generation (people perinatal exposed to high PCBs and PCDFs) compared with referents. Thirty male offspring from the Yucheng cohort were randomly selected and matched with 30 male offspring from the Yucheng' neighborhood referents with similar backgrounds. Methylation differences between the Yucheng second generation and non-exposed referents were identified using a P value < 1.06 × 10 -7 . Differential DNA methylation with epigenome-wide statistical significance was observed for 20 CpGs mapped to 11 genes, and 19 CpGs were correlated with gestational levels of PCBs or PCDF toxic equivalency (PCDF-TEQ) with the same direction of effect. Among the 11 genes, AHRR and CYP1A1 are involved in the aryl hydrocarbon receptor signaling pathway known to mediate dioxin toxicity. MYO1G, FRMD4A, ARL4C, OLFM1, and WWC3 were previously reported to be related to carcinogenesis. This is the first study examining genome-wide DNA methylation among people perinatally exposed to high concentrations of PCBs and PCDFs. We observed novel differential methylation of several genes, indicating that modifications of DNA methylation associated with perinatal PCB and PCDF exposure may persist in exposed offspring for more than 20 years. Furthermore, involvement of several carcinogesis-related genes suggested a potential in utero epigenetic mechanisms., (Copyright © 2019. Published by Elsevier Inc.)

Arf GTPase-activating proteins (Arf GAPs) control the activity of ADP-ribosylation factors (Arfs) by inducing GTP hydrolysis and participate in a diverse array of cellular functions both through mechanisms that are dependent on and independent of their Arf GAP activity. A number of these functions hinge on the remodeling of actin filaments. Accordingly, some of the effects exerted by Arf GAPs involve proteins known to engage in regulation of the actin dynamics and architecture, such as Rho family proteins and nonmuscle myosin 2. Circular dorsal ruffles (CDRs), podosomes, invadopodia, lamellipodia, stress fibers and focal adhesions are among the actin-based structures regulated by Arf GAPs. Arf GAPs are thus important actors in broad functions like adhesion and motility, as well as the specialized functions of bone resorption, neurite outgrowth, and pathogen internalization by immune cells. Arf GAPs, with their multiple protein-protein interactions, membrane-binding domains and sites for post-translational modification, are good candidates for linking the changes in actin to the membrane. The findings discussed depict a family of proteins with a critical role in regulating actin dynamics to enable proper cell function.

Arf6/ARF-6 is a crucial regulator of the endosomal phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) pool in endocytic recycling. To further characterize ARF-6 regulation, we performed an ARF-6 interactor screen in Caenorhabditis elegans and identified SAC-1, the homologue of the phosphoinositide phosphatase Sac1p in yeast, as a novel ARF-6 partner. In the absence of ARF-6, basolateral endosomes show a loss of SAC-1 staining in epithelial cells. Steady-state cargo distribution assays revealed that loss of SAC-1 specifically affected apical secretory delivery and basolateral recycling. PI(4,5)P2 levels and the endosomal labeling of the ARF-6 effector UNC-16 were significantly elevated in sac-1 mutants, suggesting that SAC-1 functions as a negative regulator of ARF-6. Further analyses revealed an interaction between SAC-1 and the ARF-6-GEF BRIS-1. This interaction outcompeted ARF-6(guanosine diphosphate [GDP]) for binding to BRIS-1 in a concentration-dependent manner. Consequently, loss of SAC-1 promotes the intracellular overlap between ARF-6 and BRIS-1. BRIS-1 knockdown resulted in a significant reduction in PI(4,5)P2 levels in SAC-1-depleted cells. Interestingly, the action of SAC-1 in sequestering BRIS-1 is independent of SAC-1's catalytic activity. Our results suggest that the interaction of SAC-1 with ARF-6 curbs ARF-6 activity by limiting the access of ARF-6(GDP) to its guanine nucleotide exchange factor, BRIS-1., (© 2018 Chen et al.)

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)P 2 , 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. This article has an associated First Person interview with the first author of the paper., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2018. Published by The Company of Biologists Ltd.)

Neuroglobin (Ngb) is expressed in the central and peripheral nervous system, cerebrospinal fluid, retina, and endocrine tissues where it is involved in binding O 2 and other gasotransmitters. Several studies have highlighted its endogenous neuroprotective function. Huntington's disease (HD), a dominant hereditary disease, is characterized by the gradual loss of neurons in discrete areas of the central nervous system. We analyzed the expression of Ngb in the brain tissue of a mouse model of HD, in order to define the role of Ngb with respect to individual cell type vulnerability in HD and to gender and age of mice. Our results showed different expressions of Ngb among neurons of a specific region and between different brain regions. We evidenced a decreased intensity of Ngb at 13 weeks of age, compared to 7 weeks of age. The double immunofluorescence and fluorescence resonance energy transfer (FRET) experiments showed that the co-localization between Ngb and huntingtin at the subcellular level was not close enough to account for a direct interaction. We also observed a different expression of Ngb in the striatum, depending on the sex and age of animals. These findings provide the first experimental evidence for an adaptive response of Ngb in HD, suggesting that Ngb may exert neuroprotective effects in HD beyond its role in reducing sensitivity to oxidative stress.

Competing Interests: The authors declare no conflict of interest.

The present study was conducted to examine whether magnesium lithospermate B (MLB) extracted from Salviae miltiorrhizae radix was renoprotective in pathways related to age-related oxidative stress in aged rats. Magnesium lithospermate B was orally administered at a dose of 2- or 8-mg/kg body weight for 16 consecutive days, and the effects were compared with those of vehicle in old and young rats. Magnesium lithospermate B administration to old rats ameliorated renal oxidative stress through reduction of reactive oxygen species. The old rats exhibited a dysregulation of the expression of proteins related to oxidative stress and inflammation in the kidneys, and MLB administration significantly reduced the protein expression of major subunits of nicotinamide adenine dinucleotide phosphate oxidase (Nox4 and p22 phox ), phospho-p38, nuclear factor-kappa B p65, cyclooxygenase-2, and inducible nitric oxide synthase. In addition, MLB-treated old rats showed lower levels of senescence-related proteins such as p16, ADP-ribosylation factor 6, p53, and p21 through effects on the mitogen-activated protein kinase pathway. Magnesium lithospermate B administration also significantly attenuated the age-related increase in serum urea nitrogen, reflecting renal dysfunction, up-regulated podocyte structural proteins, and reduced renal structural injury. Our results provide important evidence that MLB reduces the renal damage of oxidative stress in old rats. Copyright © 2017 John Wiley & Sons, Ltd., (Copyright © 2017 John Wiley & Sons, Ltd.)

Abnormalities in the ability of cells to properly degrade proteins have been identified in many neurodegenerative diseases. Recent work has implicated synaptojanin 1 (SynJ1) in Alzheimer's disease and Parkinson's disease, although the role of this polyphosphoinositide phosphatase in protein degradation has not been thoroughly described. Here, we dissected in vivo the role of SynJ1 in endolysosomal trafficking in zebrafish cone photoreceptors using a SynJ1-deficient zebrafish mutant, nrc(a14) . We found that loss of SynJ1 leads to specific accumulation of late endosomes and autophagosomes early in photoreceptor development. An analysis of autophagic flux revealed that autophagosomes accumulate because of a defect in maturation. In addition we found an increase in vesicles that are highly enriched for PI(3)P, but negative for an early endosome marker in nrc(a14) cones. A mutational analysis of SynJ1 enzymatic domains found that activity of the 5'phosphatase, but not the Sac1 domain, is required to rescue both aberrant late endosomes and autophagosomes. Finally, modulating activity of the PI(4,5)P2 regulator, Arf6, rescued the disrupted trafficking pathways in nrc(a14) cones. Our study describes a specific role for SynJ1 in autophagosomal and endosomal trafficking and provides evidence that PI(4,5)P2 participates in autophagy in a neuronal cell type., (© 2016 The Authors. BioEssays published by WILEY Periodicals, Inc.)

Members of the large Sec7 domain-containing Arf guanine nucleotide exchange factor (GEF) family have been shown to dimerize through their NH2-terminal dimerization and cyclophilin binding (DCB) and homology upstream of Sec7 (HUS) domains. However, the importance of dimerization in GEF localization and function has not been assessed. We generated a GBF1 mutant (91/130) in which two residues required for oligomerization (K91 and E130 within the DCB domain) were replaced with A and assessed the effects of these mutations on GBF1 localization and cellular functions. We show that 91/130 is compromised in oligomerization but that it targets to the Golgi in a manner indistinguishable from wild-type GBF1 and that it rapidly exchanges between the cytosolic and membrane-bound pools. The 91/130 mutant appears active as it integrates within the functional network at the Golgi, supports Arf activation and COPI recruitment, and sustains Golgi homeostasis and cargo secretion when provided as a sole copy of functional GBF1 in cells. In addition, like wild-type GBF1, the 91/130 mutant supports poliovirus RNA replication, a process requiring GBF1 but believed to be independent of GBF1 catalytic activity. However, oligomerization appears to stabilize GBF1 in cells, and the 91/130 mutant is degraded faster than the wild-type GBF1. Our data support a model in which oligomerization is not a key regulator of GBF1 activity but impacts its function by regulating the cellular levels of GBF1., (Copyright © 2016 the American Physiological Society.)

Both the Wnt/β-catenin signaling pathway and small GTPases of the ADP-ribosylation factors (ARF) family play important roles in regulating cell development, homeostasis and fate. The previous report of QS11, a small molecule Wnt synergist that binds to ARF GTPase-activating protein 1 (ARFGAP1), suggests a role for ARFGAP1 in the Wnt/β-catenin pathway. However, direct inhibition of enzymatic activity of ARFGAP1 by QS11 has not been established. Whether ARFGAP1 is the only target that contributes to QS11's Wnt synergy is also not clear. Here we present structure-activity relationship (SAR) studies of QS11 analogs in two assays: direct inhibition of enzymatic activity of purified ARFGAP1 protein and cellular activation of the Wnt/β-catenin pathway. The results confirm the direct inhibition of ARFGAP1 by QS11, and also suggest the presence of other potential cellular targets of QS11., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Primary cilia are key sensory organelles that are thought to be disassembled prior to mitosis. Inheritance of the mother centriole, which nucleates the primary cilium, in relation to asymmetric daughter cell behavior has previously been studied. However, the fate of the ciliary membrane upon cell division is unknown. Here, we followed the ciliary membrane in dividing embryonic neocortical stem cells and cultured cells. Ciliary membrane attached to the mother centriole was endocytosed at mitosis onset, persisted through mitosis at one spindle pole, and was asymmetrically inherited by one daughter cell, which retained stem cell character. This daughter re-established a primary cilium harboring an activated signal transducer earlier than the noninheriting daughter. Centrosomal association of ciliary membrane in dividing neural stem cells decreased at late neurogenesis when these cells differentiate. Our data imply that centrosome-associated ciliary membrane acts as a determinant for the temporal-spatial control of ciliogenesis., (Copyright © 2013 Elsevier Inc. All rights reserved.)

The prosurvival activity of phosphoinositide 3 kinase (PI3K)/Akt (also known as protein kinase B, PKB) pathway has been investigated in great detail in human physiology and disease. Accumulating evidence is emerging that this signaling axis also actively engages with the migratory process in motile cells, including metastatic cancer cells. Interference with the role of PI3K/Akt-mediated cell motility impairs cellular development and attenuates malignant progression of cancer metastasis. Because metastasis is responsible for 90% of mortality in cancer patients, the acceleration of cancer cell spreading observed in association with hyperactivation of the PI3K pathway, triggered for example by chemotherapy/radiotherapy in the clinic, has heightened awareness of the conflict between "good drugs" and unfavorable effects. Here, we discuss recent studies on PI3K/Akt-regulated cell motility in both physiological and pathological settings, with the aim of a better understanding of how activities of the PI3K/Akt axis initiate and transmit "migratory signals" that stimulate cell movement. We focus in particular on its direct influence on cell migration and invasion, epithelial-mesenchymal transition, and cancer metastasis.

Background: Recent findings from our laboratory have demonstrated that glucose-stimulated insulin secretion (GSIS) involves interplay between a variety of small G proteins belonging to the Rho (e.g., Cdc42 and Rac1) and ADP-ribosylation factor (e.g., Arf6) subfamilies. Using immunological, pharmacological and molecular biological approaches, we have also identified guanine nucleotide exchange factors (GEFs) for Rac1 (e.g., Tiam1) and Arf6 (e.g., ARNO) in clonal INS-1 832/13 cells, normal rat islets and human islets. As a logical extension to these studies, we investigated, herein, potential downstream signaling steps involved in Arf6/ARNO-mediated GSIS., Methods: Using a selective pharmacological inhibitor of ARNO/Arf6 signaling axis (e.g., secinH3) we assessed regulatory roles for Arf6/ARNO in promoting phospholipase D (PLD), phagocytic NADPH oxidase (Nox2), reactive oxygen species (ROS), extracellular-regulated kinases (ERK 1/2) and cofilin (actin-severing protein] signaling steps in clonal INS-1 832/13 cells., Results: Our data suggested a marked inhibition by secinH3 of glucose-induced PLD activation, ERK1/2 phosphorylation and dephosphorylation of cofilin, suggesting that Arf6/ ARNO signaling mediates PLD, ERK1/2 and cofilin activation in beta-cells. In addition, secinH3 blocked glucose-induced Nox2 activation and associated ROS generation, thus placing Nox downstream to Arf6/ARNO signaling step. Lastly, we also demonstrate a significantly higher cofilin phosphorylation (inactive) in islets derived from type 2 diabetic human donors as well as the Zucker Diabetic Fatty (ZDF) rat, a model for type 2 diabetes., Conclusion: Together, our current findings identify signaling steps downstream to ARNO/Arf6 axis leading to insulin secretion., (Copyright © 2012 S. Karger AG, Basel.)

Protein mono-ADP-ribosylation post-translationally transfers the ADP-ribose moiety from the β-NAD+ donor to various protein acceptors. This type of modification has been widely characterized and shown to regulate protein activities in animals, yeast and prokaryotes, but has never been reported in plants. In this study, using [³²P]NAD+ as the substrate, ADP-ribosylated proteins in Arabidopsis were investigated. One protein substrate of 32 kDa in adult rosette leaves was found to be radiolabeled. Heat treatment, protease sensitivity and nucleotide derivative competition assays suggested a covalent reaction of NAD+ with the 32 kDa protein. [carbonyl-¹⁴C]NAD+ could not label the 32 kDa protein, confirming that the modification was ADP-ribosylation. Poly (ADP-ribose) polymerase inhibitor failed to suppress the reaction, but chemicals that destroy mono-ADP-ribosylation on specific amino acid residues could break up the linkage, suggesting that the reaction was not a poly-ADP-ribosylation but rather a mono-ADP-ribosylation. This modification mainly existed in leaves and was enhanced by oxidative stresses. In young seedlings, two more protein substrates with the size of 45 kDa and over 130 kDa, respectively, were observed in addition to the 32 kDa protein, indicating that different proteins were modified at different developmental stages. Although the substrate proteins remain to be identified, this is the first report on the characterization of endogenously mono-ADP-ribosylated proteins in plants.

Tumor cell invasion and metastasis are the most complicated problems of cancer medicine and cancer biology, and cancer becomes a fatal disease because of cancer cell invasion and metastasis. The mechanisms of invasion and metastasis remain elusive, but tumor cell invasion still is the hotspot of research. This review summarizes the development of researching in tumor cell invasion.

Herein we report that Gts1p fused with green-fluorescent protein (GFP) is localized in the cortical actin patch besides nuclei in yeast and the cortical Gts1p changed its position together with the patch depending on the cell-cycle phase, while nuclear Gts1p accumulated predominantly in the budding phase. Whereas Gts1p does not directly bind to actin, it associated mainly with the actin-associated protein Pan1p. In the GTS1-deleted transformant gts1Delta, the number of cells containing either a fragmented vacuole or an enlarged single central vacuole increased and the uptake of the hydrophilic dye Lucifer yellow (LY) in the vacuole decreased. Further, gts1Delta transformed with a mutant Gts1p having two cysteine-to-alanine substitutions in a zinc finger resembling that of GTPase-activating proteins of ADP-ribosylation factors (ARF-GAP) neither recovered the LY uptake unlike gts1Delta transformed with the wild-type GTS1, nor reduced the average size of central vacuoles as much as the latter did. These results suggested that Gts1p in the actin patch is involved in the fluid-phase endocytosis and membrane trafficking for vacuole formation and that the putative ARF-GAP domain in Gts1p plays an important role in these functions.

Plasma membrane proteins such as receptors and ion channels allow a cell to communicate with its environment and regulate many intracellular activities. Thus, the proper control of the surface number of these proteins is essential for maintaining the structural and functional homeostasis of a cell. Internalization and recycling plays a key role in determining the surface density of receptors and channels. Whereas the clathrin-mediated internalization and its associated recycling have been the focus of research in this field, recent studies have revealed that an increasing number of receptors and channels enter a cell via clathrin-independent pathways. However, little is known about the trafficking motifs involved in controlling clathrin-independent internalization and various associated recycling pathways. By using a potassium channel as a model system, we identified a class of trafficking motifs that function along a clathrin-independent pathway to increase the surface density of a membrane protein by preventing its rapid internalization and/or facilitating its recycling via the ADP-ribosylation factor 6-dependent recycling pathway. Moreover our data suggest that these motifs may enhance the association of membrane proteins with the EFA6 family of guanine nucleotide exchange factors for ADP-ribosylation factor 6.

Metabotropic glutamate receptors (mGluRs) are G protein-coupled receptors (GPCRs) that are widely expressed throughout the brain and are involved in synaptic development, transmission, and plasticity. The endocytosis of several members of the GPCR superfamily of receptors, such as beta-adrenergic receptors, has been studied extensively. In contrast, the mechanisms regulating mGluR endocytosis and intracellular trafficking remain poorly defined. We describe here for the first time a distinct endocytic and intracellular sorting pathway utilized by mGluR7. We show that mGluR7 constitutively internalizes via a non-clathrin mediated pathway in heterologous cells and in neurons. Unlike clathrin-mediated NMDAR endocytosis, mGluR7 traffics via an Arf6-positive endosomal pathway, similar to other well-characterized proteins such as major histocompatibility complex class I (MHC I) and the GPI-anchored protein CD59. Thus constitutive endocytosis of mGluR7 in neurons is not regulated by clathrin-dependent mechanisms, and this clathrin-independent pathway ultimately determines the amount of receptor present on the plasma membrane available to bind and respond to glutamate.

The Ras superfamily is comprised of at least four large families of regulatory guanosine triphosphate-binding proteins, including the Arfs. The Arf family includes three different groups of proteins: the Arfs, Arf-like (Arls), and SARs. Several Arf family members have been very highly conserved throughout eukaryotic evolution and have orthologues in evolutionally diverse species. The different means by which Arf family members have been identified have resulted in an inconsistent and confusing array of names. This confusion is further compounded by differences in nomenclature between different species. We propose a more consistent nomenclature for the human members of the Arf family that may also serve as a guide for nomenclature in other species.

The cabbage butterfly (Pieris rapae) produces pierisin-1, an apoptosis-inducing protein against mammalian cells. In order to clarify the biological role of pierisin-1 in P. rapae, its expression during developmental stages was examined. Low levels of pierisin-1 mRNA and protein were detected in first-instar larvae. During growth until the fifth-instar larval stage, the amounts of the mRNA steadily increased to reach about 50-100 times the initial level. Then it rapidly decreased before pupation. The levels of mRNA in the pupae and the adults were as low as in the first-instar larvae. Levels of pierisin-1 protein also increased around 100 times from the first-instar to the fifth-instar larvae and then gradually decreased by over 90% during the pupal stage. Immunostaining of pierisin-1 demonstrated the protein to be mainly located in fat bodies of fifth-instar larvae and early-phase pupae. Although the staining intensity was low, fat bodies of early instars of the larvae and adults were also found to be positive. Moreover, examination of isolated fat body and other tissue samples of the insects were consistent with the above observations. Thus, the results indicate that mRNA of pierisin-1 was highly expressed in late stages of larvae, and that the protein accumulated in fat bodies where it persists during pupation.

Recruitment of the GRIP domain golgins to the trans-Golgi network is mediated by Arl1, a member of the ARF/Arl small GTPase family, through interaction between their GRIP domains and Arl1-GTP. The crystal structure of Arl1-GTP in complex with the GRIP domain of golgin-245 shows that Arl1-GTP interacts with the GRIP domain predominantly in a hydrophobic manner, with the switch II region conferring the main recognition surface. The involvement of the switch and interswitch regions in the interaction between Arl1-GTP and GRIP accounts for the specificity of GRIP domain for Arl1-GTP. Mutations that abolished the Arl1-mediated Golgi localization of GRIP domain golgins have been mapped on the interface between Arl1-GTP and GRIP. Notably, the GRIP domain forms a homodimer in which each subunit interacts separately with one Arl1-GTP. Mutations disrupting the GRIP domain dimerization also abrogated its Golgi targeting, suggesting that the dimeric form of GRIP domain is a functional unit.

Golgins are large coiled-coil proteins that play a role in Golgi structure and vesicle traffic. The Arf-like GTPase Arl1 regulates the translocation of GRIP domain-containing golgins to Golgi membranes. We report here the 1.7 A resolution structure of human Arl1-GTP in a complex with the GRIP domain of golgin-245. The structure reveals that the GRIP domain consists of an S-shaped arrangement of three helices. The domain forms a homodimer that binds two Arl1-GTPs using two helices from each monomer. The structure is consistent with golgin-245 forming parallel coiled-coils and suggests how Arl1-GTP/GRIP complexes interact with Golgi membranes via the N termini of Arl1-GTP and the C-terminal tails of the GRIP domains. In cells, bivalent association with Arl1-GTP would increase residence time of the golgins on Golgi membranes. Despite no conservation of sequence, topology, or even helical direction, several other effectors form similar interactions with small GTPases via a pair of alpha helices, suggesting a common structural basis for effector recognition.

G proteins are molecular switches that control a wide variety of physiological functions, including neurotransmission, transcriptional activation, cell migration, cell growth. and proliferation. The ability of GTPases to participate in signaling events is determined by the ratio of GTP-bound to GDP-bound forms in the cell. All known GTPases exist in an inactive (GDP-bound) and an active (GTP-bound) conformation, which are catalyzed by guanine nucleotide exchange factors and GTPase-activating proteins (GAPs), respectively. In this study, we identified and characterized a new family of bifunctional GTP-binding and GTPase-activating proteins, named GGAP. GGAPs contain an N-terminal Ras homology domain, called the G domain, followed by a pleckstrin homology (PH) domain, a C-terminal GAP domain, and a tandem ankyrin (ANK) repeat domain. Expression analysis indicates that this new family of proteins has distinct cell localization, tissue distribution, and even message sizes. GTPase assays demonstrate that GGAPs have high GTPase activity through direct intramolecular interaction of the N-terminal G domain and the C-terminal GAP domain. In the absence of the GAP domain, the N-terminal G domain has very low activity, suggesting a new model of GGAP protein regulation via intramolecular interaction like the multidomain protein kinases. Overexpression of GGAPs leads to changes in cell morphology and activation of gene transcription.

Objectives/hypothesis: We have identified families with a high incidence of tumors including head and neck squamous cell carcinoma (HNSCC). The occurrence of melanoma in these kindreds suggested that the ARF-p16 gene may be involved in carcinogenesis. We wished to determine the gene defect associated with the familial predisposition to HNSCC and to determine whether restoration of the gene may have therapeutic benefit., Study Design: Translational molecular research., Methods: Molecular techniques were used to identify mutations of the ARF-p16 gene from the affected families and to test the activity of p16 and ARF mutants. In additional, HNSCC tumor tissue was analyzed to determine whether the wild-type p16 allele was lost or maintained. ARF-expressing adenoviruses were created, and their effect on HNSCC cell lines and normal head and neck epithelial cells was determined., Results: Mutation of the ARF-p16 gene was found in two families with predisposition to develop HNSCC. Independent mutations detected in the germline DNA of both families inactivated p16, but not ARF, and the inactive mutant p16 allele segregated with disease within both families. The wild-type p16 allele was lost in HNSCC tumor tissue from both families. The efficacy of ARF in treatment of HNSCC was found to depend on retention of p53 activity within HNSCC tumor cells. Remarkably, ARF expression was found to kill cells, depending on loss of retinoblastoma activity. Because loss of retinoblastoma activity is nearly universal in tumors, ARF killed tumor cells that retained p53, but ARF spared normal cells., Conclusions: Our results support the recognition of a new clinical entity of familial head and neck cancer. We have shown that this syndrome is associated with inactivating mutations of the p16 gene that these mutations segregate with disease in two described families. Loss of the wild-type p16 allele in HNSCC tissue from both families strongly supports the role of the mutant p16 in carcinogenesis. We have also investigated the therapeutic utility of the alternate reading frame product of the p16 gene, ARF. The finding that ARF kills cells depending on loss of retinoblastoma activity and retention of p53 suggests that ARF may be effective in treatment of roughly 50% of head and neck cancers while sparing normal cells. Recognition of p16 mutations as an etiological factor in familial HNSCC provides an accessible tool for diagnosis of this syndrome. Clinical acceptance of familial head and neck cancer will ensure that patients are appropriately diagnosed and managed.