Hydrogen peroxide induces Arl1 degradation and impairs Golgi-mediated trafficking.

Reactive oxygen species (ROS)-induced oxidative stress has been associated with diseases such as amyotrophic lateral sclerosis, stroke, and cancer. While the effect of ROS on mitochondria and endoplasmic reticulum (ER) has been well documented, its consequence on the Golgi apparatus is less well understood. In this study, we characterized the Golgi structure and function in HeLa cells after exposure to hydrogen peroxide (H2O2), a reagent commonly used to introduce ROS to cells. Treatment of cells with 1 mM H2O2 for 10 min resulted in the degradation of Arl1 and dissociation of GRIP domain-containing proteins Golgin-97 and Golgin-245 from the trans-Golgi. This effect could be rescued by treatment of cells with a ROS scavenger N-acetyl cysteine or protease inhibitors. Structurally, H2O2 treatment reduced the number of cisternal membranes per Golgi stack, suggesting a loss of trans-Golgi cisternae. Functionally, H2O2 treatment inhibited both anterograde and retrograde protein transport, consistent with the loss of membrane tethers on the trans-Golgi cisternae. This study revealed membrane tethers at the trans-Golgi as novel specific targets of ROS in cells.

Inhibition of smooth muscle contraction and ARF6 activity by the inhibitor for cytohesin GEFs, secinH3, in the human prostate.

Prostate smooth muscle contraction is critical for etiology and treatment of male lower urinary tract symptoms (LUTS) and is promoted by small monomeric GTPases (RhoA and Rac). GTPases may be activated by guanosine nucleotide exchange factors (GEFs). GEFs of the cytohesin family may indirectly activate Rac, or ADP ribosylation factor (ARF) GTPases directly. Here we investigated the expression of cytohesin family GEFs and effects of the cytohesin inhibitor Sec7 inhibitor H3 (secinH3) on smooth muscle contraction and GTPase activities in human prostate tissues. Of all four cytohesin isoforms, cytohesin-1 and -2 showed the highest expression in real-time PCR. Western blot and fluorescence staining suggested that cytohesin-2 may be the predominant isoform in prostate smooth muscle cells. Contractions induced by norepinephrine, the α1-adrenoceptor agonist phenylephrine, the thromboxane A2 analog U-46619 , and endothelin-1 and -3, as well as neurogenic contractions induced by electric field stimulation (EFS), were reduced by secinH3 (30 µM). Inhibition of EFS-induced contractions appeared to have efficacy similar to that of inhibition by the α1-adrenoceptor antagonist tamsulosin (300 nM). Combined application of secinH3 plus tamsulosin caused larger inhibition of EFS-induced contractions than tamsulosin alone. Pull-down assays demonstrated inhibition of the small monomeric GTPase ARF6 by secinH3, but no inhibition of RhoA or Rac1. In conclusion, we suggest that a cytohesin-ARF6 pathway takes part in smooth muscle contraction. This may open attractive new possibilities in medical treatment of male LUTS.

Cell survival and protein secretion associated with Golgi integrity in response to Golgi stress-inducing agents.

The Golgi apparatus is part of the secretory pathway and of central importance for modification, transport and sorting of proteins and lipids. ADP-ribosylation factors, whose activation can be blocked by brefeldin A (BFA), play a major role in functioning of the Golgi network and regulation of membrane traffic and are also involved in proliferation and migration of cancer cells. Due to high cytotoxicity and poor bioavailability, BFA has not passed the preclinical stage of drug development. Recently, AMF-26 and golgicide A have been described as novel inhibitors of the Golgi system with antitumor or bactericidal properties. We provide here further evidence that AMF-26 closely mirrors the mode of action of BFA but is less potent. Using several human cancer cell lines, we studied the effects of AMF-26, BFA and golgicide A on cell homeostasis including Golgi structure, endoplasmic reticulum (ER) stress markers, secretion and viability, and found overall a significant correlation between these parameters. Furthermore, modulation of ADP-ribosylation factor expression has a profound impact on Golgi organization and survival in response to Golgi stress inducers.

Molecular characterization and expression analysis of cDNAs encoding four Rab and two Arf GTPases in the latex of Hevea brasiliensis.

In plants, Rab and Arf GTPases are key regulators of vesicle trafficking. To investigate whether these small GTPases (SG) play a role in the regulation of the regeneration of latex (the cytoplasm of the rubber-producing laticifer cell) in Hevea brasiliensis (Hevea hereafter), full-length cDNAs that encode four HbRab and two HbArf GTPases were cloned. The four HbRab proteins showed specific GTP-binding activity when expressed in Escherichia coli. Transcript expression of the six SG genes was investigated by real-time RT-PCR. All genes revealed to be expressed in each of the six Hevea tissues examined, but the expression patterns were different. Four genes, HbArf1, HbRab2, HbRab3 and HbRab4, displayed a preferential expression in latex. The expression of all genes was upregulated by the act of latex exploitation (tapping), and HbRab1 had the highest level of upregulation. Wounding markedly upregulated the expression of two SG genes (HbRab1 and HbArf2), and exogenous methyl jasmonate upregulated all six SG genes. Wounding might upregulate the expression of HbRab1 and HbArf2 through a jasmonic acid-mediated signaling pathway. None of the genes were markedly upregulated by Ethrel (an ethylene releaser and latex stimulator); instead, HbArf2 and HbRab4 were downregulated significantly after a 24 h treatment with Ethrel. This paper gives the first description of Rab and Arf GTPases in Hevea and provides clues for their involvement, HbRab1 in particular, in latex regeneration.

Reverse genetics in Candida albicans predicts ARF cycling is essential for drug resistance and virulence.

Candida albicans, the major fungal pathogen of humans, causes life-threatening infections in immunocompromised individuals. Due to limited available therapy options, this can frequently lead to therapy failure and emergence of drug resistance. To improve current treatment strategies, we have combined comprehensive chemical-genomic screening in Saccharomyces cerevisiae and validation in C. albicans with the goal of identifying compounds that can couple with the fungistatic drug fluconazole to make it fungicidal. Among the genes identified in the yeast screen, we found that only AGE3, which codes for an ADP-ribosylation factor GTPase activating effector protein, abrogates fluconazole tolerance in C. albicans. The age3 mutant was more sensitive to other sterols and cell wall inhibitors, including caspofungin. The deletion of AGE3 in drug resistant clinical isolates and in constitutively active calcineurin signaling mutants restored fluconazole sensitivity. We confirmed chemically the AGE3-dependent drug sensitivity by showing a potent fungicidal synergy between fluconazole and brefeldin A (an inhibitor of the guanine nucleotide exchange factor for ADP ribosylation factors) in wild type C. albicans as well as in drug resistant clinical isolates. Addition of calcineurin inhibitors to the fluconazole/brefeldin A combination only initially improved pathogen killing. Brefeldin A synergized with different drugs in non-albicans Candida species as well as Aspergillus fumigatus. Microarray studies showed that core transcriptional responses to two different drug classes are not significantly altered in age3 mutants. The therapeutic potential of inhibiting ARF activities was demonstrated by in vivo studies that showed age3 mutants are avirulent in wild type mice, attenuated in virulence in immunocompromised mice and that fluconazole treatment was significantly more efficacious when ARF signaling was genetically compromised. This work describes a new, widely conserved, broad-spectrum mechanism involved in fungal drug resistance and virulence and offers a potential route for single or improved combination therapies.

Increased expression of the glioma-associated antigen ARF4L after loss of the tumor suppressor PTEN. Laboratory investigation.

OBJECT: Despite recent advances in cancer immunotherapy, cellular mechanisms controlling expression of tumor-associated antigens are poorly understood. Mutations in cancer cells, such as loss of PTEN, may increase expression of tumor-associated antigens. The authors investigated the relationship between PTEN status and the expression of a glioma-associated antigen, adenosine diphosphate-ribosylation factor 4-like (ARF4L) protein. METHODS: Human glioma cell lines with confirmed PTEN status were examined by Northern blot analysis and quantitative polymerase chain reaction. Western blot analysis was used to measure ARF4L protein levels across multiple cell lines. RESULTS: The loss of PTEN was shown to lead to increased levels of ARF4L protein but no change in transcript levels. Cell lines with serial mutations, including activation of Ras and Akt pathways, also demonstrated increased levels of ARF4L protein, which decreased after treatment with rapamycin. The ARF4L transcript preferentially localized to the polysomal compartment after PTEN loss in glioma or activation of Akt in human astrocytes. CONCLUSIONS: Expression of ARF4L is controlled by the activated Akt/mTOR pathway, which is a downstream effect of the loss of PTEN function. Mutations leading to oncogenesis may impact the regulation and expression of tumor specific antigens. Screening of mutation status in glioma may be helpful in selecting patients for immunotherapy trials in the future.

ARF6 activation by Galpha q signaling: Galpha q forms molecular complexes with ARNO and ARF6.

G protein-coupled receptors (GPCRs) are widely expressed hepta-helical receptors with tightly regulated pleiotropic effects. ADP-Ribosylation Factor 6 (ARF6) plays an important role in GPCR trafficking and is the subject of intense research. However, the mechanisms underlying activation and regulation of ARF6 by GPCRs are poorly characterized. Here we report that Galpha(q) signaling leads to the activation of ARF6. Stimulation of the TPbeta receptor triggered ARF6 activation which was completely inhibited by the RGS domain of GRK2 known to specifically bind and sequester Galpha(q). Co-immunoprecipitation studies revealed that ARNO (a guanine nucleotide exchange factor for ARF6) and ARF6 formed complexes preferentially with activated Galpha(q) compared to non-activated Galpha(q). Formation of the Galpha(q) complexes with ARNO and ARF6 was detected early and was optimal after 30 min of receptor stimulation corresponding with the profile of ARF6 activation. Interestingly, binding experiments using purified proteins showed that Galpha(q) interacted directly with ARNO. Galpha(q)-dependent TPbeta receptor-mediated activation of ARF6 resulted in phosphoinositol-4,5-bisphosphate production which was potently inhibited by dominant negative mutants of ARNO and ARF6. Furthermore, our data show that the expression of ARNO and ARF6 promoted, whereas dominant negative mutants of these proteins inhibited the internalization of the TPbeta receptor. This further elucidates our previous data on the PLCbeta- and PKC-independent mechanism involved in Galpha(q)-mediated internalization of the TPbeta receptor. Taken altogether, our results support a novel model where activated Galpha(q) forms molecular complexes with ARNO and ARF6, possibly through a direct interaction with ARNO, leading to ARF6 activation.

AlFx affects the formation of focal complexes by stabilizing the Arf-GAP ASAP1 in a complex with Arf1.

Aluminum fluoride (AlFx) is known to activate directly the alpha subunit of G-proteins but not the homologous small GTP-binding proteins. However, AlFx can stabilize complexes formed between Ras, RhoA or Cdc42 and their corresponding GTPase-activating proteins (GAPs). Here, we demonstrate that Arf1GDP can be converted into an active conformation by AlFx to form a complex with the Arf-GAP ASAP1 in vitro and in vivo. Within this complex ASAP1, which GAP activity is inoperative, can still alter the recruitment of paxillin to the focal complexes, thus indicating that ASAP1 interferes with focal complexes independently of its GAP activity.

An alpha4 integrin-paxillin-Arf-GAP complex restricts Rac activation to the leading edge of migrating cells.

Formation of a stable lamellipodium at the front of migrating cells requires localization of Rac activation to the leading edge. Restriction of alpha4 integrin phosphorylation to the leading edge limits the interaction of alpha4 with paxillin to the sides and rear of a migrating cell. The alpha4-paxillin complex inhibits stable lamellipodia, thus confining lamellipod formation to the cell anterior. Here we report that binding of paxillin to the alpha4 integrin subunit inhibits adhesion-dependent lamellipodium formation by blocking Rac activation. The paxillin LD4 domain mediates this reduction in Rac activity by recruiting an ADP-ribosylation factor GTPase-activating protein (Arf-GAP) that decreases Arf activity, thereby inhibiting Rac. Finally, the localized formation of the alpha4-paxillin-Arf-GAP complex mediates the polarization of Rac activity and promotes directional cell migration. These findings establish a mechanism for the spatial localization of Rac activity to enhance cell migration.

Isolation of immortalized, INK4a/ARF-deficient cells from the subventricular zone after in utero N-ethyl-N-nitrosourea exposure.

OBJECT: Brain tumors, including gliomas, develop several months after rats are exposed in utero to N-ethyl-N-nitroso-urea (ENU). Although pathological changes cannot be detected until these animals are several weeks old, the process that eventually leads to glioma formation must begin soon after exposure given the rapid clearance of the carcinogen and the observation that transformation of brain cells isolated soon after exposure occasionally occurs. This model can therefore potentially provide useful insights about the early events that precede overt glioma formation. The authors hypothesized that future glioma cells arise from stem/progenitor cells residing in or near the subventricular zone (SVZ) of the brain. METHODS: Cells obtained from the SVZ or corpus striatum in ENU-exposed and control rats were cultured in an epidermal growth factor (EGF)-containing, chemically defined medium. Usually, rat SVZ cells cultured in this manner (neurospheres) are nestin-positive, undifferentiated, and EGF-dependent and undergo cell senescence. Consistent with these prior observations, control SVZ cells undergo senescence by the 12th to 15th doubling (20 of 20 cultures). In contrast, three of 15 cultures of cells derived from the SVZs of individual ENU-treated rats continue to proliferate for more than 60 cell passages. Each of these nestin-expressing immortalized cell lines harbored a common homozygous deletion spanning the INK4a/ARF locus and was unable to differentiate into neural lineages after exposure to specific in vitro stimuli. Nevertheless, unlike the rat C6 glioma cell line, these immortalized cell lines demonstrate EGF dependence and low clonogenicity in soft agar and did not form tumors after intracranial transplantation. CONCLUSIONS: Data in this study indicated that immortalized cells may represent glioma precursors that reside in the area of the SVZ after ENU exposure that may serve as a reservoir for further genetic and epigenetic hits that could eventually result in a full glioma phenotype.

Interaction of Arl1-GTP with GRIP domains recruits autoantigens Golgin-97 and Golgin-245/p230 onto the Golgi.

A cellular role and the mechanism of action for small GTPase Arl1 have been defined. Arl1-GTP interacts with the GRIP domains of Golgin-97 and Golgin-245, a process dependent on conserved residues of the GRIP domains that are important for Golgi targeting. The switch II region of Arl1 confers the specificity of this interaction. Arl1-GTP mediates Golgi recruitment of Golgin-97 in a switch II-dependent manner, whereas tethering Arl1-GTP onto endosomes can mediate endosomal targeting of Golgin-97. Golgin-97 and Golgin-245 are dissociated from the Golgi when Arl1 is knocked-down by its siRNA. Arl1-GTP thus functions to recruit Golgin-97 and Golgin-245 onto the Golgi via interacting with their GRIP domains.

Stimulation-induced modifications in Go proteins examined in giant fused synaptosomes.

Synaptoneurosomes (1-3 microm in diameter), prepared from rat brain stem or brain cortex, were fused with liposomes, producing a high yield of giant synaptosomes (10-60 microm in diameter). Single channel currents were measured by using the cell-attach patch-clamp technique. The membrane of the majority of these giant synaptosomes retained the cell membrane selective permeability. However, nonpermeating molecules, such as guanine nucleotides and antibodies directed against GTP-binding region in the alpha-subunit of trimeric GTP-binding proteins, were trapped in the giant synaptosomes during their preparation. Activation of Go proteins was assayed in high [K(+)]-depolarized giant synaptosomes, indicating the advantage of this preparation for tracing signal-transduction mechanisms in stimulated synaptic membranes. Stimulation-induced interactions between membrane proteins, either native or reconstituted, can be studied in the giant synaptosomes.

Signalling via ADP-ribosylation factor 6 lies downstream of phosphatidylinositide 3-kinase.

ADP-ribosylation factor (ARF) 6 regulates plasma membrane trafficking and cortical actin formation by cycling between inactive GDP and active GTP-bound conformations. Here we show that agonist stimulation of phosphatidylinositide 3-kinase (PI 3-kinase) activates a pathway that leads to ARF6 activation. We also describe experiments that propose a central role in this pathway for the PI 3-kinase-dependent plasma membrane recruitment of the cytohesin-1 family of PtdIns(3,4,5)P(3)-binding ARF-exchange factors.

Effects of S-nitroso-cysteine on proteins that regulate exocytosis in PC12 cells: inhibitory effects on translocation of synaptophysin and ADP-ribosylation of GTP-binding proteins.

S-Nitroso-cysteine (SNC) inhibits Ca2+-induced noradrenaline (NA) release from PC12 cells. Since SNC stimulated Ca2+ mobilization from intracellular Ca2+ pools and SNC-induced inhibition of NA release was not washed-out, SNC may modify exocytosis-related proteins that overcome Ca2+ mobilization. In the present study, we investigated the effects of SNC on exocytosis-related proteins in PC12 cells. Ionomycin stimulated NA release and increased the immunoreactivity of synaptophysin in the cytosol fraction. A 25-kDa synaptosome-associated protein (SNAP-25), which localizes to plasma membranes and vesicles, increased in the cytosol fraction after stimulation. The increases in these proteins by ionomycin were inhibited in PC12 cells treated with 0.6 mM SNC. Synaptobrevin and synapsin-1 in the cytosol fraction, and syntaxin and 43 kDa growth-associated protein in the membrane fraction were not affected by ionomycin or SNC. Incubation of each protein with SNC did not affect antibody immunoreactivity. [32P]ADP-ribosylation of GTP-binding proteins (Gi/Go) by pertussis toxin, but not Gs by cholera toxin, was inhibited in SNC-treated PC12 cells and by co-addition of SNC to the assay mixture. These findings suggest that 1) SNC inhibits translocation of vesicles containing synaptophysin and SNAP-25, and 2) SNC reacts with cysteine residues in Gi/Go, causing inhibition of ADP-ribosylation by pertussis toxin.