Aeromonas exoenzyme T of Aeromonas salmonicida is a bifunctional protein that targets the host cytoskeleton

Type III protein secretion has been shown recently to be important in the virulence of the fish pathogen Aeromonas salmonicida. The ADP-ribosylating toxin Aeromonas exoenzyme T (AexT) is one effector protein targeted for secretion via this system. In this study, we identified muscular and nonmuscular actin as substrates of the ADP-ribosylating activity of AexT. Furthermore, we show that AexT also functions as a GTPase-activating protein (GAP), displaying GAP activity against monomeric GTPases of the Rho family, specifically Rho, Rac, and Cdc42. Transfection of fish cells with wild type AexT resulted in depolymerization of the actin cytoskeleton and cell rounding. Point mutations within either the GAP or the ADP-ribosylating active sites of AexT (Arg-143 as well as Glu-398 and Glu-401, respectively) abolished enzymatic activity, yet did not prevent actin filament depolymerization. However, inactivation of the two catalytic sites simultaneously did. These results suggest that both the GAP and ADP-ribosylating domains of AexT contribute to its biological activity. This is the first bacterial virulence factor to be described that has a specific actin ADP-ribosylation activity and GAP activity toward Rho, Rac, and Cdc42, both enzymatic activities contributing to actin filament depolymerization.

Integrin function in T cell activation recapitulated in tumor growth

An in vitro model using highly purified freshly isolated T cells demonstrated that immobilized ligands for the integrin $\alpha4\beta1$ could cooperate to enhance mitogen signals delivered by coimmobilized anti-CD3 specfic monoclonal antibody OKT3. Costimulation through $\alpha4\beta1$ integrin lead to enhanced proliferation which depended on expression of both IL-2 as well as IL-2 receptor. The transcription factors NF-AT, AP-1, and NF-$\kappa$B, which are involved in the regulation of IL-2 as well as other cytokine genes, were weakly induced by anti-CD3 stimulation alone in electromobility shift assays, but were augmented significantly with $\alpha4\beta1$ costimulation. These results suggested that $\alpha4\beta1$ ligands delivered a growth promoting signal which could synergize with signals induced by engagement of the TCR/CD3 complex, and also suggested a dual function for integrins in both localization and subsequent delivery of a growth promoting signal for T lymphocytes. Integrin involvement in lymphocyte trafficking has been employed as a model for understanding tumor cell metastasis. Therefore we have extended the duality of integrin function in both homing and subsequent delivery of a growth promoting signal to include a role for integrins in providing growth stimulation for tumor cells. Using a gastric derived tumor line, inhibition of adhesion to substrate leads to G0/G1 cell cycle arrest, reduced cyclin A expression, and reduced phospholipid synthesis. This effect could be reversed upon $\alpha2\beta1$ integrin mediated reattachment to collagen. These observations demonstrated a role for an integrin in the growth regulation of a tumor line. The small GTP-binding protein Rho, implicated in phospholipid synthesis, can be inactivated by the ADP-ribosylation exoenzyme C3 from C. botulinum. Addition of C3 to cell cultures inhibited the growth promoting effect due to integrin mediated adhesion. Taken together, these results are consistent with a model for cooperative interaction between integrins and Rho leading to enhanced phospholipid synthesis and mitogen signaling. This model may provide a basis for understanding the phenomena of integrin costimulation in T cell activation. ^

The story of RP10: A positional cloning approach to the identification of an autosomal dominant retinitis pigmentosa gene

Retinitis pigmentosa (RP) is an inherited retinal degenerative disease that is the leading cause of inherited blindness worldwide. Characteristic features of the disease include night blindness, progressive loss of visual fields, and deposition of pigment on the retina in a bone spicule-like pattern. RP is marked by extreme genetic heterogeneity with at least 19 autosomal dominant, autosomal recessive and X-linked loci identified. RP10, which maps to chromosome 7q, was the fifth autosomal dominant RP locus identified, and accounts for the early-onset disease in two independent families. Extensive linkage and haplotype analyses have been performed in these two families which have allowed the assignment of the disease locus to a 5-cM region on chromosome 7q31.3. In collaboration with Dr. Eric Green (National Center for Human Genome Research, National Institutes of Health), a well-characterized physical map of the region was constructed which includes YAC, BAC and cosmid coverage. The entire RP10 critical region resides within a 9-Mb well-characterized YAC contig. These physical maps not only provided the resources to undertake the CAIGES (cDNA amplification for identification of genomic expressed sequences) procedure for identification of retinal candidate genes within the critical region, but also identified a number of candidate genes, including transducin-$\gamma$ and blue cone pigment genes. All candidate genes examined were excluded. In addition, a number of ESTs were mapped within the critical region. EST20241, which was isolated from an eye library, corresponded to the 3$\sp\prime$ region of the ADP-ribosylation factor (ARF) 5 gene. ARF5, with its role in vesicle transport and possible participation in the regulation of the visual transduction pathway, became an extremely interesting candidate gene. Using a primer walking approach, the entire 3.2 kb genomic sequence of the ARF5 gene was generated and developed intronic primers to screen for coding region mutations in affected family members. No mutations were found in the ARF5 gene, however, a number of additional ESTs have been mapped to the critical region, and, as the large-scale sequencing projects get underway, megabases of raw sequence data from the RP10 region are becoming available. These resources will hasten the isolation and characterization of the RP10 gene. ^

THE MECHANISM OF RETINOIC ACID REGULATION OF TISSUE TRANSGLUTAMINASE GENE EXPRESSION

Retinoic acid regulates cellular growth and differentiation by altering the expression of specific sets of genes, but the molecular mechanism by which this is achieved is unknown. We have used the rapid induction of a specific enzyme, tissue transglutaminase in mouse macrophages, human leukemia cells and a variety of other cell types to study the regulation of gene expression by retinoic acid. Soluble retinoic acid binding proteins, such as cellular Retinoic Acid Binding Protein (cRABP), have been proposed as specific mediators of retinoic acid regulation of gene expression. This thesis demonstrates the lack of cRABP in a number of cell lines which are sensitive to retinoic acid regulation of tissue transglutaminase expression. These cells are also devoid of other soluble retinoic acid binding activity. The level of retinoic acid binding activity that could have been detected (6 fmol) is far below that of most cells and tissues which are sensitive to the effects of retinoic acid on growth and differentiation. A mouse melanoma cell line, S91-C2, was found to contain an unusual retinoic acid binding protein which has a lower affinity for retinoic acid than mouse tissue cRABP and also behaves differently on gel filtration HPLC chromatography.^ The induction of tissue transglutaminase by retinoic acid in macrophages is specifically inhibited by pertussis toxin. Pertussis toxin ADP-riblosylates membrane GTP-binding proteins such as N(,i) and interferes with signalling from plasma membrane receptors to regulatory enzymes. Pertussis toxin inhibition of transglutaminase induction is due to inhibition of tissue transglutaminase mRNA accumulation and is paralleled by the ADP-ribosylation of a 41,000 dalton macrophage membrane protein. It is concluded that soluble retinoic acid binding proteins are not essential for retinoic acid induction of tissue transglutaminase and that a membrane GTP-binding protein is closely linked to the sensitive response of macrophages to retinoic acid. ^

Functional analysis of GNA -1, a Galphai superfamily member found in the filamentous fungus Neurospora crassa

Heterotrimeric GTP-binding proteins, G proteins, are integral components of eukaryotic signaling systems linking extracellular signals to intracellular responses. Through coupling to seven-transmembrane helix receptors, G proteins convey primary signaling events into multi-leveled cascades of intracellular activity by regulating downstream enzymes, collectively called effectors. The effector enzymes regulated by G proteins include adenylyl cyclase, cAMP phosphodiesterase, phospolipase C-β, mitogen-activated protein kinases, and ion channels. ^ Neurospora crassa is a multicellular, filamentous fungus that is capable of both asexual and sexual reproduction by elaboration of specialized, developmentally controlled structures that give rise to either asexual or sexual spores, respectively. N. crassa possesses at least three heterotrimeric Gα proteins (GNA-1–3) and one Gβ subunit (GNB-1). GNA-1 was the first microbial protein that could be classified in the Gαi superfamily based on its amino acid identity and demonstration that it is a substrate for ADP-ribosylation by pertussis toxin. ^ Experiments were designed to identify the signal transduction pathways and the effector enzymes regulated by GNA-1. Targeted gene-replacement of gna-1 revealed that GNA-1 controls multiple developmental pathways including both asexual and sexual reproduction, maintenance of growth, and resistance to osmotic stress. The Gαi and Gαz members of the Gαi superfamily negatively regulate adenylyl cyclase activity in mammalian cells; therefore, adenylyl cyclase and cAMP levels were measured in Δgna-1 strains and also in strains that were deleted for both gna-1 and gna-2, a second Gα in N. crassa shown to have overlapping functions with GNA-1. Direct measurements of adenylyl cyclase activity revealed that GNA-1, but not GNA-2, was responsible for GTP-stimulated adenylyl cyclase activity in N. crassa. Furthermore, anti-GNA-1 IgG could specifically inhibit GTP-stimulated adenylyl cyclase activity in wild-type strain extracts. These studies also provided evidence that N. crassa possesses feedback mechanisms that control steady-state cAMP levels through indirect regulation of cAMP-phosphodiesterase activity; mutations in gna-1 and gna-2 were additive in their effect on lowering cAMP-phosphodiesterase activity under growth conditions where steady-state cAMP levels were normal but GTP-stimulated adenylyl cyclase activity was reduced 90% in comparison to control strains. ^ Genetic and biochemical epistasis experiments utilizing a Δ gna-1 cr-1 mutant suggest that GNA-1 is essential for female fertility in a cAMP-independent pathway. Furthermore, deletion of gna-1 in a cr-1 background exacerbated many of the defects already observed in the cr-1 strain including more severe growth restriction and developmental defects. However, deletion of gna-1 had no effect on the increased thermotolerance of cr-1, which has been attributed to loss of cAMP. cr-1 possesses GNA-1 protein, and crude membrane fractions from this strain reconstituted GTP-stimulated adenylyl cyclase activity in Δgna-1 membrane fractions. These studies provide direct evidence for the involvement of Gα proteins in the regulation of adenylyl cyclase activity in eukaryotic microbes. ^

Requirement of GM2 ganglioside activator for phospholipase D activation

Sequence analysis of a heat-stable protein necessary for the activation of ADP ribosylation factor-dependent phospholipase D (PLD) reveals that this protein has a structure highly homologous to the previously known GM2 ganglioside activator whose deficiency results in the AB-variant of GM2 gangliosidosis. The heat-stable activator protein indeed has the capacity to enhance enzymatic conversion of GM2 to GM3 ganglioside that is catalyzed by β-hexosaminidase A. Inversely, GM2 ganglioside activator purified separately from tissues as described earlier [Conzelmann, E. & Sandhoff, K. (1987) Methods Enzymol. 138, 792–815] stimulates ADP ribosylation factor-dependent PLD in a dose-dependent manner. At higher concentrations of ammonium sulfate, the PLD activator protein apparently substitutes for protein kinase C and phosphatidylinositol 4,5-bisphosphate, both of which are known as effective stimulators of the PLD reaction. The mechanism of action of the heat-stable PLD activator protein remains unknown.

High-Affinity Binding Of The AP-1 Adaptor Complex to Trans-Golgi Network Membranes Devoid Of Mannose 6-Phosphate Receptors

The GTP-binding protein ADP-ribosylation factor (ARF) initiates clathrin-coat assembly at the trans-Goli network (TGN) by generating high-affinity membrane-binding sites for the AP-1 adaptor complex. Both transmembrane proteins, which are sorted into the assembling coated bud, and novel docking proteins have been suggested to be partners with GTP-bound ARF in generating the AP-1-docking sites. The best characterized, and probably the major transmembrane molecules sorted into the clathrin-coated vesicles that form on the TGN, are the mannose 6-phosphate receptors (MPRs). Here, we have examined the role of the MPRs in the AP-1 recruitment process by comparing fibroblasts derived from embryos of either normal or MPR-negative animals. Despite major alterations to the lysosome compartment in the MPR-deficient cells, the steady-state distribution of AP-1 at the TGN is comparable to that of normal cells. Golgi-enriched membranes prepared from the receptor-negative cells also display an apparently normal capacity to recruit AP-1 in vitro in the presence of ARF and either GTP or GTPγS. The AP-1 adaptor is recruited specifically onto the TGN and not onto the numerous abnormal membrane elements that accumulate within the MPR-negative fibroblasts. AP-1 bound to TGN membranes from either normal or MPR-negative fibroblasts is fully resistant to chemical extraction with 1 M Tris-HCl, pH 7, indicating that the adaptor binds to both membrane types with high affinity. The only difference we do note between the Golgi prepared from the MPR-deficient cells and the normal cells is that AP-1 recruited onto the receptor-lacking membranes in the presence of ARF1·GTP is consistently more resistant to extraction with Tris. Because sensitivity to Tris extraction correlates well with nucleotide hydrolysis, this finding might suggest a possible link between MPR sorting and ARF GAP regulation. We conclude that the MPRs are not essential determinants in the initial steps of AP-1 binding to the TGN but, instead, they may play a regulatory role in clathrin-coated vesicle formation by affecting ARF·GTP hydrolysis.

ARF Is Required for Maintenance of Yeast Golgi and Endosome Structure and Function

ADP ribosylation factor (ARF) is thought to play a critical role in recruiting coatomer (COPI) to Golgi membranes to drive transport vesicle budding. Yeast strains harboring mutant COPI proteins exhibit defects in retrograde Golgi to endoplasmic reticulum protein transport and striking cargo-selective defects in anterograde endoplasmic reticulum to Golgi protein transport. To determine whether arf mutants exhibit similar phenotypes, the anterograde transport kinetics of multiple cargo proteins were examined in arf mutant cells, and, surprisingly, both COPI-dependent and COPI-independent cargo proteins exhibited comparable defects. Retrograde dilysine-mediated transport also appeared to be inefficient in the arf mutants, and coatomer mutants with no detectable anterograde transport defect exhibited a synthetic growth defect when combined with arf1Δ, supporting a role for ARF in retrograde transport. Remarkably, we found that early and medial Golgi glycosyltransferases localized to abnormally large ring-shaped structures. The endocytic marker FM4–64 also stained similar, but generally larger ring-shaped structures en route from the plasma membrane to the vacuole in arf mutants. Brefeldin A similarly perturbed endosome morphology and also inhibited transport of FM4–64 from endosomal structures to the vacuole. Electron microscopy of arf mutant cells revealed the presence of what appear to be hollow spheres of interconnected membrane tubules which likely correspond to the fluorescent ring structures. Together, these observations indicate that organelle morphology is significantly more affected than transport in the arf mutants, suggesting a fundamental role for ARF in regulating membrane dynamics. Possible mechanisms for producing this dramatic morphological change in intracellular organelles and its relation to the function of ARF in coat assembly are discussed.

The Golgi-associated COPI-coated buds and vesicles contain β/γ-actin

It has been shown previously that the morphology and subcellular positioning of the Golgi complex is controlled by actin microfilaments. To further characterize the association between actin microfilaments and the Golgi complex, we have used the Clostridium botulinum toxins C2 and C3, which specifically inhibit actin polymerization and cause depolymerization of F-actin in intact cells by the ADP ribosylation of G-actin monomers and the Rho small GTP-binding protein, respectively. Normal rat kidney cells treated with C2 showed that disruption of the actin and the collapse of the Golgi complex occurred concomitantly. However, when cells were treated with C3, the actin disassembly was observed without any change in the organization of the Golgi complex. The absence of the involvement of Rho was further confirmed by the treatment with lysophosphatidic acid or microinjection with the constitutively activated form of RhoA, both of which induced the stress fiber formation without affecting the Golgi complex. Immunogold electron microscopy in normal rat kidney cells revealed that β- and γ-actin isoforms were found in Golgi-associated COPI-coated buds and vesicles. Taken together, the results suggest that the Rho signaling pathway does not directly regulate Golgi-associated actin microfilaments, and that β- and γ-actins might be involved in the formation and/or transport of Golgi-derived vesicular or tubular intermediates.

Multiple Roles of Arf1 GTPase in the Yeast Exocytic and Endocytic Pathways

ADP-ribosylation factors, a family of small GTPases, are believed to be key regulators of intracellular membrane traffic. However, many biochemical in vitro experiments have led to different models for their involvement in various steps of vesicular transport, and their precise role in living cells is still unclear. We have taken advantage of the powerful yeast genetic system and screened for temperature-sensitive (ts) mutants of the ARF1 gene from Saccharomyces cerevisiae. By random mutagenesis of the whole open reading frame of ARF1 by error-prone PCR, we isolated eight mutants and examined their phenotypes. arf1 ts mutants showed a variety of transport defects and morphological alterations in an allele-specific manner. Furthermore, intragenic complementation was observed between certain pairs of mutant alleles, both for cell growth and intracellular transport. These results demonstrate that the single Arf1 protein is indeed involved in many different steps of intracellular transport in vivo and that its multiple roles may be dissected by the mutant alleles we constructed.

Delivery of proteins into living cells by reversible membrane permeabilization with streptolysin-O

The pore-forming toxin streptolysin O (SLO) can be used to reversibly permeabilize adherent and nonadherent cells, allowing delivery of molecules with up to 100 kDa mass to the cytosol. Using FITC-labeled albumin, 105–106 molecules were estimated to be entrapped per cell. Repair of toxin lesions depended on Ca2+-calmodulin and on intact microtubules, but was not sensitive to actin disruption or to inhibition of protein synthesis. Resealed cells were viable for days and retained the capacity to endocytose and to proliferate. The active domains of large clostridial toxins were introduced into three different cell lines. The domains were derived from Clostridium difficile B-toxin and Clostridium sordelli lethal toxin, which glycosylate small G-proteins, and from Clostridium botulinum C2 toxin, which ADP-ribosylates actin. After delivery with SLO, all three toxins disrupted the actin cytoskeleton to cause rounding up of the cells. Glucosylation assays demonstrated that G-proteins Rho and Ras were retained in the permeabilized cells and were modified by the respective toxins. Inactivation of these G-proteins resulted in reduced stimulus-dependent granule secretion, whereas ADP-ribosylation of actin by the C. botulinum C2-toxin resulted in enhanced secretion in cells. The presented method for introducing proteins into living cells should find multifaceted application in cell biology.

Controlling small guanine–nucleotide-exchange factor function through cytoplasmic RNA intramers

ADP-ribosylation factor (ARF) GTPases and their regulatory proteins have been implicated in the control of diverse biological functions. Two main classes of positive regulatory elements for ARF have been discovered so far: the large Sec7/Gea and the small cytohesin/ARNO families, respectively. These proteins harbor guanine–nucleotide-exchange factor (GEF) activity exerted by the common Sec7 domain. The availability of a specific inhibitor, the fungal metabolite brefeldin A, has enabled documentation of the involvement of the large GEFs in vesicle transport. However, because of the lack of such tools, the biological roles of the small GEFs have remained controversial. Here, we have selected a series of RNA aptamers that specifically recognize the Sec7 domain of cytohesin 1. Some aptamers inhibit guanine–nucleotide exchange on ARF1, thereby preventing ARF activation in vitro. Among them, aptamer M69 exhibited unexpected specificity for the small GEFs, because it does not interact with or inhibit the GEF activity of the related Gea2-Sec7 domain, a member of the class of large GEFs. The inhibitory effect demonstrated in vitro clearly is observed as well in vivo, based on the finding that M69 produces similar results as a dominant-negative, GEF-deficient mutant of cytohesin 1: when expressed in the cytoplasm of T-cells, M69 reduces stimulated adhesion to intercellular adhesion molecule-1 and results in a dramatic reorganization of F-actin distribution. These highly specific cellular effects suggest that the ARF-GEF activity of cytohesin 1 plays an important role in cytoskeletal remodeling events of lymphoid cells.

Opposite actions of nitric oxide on cholinergic synapses: which pathways?

Nitric oxide (NO) produced opposite effects on acetylcholine (ACh) release in identified neuroneuronal Aplysia synapses depending on the excitatory or the inhibitory nature of the synapse. Extracellular application of the NO donor, SIN-1, depressed the inhibitory postsynaptic currents (IPSCs) and enhanced the excitatory postsynaptic currents (EPSCs) evoked by presynaptic action potentials (1/60 Hz). Application of a membrane-permeant cGMP analog mimicked the effect of SIN-1 suggesting the participation of guanylate cyclase in the NO pathway. The guanylate cyclase inhibitor, methylene blue, blocked the NO-induced enhancement of EPSCs but only reduced the inhibition of IPSCs indicating that an additional mechanism participates to the depression of synaptic transmission by NO. Using nicotinamide, an inhibitor of ADP-ribosylation, we found that the NO-induced depression of ACh release on the inhibitory synapse also involves ADP-ribosylation mechanism(s). Furthermore, application of SIN-1 paired with cGMP-dependent protein kinase (cGMP-PK) inhibitors showed that cGMP-PK could play a role in the potentiating but not in the depressing effect of NO on ACh release. Increasing the frequency of stimulation of the presynaptic neuron from 1/60 Hz to 0.25 or 1 Hz potentiated the EPSCs and reduced the IPSCs. In these conditions, the potentiating effect of NO on the excitatory synapse was reduced, whereas its depressing effect on the inhibitory synapse was unaffected. Moreover the frequency-dependent enhancement of ACh release in the excitatory synapse was greatly reduced by the inhibition of NO synthase. Our results indicate that NO may be involved in different ways of modulation of synaptic transmission depending on the type of the synapse including synaptic plasticity.

Inhibition of G-protein betagamma-subunit functions by phosducin-like protein.

Phosducin is a cytosolic protein predominantly expressed in the retina and the pineal gland that can interact with the betagamma subunits of guanine nucleotide binding proteins (G proteins) and thereby may regulate transmembrane signaling. A cDNA encoding a phosducin-like protein (PhLP) has recently been isolated from rat brain [Miles, M. F., Barhite, S., Sganga, M. & Elliott, M. (1993) Proc. Natl. Acad. Sci. USA 90, 10831-10835. Here we report the expression of PhLP in Escherichia coli and its purification. Recombinant purified PUP inhibited multiple effects of G-protein betagamma subunits. First, it inhibited the betagamma-subunit-dependent ADP-ribosylation of purified alpha(o) by pertussis toxin. Second, it inhibited the GTPase activity of purified G(o). The IC50 value of PhLP in the latter assay was 89 nM, whereas phosducin caused half-maximal inhibition at 17 nM. And finally, PhLP antagonized the enhancement of rhodopsin phosphorylation by purified betagamma subunits. The N terminus of PhLP shows no similarity to the much longer N terminus of phosducin, the region shown to be critical for phosducin-betagamma-subunit interactions. Therefore, PhLP appears to bind to G-protein betagamma subunits by an as yet unknown mode of interaction and may represent an endogenous regulator of G-protein function.

Role of guanine nucleotide-binding proteins--ras-family or trimeric proteins or both--in Ca2+ sensitization of smooth muscle.

The purpose of this study was to identify guanine nucleotide-binding proteins (G proteins) involved in the agonist- and guanosine 5'-[gamma-thio]triphosphate (GTP[gamma-S])-induced increase in the Ca2+ sensitivity of 20-kDa myosin light chain (MLC20) phosphorylation and contraction in smooth muscle. A constitutively active, recombinant val14p21rhoA.GTP expressed in the baculovirus/Sf9 system, but not the protein expressed without posttranslational modification in Escherichia coli, induced at constant Ca2+ (pCa 6.4) a slow contraction associated with increased MLC20 phosphorylation from 19.8% to 29.5% (P < 0.05) in smooth muscle permeabilized with beta-esein. The effect of val14p21rhoA.GTP was inhibited by ADP-ribosylation of the protein and was absent in smooth muscle extensively permeabilized with Triton X-100. ADP-ribosylation of endogenous p21rho with epidermal cell differentiation inhibitor (EDIN) inhibited Ca2+ sensitization induced by GTP [in rabbit mesenteric artery (RMA) and rabbit ileum smooth muscles], by carbachol (in rabbit ileum), and by endothelin (in RMA), but not by phenylephrine (in RMA), and only slowed the rate without reducing the amplitude of contractions induced in RMA by 1 microM GTP[gamma-S] at constant Ca2+ concentrations. AlF(4-)-induced Ca2+ sensitization was inhibited by both guanosine 5'-[beta-thio]diphosphate (GDP[beta-S]) and by EDIN. EDIN also inhibited, to a lesser extent, contractions induced by Ca2+ alone (pCa 6.4) in both RMA and rabbit ileum. ADP-ribosylation of trimeric G proteins with pertussis toxin did not inhibit Ca2+ sensitization. We conclude that p21rho may play a role in physiological Ca2+ sensitization as a cofactor with other messengers, rather than as a sole direct inhibitor of smooth muscle MLC20 phosphatase.