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At least 127 records · Page 7Linked to original sources
PubMed2005

The expression of the virulence-associated effector protein gene avrA is dependent on a Salmonella enterica-specific regulatory function.

Although the avrA gene is prevalent among 80% of the Salmonella enterica serovars, only a small number of them usually express the respective virulence-associated effector protein AvrA. However, under culture conditions below pH 6.0 many of the AvrA non-producer strains (e.g. S. Agona, S. Bovismorbificans, S. Virchow) begin to produce AvrA, while some others remain silent. Four phenotypical classes of S. enterica were identified under defined standard culture conditions: class 1 comprises strains with a constitutive synthesis of AvrA; class 2 comprises strains with an acid induction of AvrA; class 3 comprises strains with silent avrA genes; and the fourth class comprises strains which do not contain the avrA gene (class 0 strains). The expression of avrA was found to be controlled by a Salmonella-specific mechanism because cloned avrA genes from classes 1, 2, and 3 strains remain silent in Escherichia coli strains, while easily expressed in S. enterica strains. The expression of AvrA in classes 1, 2, and 3 strains does not coincide with the nucleotide sequences of the respective promoter or structural regions of the avrA genes, but depends directly on this Salmonella-specific regulatory system which appears to be differently modulated in the distinct Salmonella serovars.

Amino Acid Sequence↗
PubMed2001

Immunocytochemical localization of HrpA and HrpZ supports a role for the Hrp pilus in the transfer of effector proteins from Pseudomonas syringae pv. tomato across the host plant cell wall.

The Hrp pilus, composed of HrpA subunits, is an essential component of the type III secretion system in Pseudomonas syringae. We used electron microscopy (EM) and immunocytochemistry to examine production of the pilus in vitro from P. syringae pv. tomato strain DC3000 grown under hrp-inducing conditions on EM grids. Pili, when labeled with antibodies to HrpA, developed rapidly in a nonpolar manner shortly after the detection of the hrpA transcript and extended up to 5 microm into surrounding media. Structures at the base of the pilus were clearly differentiated from the basal bodies of flagella. The HrpZ protein, also secreted via the type III system, was found by immunogold labeling to be associated with the pilus in vitro. Accumulation and secretion of HrpA and HrpZ were also examined quantitatively after the inoculation of wild-type DC3000 and hrpA and hrpZ mutants into leaves of Arabidopsis thaliana. The functional pilus crossed the plant cell wall to generate tracks of immunogold labeling for HrpA and HrpZ. Mutants that produced HrpA but did not assemble pili were nonpathogenic, did not secrete HrpA protein, and were compromised for the accumulation of HrpZ. A model is proposed in which the rapidly elongating Hrp pilus acts as a moving conveyor, facilitating transfer of effector proteins from bacteria to the plant cytoplasm across the formidable barrier of the plant cell wall.

Arabidopsis↗
PubMed2006

The Salmonella effector protein PipB2 is a linker for kinesin-1.

Understanding the mechanisms of Salmonella virulence is an important challenge. The capacity of this intracellular bacterial pathogen to cause diseases depends on the expression of virulence factors including the second type III secretion system (TTSS-2), which is used to translocate into the eukaryotic cytosol a set of effector proteins that divert the biology of the host cell and shape the bacterial replicative niche. Yet little is known about the eukaryotic functions affected by individual Salmonella effectors. Here we report that the TTSS-2 effector PipB2 interacts with the kinesin light chain, a subunit of the kinesin-1 motor complex that drives anterograde transport along microtubules. Translocation of PipB2 is both necessary and sufficient for the recruitment of kinesin-1 to the membrane of the Salmonella-containing vacuole. In vivo, PipB2 contributes to the attenuation of Salmonella mutant strains in mice. Taken together, our data indicate that the TTSS-2-mediated fine-tuning of kinesin-1 activity associated with the bacterial vacuole is crucial for the virulence of Salmonella.

Animals↗
PubMed1989

G protein-effector coupling: interactions of recombinant inhibitory gamma subunit with transducin and phosphodiesterase.

A bacterial expression vector for the inhibitory gamma subunit of retinal rod phosphodiesterase has been constructed by inserting a mouse gamma cDNA into pUC19. Escherichia coli 222 transformed with this plasmid produces a 12-kDa recombinant protein consisting of 18 additional amino acids attached to the amino terminus of gamma. The fusion protein, designated beta-gal-gamma, has been refolded into an active form in formic acid and partially purified by gel filtration chromatography. Despite a large extended sequence at the amino terminus, beta-gal-gamma is able to inhibit the activity of trypsin-activated phosphodiesterase, bind tightly to the catalytic alpha beta subunits, and interact with the alpha subunit of transducin in a nucleotide-dependent manner. The availability of large quantities of active bacterial gamma, together with the ability to change its primary structure by site-directed mutagenesis, promises to provide considerable new information on the interaction between transducin and phosphodiesterase, as well as insights into the molecular mechanism of G protein-effector coupling.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1997

G proteins, effectors and GAPs: structure and mechanism.

G proteins from a diverse family of regulatory GTPases which, in the GTP-bound state, bind to and activate downstream effectors. Structures of Ras homologs bound to effector domains have revealed mechanisms by which G proteins couple GTP binding to effector activation and achieve specificity. Complexes between structurally unrelated GTPase-activating proteins with complementary G proteins suggest common mechanisms by which GTP hydrolysis is stimulated via direct interactions with conformationally labile switch regions of the G protein.

Binding Sites↗
PubMed2005

Crystal structures and functional studies of T4moD, the toluene 4-monooxygenase catalytic effector protein.

Toluene 4-monooxygenase (T4MO) is a four-component complex that catalyzes the regiospecific, NADH-dependent hydroxylation of toluene to yield p-cresol. The catalytic effector (T4moD) of this complex is a 102-residue protein devoid of metals or organic cofactors. It forms a complex with the diiron hydroxylase component (T4moH) that influences both the kinetics and regiospecificity of catalysis. Here, we report crystal structures for native T4moD and two engineered variants with either four (DeltaN4-) or 10 (DeltaN10-) residues removed from the N-terminal at 2.1-, 1.7-, and 1.9-A resolution, respectively. The crystal structures have C-alpha root-mean-squared differences of less than 0.8 A for the central core consisting of residues 11-98, showing that alterations of the N-terminal have little influence on the folded core of the protein. The central core has the same fold topology as observed in the NMR structures of T4moD, the methane monooxygenase effector protein (MmoB) from two methanotrophs, and the phenol hydroxylase effector protein (DmpM). However, the root-mean-squared differences between comparable C-alpha positions in the X-ray structures and the NMR structures vary from approximately 1.8 A to greater than 6 A. The X-ray structures exhibit an estimated overall coordinate error from 0.095 (0.094) A based on the R-value (R free) for the highest resolution DeltaN4-T4moD structure to 0.211 (0.196) A for the native T4moD structure. Catalytic studies of the DeltaN4-, DeltaN7-, and DeltaN10- variants of T4moD show statistically insignificant changes in k(cat), K(M), k(cat)/K(M), and K(I) relative to the native protein. Moreover, there was no significant change in the regiospecificity of toluene oxidation with any of the T4moD variants. The relative insensitivity to changes in the N-terminal region distinguishes T4moD from the MmoB homologues, which each require the approximately 33 residue N-terminal region for catalytic activity.

Amino Acid Sequence↗
PubMed2006

Identification of an effector protein and gain-of-function mutants that activate Pfmrk, a malarial cyclin-dependent protein kinase.

Cyclin-dependent protein kinases (CDKs) are key regulators of cell cycle control. In humans, CDK7 performs dual roles as the CDK activating kinase (CAK) responsible for regulating numerous CDKs and as the RNA polymerase II carboxyl-terminal domain (CTD) kinase involved in the regulation of transcription. Binding of an effector protein, human MAT1, stimulates CDK7 kinase activity and influences substrate specificity. In Plasmodium falciparum, CDKs and their roles in regulating growth and development are poorly understood. In this study, we characterized the regulatory mechanisms of Pfmrk, a putative homolog of human CDK7. We identified an effector, PfMAT1, which stimulates Pfmrk kinase activity in a cyclin-dependent manner. The addition of PfMAT1 stimulated RNA polymerase II CTD phosphorylation and had no effect on the inability of Pfmrk to phosphorylate PfPK5, a putative CDK1 homolog, which suggests that Pfmrk may be a CTD kinase rather than a CAK. In an attempt to abrogate the requirement for PfMAT1 stimulation, we mutated amino acids within the active site of Pfmrk. We found that two independent mutants, S138K and F143L, yielded a 4-10-fold increase in Pfmrk activity. Significant kinase activity of these mutants was observed in the absence of either cyclin or PfMAT1. Finally, we observed autophosphorylation of Pfmrk that is unaffected by the addition of either cyclin or PfMAT1.

Amino Acid Sequence↗
PubMed2003

Human homologue of Drosophila CNK interacts with Ras effector proteins Raf and Rlf.

Connector enhancer of KSR (CNK) is a multidomain protein that participates in Ras signaling in Drosophila eye development. In this report we identify the human homologue of CNK, termed CNK2A, and a truncated alternatively spliced variant, CNK2B. We characterize CNK2 phosphorylation, membrane localization, and interaction with Ras effector molecules. Our results show that MAPK signaling appears to play a role in the phosphorylation of CNK2 in vivo. CNK2 is found in both membrane and cytoplasmic fractions of the cell. In MDCK cells, full-length CNK2 is localized to the lateral plasma membrane. Consistent with previous reports, we show CNK2 interacts with Raf. CNK2 interaction was mapped to the regulatory and kinase domains of Raf, as well as to the carboxyl-terminal half of CNK2. CNK2 also interacts with the Ral signaling components, Ral GTPase, and the RalGDS family member Rlf. CNK2 interaction was mapped to the GEF domain of Rlf. The ability of CNK2 to interact with both Ras effector proteins Raf and Rlf suggests that CNK2 may integrate signals between MAPK and Ral pathways through a complex interplay of components.

Adaptor Proteins, Signal Transducing↗
PubMed2006

Legionella pneumophila exploits PI(4)P to anchor secreted effector proteins to the replicative vacuole.

The causative agent of Legionnaires' disease, Legionella pneumophila, employs the intracellular multiplication (Icm)/defective organelle trafficking (Dot) type IV secretion system (T4SS) to upregulate phagocytosis and to establish a replicative vacuole in amoebae and macrophages. Legionella-containing vacuoles (LCVs) do not fuse with endosomes but recruit early secretory vesicles. Here we analyze the role of host cell phosphoinositide (PI) metabolism during uptake and intracellular replication of L. pneumophila. Genetic and pharmacological evidence suggests that class I phosphatidylinositol(3) kinases (PI3Ks) are dispensable for phagocytosis of wild-type L. pneumophila but inhibit intracellular replication of the bacteria and participate in the modulation of the LCV. Uptake and degradation of an icmT mutant strain lacking a functional Icm/Dot transporter was promoted by PI3Ks. We identified Icm/Dot-secreted proteins which specifically bind to phosphatidylinositol(4) phosphate (PI(4)P) in vitro and preferentially localize to LCVs in the absence of functional PI3Ks. PI(4)P was found to be present on LCVs using as a probe either an antibody against PI(4)P or the PH domain of the PI(4)P-binding protein FAPP1 (phosphatidylinositol(4) phosphate adaptor protein-1). Moreover, the presence of PI(4)P on LCVs required a functional Icm/Dot T4SS. Our results indicate that L. pneumophila modulates host cell PI metabolism and exploits the Golgi lipid second messenger PI(4)P to anchor secreted effector proteins to the LCV.

Animals↗
PubMed1998

Genes encoding putative effector proteins of the type III secretion system of Salmonella pathogenicity island 2 are required for bacterial virulence and proliferation in macrophages.

The type III secretion system of Salmonella pathogenicity island 2 (SPI-2) is required for systemic infection of this pathogen in mice. Cloning and sequencing of a central region of SPI-2 revealed the presence of genes encoding putative chaperones and effector proteins of the secretion system. The predicted products of the sseB, sseC and sseD genes display weak but significant similarity to amino acid sequences of EspA, EspD and EspB, which are secreted by the type III secretion system encoded by the locus of enterocyte effacement of enteropathogenic Escherichia coli. The transcriptional activity of an sseA::luc fusion gene was shown to be dependent on ssrA, which is required for the expression of genes encoding components of the secretion system apparatus. Strains carrying nonpolar mutations in sseA, sseB or sseC were severely attenuated in virulence, strains carrying mutations in sseF or sseG were weakly attenuated, and a strain with a mutation in sseE had no detectable virulence defect. These phenotypes were reflected in the ability of mutant strains to grow within a variety of macrophage cell types: strains carrying mutations in sseA, sseB or sseC failed to accumulate, whereas the growth rates of strains carrying mutations in sseE, sseF or sseG were only modestly reduced. These data suggest that, in vivo, one of the functions of the SPI-2 secretion system is to enable intracellular bacterial proliferation.

Acetyltransferases↗
PubMed2006

The Rab4A effector protein Rabip4 is involved in migration of NIH 3T3 fibroblasts.

The small GTP-binding protein Rab4 has been involved in the recycling of alphavbeta3 integrins in response to platelet-derived growth factor (PDGF) stimulation suggesting a role for Rab4 in cell adhesion and migration. In this study, we explored the role of Rabip4 and Rabip4', two Rab4 effector proteins, in migration of NIH 3T3 fibroblasts. In these cells, Rabip4 and Rabip4', collectively named Rabip4s, were partially co-localized with the early endosomal marker EEA1. PDGF treatment re-distributed endogenous Rabip4s toward the cell periphery where they colocalized with F-actin. In cells expressing green fluorescent protein (GFP)-Rabip4 or GFP-Rabip4', constitutive appearance of GFP-Rabip4s at the cell periphery was accompanied by local increase in cortical F-actin in membrane ruffles at the leading edge. The expression of GFP-Rabip4 induced an increased migration compared with control cells expressing GFP alone, even in the absence of PDGF stimulation. On the contrary, in cells expressing a mutated form of Rabip4s unable to interact with Rab4, lack of typical leading edge was observed. Furthermore, PDGF treatment did not stimulate the migration of these cells. Furthermore, down-regulation of the expression of Rabip4s inhibited PDGF-stimulated cell migration. Endogenous Rabip4s were localized with alphav integrins at the leading edge following PDGF treatment, whereas in cells expressing GFP-Rabip4s, alphav integrins, together with GFP-Rabip4s, were constitutively localized at the leading edge. In contrast, reduction in Rabip4s expression levels using small interfering RNA was associated with impaired PDGF-induced translocation of alphav integrins toward the leading edge. Taken together, our data provide evidence that Rabip4s, possibly via their interaction with Rab4, regulate integrin trafficking and are involved in the migration of NIH 3T3 fibroblasts.

Actins↗
PubMed2005

Rab15 effector protein: a novel protein for receptor recycling from the endocytic recycling compartment.

Sorting endosomes and the endocytic recycling compartment are critical intracellular stores for the rapid recycling of internalized membrane receptors to the cell surface in multiple cell types. However, the molecular mechanisms distinguishing fast receptor recycling from sorting endosomes and slow receptor recycling from the endocytic recycling compartment remain poorly understood. We previously reported that Rab15 differentially regulates transferrin receptor trafficking through sorting endosomes and the endocytic recycling compartment, suggesting a role for distinct Rab15-effector interactions at these endocytic compartments. In this study, we identified the novel protein Rab15 effector protein (REP15) as a binding partner for Rab15-GTP. REP15 is compartment specific, colocalizing with Rab15 and Rab11 on the endocytic recycling compartment but not with Rab15, Rab4, or early endosome antigen 1 on sorting endosomes. REP15 interacts directly with Rab15-GTP but not with Rab5 or Rab11. Consistent with its localization, REP15 overexpression and small interfering RNA-mediated depletion inhibited transferrin receptor recycling from the endocytic recycling compartment, without affecting receptor entry into or recycling from sorting endosomes. Our data identify REP15 as a compartment-specific protein for receptor recycling from the endocytic recycling compartment, highlighting that the rapid and slow modes of transferrin receptor recycling are mechanistically distinct pathways.

Adaptor Proteins, Signal Transducing↗
PubMed2006

The Agrobacterium VirE3 effector protein: a potential plant transcriptional activator.

During the infection of plants, Agrobacterium tumefaciens introduces several Virulence proteins including VirE2, VirF, VirD5 and VirE3 into plant cells in addition to the T-DNA. Here, we report that double mutation of virF and virE3 leads to strongly diminished tumor formation on tobacco, tomato and sunflower. The VirE3 protein is translated from a polycistronic mRNA containing the virE1, virE2 and virE3 genes, in Agrobacterium. The VirE3 protein has nuclear localization sequences, which suggests that it is transported into the plant cell nucleus upon translocation. Indeed we show here that VirE3 interacts in vitro with importin-alpha and that a VirE3-GFP fusion protein is localized in the nucleus. VirE3 also interacts with two other proteins, viz. pCsn5, a component of the COP9 signalosome and pBrp, a plant specific general transcription factor belonging to the TFIIB family. We found that VirE3 is able to induce transcription in yeast when bound to DNA through the GAL4-BD. Our data indicate that the translocated effector protein VirE3 is transported into the nucleus and there it may interact with the transcription factor pBrp to induce the expression of genes needed for tumor development.

Agrobacterium tumefaciens↗
PubMed1996

Expression pattern of candidate cell death effector proteins Bax, Bcl-2, Bcl-X, and c-Jun in sensory and motor neurons following sciatic nerve transection in the rat.

Numerous studies have demonstrated a prolonged expression of c-Jun transcription factor in neurons following axotomy, and it has been hypothesized that c-Jun may be causally involved in neuroregeneration in vivo. By contrast, there is growing evidence from in vitro studies that induction of c-Jun may be necessary for neuronal cell death induced by growth factor starvation. It has been demonstrated that protein levels of cell death repressor Bcl-2 and cell death promotor Bax determine the threshold for neuronal cell death and that their expression is dynamically modulated at the onset of neurodegeneration. In the present study, we investigated by double-immunolabeling methods activation of c-Jun transcription factor and expression of members of the Bcl-2 family of cell death effector proteins in axotomized neurons. Six days after transection of the sciatic nerve in young rats, when axotomized neurons start to degenerate, strong nuclear Jun immunostaining in spinal cord motoneurons was associated with intense cytoplasmic Bax labeling and signs of neuronal atrophy. Bcl-2 and Bcl-X proteins were present only at moderate to low levels. In situ end-labeling by terminal transferase revealed nuclear DNA fragmentation in scattered motoneurons of the ipsilateral ventral horn (1 or 2 labeled nuclei per section). In the L5 dorsal root ganglia (DRG) levels of Bax, Bcl-2, and Bcl-X proteins were highly variable. High levels of Bax immunoreactivity together with intense Jun immunofluorescence were frequently observed in small-diameter sensory neurons. RT-PCR analysis revealed expression of exclusively the anti-apoptotic bcl-xL mRNA isoform in rat DRG which decreased significantly following sciatic nerve transection. These findings indicate that the high susceptibility of central neurons and small-sized DRG neurons to axotomy-induced cell death might be related to their low ratio of cell death repressor Bcl-2 and Bcl-XL to cell death promotor Bax expression. It should be noted, however, that numerous strongly Jun-positive DRG neurons contained low levels of Bax or high levels of Bcl-2 and Bcl-X immunoreactivity. Thus, high levels of c-Jun protein in axotomized neurons do not necessarily suggest a destination to die, and other factors may determine the outcome of axotomy.

Animals↗
PubMed1985

The nature of neutralization reaction between effector protein and monoclonal antibody: a quantitative study of neutralization characteristics of anti-interferon antibodies.

Several monoclonal antibodies to human interferon-alpha and beta were examined quantitatively for neutralization of antigenically homogeneous interferons. The extent of neutralization increased with antibody concentration in each case with a rate considerably lower than in the case of conventional (polyclonal) antibodies, and there often appeared to be a limit to the maximum interferon titre that can be neutralized, even using very high antibody concentrations (there were no such limits with conventional antibodies). This suggests that the interferon in a 1:1 interferon-antibody complex in general retains some activity, to the degree characteristic of that antibody; namely, each monoclonal antibody is considered to have a characteristic efficacy of neutralization, rather than being either neutralizing or non-neutralizing in an all-or-one fashion. The antibody dose-dependence curves were interpreted to be governed by two independent parameters of the antibody: the efficacy of neutralization and the affinity. The former is reflected by the maximum interferon titre neutralizable by high antibody doses and the latter by the minimum antibody dose that can effect detectable neutralization. Thus, quantitative neutralization tests of monoclonal antibodies to an effector protein would give useful information for classifying them as to their affinities and as to whether the epitopes they recognize are important for the biological activity of the effector.

Antibodies, Monoclonal↗
PubMed2005

Suppression of long-term facilitation by Rab3-effector protein interaction.

Long-term facilitation (LTF) in Aplysia is achieved by the modulation of presynaptic release. However, the underlying mechanism that might be related with the regulation of synaptic vesicle release remains unknown. Since Rab3, a neuronal GTP-binding protein, is known to be a key regulator of synaptic vesicle fusion, we investigated the involvement of Rab3 in LTF. To address this issue, we examined the effect of overexpression of wild type Aplysia Rab3 (apRab3) and its mutant forms on LTF. Overexpression of either apRab3 Q80L, a constitutively active apRab3 mutant, or wild type apRab3 completely inhibited LTF. This inhibitory role of apRab3 appears to be mediated by an interaction with an effector molecule(s), possibly Rim. Expression of apRab3 Q80L, V54E double mutant, which do not bind effector molecules such as Rim or Rabphilin, had no effect on LTF. Furthermore, expression of apRab3 Q80L, F18L, D19E triple mutant, which has reduced binding activity with Rim but normally binds with Rabphilin, enhanced evoked basal synaptic release, and the increase in synaptic strength occluded LTF. In conclusion, our data suggest that apRab3 may act as a negative clamp of LTF through the interaction with effector protein(s), possibly Rim.

Amino Acid Sequence↗
PubMed1994

A putative sirolimus (rapamycin) effector protein.

Sirolimus (rapamycin), a new immunosuppressive drug, inhibits proliferation of a wide spectrum of T and B cells. The immunosuppressive mechanism of sirolimus is still unclear. We recently isolated a membrane associated protein with an apparent molecular weight of 210 kDa, p210, from cultured Molt 4 cells and BJAB cells and from normal human T cells using an affinity matrix method. The p210 binds to sirolimus:FKBP12 complex, but only at background levels to FKBP12 alone, to FK506:FKBP12 complex, or sirolimus-biotin alone. Among the sirolimus analogs tested, the binding ability of p210 to drug:FKBP12 complexes correlates with the immunosuppressive activity of the drugs, suggesting that p210 is the sirolimus effector protein.

B-Lymphocytes↗
PubMed1997

A secreted effector protein of Salmonella dublin is translocated into eukaryotic cells and mediates inflammation and fluid secretion in infected ileal mucosa.

Enteritis induced by non-typhoid pathogenic Salmonella is characterized by fluid secretion and inflammatory responses in the infected ileum. The inflammatory response provoked by Salmonella initially consists largely of a neutrophil (PMN) migration into the intestinal mucosa and the gut lumen. The interactions between Salmonella and intestinal epithelial cells are known to play an essential role in inducing the inflammatory response. Upon interaction with epithelial cells salmonellae are able to elicit transepithelial signalling to neutrophils. This signalling is recognized as a key virulence feature underlying Salmonella-induced enteritis. However, the nature and mechanism of such signalling has not been clarified to date. Here, we characterize SopB, a novel secreted effector protein of Salmonella dublin, and present data implying that SopB is translocated into eukaryotic cells via a sip-dependent pathway to promote fluid secretion and inflammatory responses in the infected ileum.

Amino Acid Sequence↗