TaRBP1 stabilizes TaGLTP and negatively regulates stripe rust resistance in wheat

The dynamic balance and distribution of sphingolipid metabolites modulate the level of programmed cell death and plant defence. However, current knowledge is still limited regarding the molecular mechanism underlying the relationship between sphingolipid metabolism and plant defence. In this study, we identified a wheat RNA-binding protein 1 (TaRBP1) and TaRBP1 mRNA accumulation significantly decreased in wheat after infection by Puccinia striiformis f. sp. tritici (Pst). Knockdown of TaRBP1 via virus-induced gene silencing conferred strong resistance to Pst by enhancing host plant reactive oxygen species (ROS) accumulation and cell death, indicating that TaRBP1 may act as a negative regulator in response to Pst. TaRBP1 formed a homopolymer and interacted with TaRBP1 C-terminus in plants. Additionally, TaRBP1 physically interacted with TaGLTP, a sphingosine transfer protein. Knockdown of TaGLTP enhanced wheat resistance to the virulent Pst CYR31. Sphingolipid metabolites showed a significant accumulation in TaGLTP-silenced wheat and TaRBP1-silenced wheat, respectively. In the presence of the TaRBP1 protein, TaGLTP failed to be degraded in a 26S proteasome-dependent manner in plants. Our results reveal a novel susceptible mechanism by which a plant fine-tunes its defence responses by stabilizing TaGLTP accumulation to suppress ROS and sphingolipid accumulation during Pst infection.     

Molecular sequestration stabilizes CAP-DNA complexes during polyacrylamide gel electrophoresis.

The gel electrophoresis mobility shift assay is widely used for qualitative and quantitative characterization of protein complexes with nucleic acids. Often it is found that complexes that are short-lived in free solution (t1/2 of the order of minutes) persist for hours under the conditions of gel electrophoresis. We have investigated the influence of polyacrylamide gels on the pseudo first-order dissociation kinetics of complexes containing the E.coli cyclic AMP receptor protein (CAP) and lactose promoter DNA. Within the gel matrix, kdiss decreased with increasing [polyacrylamide] and the order of the reaction was changed. In free solution, kdiss was proportional to [DNA]2, while in 5% gels, kdiss was proportional to [DNA]0.3. In gels of [polyacrylamide] > or = 10%, kdiss was nearly independent of [DNA] until fragment concentrations exceeded 0.1 microM. Even in the absence of competing DNA, kdiss(gel) < kdiss(solution). These results suggest that the lifetime of CAP-DNA complexes in free solution is limited by their encounter frequency with molecules of DNA or with protein-DNA complexes; some or all of the stabilization observed in gels may be due to a reduction in this frequency.     

HIV-1 Nef stabilizes the association of adaptor protein complexes with membranes

The maximal virulence of HIV-1 requires Nef, a virally encoded peripheral membrane protein. Nef binds to the adaptor protein (AP) complexes of coated vesicles, inducing an expansion of the endosomal compartment and altering the surface expression of cellular proteins including CD4 and class I major histocompatibility complex. Here, we show that Nef stabilizes the association of AP-1 and AP-3 with membranes. These complexes remained with Nef on juxtanuclear membranes despite the treatment of cells with brefeldin A, which induced the release of ADP-ribosylation factor 1 (ARF1) from these membranes to the cytosol. Nef also induced a persistent association of AP-1 and AP-3 with membranes despite the expression of dominant-negative ARF1 or the overexpression of an ARF1-GTPase activating protein. Mutational analysis indicated that the direct binding of Nef to the AP complexes is essential for this stabilization. The leucine residues of the EXXXLL motif found in Nef were required for binding to AP-1 and AP-3 in vitro and for the stabilization of these complexes on membranes in vivo, whereas the glutamic acid residue of this motif was required specifically for the binding and stabilization of AP-3. These data indicate that Nef mediates the persistent attachment of AP-1 and AP-3 to membranes by an ARF1-independent mechanism. The stabilization of these complexes on membranes may underlie the pleiotropic effects of Nef on protein trafficking within the endosomal system.     

Polyethylenimine effectively induces,stabilizes, and regulates intramolecular G-quadruplexes

The interaction between polyethylenimine (PEI) and telomeric DNA was studied. The results demonstrated that PEI could effectively induce, stabilize, and regulate intramolecular G-quadruplexes. The mechanism for these results has been discussed, and was estimated to be condensation effect of PEI.     

The plant-specific actin binding protein SCAB1 stabilizes actin filaments and regulates stomatal movement in Arabidopsis

Microfilament dynamics play a critical role in regulating stomatal movement; however, the molecular mechanism underlying this process is not well understood. We report here the identification and characterization of STOMATAL CLOSURE-RELATED ACTIN BINDING PROTEIN1 (SCAB1), an Arabidopsis thaliana actin binding protein. Plants lacking SCAB1 were hypersensitive to drought stress and exhibited reduced abscisic acid-, H(2)O(2)-, and CaCl(2)-regulated stomatal movement. In vitro and in vivo analyses revealed that SCAB1 binds, stabilizes, and bundles actin filaments. SCAB1 shares sequence similarity only with plant proteins and contains a previously undiscovered actin binding domain. During stomatal closure, actin filaments switched from a radial orientation in open stomata to a longitudinal orientation in closed stomata. This switch took longer in scab1 plants than in wild-type plants and was correlated with the delay in stomatal closure seen in scab1 mutants in response to drought stress. Our results suggest that SCAB1 is required for the precise regulation of actin filament reorganization during stomatal closure.     

Interaction of Wnt-1 proteins with the binding protein BiP.

The mouse Wnt-1 gene, a target for insertional activation in mouse mammary tumor virus-induced mammary tumors, encodes poorly secreted, cysteine-rich glycoproteins required for proper central nervous system development. We have been analyzing the biosynthesis of Wnt-1 proteins in several cell lines that express Wnt-1 cDNA from heterologous promoters. A protein of 78 kDa was found to be associated with the intracellular forms of Wnt-1 proteins in mammalian and avian cells by using multiple antisera against Wnt-1 proteins. We have identified p78 as the binding protein BiP with anti-BiP antisera and by its release from Wnt-1 immunoprecipitates upon incubation with MgCl2 and ATP. Experiments with a Wnt-1 mutant that lacks the sequence encoding the signal peptide indicates that Wnt-1 proteins must enter the secretory pathway in order to interact with BiP. We demonstrate that Wnt-1 proteins are associated with BiP in cells in which active Wnt-1 proteins are produced, such as a cultured mammary epithelial cell line and Wnt-1 transgenic mouse mammary tumor cells. The association of Wnt-1 proteins with BiP may be a factor in determining the efficiency of secretion of Wnt-1 gene products.     

Mutational analysis of mouse Wnt-1 identifies two temperature-sensitive alleles and attributes of Wnt-1 protein essential for transformation of a mammary cell line.

The proto-oncogene Wnt-1 encodes a cysteine-rich, secretory glycoprotein implicated in virus-induced mouse mammary cancer and intercellular signaling during vertebrate neural development. To attempt to correlate structural motifs of Wnt-1 protein with its function, 12 mutations were introduced singly and in several combinations into the coding sequence of Wnt-1 cDNA by site-directed mutagenesis. Mutant alleles in a retroviral vector were tested for their ability to transform the mouse mammary epithelial cell line C57MG in two ways: by direct infection of C57MG cells and by infection of NIH3T3 cells that serve as donors of Wnt-1 protein to adjacent C57MG cells in a secretion-dependent (paracrine) assay. In addition, the synthesis and secretion of mutant proteins were monitored in multiple cell types by immunological assays. Deletion of the signal peptide demonstrated that transformation in both direct and paracrine assays depends upon entry of Wnt-1 protein into the endoplasmic reticulum. Changes in potential proteolytic processing sites (two basic dipeptides and a probable signal peptidase cleavage site) did not adversely impair biological activity or protein processing and uncovered a second site for cleavage by signal peptidase. Replacement of each of the four asparagine-linked glycosylation sites did not affect transforming activity at normal temperatures, but one glycosylation site mutant was found to be temperature-sensitive for transformation. An allele encoding a protein that lacks all four glycosylation sites was also transformation competent. In two of four cases, substitution of serine for a cysteine residue impaired transforming activity at the usual temperature, and transformation was temperature sensitive in a third case, implying that at least some of the highly conserved cysteine residues are important for Wnt-1 function.     

The midbrain-hindbrain phenotype of Wnt-1-/Wnt-1- mice results from stepwise deletion of engrailed-expressing cells by 9.5 days postcoitum.

Mice homozygous for null alleles of the putative signaling molecule Wnt-1 have a reproducible phenotype: loss of the midbrain and adjacent cerebellar component of the metencephalon. By examining embryonic expression of the mouse engrailed (En) genes, from 8.0 to 9.5 days postcoitum, we demonstrate that Wnt-1 primarily regulates midbrain development. The midbrain itself is required for normal development of the metencephalon. Thus, the observed neonatal phenotype is explained by a series of early events, within 48 hr of neural plate induction, that leads to a complete loss of En domains in the anterior central nervous system. Wnt-1 and a related gene, Wnt-3a, are coexpressed from early somite stages in dorsal aspects of the myelencephalon and spinal cord. We suggest that functional redundancy between these two genes accounts for the lack of a caudal central nervous system phenotype.     

Vps45p stabilizes the syntaxin homologue Tlg2p and positively regulates SNARE complex formation.

Sec1p-like/Munc-18 (SM) proteins bind to t-SNAREs and inhibit ternary complex formation. Paradoxically, the absence of SM proteins does not result in constitutive membrane fusion. Here, we show that in yeast cells lacking the SM protein Vps45p, the t-SNARE Tlg2p is down-regulated, to undetectable levels, by rapid proteasomal degradation. In the absence of Vps45p, Tlg2p can be stabilized through abolition of proteasome activity. Surprisingly, the stabilized Tlg2p was targeted to the correct intracellular location. However, the stabilized Tlg2p is non-functional and unable to bind its cognate SNARE binding partners, Tlg1p and Vti1p, in the absence of Vps45p. A truncation mutant lacking the first 230 residues of Tlg2p no longer bound Vps45p but was able to form complexes with Tlg1p and Vti1p in the absence of the SM protein. These data provide us with two valuable insights into the function of SM proteins. First, SM proteins act as chaperone-like molecules for their cognate t-SNAREs. Secondly, SM proteins play an essential role in the activation process allowing their cognate t-SNARE to participate in ternary complex formation.     

Homeostatic regulation of free retinol-binding protein and free thyroxine pools of plasma by their plasma carrier proteins in chicken.

The mechanism underlying homeostatic regulation of the plasma levels of free retinol-binding protein and free thyroxine, the systemic distribution of which is of great importance, has been investigated. A simple method has been developed to determine the rate of dissociation of a ligand from the binding protein. Analysis of the dissociation process of retinol-binding protein from prealbumin-2 reveals that the free retinol-binding protein pool undergoes massive flux, and that prealbumin-2 participates in homeostatic regulation of the free retinol-binding protein pool. Studies on the dissociation process of thyroxine from its plasma carrier proteins show that the various plasma carrier proteins share two roles. Of the two types of protein, the thyroxine-binding globulin (the high affinity binding protein) contributes only 27% of the free thyroxine in a rapid transition process, despite its being the major binding protein. But prealbumin-2, which has lower affinity towards thyroxine, participates mainly in a rapid flux of the free thyroxine pool. Thus thyroxine-binding globulin acts predominantly as a plasma reservoir of thyroxine, and also probably in the 'buffering' action on plasma free thyroxine level, in the long term, while prealbumin-2 participates mainly in the maintenance of constancy of free thyroxine levels even in the short term. The existence of these two types of binding protein facilitates compensation for the metabolic flux of the free ligand and maintenance of the thyroxine pool within a very narrow range.     

Stromelysin-1 and mesothelin are differentially regulated by Wnt-5a and Wnt-1 in C57mg mouse mammary epithelial cells

Background  

The Wnt signal transduction pathway is important in a wide variety of developmental processes as well as in the genesis of human cancer. Vertebrate Wnt pathways can be functionally separated into two classes, the canonical Wnt/beta-catenin pathway and the non-canonical Wnt/Ca²⁺ pathway. Supporting differences in Wnt signaling, gain of function of Wnt-1 in C57mg mouse mammary epithelial cells leads to their morphological transformation while loss of function of Wnt-5a leads to the same transformation. Many downstream target genes of the Wnt/beta-catenin pathway have been identified. In contrast, little is known about the Wnt/Ca²⁺ pathway and whether it regulates gene expression.     

ATM regulates ionizing radiation-induced disruption of HDAC1:PP1:Rb complexes

Ionizing radiation elicits signaling events that coordinate DNA repair and interruption of cell cycle progression. We previously demonstrated that ionizing radiation (IR) of cells activates nuclear protein phosphatase-1 (PP1) by promoting dephosphorylation of Thr320, an inhibitory site in the enzyme and that the ATM kinase is required for this response. We sought to identify potential targets of IR-activated PP1. Untreated and IR-treated Jurkat cells were labeled with (32)P orthophosphate, and nuclear extracts were subjected to microcystin affinity chromatography to recover phosphatase complexes that were analyzed by 2D-PAGE and mass spectrometry. Several proteins associated with protein phosphatases demonstrated a significant decrease in (32)P intensity following IR, and one of these was identified as HDAC1. Co-immunoprecipitation revealed complexes containing PP1 with HDAC1 and Rb in cell extracts. In response to IR, there was an ATM-dependent activation of PP1, dephosphorylation of HDAC1, dissociation of HDAC1-PP1-Rb complexes and increased HDAC1 activity. These results suggest that IR regulates HDAC1 phosphorylation and activity through ATM-dependent activation of PP1.     

Inhibition of receptor binding stabilizes Newcastle disease virus HN and F protein-containing complexes

Receptor binding of paramyxovirus attachment proteins and the interactions between attachment and fusion (F) proteins are thought to be central to activation of the F protein activity; however, mechanisms involved are unclear. To explore the relationships between Newcastle disease virus (NDV) HN and F protein interactions and HN protein attachment to sialic acid receptors, HN and F protein-containing complexes were detected and quantified by reciprocal coimmunoprecipitation from extracts of transfected avian cells. To inhibit HN protein receptor binding, cells transfected with HN and F protein cDNAs were incubated with neuraminidase from the start of transfection. Under these conditions, no fusion was observed, but amounts of HN and F protein complexes increased twofold over amounts detected in extracts of untreated cells. Stimulation of attachment by incubation of untransfected target cells with neuraminidase-treated HN and F protein-expressing cells resulted in a twofold decrease in amounts of HN and F protein complexes. In contrast, high levels of complexes containing HN protein and an uncleaved F protein (F-K115Q) were detected, and those levels were unaffected by neuraminidase treatment of cell monolayers or by incubation with target cells. These results suggest that HN and F proteins reside in a complex in the absence of receptor binding. Furthermore, the results show that not only receptor binding but also F protein cleavage are necessary for disassociation of the HN and F protein-containing complexes.     

过量表达Wnt-1基因诱导P19细胞的神经分化

Ｗｎｔ－１基因在小鼠神经发育过程中起着重要的作用。该基因在胚胎性癌细胞Ｐ１９细胞经分化过程中存在瞬时性表达。利用克隆到的Ｗｎｔ－１基因转染Ｐ１９细胞,可使细胞不经视黄酸诱导,自发向神经细胞方向分化。     

Escherichia coli thioredoxin stabilizes complexes of bacteriophage T7 DNA polymerase and primed templates

The DNA polymerase activity induced after bacteriophage T7 infection of Escherichia coli is found in a complex of two proteins, the T7 gene 5 protein and a host protein, thioredoxin. Gene 5 protein is a DNA polymerase and a 3' to 5' exonuclease. Thioredoxin binds tightly to the gene 5 protein and increases the processivity of polymerization some 1000-fold. Gene 5 protein forms a short-lived complex with the primer-template, poly(dA).oligo(dT), in the absence of Mg2+ and nucleotides. Thioredoxin increases the half-life of the preformed primer-template-polymerase complex from less than a second to approximately 5 min. The dissociation is accelerated by excess single-stranded DNA in an apparent second order reaction, indicating direct transfer of polymerase between DNA fragments. Thioredoxin also reduces the equilibrium dissociation constant, Kd, of the gene 5 protein -poly(dA).oligo(dT) complex 20- to 80-fold. The salt dependence of Kd indicates that thioredoxin stabilizes the primer-template-polymerase complex mainly through additional charge-charge interactions, increasing the estimated number of interactions from 2 to 7. The affinity of gene 5 protein for single-stranded DNA is at least 1000-fold higher than for double-stranded DNA and is little affected by thioredoxin. Under conditions of steady state synthesis the effect of thioredoxin on the polymerization rate is determined by two competing factors, an increase in processivity and a decrease of the dissociation rate of polymerase and replicated template.     

Sphingosine-1-phosphate signaling regulates lamellipodia localization of cortactin complexes in endothelial cells

Sphingosine-1-phosphate (S1P), a serum-borne lipid mediator, was demonstrated to be a potent chemoattractant of endothelial cells. It was recently shown that the colocalization of cortactin and actin related protein 2/3 (Arp2/3) in the lamellipodia is critical to S1P-induced endothelial chemotaxis. In this report, we describe that S1P-stimulated cortactin translocation to the cell periphery to form lamellipodia is specifically mediated by the endothelial S1P1 G-protein coupled receptor, and is regulated by G_i-mediated Akt-dependent S1P1 receptor phosphorylation and Cdc42/Rac activation pathways. In contrast to Src-dependent fibroblast growth factor-induced cortactin translocation, tyrosine phosphorylation cascades are not required for S1P-mediated lamellipodia formation and chemotaxis. Furthermore, we also demonstrate that S1P signaling, via the G_i/Akt/S1P1 phosphorylation/Rac pathway, regulates the cortactin–Arp2/3 complex formation, which ultimately results in membrane ruffling, formation of the lamellipodia and endothelial migration.J.F. Lee and H. Ozaki contributed equally to this work