Down-regulation of the amyloid protein precursor of Alzheimer's disease by antisense oligonucleotides reduces neuronal adhesion to specific substrata

The hallmark of Alzheimer's disease is the cerebral deposition of amyloid which is derived from the amyloid precursor protein (APP). The function of APP is unknown but there is increasing evidence for the role of APP in cell-cell and/or cell-matrix interactions. Primary cultures of murine neurons were treated with antisense oligonucleotides to down-regulate APP. This paper presents evidence that APP mediates a substrate-specific interaction between neurons and extracellular matrix components collagen type I, laminin and heparan sulphate proteoglycan but not fibronectin or poly-L-lysine. It remains to be determined whether this effect is the direct result of APP-matrix interactions, or whether an intermediary pathway is involved. (C) 1997 Elsevier Science B.V.

Crystallization and preliminary X-ray diffraction studies of FHA domains of Dun1 and Rad53 protein kinases

Forkhead-associated (FHA) domains are modular protein–protein interaction domains of ~130 amino acids present in numerous signalling proteins. FHA-domain-dependent protein interactions are regulated by phosphorylation of target proteins and FHA domains may be multifunctional phosphopeptide-recognition modules. FHA domains of the budding yeast cell-cycle checkpoint protein kinases Dun1p and Rad53p have been crystallized. Crystals of the Dun1-FHA domain exhibit the symmetry of the space group P6122 or P6522, with unit-cell parameters a = b = 127.3, c = 386.3 Å; diffraction data have been collected to 3.1 Å resolution on a synchrotron source. Crystals of the N-terminal FHA domain (FHA1) of Rad53p diffract to 4.0 Å resolution on a laboratory X-ray source and have Laue-group symmetry 4/mmm, with unit-cell parameters a = b = 61.7, c = 104.3 Å.

Genotype-by-management interactions for grain yield and grain protein concentration of wheat

The magnitude of genotype-by-management (G x M) interactions for grain yield and grain protein concentration was examined in a multi-environment trial (MET) involving a diverse set of 272 advanced breeding lines from the Queensland wheat breeding program. The MET was structured as a series of management-regimes imposed at 3 sites for 2 years. The management-regimes were generated at each site-year as separate trials in which planting time, N fertiliser application rate, cropping history, and irrigation were manipulated. irrigation was used to simulate different rainfall regimes. From the combined analysis of variance, the G x M interaction variance components were found to be the largest source of G x E interaction variation for both grain yield (0.117 +/- 0.005 t(2) ha(-2); 49% of total G x E 0.238 +/- 0.028 t(2) ha(-2)) and grain protein concentration (0.445 +/- 0.020%(2); 82% of total G x E 0.546 +/- 0.057%(2)), and in both cases this source of variation was larger than the genotypic variance component (grain yield 0.068 +/- 0.014 t(2) ha(-2) and grain protein 0.203 +/- 0.026%(2)). The genotypic correlation between the traits varied considerably with management-regime, ranging from -0.98 to -0.31, with an estimate of 0.0 for one trial. Pattern analysis identified advanced breeding lines with improved grain yield and grain protein concentration relative to the cultivars Hartog, Sunco and Meteor. It is likely that a large component of the previously documented G x E interactions for grain yield of wheat in the northern grains region are in part a result of G x M interactions. The implications of the strong influence of G x M interactions for the conduct of wheat breeding METs in the northern region are discussed. (C) 2001 Elsevier Science B.V. All rights reserved.

A novel 14-kilodalton protein interacts with the mitogen-activated protein kinase scaffold MP1 on a late endosomal/lysosomal compartment

We have identified a novel, highly conserved protein of 14 kD copurifying with late endosomes/lysosomes on density gradients. The protein, now termed p14, is peripherally associated with the cytoplasmic face of late endosomes/lysosomes in a variety of different cell types. In a two-hybrid screen with p14 as a bait, we identified the mitogen-activated protein kinase (MAPK) scaffolding protein MAPK/extracellular signal-regulated kinase (ERK) kinase (MEK) partner 1 (MP1) as an interacting protein. We confirmed the specificity of this interaction in vitro by glutathione S-transferase pull-down assays and by coimmunoprecipitation, cosedimentation on glycerol gradients, and colocalization. Moreover, expression of a plasma membrane-targeted p14 causes mislocalization of coexpressed MP1. In addition, we could reconstitute protein complexes containing the p14-MP1 complex associated with ERK and MEK in vitro. The interaction between p14 and MP1 suggests a MAPK scaffolding activity localized to the cytoplasmic surface of late endosomes/lysosomes, thereby combining catalytic scaffolding and subcellular compartmentalization as means to modulate MAPK signaling within a cell.

Copper transfer from the Cu(I) chaperone, CopZ, to the repressor, Zn(II)CopY: Metal coordination environments and protein interactions

Extracellular copper regulates the DNA binding activity of the CopY repressor of Enterococcus hirae and thereby controls expression of the copper homeostatic genes encoded by the cop operon. CopY has a CxCxxxxCxC metal binding motif. CopZ, a copper chaperone belonging to a family of metallochaperones characterized by a MxCxxC metal binding motif, transfers copper to CopY. The copper binding stoichiometries of CopZ and CopY were determined by in vitro metal reconstitutions. The stoichiometries were found to be one copper(l) per CopZ and two copper(l) per CopY monomer. X-ray absorption studies suggested a mixture of two- and three-coordinate copper in Cu(1)CopZ, but a purely three-coordinate copper coordination with a Cu-Cu interaction for Cu(1)(2)CopY. The latter coordination is consistent with the formation of a compact binuclear Cu(l)-thiolate core in the CxCxxxxCxC binding motif of CopY. Displacement of zinc, by copper. from CopY was monitored with 2,4-pyridylazoresorcinol. Two copper(l) ions were required to release the single zinc(II) ion bound per CopY monomer. The specificity of copper transfer between CopZ and CopY was dependent on electrostatic interactions. Relative copper binding affinities of the proteins were investigated using the chelator, diethyldithiocarbamic acid (DDC). These data suggest that CopY has a higher affinity for copper than CopZ. However, this affinity difference is not the sole factor in the copper exchange: a charge-based interaction between the two proteins is required for the transfer reaction to proceed. Gain-of-function mutation of a CopZ homologue demonstrated the necessity of four lysine residues on the chaperone for the interaction with CopY. Taken together, these results suggest a mechanism for copper exchange between CopZ and CopY.

The Wilms' Tumour Suppressor Protein, WT1, Undergoes CRM1-independent Nucleocytoplasmic Shuttling

The Wilms' tumour suppressor gene (WT1) encodes a zinc finger-containing nuclear protein essential for kidney and urogenital development. Initially considered a transcription factor, there is mounting evidence that WT1 has a role in post-transcriptional processing. Using the interspecies heterokaryon assay, we have demonstrated that WT1 can undergo nucleocytoplasmic shuttling. We have also mapped the region responsible for nuclear export to residues 182-324. Our data add further complexity to the role of WT1 in trancriptional and post-transcriptional regulation. (C) 2003 Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies.

Identification of the essential histidine residue for high-affinity binding of AlbA protein to albicidin antibiotics

The albA gene from Klebsiella oxytoca encodes a protein that binds albicidin phytotoxins and antibiotics with high affinity. Previously, it has been shown that shifting pH from 6 to 4 reduces binding activity of AlbA by about 30%, indicating that histidine residues might be involved in substrate binding. In this study, molecular analysis of the albA coding region revealed sequence discrepancies with the albA sequence reported previously, which were probably due to sequencing errors. The albA gene was subsequently cloned from K oxytoca ATCC 13182(T) to establish the revised sequence. Biochemical and molecular approaches were used to determine the functional role of four histidine residues (His(78), HiS(125), HiS(141) and His(189)) in the corrected sequence for AlbA. Treatment of AlbA with diethyl pyrocarbonate (DEPC), a histidine-specific alkylating reagent, reduced binding activity by about 95%. DEPC treatment increased absorbance at 240-244 nm by an amount indicating conversion to N-carbethoxyhistidine of a single histidine residue per AlbA molecule. Pretreatment with albicidin protected AlbA against modification by DEPC, with a 1 : 1 molar ratio of albicidin to the protected histidine residues. Based on protein secondary structure and amino acid surface probability indices, it is predicted that HiS125 might be the residue required for albicidin binding. Mutation of HiS125 to either alanine or leucine resulted in about 32% loss of binding activity, and deletion of HiS125 totally abolished binding activity. Mutation of HiS125 to arginine and tyrosine had no effect. These results indicate that HiS125 plays a key role either in an electrostatic interaction between AlbA and albicidin or in the conformational dynamics of the albicidin-binding site.

Effects of phytase supplementation of diets with two tiers of nutrient specifications on growth performance and protein efficiency ratios of broiler chickens

in two feeding experiments male and mixed-sex broiler chicks were offered diets based on sorghum and a wheat-sorghum blend with two tiers of nutrient specifications, without and with microbial phytase (600 and 800 FTU/kg), from 7-25 and 1-42 days post-hatch, respectively. The nutrient specifications for protein, amino acids, energy density and phosphorus (P) of standard diets were reduced to formulate the modified diets on a least-cost basis. Calculated differences in nutrient specifications between standard and modified diets ranged from 14.3 to 17.1 g/kg crude protein, 0.24 to 0.40 MJ/kg apparent metabolisable energy (AME) and 1.06 to 1.20 g/kg available P. In both experiments, reduced nutrient specifications had a negative impact on growth rates and feed efficiency and phytase supplementation had a positive influence on growth performance and protein efficiency ratios (PER). Phytase addition to the less expensive, modified diets either partially or entirely compensated for reduced growth performance and, consequently, feed costs per kg of live weight gain were reduced. In Experiment 1, phytase increased (p<0.001) nitrogen-corrected AME (AMEn) from 15.39 to 15.89 MJ/kg dry matter. For nitrogen (N) retention there was an interaction (p<0.05) between diet type and phytase as the effects of phytase on N retention were more pronounced in the modified diets, with an increase from 0.512 to 0.561. These results demonstrate the positive effects of phytase on protein and energy utilisation, in addition to its established liberation of phytate-bound P and illustrate the feasibility of assigning nutrient replacement values to the feed enzyme for consideration in least-cost ration formulations. Further work is, however, required to define the most appropriate reductions in nutrient specifications in association with phytase supplementation.

Inhibition of protein interactions with the beta(2) sliding clamp of Escherichia coli DNA polymerase III by peptides from beta(2)-binding proteins

The sliding clamp of the Escherichia coli replisome is now understood to interact with many proteins involved in DNA synthesis and repair. A universal interaction motif is proposed to be one mechanism by which those proteins bind the E. coli sliding clamp, a homodimer of the beta subunit, at a single site on the dimer. The numerous beta(2)-binding proteins have various versions of the consensus interaction motif, including a related hexameric sequence. To determine if the variants of the motif could contribute to the competition of the beta-binding proteins for the beta(2) site, synthetic peptides derived from the putative beta(2)-binding motifs were assessed for their abilities to inhibit protein-beta(2) interactions, to bind directly to beta(2), and to inhibit DNA synthesis in vitro. A hierarchy emerged, which was consistent with sequence similarity to the pentameric consensus motif, QL(S/D)LF, and peptides containing proposed hexameric motifs were shown to have activities comparable to those containing the consensus sequence. The hierarchy of peptide binding may be indicative of a competitive hierarchy for the binding of proteins to beta(2) in various stages or circumstances of DNA replication and repair.

N4WBP5A (Ndfip2), a Nedd4-interacting protein, localizes to multivesicular bodies and the Golgi, and has a potential role in protein trafficking

N4WBP5A (Ndfip2) belongs to an evolutionarily conserved group of Nedd4-interacting proteins with two homologues in mammalian species. We have previously shown that N4WBP5A expression in Xenopus oocytes results in increased cell-surface expression of the epithelial sodium channel. N4WBPs are characterized by one or two amino terminal PPxY motifs and three transmembrane domains. Here we show that both PPxY motifs of N4WBP5A mediate interaction with WW domains of Nedd4 and that N4WBP5A can physically interact with the WW domains of several Nedd4-family proteins. N4WBP5A is ubiquitinated and ubiquitination does not significantly affect the turnover of N4WBP5A protein. Ubiquitination of N4WBP5A is enhanced by Nedd4 and Nedd4-2 expression. N4WBP5A localizes to the Golgi, vesicles associated with the Golgi complex and to multivesicular bodies. We show that the ectopic expression of N4WBP5A inhibits receptor-mediated endocytosis of labelled epidermal growth factor. N4WBP5A overexpression inhibits accumulation of EGF in large endocytic/lysosomal vesicles suggestive of a role for N4WBP5A in protein trafficking. We propose that N4WBP5A acts as an adaptor to recruit Nedd4 family ubiquitin-protein ligases to the protein trafficking machinery.

Adherence of Plasmodium falciparum infected erythrocytes to CHO-745 cells and inhibition of binding by protein A in the presence of human serum

Adhesion of erythrocytes infected with the malaria parasite Plasmodium falciparum to human host receptors is a process associated with severe malarial pathology. A number of in vitro cell lines are available as models for these adhesive processes, including Chinese hamster ovary (CHO) cells which express the placental adhesion receptor chondroitin-4-sulphate (CSA) on their surface. CHO-745 cells, a glycosaminoglycan-negative mutant CHO cell line lacking CSA and other reported P. falciparum adhesion receptors, are often used for recombinant expression of host receptors and for receptor binding studies. In this study we show that P. falciparum-infected erythrocytes can be easily selected for adhesion to an endogenous receptor on the surface of CHO-745 cells, bringing into question the validity of using these cells as a tool for P. falciparum adhesin expression studies. The adhesive interaction between CHO-745 cells and parasitized erythrocytes described here is not mediated by the known P. falciparum adhesion receptors CSA, CD36, or ICAM-1. However, we found that CHO-745-selected parasitized erythrocytes bind normal human IgM and that adhesion to CHO-745 cells is inhibited by protein A in the presence of serum, but not in its absence, indicating a non-specific inhibitory effect. Thus, protein A, which has been used as an inhibitor for a recently described interaction between infected erythrocytes and the placenta, may not be an appropriate in vitro inhibitor for understanding in vivo adhesive interactions. (c) 2005 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

Selective modulation of neuronal nicotinic acetylcholine receptor channel subunits by G(o)-protein subunits

protein modulation of neuronal nicotinic acetylcholine receptor ( nAChR) channels in rat intrinsic cardiac ganglia was examined using dialyzed whole-cell and excised membrane patch-recording configurations. Cell dialysis with GTP gamma S increased the agonist affinity of nAChRs, resulting in a potentiation of nicotine-evoked whole-cell currents at low concentrations. ACh- and nicotine-evoked current amplitudes were increased approximately twofold in the presence of GTP gamma S. In inside-out membrane patches, the open probability (NPo) of nAChR-mediated unitary currents was reversibly increased fourfold after bath application of 0.2mM GTP gamma S relative to control but was unchanged in the presence of GDP gamma S. The modulation of nAChR-mediated whole- cell currents was agonist specific; currents evoked by the cholinergic agonists ACh, nicotine, and 1,1-dimethyl-4-phenylpiperazinium iodide, but not cytisine or choline, were potentiated in the presence of GTP gamma S. The direct interaction between G-protein subunits and nAChRs was examined by bath application of either G(o)alpha or G beta gamma subunits to inside-out membrane patches and in glutathione S-transferase pull-down and coimmunoprecipitation experiments. Bath application of 50 nM G beta gamma increased the open probability of ACh- activated single-channel currents fivefold, whereas G(o)alpha( 50 nM) produced no significant increase in NPo. Neuronal nAChR subunits alpha 3-alpha 5 and alpha 2 exhibited a positive interaction with G(o)alpha and G beta gamma, whereas beta 4 and alpha 7 failed to interact with either of the G-protein subunits. These results provide evidence for a direct interaction between nAChR and G-protein subunits, underlying the increased open probability of ACh-activated single-channel currents and potentiation of nAChR-mediated whole-cell currents in parasympathetic neurons of rat intrinsic cardiac ganglia.

The human stress-activated protein kinase-interacting 1 gene encodes JNK-binding proteins

The orthologous proteins of the stress-activated protein kinase-interacting 1 (Sin1) family have been implicated in several different signal transduction pathways. In this study, we have investigated the function of the full-length human Sin1 protein and a C-terminally truncated isoform, Sin 1 alpha, which is produced by alternative splicing. Immunoblot analysis using an anti-Sin 1 polyclonal antibody showed that full-length Sin I and several smaller isoforms are widely expressed. Sin 1 was demonstrated to bind to c-Jun N-terminal kinase (JNK) in vitro and in vivo, while no interaction with p38- or ERK1/2-family MAPKs was observed. The Sin1 alpha isoform could also form a complex with JNK in vivo. Despite localizing in distinct compartments within the cell, both Sin1 and Sin1 alpha co-localized with JNK, suggesting that the Sin1 proteins could recruit JNK. Over-expression of full-length Sin1 inhibited the activation of JNK by UV-C in DG75 cells, as well as basal JNK-activity in HEK293 cells. These data suggest that the human Sin1 proteins may act as scaffold molecules in the regulation of signaling by JNK. (c) 2004 Elsevier Inc. All rights reserved.

Milling quality and protein properties of autumn-sown Chinese wheats evaluated through multi-location trials

Eight milling quality and protein properties of autumn-sown Chinese wheats were investigated using 59 cultivars and advanced lines grown in 14 locations in China from 1995 to 1998. Wide ranges of variability for all traits were observed across genotypes and locations. Genotype, location, year, and their interactions all significantly influenced most of the quality parameters. Kernel hardness, Zeleny sedimentation value, and mixograph development time were predominantly influenced by the effects of genotype. Genotype, location and genotype x location interaction were all important sources of variation for thousand kernel weight, test weight, protein content, and falling number, whereas genotype x location interaction had the largest effect on flour yield. Most of the genotypes were characterized by weak gluten strength with Zeleny sedimentation values less than 40 ml and mixograph development time shorter than 3 min. Eight groups of genotypes were recognized based on the average quality performance, grain hardness and gluten strength were the two parameters that determined the grouping, with contributions from protein content. Genotypes such as Zhongyou 16 and Annong 8903 displayed good milling quality, high grain hardness, protein content and strong gluten strength with high sedimentation value and long mixograph development time. Genotypes such as Lumai 15 and Yumai 18 were characterized by low grain hardness, protein content and weak gluten strength. Genotypes such as Yannong 15 and Chuanmai 24 were characterized by strong gluten strength with high sedimentation value and long mixograph development time, but low grain hardness and protein content lower than 12.3%. Genotypes such as Jingdong 6 and Xi'an 8 had weak gluten strength, but with high grain hardness and protein content higher than 12.2%. Five groups of locations were identified, and protein content and gluten strength were the two parameters that determined the grouping. Beijing, Shijiazhuang, Nanyang, Zhumadian and Nanjing produced wheats with medium to strong gluten strength and medium protein content, although there was still a large variation for most of the traits investigated between the locations. Wheat produced in Yantai was characterized by strong gluten strength, but with low protein content. Jinan, Anyang and Linfen locations produced wheats with medium to weak gluten strength and medium to high protein content. Wheats produced in Yangling, Zhenzhou, and Chengdu were characterized by weak gluten strength with medium to low protein content, whereas wheats produced in Xuzhou and Wuhan were characterized by weak gluten strength with low protein content. Industrial grain quality could be substantially improved through integrating knowledge of geographic genotype distribution with key location variables that affected end-use quality.

Cdk1/Erk2- and Plk1-dependent phosphorylation of a centrosome protein, Cep55, is required for its recruitment to midbody and cytokinesis

Centrosomes in mammalian cells have recently been implicated in cytokinesis; however, their role in this process is poorly defined. Here, we describe a human coiled-coil protein, Cep55 (centrosome protein 55 kDa), that localizes to the mother centriole during interphase. Despite its association with gamma-TuRC anchoring proteins CG-NAP and Kendrin, Cep55 is not required for microtubule nucleation. Upon mitotic entry, centrosome dissociation of Cep55 is triggered by Erk2/Cdk1-dependent phosphorylation at S425 and S428. Furthermore, Cep55 locates to the midbody and plays a role in cytokinesis, as its depletion by siRNA results in failure of this process. S425/428 phosphorylation is required for interaction with Plk1, enabling phosphorylation of Cep55 at S436. Cells expressing phosphorylation-deficient mutant forms of Cep55 undergo cytokinesis failure. These results highlight the centrosome as a site to organize phosphorylation of Cep55, enabling it to relocate to the midbody to function in mitotic exit and cytokinesis.