cAMP-dependent protein kinase regulates ubiquitin-proteasome-mediated degradation and subcellular localization of the nuclear receptor coactivator GRIP1

Nuclear receptors and their coactivators are key regulators of numerous physiological functions. GRIP1 (glucocorticoid receptor-interacting protein) is a member of the steroid receptor coactivator family. Here, we show that GRIP1 is regulated by cAMP-dependent protein kinase (PKA) that induces its degradation through the ubiquitin-proteasome pathway. GRIP1 was down-regulated in transiently transfected COS-1 cells after treatment with 8-para-chlorophenylthio-cAMP or forskolin and 3-isobutyl-1-methylxanthine and in adrenocortical Y1 cells after incubation with adrenocorticotropic hormone. Pulse-chase experiments with transiently transfected COS-1 cells demonstrated that the half-life of GRIP1 was markedly reduced in cells overexpressing the PKA catalytic subunit, suggesting that activation of PKA increases the turnover of GRIP1 protein. The proteasome inhibitors MG132 and lactacystin abolished the PKA-mediated degradation of GRIP1. Using ts20 cells, a temperature-sensitive cell line that contains a thermolabile ubiquitin-activating E1 enzyme, it was confirmed that PKA-mediated degradation of GRIP1 is dependent upon the ubiquitin-proteasome pathway. Coimmunoprecipitation studies of COS-1 cells transfected with expression vectors encoding GRIP1 and ubiquitin using anti-GRIP1 and anti-ubiquitin antibodies showed that the ubiquitination of GRIP1 was increased by overexpression of PKA. Finally, we show that PKA regulates the intracellular distribution pattern of green fluorescent protein-GRIP1 and stimulates recruitment of GRIP1 to subnuclear foci that are colocalized with the proteasome. Taken together, these data demonstrate that GRIP1 is ubiquitinated and degraded through activation of the PKA pathway. This may represent a novel regulatory mechanism whereby hormones down-regulate a nuclear receptor coactivator.     

Biological effects of Trichoderma harzianum peptaibols on mammalian cells

Trichoderma species isolated from water-damaged buildings were screened for toxicity by using boar sperm cells as indicator cells. The crude methanolic cell extract from Trichoderma harzianum strain ES39 inhibited the boar sperm cell motility at a low exposure concentration (50% effective concentration, 1 to 5 microg [dry weight] ml of extended boar semen(-1)). The same exposure concentration depleted the boar sperm cells of NADH(2). Inspection of the exposed boar sperm cells by transmission electron microscopy revealed damage to the plasma membrane. By using the black lipid membrane technique, it was shown that the semipurified metabolites (eluted from a SepPak C(18) cartridge) of T. harzianum strain ES39 induced voltage-dependent conductivity. The high-performance liquid chromatography-purified metabolites of T. harzianum strain ES39 dissipated the mitochondrial membrane potential (Deltapsi(m)) of human lung epithelial carcinoma cells (cell line A549). The semipurified metabolites (eluted from a SepPak C(18) cartridge) of T. harzianum strain ES39 were analyzed by mass spectrometry (MS). Matrix-assisted laser desorption ionization and nanoflow electrospray ionization MS revealed five major peptaibols, each of which contained 18 residues and had a mass ranging from 1,719 to 1,775 Da. Their partial amino acid sequences were determined by collision-induced dissociation tandem MS.     

Metal ion complexes of macrocyclic polyamines enhance both the phosphate hydrolysis and imidazole ring opening of RNA 5'-cap structure

The cleavage of P1-(7-methylguanosyl-5') P3-(guanosyl-5') triphosphate, a RNA 5'-cap model, by 2-hydroxyethyl- (6a-6c) and 2-aminoethyl- (7a-7c) substituted macrocycles in the presence and absence of Zn2+ and Cu2+ ions has been studied at pH 7.2 and 60 degrees. In the presence of the metal ions, hydrolysis of the phosphate group is enhanced. The mono- and dinuclear Zn2+ complexes promote solely the phosphate hydrolysis, whereas the corresponding Cu2+ complexes accelerate both the phosphate hydrolysis and the imidazole ring opening of the 7-methylguanine base. In the absence of the metal ions, the macrocycles mainly promote breakdown of the 7-methylguanine base, most probably by enhancing the nucleophilic attack of hydroxide ion on the C(8)-atom by shielding the repulsive negative charge on the phosphate moiety. The 2-hydroxyethyl and 2-aminoethyl side arms exhibit a two- to three-fold rate acceleration. Opening of the imidazole ring eventually results in cleavage of the triphosphate bridge.     

Transglutaminase 1 mutations in autosomal recessive congenital ichthyosis: private and recurrent mutations in an isolated population.

Autosomal recessive congenital ichthyosis (ARCI) is a rare, heterogenous keratinization disorder of the skin, classically divided into two clinical subtypes, lamellar ichthyosis (LI) and nonbullous congenital ichthyosiformis erythroderma (CIE). Recently, strong evidence for the involvement of the transglutaminase 1 gene (TGM1) in LI has evolved. We have studied ARCI in the isolated Finnish population, in which recessive disorders are often caused by single mutations enriched by a founder effect. Surprisingly, five different mutations of TGM1 (Arg141His, Arg142Cys, Gly217Ser, Val378Leu, and Arg395Leu) were found in Finnish ARCI patients. In addition to affected LI patients, we also identified TGM1 mutations in CIE patients. Moreover, haplotype analysis of the chromosomes carrying the most common mutation, a C-->T transition changing Arg142 to Cys, revealed that the same mutation has been introduced twice in the Finnish population. In addition to this Arg142Cys mutation, three other mutations, in Arg141 and Arg142, have been described elsewhere, in other populations. These findings suggest that this region of TGM1 is more susceptible to mutation. The corresponding amino acid sequence is conserved in other transglutaminases, but, for example, coagulation factor XIII (FXIII) mutations do not cluster in this region. Protein modeling of the Arg142Cys mutation suggested disruption or destabilization of the protein. In transfection studies, the closely related transglutaminase FXIII protein with the corresponding mutation was shown to be susceptible to degradation in COS cells, further supporting evidence of the destabilizing effect of the Arg142Cys mutation in TGM1.     

Mechanical Properties of Mammalian and Fish Gelatins as a Function of the Contents of α-Chain, β-Chain,and Low and High Molecular Weight Fractions

Small-strain oscillatory measurements and size-exclusion chromatography coupled to multiangle laser light scattering were used to study the mechanical properties and the molecular weight distribution, respectively, of acid porcine skin gelatins (type A), lime bovine bone gelatins (type B), and cold water fish gelatins, while principal component analysis (PCA) and partial least squares regression were used to relate the mechanical properties with the molecular weight distribution. The present study suggests a linear relationship between the mechanical properties and the fractions of low molecular weight (LMW) molecules, α-chains, β-chains, and high molecular weight (HMW) molecules. The Bloom value for mammalian gelatin was positively correlated with the fractions of α-chains, β-chains, and HMW molecules and negatively correlated with the fraction of LMW molecules. The dynamic storage modulus for cold water fish gelatin was positively correlated with the fractions of β-chains and HMW molecules and negatively correlated with the fractions of LMW molecules and α-chains.     

Bacillus subtilis and B. mojavensis strains connected to food poisoning produce the heat stable toxin amylosin

Aim:  To screen and characterize toxic, heat-stable substances produced by food borne strains from Bacillus subtilis group.
Methods and Results:  Using the boar sperm motility inhibition assay, six isolates from two outbreaks, out of the 94 isolates from 26 foods, were found to produce ethanol-soluble heat-stable substances that were toxic to sperm cells by depleting the mitochondrial membrane potentials. The toxic isolates were identified as Bacillus subtilis and B mojavensis. Colon carcinoma cells (Caco-2) were used to model the contact with the human digestive tract. The extract of B. subtilis F 2564/96 depolarized the mitochondria in intact Caco-2 cells similarly as in sperm cells. The substance responsible for these effects was purified using HPLC and identified by electron spray ionization ion trap mass spectrometry analysis as amylosin. The temperature requirement for amylosin production was 21–37°C for B. subtilis and 11–21°C for B. mojavensis . Both species produced amylosin in air as well as in 7–8% CO₂ with 8–9% O_2.
Conclusions:  Food borne illness related strains of B. subtilis and B. mojavensis, produced the heat-stable toxin amylosin.
Significance and Impact of the Study:  This is the first report that suggests a role for the heat-stable, ion-channel forming toxin amylosin, as a virulence factor in food borne Bacillus .     

Annexin A2 recognises a specific region in the 3'-UTR of its cognate messenger RNA

Annexin A2 is a multifunctional Ca(2+)- and lipid-binding protein. We previously showed that a distinct pool of cellular Annexin A2 associates with mRNP complexes or polysomes associated with the cytoskeleton. Here we report in vitro and in vivo experiments showing that Annexin A2 present in this subset of mRNP complexes interacts with its cognate mRNA and c-myc mRNA, but not with beta(2)-microglobulin mRNA translated on membrane-bound polysomes. The protein recognises sequence elements within the untranslated regions, but not within the coding region, of its cognate mRNA. Alignment of the Annexin A2-binding 3'-untranslated regions of annexin A2 mRNA from several species reveals a five nucleotide consensus sequence 5'-AA(C/G)(A/U)G. The Annexin A2-interacting region of the 3'-untranslated region can be mapped to a sequence of about 100 nucleotides containing two repeats of the consensus sequence. The binding elements appear to involve both single and double stranded regions, indicating that a specific higher order mRNA structure is required for binding to Annexin A2. We suggest that this type of interaction is representative for a group of mRNAs translated on cytoskeleton-bound polysomes.     

Anti-carcinoembryonic antigen single-chain variable fragment antibody variants bind mouse and human neonatal Fc receptor with different affinities that reveal distinct cross-species differences in serum half-life

Serum half-life of IgG is controlled by the neonatal Fc receptor (FcRn) that interacts with the IgG Fc region and may be increased or decreased as a function of altered FcRn binding. Preclinical evaluations of modified IgGs are frequently carried out in mice, but such IgGs may bind differently to mouse and human FcRn (mFcRn and hFcRn). Here, we report a detailed characterization of a matched set of mouse-human chimeric T84.66 scFv-Fc variants with specificity for the tumor carcinoembryonic antigen and mutations in the FcRn-binding site. Binding to soluble mFcRn and hFcRn was measured using in vitro assays, and the results were compared with blood clearance in vivo in normal (mFcRn bearing) and hFcRn transgenic mice. All variants bound better to mFcRn than to hFcRn. The loss of affinity varied among the mutants, however, and also the hierarchy of binding differed depending on the receptor. The mutations had no major impact on binding to the classical Fcγ receptors. Importantly, the trend of blood clearance in both strains of mice correlated with the hierarchy of binding obtained using soluble FcRn. Consequently, in vitro interaction analysis of engineered IgGs regarding their cross-species FcRn binding ability provides information for prediction of in vivo pharmacokinetics.