The Oligomeric States of the Purified Sigma-1 Receptor Are Stabilized by Ligands

Sigma-1 receptor (S1R) is a mammalian member of the ERG2 and sigma-1 receptor-like protein family (pfam04622). It has been implicated in drug addiction and many human neurological disorders, including Alzheimer and Parkinson diseases and amyotrophic lateral sclerosis. A broad range of synthetic small molecules, including cocaine, (+)-pentazocine, haloperidol, and small endogenous molecules such as N,N-dimethyltryptamine, sphingosine, and steroids, have been identified as regulators of S1R. However, the mechanism of activation of S1R remains obscure. Here, we provide evidence in vitro that S1R has ligand binding activity only in an oligomeric state. The oligomeric state is prone to decay into an apparent monomeric form when exposed to elevated temperature, with loss of ligand binding activity. This decay is suppressed in the presence of the known S1R ligands such as haloperidol, BD-1047, and sphingosine. S1R has a GXXXG motif in its second transmembrane region, and these motifs are often involved in oligomerization of membrane proteins. Disrupting mutations within the GXXXG motif shifted the fraction of the higher oligomeric states toward smaller states and resulted in a significant decrease in specific (+)-[³H]pentazocine binding. Results presented here support the proposal that S1R function may be regulated by its oligomeric state. Possible mechanisms of molecular regulation of interacting protein partners by S1R in the presence of small molecule ligands are discussed.     

Hsp90 cochaperones p23 and FKBP4 physically interact with hAgo2 and activate RNA interference–mediated silencing in mammalian cells

Argonaute proteins and small RNAs together form the RNA-induced silencing complex (RISC), the central effector of RNA interference (RNAi). The molecular chaperone Hsp90 is required for the critical step of loading small RNAs onto Argonaute proteins. Here we show that the Hsp90 cochaperones Cdc37, Aha1, FKBP4, and p23 are required for efficient RNAi. Whereas FKBP4 and p23 form a stable complex with hAgo2, the function of Cdc37 in RNAi appears to be indirect and may indicate that two or more Hsp90 complexes are involved. Our data also suggest that p23 and FKBP4 interact with hAgo2 before small RNA loading and that RISC loading takes place in the cytoplasm rather than in association with RNA granules. Given the requirement for p23 and FKBP4 for efficient RNAi and that these cochaperones bind to hAgo2, we predict that loading of hAgo2 is analogous to Hsp90-mediated steroid hormone receptor activation. To this end, we outline a model in which FKBP4, p23, and Aha1 cooperatively regulate the progression of hAgo2 through the chaperone cycle. Finally, we propose that hAgo2 and RNAi can serve as a robust model system for continued investigation into the Hsp90 chaperone cycle.     

Ribonucleic acid is a component of the oligomeric, transformed mouse AtT-20 cell glucocorticoid receptor

The glucocorticoid receptor from mouse AtT-20 pituitary tumor cells exists in three forms. The largest form is an untransformed (non-DNA-binding), oligomeric species (9.1 S, 8.3 nm, Mr 319 000). Two transformed (DNA-binding) forms can be generated. One is an oligomeric protein (5.2 S, 6-8.3 nm, Mr 132 000-182 000), while the other is the monomeric, hormone-binding subunit (3.8 S, 6 nm, Mr 96 000). The composition of the oligomeric, transformed receptor and its relationship to the monomeric protein were examined. The 3.8S monomer can be isolated from DEAE-cellulose (0.12 M step elution) in a form that continues to sediment at about 3.8 S on molybdate-containing sucrose gradients and at about 4.2 S on molybdate-free gradients. Addition of a non-hormone-binding component isolated from the same DEAE-cellulose column (0.5 M KCl step) can apparently interact with the 3.8-4.2 S monomer, increasing its sedimentation coefficient to 5.2 S (on molybdate-containing gradients) or 6.6 S (on low-salt, molybdate-free gradients). This factor is a macromolecule (nondialyzable) and is heat-stable (100 degrees C, 20 min). A dose-dependent shift to the higher sedimentation coefficient is observed when increasing quantities of the 0.5 M step material are added to the receptor monomer. This activity is abolished when the 0.5 M step material is treated with ribonuclease A. Further, when RNA is purified from the 0.5 M step by phenol/chloroform extraction, its ability to increase the S value of the monomer is retained. Ribonuclease treatment of the untransformed, 9.1S, oligomeric complex does not cause a significant decrease in sedimentation rate, while the same treatment of the 5.2S, oligomeric, transformed receptor (obtained after Sephadex G-25 transformation) causes a decrease in sedimentation rate to about 3.8 S. The addition of bovine liver mRNA and rRNA does not cause a shift in sedimentation rate of the receptor monomer to a discrete, higher sedimenting receptor form. However, the addition of total rabbit liver tRNA or three distinct tRNA species causes a shift in sedimentation to a similar, but not identical, form as that with the 0.5 M step material. We propose that the 5.2S, oligomeric transformed glucocorticoid receptor is composed of one monomeric hormone-binding, protein subunit (Mr 96 000) and a low molecular weight RNA (Mr 36 000). This interaction may be important for the role of the receptor in regulating gene expression.     

PGE2 stimulates human brain natriuretic peptide expression via EP4 and p42/44 MAPK

Brain natriuretic peptide (BNP) produced by cardiac myocytes has antifibrotic and antigrowth properties and is a marker of cardiac hypertrophy. We previously showed that prostaglandin E2 (PGE2) is the main prostaglandin produced in myocytes treated with proinflammatory stimuli and stimulates protein synthesis by binding to its EP4 receptor. We hypothesized that PGE2, acting through EP4, also regulates BNP gene expression. We transfected neonatal ventricular myocytes with a plasmid encoding the human BNP (hBNP) promoter driving expression of a luciferase reporter gene. PGE2 increased hBNP promoter activity 3.5-fold. An EP4 antagonist reduced the stimulatory effect of PGE2 but not an EP1 antagonist. Because EP4 signaling can involve adenylate cyclase, cAMP, and protein kinase A (PKA), we tested the effect of H-89, a PKA inhibitor, on PGE2 stimulation of the hBNP promoter. H-89 at 5 muM decreased PGE2 stimulation of BNP promoter activity by 100%. Because p42/44 MAPK mediates the effect of PGE2 on protein synthesis, we also examined the role of MAPKs in the regulation of BNP promoter activity. PGE2 stimulation of the hBNP promoter was inhibited by a MEK1/2 inhibitor and a dominant-negative mutant of Raf, indicating that p42/44 MAPK was involved. In contrast, neither a p38 MAPK inhibitor nor a JNK inhibitor reduced the stimulatory effect of PGE2. Involvement of small GTPases was also studied. Dominant-negative Rap inhibited PGE2 stimulation of the hBNP promoter, but dominant-negative Ras did not. We concluded that PGE2 stimulates the BNP promoter mainly via EP4, PKA, Rap, and p42/44 MAPK.     

Molecular Characterization of a Theta Replication Plasmid and Its Use for Development of a Two-Component Food-Grade Cloning System for Lactococcus lactis

pCD4, a small, highly stable theta-replicating lactococcal plasmid, was used to develop a food-grade cloning system. Sequence analysis revealed five open reading frames and two putative cis-acting regions. None appears to code for undesirable phenotypes with regard to food applications. Functional analysis of the replication module showed that only the cis-acting ori region and the repB gene coding for the replication initiator protein were needed for the stable replication and maintenance of pCD4 derivatives in Lactococcus lactis. A two-component food-grade cloning system was derived from the pCD4 replicon. The vector pVEC1, which carries the functional pCD4 replicon, is entirely made up of L. lactis DNA and has no selection marker. The companion pCOM1 is a repB-deficient pCD4 derivative that carries an erythromycin resistance gene as a dominant selection marker. The pCOM1 construct can only replicate in L. lactis if trans complemented by the RepB initiator provided by pVEC1. Since only the cotransformants that carry both pVEC1 and pCOM1 can survive on plates containing erythromycin, pCOM1 can be used transiently to select cells that have acquired pVEC1. Due to the intrinsic incompatibility between these plasmids, pCOM1 can be readily cured from the cells grown on an antibiotic-free medium after the selection step. The system was used to introduce a phage resistance mechanism into the laboratory strain MG1363 of L. lactis and two industrial strains. The introduction of the antiphage barrier did not alter the wild-type plasmid profile of the industrial strains. The phenotype was stable after 100 generations and conferred an effective resistance phenotype against phages of the 936 and c2 species.     

Structural basis for cargo regulation of COPII coat assembly

Using cryo-electron microscopy, we have solved the structure of an icosidodecahedral COPII coat involved in cargo export from the endoplasmic reticulum (ER) coassembled from purified cargo adaptor Sec23-24 and Sec13-31 lattice-forming complexes. The coat structure shows a tetrameric assembly of the Sec23-24 adaptor layer that is well positioned beneath the vertices and edges of the Sec13-31 lattice. Fitting the known crystal structures of the COPII proteins into the density map reveals a flexible hinge region stemming from interactions between WD40 beta-propeller domains present in Sec13 and Sec31 at the vertices. The structure shows that the hinge region can direct geometric cage expansion to accommodate a wide range of bulky cargo, including procollagen and chylomicrons, that is sensitive to adaptor function in inherited disease. The COPII coat structure leads us to propose a mechanism by which cargo drives cage assembly and membrane curvature for budding from the ER.     

Consensus-Degenerate Hybrid Oligonucleotide Primers for Amplification of Priming Glycosyltransferase Genes of the Exopolysaccharide Locus in Strains of the Lactobacillus casei Group

A primer design strategy named CODEHOP (consensus-degenerate hybrid oligonucleotide primer) for amplification of distantly related sequences was used to detect the priming glycosyltransferase (GT) gene in strains of the Lactobacillus casei group. Each hybrid primer consisted of a short 3′ degenerate core based on four highly conserved amino acids and a longer 5′ consensus clamp region based on six sequences of the priming GT gene products from exopolysaccharide (EPS)-producing bacteria. The hybrid primers were used to detect the priming GT gene of 44 commercial isolates and reference strains of Lactobacillus rhamnosus, L. casei, Lactobacillus zeae, and Streptococcus thermophilus. The priming GT gene was detected in the genome of both non-EPS-producing (EPS⁻) and EPS-producing (EPS⁺) strains of L. rhamnosus. The sequences of the cloned PCR products were similar to those of the priming GT gene of various gram-negative and gram-positive EPS⁺ bacteria. Specific primers designed from the L. rhamnosus RW-9595M GT gene were used to sequence the end of the priming GT gene in selected EPS⁺ strains of L. rhamnosus. Phylogenetic analysis revealed that Lactobacillus spp. form a distinctive group apart from other lactic acid bacteria for which GT genes have been characterized to date. Moreover, the sequences show a divergence existing among strains of L. rhamnosus with respect to the terminal region of the priming GT gene. Thus, the PCR approach with consensus-degenerate hybrid primers designed with CODEHOP is a practical approach for the detection of similar genes containing conserved motifs in different bacterial genomes.     

Molecular characterization of a theta replication plasmid and its use for development of a two-component food-grade cloning system for Lactococcus lactis

pCD4, a small, highly stable theta-replicating lactococcal plasmid, was used to develop a food-grade cloning system. Sequence analysis revealed five open reading frames and two putative cis-acting regions. None appears to code for undesirable phenotypes with regard to food applications. Functional analysis of the replication module showed that only the cis-acting ori region and the repB gene coding for the replication initiator protein were needed for the stable replication and maintenance of pCD4 derivatives in Lactococcus lactis. A two-component food-grade cloning system was derived from the pCD4 replicon. The vector pVEC1, which carries the functional pCD4 replicon, is entirely made up of L. lactis DNA and has no selection marker. The companion pCOM1 is a repB-deficient pCD4 derivative that carries an erythromycin resistance gene as a dominant selection marker. The pCOM1 construct can only replicate in L. lactis if trans complemented by the RepB initiator provided by pVEC1. Since only the cotransformants that carry both pVEC1 and pCOM1 can survive on plates containing erythromycin, pCOM1 can be used transiently to select cells that have acquired pVEC1. Due to the intrinsic incompatibility between these plasmids, pCOM1 can be readily cured from the cells grown on an antibiotic-free medium after the selection step. The system was used to introduce a phage resistance mechanism into the laboratory strain MG1363 of L. lactis and two industrial strains. The introduction of the antiphage barrier did not alter the wild-type plasmid profile of the industrial strains. The phenotype was stable after 100 generations and conferred an effective resistance phenotype against phages of the 936 and c2 species.     

Improved methods for mutacin detection and production

Studies of mutacins have always been hampered by the difficulties in obtaining active liquid preparations of these substances. In order to be commercially produced, good mutacin yields have to be obtained, preferably in inexpensive media. The results presented here indicate that mutacins can be produced in supplemented cheese whey permeate. The influence of carbon and nitrogen supplements on mutacin production varied according to the producer strain. The use of CaCO3 as a buffer in batch cultures resulted in improved yields of mutacin in the supernatants. Antimicrobial activity assays were improve by acidification of the diluent (pH 2) and were less variable in peptone water (0.5%). The culture medium consisting of cheese whey permeate (6% w/v), yeast extract (2% w/v) and CaCO3 (1% w/v) was found to be an inexpensive medium for the efficient production of mutacins.