Yeast One-Hybrid Gγ Recruitment System for Identification of Protein Lipidation Motifs

Fatty acids and isoprenoids can be covalently attached to a variety of proteins. These lipid modifications regulate protein structure, localization and function. Here, we describe a yeast one-hybrid approach based on the Gγ recruitment system that is useful for identifying sequence motifs those influence lipid modification to recruit proteins to the plasma membrane. Our approach facilitates the isolation of yeast cells expressing lipid-modified proteins via a simple and easy growth selection assay utilizing G-protein signaling that induces diploid formation. In the current study, we selected the N-terminal sequence of Gα subunits as a model case to investigate dual lipid modification, i.e., myristoylation and palmitoylation, a modification that is widely conserved from yeast to higher eukaryotes. Our results suggest that both lipid modifications are required for restoration of G-protein signaling. Although we could not differentiate between myristoylation and palmitoylation, N-terminal position 7 and 8 play some critical role. Moreover, we tested the preference for specific amino-acid residues at position 7 and 8 using library-based screening. This new approach will be useful to explore protein-lipid associations and to determine the corresponding sequence motifs.     

Identification of Novel Short Peptide Inhibitors of Soluble 37/48 kDa Oligomers of Amyloid β42

Since the soluble oligomers of 42-residue amyloid β (Aβ42) might be neurotoxins at an early stage of Alzheimer’s disease (AD), inhibition of soluble Aβ42 oligomerization should be effective in the treatment of AD. We have found by phage display that a 7-residue peptide, SRPGLRR, exhibited inhibitory activity against soluble 37/48 kDa oligomers of Aβ42. In the present study, we newly prepared 3- and 4-residue random peptides libraries and performed pannings of them against soluble Aβ42 to search for important factors in the inhibition of Aβ42 oligomerization. After the fifth round, arginine-containing peptides were enriched in both libraries. SDS–PAGE and size-exclusion chromatography indicated that the inhibitory activities of 4-residue peptides against the soluble 37/48 kDa oligomers of Aβ42 were higher than those of the 3-residue peptides, and RFRK exhibited strong inhibitory activity as well as SRPGLRR. These short peptides should be useful for the suppression of soluble Aβ42 oligomer formation.     

Inhibitory Effects of Bombusae concretio Salicea on Neuronal Secretion of Alzheimer's β-Amyloid Peptides, a Neurodegenerative Peptide

Alzheimer's disease (AD) is characterized by the age-related deposition of -amyloid (A) 40/42 peptide aggregates in vulnerable brain regions. Multiple levels of evidence implicate a central role for A in the pathophysiology of AD. A is generated by the regulated cleavage of a = 700 amino acid A precursor protein (APP). Full-length APP can undergo proteolytic cleavage either within the A domain to generate secreted sAPP or at the N-terminal and C-terminal domain(s) of A to generate amyloidogenic A peptides. Several epidemiological studies have reported that estrogen replacement therapy protects against the development of AD in postmenopausal women. The aim of this study was to elucidate the antioxidant neuroprotective mechanism of Bombusae concretio Salicea (BC). BC was effective protectants against oxidative glutamate toxicity in the murine neuroblastoma cells (N2a) and human neuroblastoma cells (SK-N-MC). BC exhibited similar protective properties against oxidative glutamate toxicity and H₂O₂ toxicity. BC exhibited an antioxidant activity at approximately 20 g/ml. BC of 5 g/ml was ineffective in preventing the oxidative modification of LDL. The half-maximal effective concentration for BC was 16 g/ml. These results suggested that BC supplementation in elderly men may be protective in the treatment of Alzheimer's disease (AD). We report here that treatment with BC increases the secretion of the nonamyloidogenic APP fragment, sAPP and decreases the secretion of A peptides from N2a cells and rat primary cerebrocortical neurons. These results raise the possibility that BC supplementation in elderly men may be protective in the treatment of AD.     

Enhanced Production of β-Carotene by Recombinant Industrial Wine Yeast Using Grape Juice as Substrate

In this study, both recombinant Saccharomyces cerevisiae T73-63 and FY-09 derived from the industrial wine yeast T73-4 and laboratory yeast FY1679-01B, respectively, were constructed and compared for their β-carotene production in real grape juice. The results showed that highest β-carotene content (5.89 mg/g) was found in strain T73-63, which was 2.1 fold higher than that of strain FY-09. Although the cell growth was inhibited by the metabolic burden induced by the production of heterogeneous β-carotene, the pigment yield in T73-63 was still 1.7 fold higher than that of FY-09. Furthermore, high contents of ergosterol and fatty acid were also observed in T73-63. These results suggest that industrial wine yeast has highly active metabolic flux in mevalonate pathway, which leads to more carbon flux into carotenoid branch compared to that of laboratory yeast. The results of this study collectively suggest that in the application of recombinant strains to produce carotenoid using agro-industrial by-products as substrate, the suitable host strains should have active mevalonate pathway. For this purpose, the industrial wine yeast is a suitable candidate.     

Products of acylase and β-Lactamase hydrolysis of some penicillins

The products of acylase of acylase and β-lactamase hydrolysis of 7 penicillins were determined by paper chromatography. The procedures used allowed us to detect also the product without antibiotic activity. When benzylpenicillin, phenoxymethylpenicillin, and ampicillin were hydrolyzed by cell suspension ofAlcaligenes faecalis of a high acylase activity, penicic acid and another unidentified metabolite, were found beside 6—APA. The hydrolysis of benzylpenicillin byEscherichia coli produced the same metabolites with prevailing benzylpenicilloic acid. The way of formation of penicic acid is discussed. Purified β-lactamase and cell suspension of the test or penicillin-resistent strain ofStaphylococcus aureus produced in all penicillins tested the same metabolite-corresponding penicilloic acid.     

The Tn3 β-Lactamase Gene Acts as a Hotspot for Meiotic Recombination in Yeast

Although genetic distances are often assumed to be proportional to physical distances, chromosomal regions with unusually high (hotspots) or low (coldspots) levels of meiotic recombination have been described in a number of genetic systems. In general, the DNA sequences responsible for these effects have not been determined. We report that the 5' region of the beta-lactamase (ampR) gene of the bacterial transposon Tn3 is a hotspot for meiotic recombination when inserted into the chromosomes of the yeast Saccharomyces cerevisiae. When these sequences are homozygous, both crossing over and gene conversion are locally stimulated. The 5' end of the beta-lactamase gene is about 100-fold "hotter" for crossovers than an average yeast DNA sequence.     

Identification of the Metallo-β-Lactamase Gene from Clinical Isolates of Bacteroides fragilis

Bacteroides fragilis is one of the organisms known to produce carbapenem-hydrolysing metallo-β-lactamase, which can confer resistance to a wide variety of β-lactams. The purpose of this study was to identify carbapenem-hydrolysing metallo-β-lactamase-producing B. fragilis strains by means of PCR assay, nucleotide sequencing and enzyme inhibition studies. Ten β-lactam-resistant B. fragilis isolates were investigated. Four imipenem-resistant strains among the 10 isolates gave positive reactions in the PCR assay. The nucleotide sequences of the PCR products from two imipenem-resistant strains shared >98% similarity with the metallo-β-lactamase gene from B. fragilis TAL 3636, which was used as a control. The amino acid sequence homology between the two imipenem-resistant strains and B. fragilis TAL 3636 was 99.2%. These strains produced high amounts of Zn²⁺-dependent β-lactamases which were inactivated by EDTA.     

Identification of residues involved in homodimer formation located within a β-strand region of the N-terminus of a Yeast G protein-coupled receptor

G protein-coupled receptors (GPCRs) are members of a superfamily of cell surface signaling proteins that play critical roles in many physiological functions; malfunction of these proteins is associated with multiple diseases. Understanding the structure-function relationships of these proteins is important, therefore, for GPCR-based drug discovery. The yeast Saccharomyces cerevisiae tridecapeptide pheromone α-factor receptor Ste2p has been studied as a model to explore the structure-function relationships of this important class of cell surface receptors. Although transmembrane domains of GPCRs have been examined extensively, the extracellular N-terminus and loop regions have received less attention. We have used substituted cysteine accessibility method to probe the solvent accessibility of single cysteine residues engineered to replace residues Gly20 through Gly33 of the N-terminus of Ste2p. Unexpectedly, our analyses revealed that the residues Ser22, Ile24, Tyr26, and Ser28 in the N-terminus were solvent inaccessible, whereas all other residues of the targeted region were solvent accessible. The periodicity of accessibility from residues Ser22-Ser28 is indicative of an underlying structure consistent with a β-strand that was predicted computationally in this region. Moreover, a number of these Cys-substituted Ste2p receptors (G20C, S22C, I24C, Y26C, S28C and Y30C) were found to form increased dimers compared to the Cys-less Ste2p. Based on these data, we propose that part of the N-terminus of Ste2p is structured and that this structure forms a dimer interface for Ste2p molecules. Dimerization mediated by the N-terminus was affected by ligand binding, indicating an unanticipated conformational change in the N-terminus upon receptor activation.     

Identification of residues involved in homodimer formation located within a β-strand region of the N-terminus of a Yeast G protein-coupled receptor

G protein-coupled receptors (GPCRs) are members of a superfamily of cell surface signaling proteins that play critical roles in many physiological functions; malfunction of these proteins is associated with multiple diseases. Understanding the structure–function relationships of these proteins is important, therefore, for GPCR-based drug discovery. The yeast Saccharomyces cerevisiae tridecapeptide pheromone α-factor receptor Ste2p has been studied as a model to explore the structure–function relationships of this important class of cell surface receptors. Although transmembrane domains of GPCRs have been examined extensively, the extracellular N-terminus and loop regions have received less attention. We have used substituted cysteine accessibility method to probe the solvent accessibility of single cysteine residues engineered to replace residues Gly20 through Gly33 of the N-terminus of Ste2p. Unexpectedly, our analyses revealed that the residues Ser22, Ile24, Tyr26, and Ser28 in the N-terminus were solvent inaccessible, whereas all other residues of the targeted region were solvent accessible. The periodicity of accessibility from residues Ser22–Ser28 is indicative of an underlying structure consistent with a β-strand that was predicted computationally in this region. Moreover, a number of these Cys-substituted Ste2p receptors (G20C, S22C, I24C, Y26C, S28C and Y30C) were found to form increased dimers compared to the Cys-less Ste2p. Based on these data, we propose that part of the N-terminus of Ste2p is structured and that this structure forms a dimer interface for Ste2p molecules. Dimerization mediated by the N-terminus was affected by ligand binding, indicating an unanticipated conformational change in the N-terminus upon receptor activation.     

Identification of the β-Lactamase Inhibitor Protein-II (BLIP-II) Interface Residues Essential for Binding Affinity and Specificity for Class A β-Lactamases

The interactions between β-lactamase inhibitory proteins (BLIPs) and β-lactamases have been used as model systems to understand the principles of affinity and specificity in protein-protein interactions. The most extensively studied tight binding inhibitor, BLIP, has been characterized with respect to amino acid determinants of affinity and specificity for binding β-lactamases. BLIP-II, however, shares no sequence or structural homology to BLIP and is a femtomolar to picomolar potency inhibitor, and the amino acid determinants of binding affinity and specificity are unknown. In this study, alanine scanning mutagenesis was used in combination with determinations of on and off rates for each mutant to define the contribution of residues on the BLIP-II binding surface to both affinity and specificity toward four β-lactamases of diverse sequence. The residues making the largest contribution to binding energy are heavily biased toward aromatic amino acids near the center of the binding surface. In addition, substitutions that reduce binding energy do so by increasing off rates without impacting on rates. Also, residues with large contributions to binding energy generally exhibit low temperature factors in the structures of complexes. Finally, with the exception of D206A, BLIP-II alanine substitutions exhibit a similar trend of effect for all β-lactamases, i.e., a substitution that reduces affinity for one β-lactamase usually reduces affinity for all β-lactamases tested.     

Inhibitory Effect of Methyl Methanethiosulfinate on β-Glucuronidase Activity

Minced or cut-up leaves of Chinese chive (Allium tuberosum Rottler) contain thiosulfinates and their disproportionate reaction products. Among these organosulfur compounds, methyl methanethiosulfinate was found to be an uncompetitive inhibitor of β-glucuronidase. Approximately 80% of the enzyme activity was inhibited by methyl methanethiosulfinate at 50 µM, the IC₅₀ value being comparable to 3.6 µM.     

An Inducible System for the Hydrolysis and Transport of β-Glucosides in Yeast : I. Characteristics of the β-glucosidase activity of intact and of lysed cells

A strain of bakers'' yeast was isolated which could utilize cellobiose and other β-D-glucosides quantitatively as carbon and energy sources for growth. Cellobiose-grown cells contained a largely cryptic enzyme active against the chromogenic substrate p-nitrophenyl-β-D-glucoside. The patent (intact cell) activity of such cells was inhibited by azide and, competitively, by cellobiose; neither agent inhibited the β-glucosidase activity of lysed cells or of extracts. The enzyme induced by growth in cellobiose medium had no affinity for cellobiose as either substrate or inhibitor; its substrate specificity classifies it as an aryl-β-glucosidase. It was concluded that growth in cellobiose also induced the formation of a stereospecific and energy-dependent system whose function determined the rate at which intact cells could hydrolyze substrates of the intracellular β-glucosidase.     

Recovery of β-Galactosidase from E. Coli Double Lysogen Using Aqueous Two Phase System

An aqueous two phase system (ATPS) was employed to recover -galactosidase from the lytic broth of the phage double lysogen system, where production of an intracellular product and cell disruption are carried out sequentially in a container. Partition of -galactosidase was more favorable to PEG phase comparing to total proteins, which was enhanced with Na₂SO₄. Recovery of -galactosidase was more than 90% in most cases, which was much more the recovery of total proteins.     

Simultaneous Identification and Quantification of 20 β-Receptor Agonists in Feed Using Gas Chromatography-Tandem Mass Spectrometry

“Lean meat powder” is a class of toxic chemicals that have structures similar to that of β-adrenergic receptor agonists. At least 16 chemicals from this class have been specifically banned by the 176^th bulletin of the Chinese Department of Agriculture on breeding animals, and methods for monitoring the illicit use of β-agonists in animal feed are required. Herein, a method to quantify 20 β-agonists in feed, via analyte derivatization followed by gas chromatography-tandem mass spectrometry, has been developed. The optimized method has a good linear correlation (calibration coefficient > 0.99) between the quantitative ion peak area and the concentration of β-agonists over a large working range (0.05–1 mg/kg). The limit of detection (LOD) was 0.01 mg/kg, the recoveries for three β-agonists spikes (0.05, 0.1, and 1 µg/g) in feed ranged from 75.6 to 102.4%, repeatability ranged from 1.2 to 9.4% for all of the compounds, and intermediate precisions were lower than 13.8%. This precise, accurate method was applied to quantify 20 β-agonists in actual feed samples and represents an excellent complement to existing quantification methods.     

A Further Separation and Some Properties of β-Lactamase I

A carbohydrate binding protein was found in mid-lactating rat mammary gland. This rat mammary gland lectin agglutinated trypsinized rabbit erythrocytes and the hemagglutination was inhibited by the addition of β-d-galactosides such as lactose, melibiose, UDP-galactose and thio-d-galactoside. The lectin was partially purified by affinity chromatography on a column of Sepharose 4B to which asialo-fetuin had been covalently linked. Rat mammary gland lectin is a glycoprotein with a molecular weight of 14,800, estimated from SDS-PAGE, or 16,800 from gel filtration.

The occurrence of two glycoproteins, C4-casein and α-lactalbumin, is known in rat milk. Bovine κ-casein is a well-characterized glycoprotein. These glycoproteins were found to be bound by the rat mammary gland lectin, when they were desialylated by the action of neuraminidase. Neuraminidase-untreated α-lactalbumin also bound to the lectin but to a lesser extent. The level of the lectin in rat mammary gland was greatly reduced during regression of the gland after weaning.     

Peptide mapping of β-casein by capillary zone electrophoresis

A method to the study of -casein proteolysis by aspartic proteinases is developed. The 3% trichloroacetic acid-soluble peptides of -casein digested with cardosin A were separated by capillary zone electrophoresis under different experimental conditions in an uncoated fused silica capillary. The best separation was at pH 3.01, 30 kV and 30 °C.     

Identification of paxilin domains interacting with β-catenin

Barrier-protective agonists induce association of focal adhesions (FA) and adherens junctions (AJ) in endothelial cells. Here we identified specific domains of FA protein paxillin interacting with AJ protein and examined regulation of paxillin domain interactions with β-catenin by Rac GTPase. Co-expression of paxillin LD-1,2; LD-3,4; LIM-1,2; and LIM-3,4 domains with β-catenin showed exclusive interaction of LIM-1,2 and LIM-3,4 with β-catenin, which was enhanced by agonist-induced Rac activation or expression of activated Rac mutant. These results demonstrate a novel function of paxillin LIM domains in targeting β-catenin in a Rac-dependent manner, which may play a role in Rac-dependent control of FA-AJ interactions and monolayer integrity.     

Identification of β-aggregate sites in protein chains

The problem of amyloidoses is pressing and have recently attracted special attention throughout the world because of epidemics of prion diseases such as mad cow disease and human Creutzfeldt-Jacob disease. These diseases result from the conversion of a native protein or peptide into a highly stable pathological form. Molecules having a pathological conformation aggregate to form amyloid fibrils, capable of unlimited growth. It is important to study the molecular mechanisms of prion diseases and to identify the protein regions responsible for their development. The review considers theoretical and experimental works focusing on the formation of amyloid fibrils.     

Yeast Two-Hybrid Analysis of PriB-Interacting Proteins in Replication Restart Primosome: A Proposed PriB–SSB Interaction Model

PriB is one of the components of the replication restart primosome. The activity mediated by the primosomal proteins, including PriB, is required for reinitiating chromosomal DNA replication in bacteria after DNA damage. As such, the study of the interactions between PriB and members in the primosome is essential to better understand their mechanism of action. In this study, we investigated PriB-interacting proteins in the primosome through the yeast two-hybrid system. Yeast two-hybrid analysis using two strains, Y187 and AH109, revealed that PriB interacts with PriA, DnaT, SSB, and itself and does not interact with DnaA, DnaB, DnaC, or DnaG. In addition, mutational analysis showed that PriB may bind to SSB via K82, K84, and K89 in the L₄₅ loop. Based on these preliminary data, we proposed a PriB–SSB interaction model. Further work can focus on determination of how PriB binds to the PriA–SSB complex in replication restart.