Proteolytic processing of certain CaaX motifs can occur in the absence of the Rce1p and Ste24p CaaX proteases

The CaaX motif directs C‐terminal protein modifications that include isoprenylation, proteolysis and carboxylmethylation. Proteolysis is generally believed to require either Rce1p or Ste24p. While investigating the substrate specificity of these proteases, using the yeast a‐factor mating pheromone as a reporter, we observed Rce1p‐ and Ste24p‐independent mating (RSM) when the CKQQ CaaX motif was used in lieu of the natural a‐factor CVIA motif. Uncharged or negatively charged amino acid substitutions at the a₁ position of the CKQQ motif prevented RSM. Alanine substitutions at the a₂ and X positions enhanced RSM. Random mutagenesis of the CaaX motif provided evidence that RSM occurs with approximately 1% of all possible CaaX motif permutations. Combined mutational and genetic data indicate that RSM‐promoting motifs have a positively charged amino acid at the a₁ position. Two of nine naturally occurring yeast CaaX motifs conforming to this pattern promoted RSM. The activity of the isoprenylcysteine carboxyl methyltransferase Ste14p was required for RSM, indicating that RSM‐promoting CaaX motifs are indeed proteolysed. RSM was enhanced by the overexpression of Axl1p or Ste23p, suggesting a role for these M16A subfamily metalloproteases in this process. We have also determined that an N‐terminal extension of the a‐factor precursor, which is typically removed by the yeast M16A enzymes, is required for optimal RSM. These observations suggest a model that involves targeting of the a‐factor precursor to the peptidosome cavity of M16A enzymes where subsequent interactions between RSM‐promoting CaaX motifs and the active site of the M16A enzyme lead to proteolytic cleavage. Copyright © 2009 John Wiley & Sons, Ltd.     

Pheromone responsiveness is regulated by components of the Gpr1p‐mediated glucose sensing pathway in Saccharomyces cerevisiae

Many fungi have evolved mechanisms to assess environmental nutrient availability prior to the energy‐intensive process of mating. In this study, we examined one such system in Saccharomyces cerevisiae, involving a glucose‐sensing pathway mediated by Gpr1p and the pheromone‐induced mating pathway. Initially we observed that the mating pathway in MATa cells is sensitive to environmental glucose depletion. This phenomenon can be partially reversed with a high glucose spike, but not with the addition of low levels of glucose. Deletion of the low‐affinity glucose receptor, Gpr1p, eliminated this glucose‐induced recovery of pheromone responsiveness. We then determined the impact of GPR1 deletion on the mating pathway and observed that, in all end points studied, the mating pathway response to pheromone is reduced in the absence of Gpr1p. Similarly, elimination of the Gα for Gpr1p, Gpa2p, resulted in reduction in pheromone sensitivity in all assays studied. The negative effect of removing Gpr1p on mating pathway activation could be recovered by overexpressing the mating receptor, Ste2p. Furthermore, Ste2p levels are reduced in the absence of glucose and GPR1. These data suggest that activity of the GPCR‐mediated mating pathway in S. cerevisiae is modulated by extracellular glucose concentrations through the only other GPCR in MATa cells, Gpr1p. Copyright © 2014 John Wiley & Sons, Ltd.     

Candida glabrata Ste20 is involved in maintaining cell wall integrity and adaptation to hypertonic stress, and is required for wild-type levels of virulence

The conserved family of fungal Ste20 p21-activated serine-threonine protein kinases regulate several signalling cascades. In Saccharomyces cerevisiae Ste20 is involved in pheromone signalling, invasive growth, the hypertonic stress response, cell wall integrity and binds Cdc42, a Rho-like small GTP-binding protein required for polarized morphogenesis. We have cloned the STE20 homologue from the fungal pathogen Candida glabrata and have shown that it is present in a single copy in the genome. Translation of the nucleotide sequence predicts that C. glabrata Ste20 contains a highly conserved p21-activated serine-threonine protein kinase domain, a binding site for G-protein beta subunits and a regulatory Rho-binding domain that enables the kinase to interact with Cdc42 and/or Rho-like small GTPases. C. glabrata Ste20 has 53% identity and 58% predicted amino acid similarity to S. cerevisiae Ste20 and can complement both the nitrogen starvation-induced filamentation and mating defects of S. cerevisiae ste20 mutants. Analysis of ste20 null and disrupted strains suggest that in C. glabrata Ste20 is required for a fully functional hypertonic stress response and intact cell wall integrity pathway. C. glabrata Ste20 is not required for nitrogen starvation-induced filamentation. Survival analysis revealed that C. glabrata ste20 mutants, while still able to cause disease, are mildly attenuated for virulence compared to reconstituted STE20 cells.     

Separate roles for N- and C-termini of the STE4 (β) subunit of the Saccharomyces cerevisiae G protein in the mediation of the growth arrest. Lack of growth-arresting activity of mammalian βγ complexes

Mating pheromone signal transduction in Saccharomyces cerevisiae involves a G protein composed to Scg1p (Gpa1p), Ste4p and Ste18p subunits, homologous to the α, β and γ subunits of mammalian G proteins. Growth arrest in G1 phase is activated by the Ste4p/Ste18p complex via a downstream pathway and it is negatively controlled by the Scg1p subunit. Here we explored whether mammalian β or γ subunits could functionally substitute for their yeast homologues. While no evidence was obtained for functional replacement of Ste4p and Ste18p, we found that overexpression of Ste18p potentiated the effect of hybrid proteins in which the N terminus of the Ste4p subunit was replaced by that of the mammalian β, ste4 mutants having deletions in the N terminus showed a decreased activity in signalling to the downstream effector of the pheromone response. This defect was totally cured by overexpression of Ste18p, indicating that the truncated forms of Ste4p have retained their ability to form an active complex with Ste18p. Removal of six amino acids from the C terminus of Ste4p rendered a completely inactive subunit and this defect persisted in hybrids where the C terminus was placed by that of the β subunit, indicating that the C terminus of Ste4p is essential to trigger the effector of the yeast pheromone response pathway.     

Antioxidant peptides obtained from goose egg white proteins by enzymatic hydrolysis

In this study, antioxidant peptides from goose egg white proteins produced using various enzymes were purified and characterised. Two peptides were named as p14 and p16, showing the highest scavenging activity of 1,1‐diphenyl‐2‐picrylhydrazyl (DPPH) radical and the highest metal ion chelating activity, respectively. The sequences of p14 and p16 were identified to be STMMEERRMKVY (1560.72 Da) and DVFRELRVQ (1161.62 Da), respectively. The sequence of p14 has a similarity of 75% to ovalbumin from Meleagris gallopavo and the sequence of p16 has a similarity of 67% to ovalbumin from Taeniopygia guttata. IC₅₀ values of p14 and p16 were determined, and results showed that DPPH radical scavenging activity was 81.6 and 205.5 μm , 2,2′‐azino‐bis(3‐ethylbenzothiazoline‐6‐sulphonicacid)(ABTS) radical scavenging was 88.4 and 153.8 μm , hydroxyl radical scavenging was 85.5 and 116.3 μm and metal ion chelating was 170.6 and 117.9 μm , respectively. The two identified peptides from goose egg white hydrolysates act as potent natural antioxidant agents.     

Specific negative effects resulting from elevated levels of the recombinational repair protein Rad54p in Saccharomyces cerevisiae

RAD54 is an important gene in the RAD52 group that controls recombinational repair of DNA damage in Saccharomyces cerevisiae. Rad54p is a DNA‐dependent ATPase and shares seven conserved sequence motifs with proteins of the Swi2p/Snf2p family. Genetic analysis of mutations in motif IA, the putative ATP‐binding fold of Rad54p, demonstrated the functional importance of this motif. Overexpression of these mutant proteins resulted in strong, dominant‐negative effects on cell survival. High levels of full‐length wild‐type Rad54p or specific parts of Rad54p also resulted in negative effects, dependent on the ploidy of the host cell. This differential effect was not under a /α mating‐type control. Deletion of the RAD54 gene led to a small but significant increase in the mutation rate. However, the negative overexpression effects in haploid cells could not be explained by an accumulation of (recessive) lethal mutations. All negative overexpression effects were found to be enhanced under genotoxic stress. We suggest that the negative overexpression effects are the result of unbalanced protein–protein interactions, indicating that Rad54p is involved in multiple interactions, dependent on the physiological situation. Diploid wild‐type cells contained an estimated 7000 Rad54p molecules/cell, whereas haploid cells about 3500/cell. Rad54p levels were highest in actively growing cells compared to stationary phase cells. Rad54 protein levels were found to be elevated after DNA damage. Copyright © 1999 John Wiley & Sons, Ltd.     

The yeast Saccharomyces cerevisiae Pdr16p restricts changes in ergosterol biosynthesis caused by the presence of azole antifungals

Pdr16p belongs to the family of phosphatidylinositol transfer proteins in yeast. The absence of Pdr16p results in enhanced susceptibility to azole antifungals in Saccharomyces cerevisiae. In the major fungal human pathogen Candida albicans, CaPDR16 is a contributing factor to clinical azole resistance. The current study was aimed at better understanding the function of Pdr16p, especially in relation to azole resistance in S. cerevisiae. We show that deletion of the PDR16 gene increased susceptibility of S. cerevisiae to azole antifungals that are used in clinical medicine and agriculture. Significant differences in the inhibition of the sterol biosynthetic pathway were observed between the pdr16Δ strain and its corresponding wild‐type (wt) strain when yeast cells were challenged by sub‐inhibitory concentrations of the azoles miconazole or fluconazole. The increased susceptibility to azoles, and enhanced changes in sterol biosynthesis upon exposure to azoles of the pdr16Δ strain compared to wt strain, are not the results of increased intracellular concentration of azoles in the pdr16Δ cells. We also show that overexpression of PDR17 complemented the azole susceptible phenotype of the pdr16Δ strain and corrected the enhanced sterol alterations in pdr16Δ cells in the presence of azoles. Pdr17p was found previously to be an essential part of a complex required for intermembrane transport of phosphatidylserine at regions of membrane apposition. Based on these observations, we propose a hypothesis that Pdr16p assists in shuttling sterols or their intermediates between membranes or, alternatively, between sterol biosynthetic enzymes or complexes. Copyright © 2013 John Wiley & Sons, Ltd.     

Cell wall structure suitable for surface display of proteins in Saccharomyces cerevisiae

A display system for adding new protein functions to the cell surfaces of microorganisms has been developed, and applications of the system to various fields have been proposed. With the aim of constructing a cell surface environment suitable for protein display in Saccharomyces cerevisiae, the cell surface structures of cell wall mutants were investigated. Four cell wall mutant strains were selected by analyses using a GFP display system via a GPI anchor. β‐Glucosidase and endoglucanase II were displayed on the cell surface in the four mutants, and their activities were evaluated. mnn2 deletion strain exhibited the highest activity for both the enzymes. In particular, endoglucanase II activity using carboxymethylcellulose as a substrate in the mutant strain was 1.9‐fold higher than that of the wild‐type strain. In addition, the activity of endoglucanase II released from the mnn2 deletion strain by Zymolyase 20T treatment was higher than that from the wild‐type strain. The results of green fluorescent protein (GFP) and endoglucanase displays suggest that the amounts of enzyme displayed on the cell surface were increased by the mnn2 deletion. The enzyme activity of the mnn2 deletion strain was compared with that of the wild‐type strain. The relative value (mnn2 deletion mutant/wild‐type strain) of endoglucanase II activity using carboxymethylcellulose as a substrate was higher than that of β‐glucosidase activity using p‐nitrophenyl‐β‐glucopyranoside as a substrate, suggesting that the cell surface environment of the mnn2 deletion strain facilitates the binding of high‐molecular‐weight substrates to the active sites of the displayed enzymes. Copyright © 2014 John Wiley & Sons, Ltd.     

Homolog of a-factor receptor gene in Saccharomyces exiguus

Homologs of Saccharomyces cerevisiae STE3, a -factor receptor gene were detected from S. exiguus NFRI 3539 by low stringency Southern hybridization. This strain might have at least two types of homolog. One of these homologs, designated as e-STE3 was cloned. Its nucleotide sequence revealed 60% identity to STE3. The putative protein coding region consisted of 453 amino acid residues. The amino acid sequence identity between STE3 and e-STE3 was 62%, and that of the N-terminal 303 amino acid residues considered to be the pheromone binding domain was 79%. The e-STE3 sequence submitted to the DDBJ/EMBL/GenBank data libraries is available under Accession Number AB003086. © 1998 John Wiley & Sons, Ltd.     

Jaboticaba berry peel intake prevents insulin‐resistance‐induced tau phosphorylation in mice

The hyperphosphorylation of microtubule‐associated protein tau (tau) in the hippocampus can be caused by central and peripheral insulin resistance and these alterations are related to the development of tauopathies, such as Alzheimer's disease. In this study, we used a high‐fat diet to induce obesity and insulin resistance in adult Swiss mice and checked whether supplementation with Myrciaria jaboticaba berry peel for 10 weeks could improve insulin sensitivity, learning/memory performance, and prevent tau phosphorylation in the hippocampus. Furthermore, adipocytokines, inflammatory markers, and oxidative stress were assessed. Myrciaria jaboticaba peel has phenolic compounds (e.g., cyanidin, ellagic acid), dietary fiber and carotenoids, which contribute to great antioxidant capacity. Supplementation of the high‐fat diet with 4% M. jaboticaba peel prevented fat weight gain and reduced peripheral insulin resistance. The treated group also showed lower tau phosphorylation in the hippocampus corroborating better learning/memory performance in the Morris water maze test. Maintenance of neuronal viability, lower levels of hippocampal inflammatory markers, and improved brain antioxidant defenses were also related to the consumption of M. jaboticaba peel. These findings contribute to a better understanding of how a high‐fat diet supplemented with jaboticaba berry peel counteracts the impairment of cognitive functions caused by high‐fat diet intake and diet‐induced insulin resistance.     

PURIFICATION AND KINETIC CHARACTERIZATION OF POLYPHENOL OXIDASE FROM TOMATO FRUITS (LYCOPERSICON ESCULENTUM CV. MUCHAMIEL)

Two different polyphenol oxidase (PPO) fractions, soluble and particulate, were purified from unripe tomato fruits (Lycopersicon esculentum M. cv. Muchamiel). The PPO present in the soluble fraction was purified fivefold with a 43.5% yield after ammonium sulfate fractionation. PPO in the particulate was purified 4.56‐fold with a 23% yield using the nonionic detergent Triton X‐114. A strong correlation between tomato fruit PPO activity and the physiological disorder blossom‐end rot (BER) was found, with a large increase of the PPO activity in the particulate fraction. Kinetic characterization, including kinetic parameters, pH and temperature profiles, substrate specificity and inhibitors showed similarities in both the soluble and the particulate enzyme(s). However, thermal stability of the particulate enzyme was significantly higher than stability of the soluble PPO, thus indicating possible structural differences. Cupric ions were activators, probably because of their ability to reactivate PPO partly denatured during purification.     

Isolation and characterisation of lactic acid bacteria from jiang-gua (fermented cucumbers), a traditional fermented food in Taiwan

BACKGROUND: Jiang‐gua (fermented cucumbers) is a popular traditional fermented food in Taiwan. The microflora of lactic acid bacteria (LAB) in jiang‐gua have not been investigated in detail. In this study, LAB from jiang‐gua were isolated, characterised and identified. RESULTS: A total of 103 LAB were isolated; 70 cultures were isolated from jiang‐gua samples and 33 cultures were isolated from its raw substrate, cucumber. These isolates were mainly characterised phenotypically and then divided into seven groups (A‐G) by restriction fragment length polymorphism analysis and sequencing of 16S ribosomal DNA. The isolates were identified as Enterococcus casseliflavus, Leuconostoc lactis, Leuconostoc mesenteroides, Lactobacillus pentosus, Lactobacillus plantarum, Lactobacillus paraplantarum, Lactococcus lactis subsp. lactis, Weissella cibaria and Weissella hellenica. The antibacterial activities of the isolates were determined and 11 Lc. lactis subsp. lactis strains showed inhibitory activity against the indicator strain Lactobacillus sakei JCM 1157^T. CONCLUSION: Heterofermentative W. cibaria and Leu. lactis were the major LAB found in jiang‐gua samples without soy sauce. In soy sauce‐added samples, homofermentative L. pentosus and L. plantarum were the most abundant LAB. In addition, the results also suggested that HhaI and RsaI restriction enzymes could be applied to distinguish W. hellenica and Weissella paramesenteroides. Copyright © 2012 Society of Chemical Industry     

Properties and substrate specificities of proteolytic enzymes from the edible basidiomycete Grifola frondosa

Highly active proteolytic enzymes are found in the fruiting bodies of Grifola frondosa. The general properties and substrate specificities of these proteases from G. frondosa (ProGF) were studied. The optimal pH for ProGF activity was pH 3 or 7 using hemoglobin or Hammersten casein as a substrate, respectively. The ProGF exhibited over 70% of maximal activity within the pH range of 4.5–8.5. In terms of temperature, the ProGF were maximally active at 55 °C, while over 80% of maximal activity was observed within the range of 50–75 °C. These proteases were substrate-specific, mainly cleaving at Ala¹⁴-Leu¹⁵, Tyr¹⁶-Leu¹⁷, and Pro²⁸-Lys²⁹ bonds, with occasional cleavage of Phe²⁴-Phe²⁵ bonds in the oxidized insulin B-chain. The ProGF also liberated hydrophobic amino acids, such as valine, leucine, and phenylalanine, using the oxidized insulin B-chain as a substrate. When soy protein was used as a substrate, valine, leucine, phenylalanine, and tyrosine were selectively released from the hydrolysate. Thus, over the time course of incubation, the peptide concentration increased as the average peptide chain length decreased. These results indicate that the ProGF include both endopeptidases recognizing leucine, phenylalanine, and lysine at the P1′ position, and aminopeptidases preferentially releasing hydrophobic and aromatic amino acids such as valine, leucine, phenylalanine, and tyrosine.     

XIV. Yeast sequencing reports. Sequence analysis of a 30 kb DNA segment from yeast chromosome XIV carrying a ribosomal protein gene cluster,the genes encoding a plasma membrane protein and a subunit of replication factor C,and a novel putative serine/threonine protein kinase gene

We have determined the nucleotide sequence of a 30 kb fragment of chromosome XIV of Saccharomyces cerevisiae. The sequence revealed the presence of 19 open reading frames (ORFs) longer than 300 bp. NO422 and NO425 correspond to the split ribosomal protein genes encoding S16A and rp28, respectively, NO450 displays a striking similarity with serine/threonine protein kinase genes, in particular with STE20, and therefore may encode a novel member of this protein family. NO453 is the longest ORF in this DNA segment, having a size of 4908 bp, but its function is not yet known. NO530 encodes the plasma membrane protein Mid1p and NO533 corresponds to the gene coding for a 40 kDa subunit of replication factor C. The remaining ORFs show weak or no homology with proteins in the data bases. The sequence has been submitted to the EMBL data library under Accession Number U23084.     

Increased basal transepidermal water loss leads to elevation of some but not all stratum corneum serine proteases

There are indications of elevation of some inflammatory serine proteases in barrier damaged skin (e.g. plasmin and urokinase). Moreover, many other serine protease activities are present such as desquamatory enzymes as well as a newly detected tryptase‐like serine protease. However, the activities of these proteases have never been correlated with stratum corneum (SC) barrier function. The activity of extractable key serine proteases (SC trypsin‐like kallikreins, SC chymotrypsin‐like kallikreins, SC tryptase‐like serine protease, urokinase and plasmin) was measured from the outermost layers of SC obtained from facial tape strippings in clinically normal subjects. The protein content of the tape strippings was quantified by absorption measurements with the novel infrared densitometer SquameScan^TM 850A and the protease activities by the use of fluorogenic peptide substrates. SC barrier function, SC hydration and skin surface pH were measured using AquaFlux^TM, NOVA dermal phase meter and Skin‐pH‐Meter^®, respectively. As expected, SC hydration was reduced with increased transepidermal water loss (TEWL) values indicative of barrier impairment. Surprisingly, SC chymotrypsin‐like activity showed no correlation with hydration or TEWL, whereas all other enzymes positively correlated with impaired barrier function and some were statistically significant: SC trypsin‐like kallikreins (R² = 0.66, P < 0.01), SC tryptase‐like enzyme (R² = 0.95, P < 0.001), plasmin (R² = 0.86, P < 0.001) and urokinase (R² = 0.50, P < 0.05). All enzymes except urokinase also negatively correlated with SC hydration. Elevated levels of SC serine proteases have been associated with some dermatological disorders, such as atopic dermatitis, psoriasis and rosacea but these results indicate that these enzymes are also elevated with milder forms of barrier disruption, which is not clinically evident as irritated skin. As these proteases are elevated in the SC, they will also be elevated in the epidermis where they can be involved in neurogenic inflammation and epidermal barrier impairment via activation of the protease‐activated receptors. These results highlight the need for using serine protease inhibitors especially for urokinase and plasmin, SC tryptase‐like serine protease and possibly SC trypsin‐like kallikreins even in milder forms of barrier damage.     

XV. Yeast sequencing reports. Isolation and DNA sequence of the STE13 gene encoding dipeptidyl aminopeptidase

We have isolated a mutant which exhibits partial constitutivity for a -specific gene expression in α cells. The wild-type gene was cloned and demonstrated to be allelic to the STE13 gene, which encodes the dipeptidyl aminopeptidase involved in processing of the α-factor prepropheromone. Thus, the mating defect of the ste13 mutations in α cells may result both from the production of incompletely processed α-factor and from the increased expression of a -specific genes. The STE13 open reading frame of 931 amino acids contains a putative membrane-spanning segment near its amino terminus and is 31% identical to a second yeast dipeptidyl aminopeptidase (DAP2). A null mutant of STE13 has been constructed: it is viable and sporulation-proficient. The sequence has been deposited in the GenBank data library under Accession Number L21944.     

Cover Caption

On the cover: Sprouts grown from seeds treated with sodium hypochlorite and calcinated calcium, from “The Effect of Calcinated Calcium and Chlorine Treatments on Escherichia coli O157:H7 87‐23 Population Reduction in Radish Sprouts” by Lilia Fransisca, Bin Zhou, Heekyung Park, and Hao Feng; p M404.     

PURIFICATION AND CHARACTERIZATION OF BACILLUS SUBTILIS JM-3 PROTEASE FROM ANCHOVY SAUCE

Bacillus subtilis JM‐3 was isolated from anchovy sauce naturally fermented in an underground cellar at 15 ± 3C for 3 years. The activity of the B. subtilis protease was highest in the 40–60% ammonium sulfate fraction. The yield of the purified protease was 5.3%, and its purification ratio was 35.6 folds. The molecular weight of the B. subtilis protease was 17.1 kDa, and its K_m and V_maxvalues were 1.75 μg/mL and 318 μM 1/min, respectively. The optimal temperature for protease activity was 60C, but optimal stability temperature was 30C. The optimal pH for protease activity and stability was 5.5. Therefore, the B. subtilis JM‐3 protease was classified as an acid protease. The relative activities of the B. subtilis JM‐3 protease were 69, 21 and 1.3% at 10, 20 and 30% NaCl concentrations, respectively. The best substrate for the B. subtilis JM‐3 protease was benzyloxycarbonyl‐glycine‐p‐nitrophenyl ester followed by bovine serum albumin. p‐Toluene‐sulfonyl‐L‐lysine chloromethylketone was the strongest inhibitor followed by soybean trypsin inhibitor, but N‐ethylmaleimide did not inhibit this enzyme. The B. subtilis JM‐3 protease was therefore presumed to be a trypsin‐like serine protease.     

Metabolism of the Hepatotoxic Compound Sophoraflavanone G in Rat Liver Microsomes

Our study aimed at investigating the metabolic characteristics of sophoraflavanone G (SFG), one of the hepatotoxic constituents of Sophora flavescens, in rat liver microsomes (RLMs). SFG was metabolized to 3 phase I metabolites, di‐hydroxylated SFG (M1), mono‐hydroxylated SFG (M2), dehydrogenated product of mono‐hydroxylated SFG (M3) and 3 SFG glucuronides (M4, M5, and M6) by RLMs. The formation kinetics of M2 conformed to biphasic kinetics in RLMs. The formation kinetics of M4 and M5 best‐fitted the Hill equation kinetics. Chemical inhibition studies found that CYP1A2 and CYP2E1 were the major enzymes responsible for the formation of M2, and the formation of M4 and M5 may be catalyzed by multiple UGT1A isoforms.