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PubMed1999

Protein structure prediction in a 210-type lattice model: parameter optimization in the genetic algorithm using orthogonal array.

We have applied the orthogonal array method to optimize the parameters in the genetic algorithm of the protein folding problem. Our study employed a 210-type lattice model to describe proteins, where the orientation of a residue relative to its neighboring residue is described by two angles. The statistical analysis and graphic representation show that the two angles characterize protein conformations effectively. Our energy function includes a repulsive energy, an energy for the secondary structure preference, and a pairwise contact potential. We used orthogonal array to optimize the parameters of the population, mating factor, mutation factor, and selection factor in the genetic algorithm. By designing an orthogonal set of trials with representative combinations of these parameters, we efficiently determined the optimal set of parameters through a hierarchical search. The optimal parameters were obtained from the protein crambin and applied to the structure prediction of cytochrome B562. The results indicate that the genetic algorithm with the optimal parameters reduces the computing time to reach a converged energy compared to nonoptimal parameters. It also has less chance to be trapped in a local energy minimum, and predicts a protein structure which is closer to the experimental one. Our method may also be applicable to many other optimization problems in computational biology.

Algorithms↗

PubMedDate not supplied

A novel protease from yeast with specificity towards paired basic residues.

Paired basic residues have been observed as sites of proteolytic processing of prohormones in a wide range of eukaryotic species. This strongly suggests that proteases exhibiting specificity towards paired basic residues may be involved in prohormone processing, but candidate enzymes have not so far been identified. Yeast Saccharomyces cerevisiae alpha-cells synthesize and secrete alpha-mating factor, a peptide of 13 amino acids, the processing of which from a larger precursor involves cleavage at paired basic residues (-Lys-Arg-). We have therefore used them as a simple model system for the study of prohormone processing and report here the identification, in cell lysates, of a novel protease which specifically recognizes and cleaves the peptide bonds between consecutive basic residues. The purified enzyme, which we have called propheromone -convertase Y, has a molecular weight (MW) of around 43,000. It cleaves various peptide substrates at paired basic residues, but not at single basic residues, implying it is distinct from trypsin-like proteases. Its unique substrate specificity suggests the enzyme may be involved in propheromone processing in vivo.

Chromatography, High Pressure Liquid↗

PubMed1990

Structural model of ATP-binding proteins associated with cystic fibrosis, multidrug resistance and bacterial transport.

The ATP-binding cassette (ABC) superfamily of transport systems now includes over thirty proteins that share extensive sequence similarity and domain organization. This superfamily includes the well characterized periplasmic binding protein-dependent uptake systems of prokaryotes, bacterial exporters, and eukaryotic proteins including the P-glycoprotein associated with multidrug resistance in tumours (MDR), the STE6 gene product that mediates export of yeast a-factor mating pheromone, pfMDR that is implicated in chloroquine resistance of the malarial parasite, and the product of the cystic fibrosis gene (CFTR). Here we present a tertiary structure model of the ATP-binding cassettes characteristic of this class of transport system, based on similarities between the predicted secondary structures of members of this family and the previously determined structure of adenylate kinase. This model has implications for both the molecular basis of transport and cystic fibrosis and provides a framework for further experimentation.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1996

Production of a recombinant bovine enterokinase catalytic subunit in the methylotrophic yeast Pichia pastoris.

We describe the heterologous expression of a 26.3 kD protein containing the catalytic domain of bovine enterokinase (EKL) in the methylotrophic yeast Pichia pastoris. A highly active protein is secreted and glycosylated, and it has the native amino-terminus of EKL. The cDNA encoding EKL was cloned with the KEX2 protease cleavage site following the alpha mating factor prepro secretion signal from Saccharomyces cerevisiae. The secreted EKL was easily purified from the few native proteins found in the P. pastoris fermentation supernatant, using ion exchange and affinity chromatography. The yield of the purified EKL was 6.3 mg per liter of fermentation culture. This is significantly higher than previous reports of expressions in E. coli and COS cells. The ability of this highly specific protease to cleave immediately after the carboxyl-terminal residue of the (Asp)4-Lys recognition sequence allows regeneration of native amino-terminal residues of recombinant proteins. Its application is demonstrated by the removal of thioredoxin (TrxA), and polyhistidine fusion partners from proteins of interest.

Animals↗

PubMed2003

Expression and characterization of human interferon-beta1 in the methylotrophic yeast Pichia pastoris.

We describe the heterologous expression of a human interferon-beta1 in the methylotrophic yeast Pichia pastoris. Biologically active recombinant human interferon-beta1 (rHuIFN-beta1) was secreted from shake-flask-grown P. pastoris cells into the medium using the Saccharomyces cerevisiae alpha-mating factor prepro-leader sequence at the level of (1-3) x 10(5) i.u. (international units)/ml (6-12 mg/litre). An rHuIFN-beta1 with an N-terminal sequence identical with that of native HuIFN-beta1 was purified and the specific activity was determined (2-3 x 10(7) i.u./mg). It was found that the secreted recombinant protein was partially N-glycosylated.

Amino Acid Sequence↗

PubMed2006

A common genetic system for functional studies of pitrilysin and related M16A proteases.

Pitrilysin is a bacterial protease that is similar to the mammalian insulin-degrading enzyme, which is hypothesized to protect against the onset of Alzheimer's disease, and the yeast enzymes Axl1p and Ste23p, which are responsible for production of the a-factor mating pheromone in Saccharomyces cerevisiae. The lack of a phenotype associated with pitrilysin deficiency has hindered studies of this enzyme. Herein, we report that pitrilysin can be heterologously expressed in yeast such that it functionally substitutes for the shared roles of Axl1p and Ste23p in pheromone production, resulting in a readily observable phenotype. We have exploited this phenotype to conduct structure-function analyses of pitrilysin and report that residues within four sequence motifs that are highly conserved among M16A enzymes are essential for its activity. These motifs include the extended metalloprotease motif, a second motif that has been hypothesized to be important for the function of M16A enzymes, and two others not previously recognized as being important for pitrilysin function. We have also established that the two self-folding domains of pitrilysin are both required for its proteolytic activity. However, pitrilysin does not possess all the enzymatic properties of the yeast enzymes since it cannot substitute for the role of Axl1p in the repression of haploid invasive growth. These observations further support the utility of the yeast system for structure-function and comparative studies of M16A enzymes.

Amino Acid Motifs↗

PubMed1989

Purification and characterization of recombinant human interleukin 4. Biological activities, receptor binding and the generation of monoclonal antibodies.

A synthetic gene coding for human interleukin 4 (IL-4) was cloned and expressed in Saccharomyces cerevisiae (baker's yeast) as a C-terminal fusion protein with the yeast prepro alpha-mating factor sequence, resulting in secretion of mature IL-4 into the culture medium (0.6-0.8 micrograms/ml). A protocol was developed for purification of this protein. Crude cell-free conditioned medium was passed over a concanavalin A-Sepharose affinity column; bound proteins were eluted and further purified by S-Sepharose Fast Flow cation exchange and C18 reverse-phase h.p.l.c. Highly purified IL-4 was obtained by this method (0.3-0.4 mg per litre of culture) with a recovery of 51%. Thermospray liquid chromatography-mass spectrometry showed the C-terminal N-glycosylation site to be largely unmodified, and also showed that the N-terminus of the purified recombinant IL-4 (rIL-4) was authentic. Thiol titration revealed no free cysteine residues, implying that there are three disulphide groups, the positions of which remain to be determined. We have characterized the biological activities of the purified rIL-4. This material is active in B-cell co-stimulator assays, T-cell proliferation assays and in the induction of cell-surface expression of CD23 (the low-affinity receptor for IgE) on tonsillar B-cells. Half-maximal biological activity of the rIL-4 was achieved at a concentration of 120 pM. We have radioiodinated rIL-4 without loss of biological activity and performed equilibrium binding studies on Raji cells, a human B-cell line. The 125I-rIL-4 bound specifically to a single class of binding studies on Raji cells, a human B-cell line. The 125I-rIL-4 bound specifically to a single class of binding site with high affinity (Kd = 100 pM) and revealed 1100 receptors per cell. Receptor-ligand cross-linking studies demonstrated a single cell-surface receptor with an apparent molecular mass of 124 kDa. Two monoclonal antibodies have been raised to the human rIL-4, one of which blocks both the biological activity of rIL-4 and binding to its receptor.

Amino Acid Sequence↗

PubMed1998

Species-specificity in endoplasmic reticulum signal peptide utilization revealed by proteins from Trypanosoma brucei and Leishmania.

N-Terminal signal peptides direct secretory and most membrane proteins into the exocytic pathway at the endoplasmic reticulum. Signal sequences can function across kingdoms. However, our attempts at translocating variant surface glycoprotein (VSG) 117, VSG MVAT7, VSG 221 and BiP from Trypanosoma brucei and gp63 from Leishmania chagasi into canine pancreas microsomes failed. On replacing the signal peptide of VSG 117 with that from yeast prepro-alpha-mating factor (ppalphaMF) the chimaeric protein was imported, indicating that the signal sequence of VSG 117 was incompatible with the protein-import machinery of mammalian microsomes. Replacement of the gp63-h-region with a hybrid composed of the N-terminal nine residues from the h-region of gp67 from Autographa californica nuclear polyhedrosis virus and the C-terminal 10 residues from the h-region of gp63 from L. major produced a functional signal peptide. Thus, the h-region of kinetoplastid signal peptides appears to be the subdomain that is non-functional at the mammalian translocon. The calculated biophysical properties and computed discriminant scores (predictive of importability of signal peptides into mammalian microsomes) of the kinetoplastid signal sequences nevertheless are similar to those of ppalphaMF and Escherichia coli beta-lactamase both of which were imported. These signal peptides are the first collection from one biological family that have been found to fail to function across a species barrier. They indicate that signal peptides are not as universally interchangeable as previously believed. Intriguingly, endoplasmic reticulum signal peptides from Leishmania and Crithidia fasciculata are reminiscent of signal peptides from Gram-positive bacteria.

Amino Acid Sequence↗

PubMed2002

High-level expression of recombinant house dust mite allergen Der p 1 in Pichia pastoris.

BACKGROUND: The major house dust mite Der p 1 allergen is associated with allergic disease. Heterologous over-expression of biologically active Der p 1 was previously attempted but with limited success. OBJECTIVE: The aim of this study was to establish an efficient system for the production of recombinant Der p 1. METHODS: The proform of Der p 1 was expressed in Pichia pastoris as a fusion with the alpha mating factor signal sequence. The recombinant product was purified from culture medium and compared to Der p 1 isolated from mite culture, in terms of enzymatic activity as well as IgE binding capacity. RESULTS: ProDer p 1 was efficiently secreted into culture medium as a hyperglycosylated protein of 40-60 kDa. Postpurification dialysis in acidic buffer was required for the autocatalytic processing of Der p 1. During this treatment, the prosequence was efficiently removed to give highly glycosylated recombinant mature Der p 1. Competition ELISA experiments as well as cysteine proteinase activity assays indicated that recombinant processed Der p 1 was similar to natural Der p 1 isolated from mite cultures in terms of IgE binding and enzymatic activities. However, the histamine releasing activity of recombinant Der p 1 was slightly weaker than that of natural Der p 1. CONCLUSION: This efficient system for recombinant Der p 1 expression leads the way for the design of new diagnostics for house dust mite allergy, epitope mapping, allergen engineering, structural and immunological studies and new immunotherapeutic treatments.

Antigens, Dermatophagoides↗

PubMed2002

Characterization of a fungal protein kinase from Cryphonectria parasitica and its transcriptional upregulation by hypovirus.

The chestnut blight fungus Cryphonectria parasitica and its hypovirus comprise useful model system to study the mechanisms of hypoviral infection. We used degenerate primers based on fungal protein kinases to isolate a gene, cppk1, which encodes a novel Ser/Thr protein kinase of C. parasitica. The gene showed highest homology to ptk1, a Ser/Thr protein kinase from Trichoderma reesei. The encoded protein had a predicted mass of 70.5 kDa and a pI of 7.45. Northern blot analyses revealed that the cppk1 transcript was expressed from the beginning of culture, with a slight increase by 5 days of culture. However, its expression was specifically affected by the presence of virus, and it was transcriptionally upregulated in the fungal strain infected with the hypovirus. A kinase assay using Escherichia coli-derived CpPK1 revealed CpPK1-specific phosphorylated proteins with estimated masses of 50 kDa and 44 kDa. In addition, the phosphorylation of both proteins was higher in a cell-free extract from the hypovirulent strain. The increased expression of cppk1 by the introduction of an additional copy results in a subset of viral symptoms of reduced pigmentation and conidiation in a virus-free isolate. cppk1 overexpression also causes the downregulation of mating factor genes Mf2/1 and Mf2/2, resulting in female sterility. The present study suggests that the hypovirus disturbs fungal signalling by transcriptional upregulation of cppk1, which results in reduced pigmentation and conidiation and female sterility.

Base Sequence↗

PubMed1999

Production and detailed characterization of biologically active olive pollen allergen Ole e 1 secreted by the yeast Pichia pastoris.

The glycoprotein Ole e 1 is a significant aeroallergen from the olive tree (Olea europaea) pollen, with great clinical relevance in the Mediterranean area. To produce a biologically active form of recombinant Ole e 1, heterologous expression in the methylotrophic yeast Pichia pastoris was carried out. A cDNA encoding Ole e 1, fused to a Saccharomyces cerevisiae alpha-mating factor prepropeptide using the pPIC9 vector, was inserted into the yeast genome under the control of the AOX1 promoter. After induction with methanol, the protein secreted into the extracellular medium was purified by ion-exchange and size-exclusion chromatography. The structure of the isolated recombinant Ole e 1 was determined by chemical and spectroscopic techniques, and its immunological properties analysed by blotting and ELISA inhibition with Ole e 1-specific monoclonal antibodies and IgE from sera of allergic patients. The allergen was produced at a yield of 60 mg per litre of culture as a homogeneous glycosylated protein of around 18.5 kDa. Recombinant Ole e 1 appears to be properly folded, as it displays spectroscopic properties (CD and fluorescence) and immunological reactivities (IgG binding to monoclonal antibodies sensitive to denaturation and IgE from sera of allergic patients) indistinguishable from those of the natural protein. This approach gives high-yield production of homogeneous and biologically active allergen, which should be useful for scientific and clinical purposes.

Allergens↗

PubMed1986

Secretion and processing of insulin precursors in yeast.

A series of dibasic insulin precursors including proinsulin was expressed and secreted from Saccharomyces cerevisiae. Recombinant plasmids were constructed to encode fusion proteins consisting of a modified mating factor alpha 1 leader sequence and an insulin precursor. The leader sequence serves to direct the fusion protein into the secretory pathway of the cell and to expose it to the Lys-Arg processing enzyme system. The secreted peptides were purified from the fermentation broth and characterized by sequencing and amino acid analysis. Processing at one or both dibasic sequences was shown in proinsulin and in other insulin precursors containing a short spacer peptide in place of the C peptide. In contrast, no processing was observed in the absence of a spacer peptide in the insulin precursor molecule, e.g., B-Lys-Arg-A (where A and B are the A and B chain of human proinsulin, respectively). This type of single-chain insulin precursors isolated from such constructions could be enzymatically converted into insulin by treatment with trypsin and carboxypeptidase B. The above results suggest that the C-peptide region of proinsulin serves to direct the trypsin-like converting enzyme to process at the two dibasic sequences. We propose that in hormone precursors in general the spacer peptides serve to expose dibasic sequences for processing.

Amino Acid Sequence↗

PubMed1990

G protein gamma subunits contain a 20-carbon isoprenoid.

A small subset of cellular proteins are covalently modified by the addition of isoprenoid groups. These include p21ras, fungal mating factors, and nuclear lamins, which are isoprenylated at carboxyl-terminal cysteine residues with a 15-carbon farnesyl group. The similarity of the carboxyl-terminal sequences of these proteins with the alpha and gamma subunits of signal-transducing guanine nucleotide-binding regulatory proteins (G proteins) prompted examination of isoprenylation of G protein subunits. PC-12 cells were incubated with the isoprenoid precursor [3H]mevalonolactone. The beta and gamma subunits were isolated by specific association with an affinity column of immobilized alpha subunits. The gamma subunits were radiolabeled, and the tritiated lipid released from them by treatment with methyl iodide comigrated chromatographically with the 20-carbon isoprenoid geranylgeraniol. Label was not detected in G protein alpha or beta subunits. Isoprenylation of gamma subunits by the geranylgeranyl group is presumed to contribute to the association of G proteins with membranes.

Adrenal Gland Neoplasms↗

PubMed1990

The gamma subunit of transducin is farnesylated.

Protein prenylation with farnesyl or geranylgeranyl moieties is an important posttranslational modification that affects the activity of such diverse proteins as the nuclear lamins, the yeast mating factor mata, and the ras oncogene products. In this article, we show that whole retinal cultures incorporate radioactive mevalonic acid into proteins of 23-26 kDa and one of 8 kDa. The former proteins are probably the "small" guanine nucleotide-binding regulatory proteins (G proteins) and the 8-kDa protein is the gamma subunit of the well-studied retinal heterotrimeric G protein (transducin). After deprenylating purified transducin and its subunits with Raney nickel or methyl iodide/base, the adducted prenyl group can be identified as an all-trans-farnesyl moiety covalently linked to a cysteine residue. Thus far, prenylation reactions have been found to occur at cysteine in a carboxyl-terminal consensus CAAX sequence, where C is the cysteine, A is an aliphatic amino acid, and X is undefined. Both the alpha and gamma subunits of transducin have this consensus sequence, but only the gamma subunit is prenylated. Therefore, the CAAX motif is not necessary and sufficient to direct prenylation. Finally, since transducin is the best understood G protein, both structurally and mechanistically, the discovery that it is farnesylated should allow for a quantitative understanding of this post-translational modification.

Animals↗

PubMed1993

Genetic evidence for the roles of the bud-site-selection genes BUD5 and BUD2 in control of the Rsr1p (Bud1p) GTPase in yeast.

Yeast cells normally display either an axial (for MATa or MAT alpha cells) or bipolar (for MATa/alpha cells) pattern of bud-site selection. The RSR1 gene, which was previously identified as a multicopy suppressor of Ts- mutations in the bud-emergence gene CDC24, encodes a GTPase of the Ras family that is required for both budding patterns. Mutations in Rsr1p that presumably block its ability to bind or hydrolyze GTP cause a randomized budding phenotype, suggesting that regulators of Rsr1p will prove to be required for proper bud positioning. The BUD5 gene product is required for proper bud-site selection and contains similarity to GDP-dissociation stimulators (GDS) for Ras-type proteins, suggesting that Bud5p may be a GDS for Rsr1p. Here I report that BUD5 is required for wild-type RSR1, but not for mutationally activated rsr1val12, to serve as a multicopy suppressor of cdc24, indicating that Bud5p functions as a GDS for Rsr1p in vivo. To identify the GAP (GTPase-activating protein) for Rsr1p, a genetic selection was designed based on the observation that mutationally activated rsr1val12, but not wild-type RSR1, can serve as a multicopy suppressor of yeast RAS2(Ts) mutants. Mutants were selected that allowed wild-type RSR1 to act as a multicopy suppressor of RAS2(Ts). Two such mutations proved to be in the BUD2 gene, suggesting that Bud2p functions as a GAP for Rsr1p in vivo.

Cell Cycle Proteins↗

PubMed1994

Reconstitution of a yeast protein kinase cascade in vitro: activation of the yeast MEK homologue STE7 by STE11.

The mating-factor response pathway of Saccharomyces cerevisiae employs a set of protein kinase similar to kinases that function in signal transduction pathways of metazoans. We have purified the yeast protein kinases encoded by STE11, STE7, and FUS3 as fusions to glutathione S-transferase (GST) and reconstituted a kinase cascade in which STE11 phosphorylates and activates STE7, which in turn phosphorylates the mitogen-activated protein kinase FUS3. GST-STE11 is active even when purified from cells that have not been treated with alpha-factor. This observation raises the possibility that STE11 activity is governed by an inhibitor which is regulated by pheromone. We also identify a STE11-dependent phosphorylation site in STE7 which is required for activity of STE7. Conservation of this site in the mammalian STE7 homologue MEK and other STE7 relatives suggests that this may be a regulatory phosphorylation site in all MAP kinase kinases.

Amino Acid Sequence↗

PubMed1997

Rer1p as common machinery for the endoplasmic reticulum localization of membrane proteins.

Rer1p, a Golgi membrane protein, is required for the correct localization of an endoplasmic reticulum (ER) membrane protein, Sec12p, by a retrieval mechanism from the cis-Golgi to the ER. To test whether or not the role of Rer1p is common to multiple ER membrane proteins, we examined the localization of two other ER membrane proteins, Sec71p and Sec63p, in the wild-type and rer1 mutant yeast cells, using their fusions with an alpha-mating factor precursor (Mfalpha1p). Although Sec71p and Sec63p have completely different topology from Sec12p, their Mfalpha1p fusion proteins were also mislocalized to the trans-Golgi in the rer1 mutant. Overexpression of these fusions caused their mislocalization to the trans-Golgi even in the wild-type cells, and this mislocalization was partially suppressed by the co-overexpression of Rer1p. Either Sec71p or an artificial chimeric protein whose ER localization depends on Rer1p gave a competitive effect on the localization of the Mfalpha1-Sec71p fusion, which was abolished in rer1. Thus, Rer1p appears to be one of the common limiting components in the retrieval machinery for ER membrane proteins. The results also suggest that Sec71p and Sec63p depend on ER-Golgi recycling, at least partly, for ER localization. We also examined the effect of a mutation in alpha-COP, a subunit of yeast coatomer, on the localization of these ER membrane proteins. The Mfalpha1p fusions of Sec12p, Sec71p, and Sec63p were all more or less mislocalized in ret1-1. These observations imply that the roles of Rer1p and coatomer are much more general than thought before.

Endoplasmic Reticulum↗

PubMed1995

Secretion of biologically active recombinant fibrinogen by yeast.

Fibrinogen (340 kDa) is a plasma protein that plays an important role in the final stages of blood clotting. Human fibrinogen is a dimer with each half-molecule composed of three different polypeptides (A alpha, 67 kDa; B beta, 57 kDa; gamma, 47 kDa). To understand the mechanism of fibrinogen chain assembly and secretion and to obtain a system capable of producing substantial amounts of fibrinogen for structure-function studies, we developed a recombinant system capable of secreting fibrinogen. An expression vector (pYES2) was constructed with individual fibrinogen chain cDNAs under the control of a Gal-1 promoter fused with mating factor F alpha 1 prepro secretion signal (SS) cascade. In addition, other constructs were prepared with combinations of cDNAs encoding two chains or all three chains in tandem. Each chain was under the control of the Gal-1 promoter. These constructs were used to transform Saccharomyces cerevisiae (INVSC1; Mat alpha his3-delta 1 leu2 trp1-289 ura3-52) in selective media. Single colonies from transformed yeast cells were grown in synthetic media with 4% raffinose to a density of 1 x 10(8) cells/ml and induced with 2% galactose for 16 h. Yeast cells expressing all three chains contained fibrinogen precursors and nascent fibrinogen and secreted about 30 micrograms/ml of fibrinogen into the culture medium. The B beta and gamma chains, but not A alpha, were glycosylated. Glycosylation of B beta and gamma chains was inhibited by treatment of transformed yeast cells with tunicamycin. Intracellular B beta and gamma chains, but not the A alpha chains in secreted fibrinogen, were cleaved by endoglycosidase H. Carbohydrate analysis indicated that secreted recombinant fibrinogen contained N-linked asialo-galactosylated biantennary oligosaccharide. Recombinant fibrinogen yielded the characteristic plasmin digestion products, fragments D and E, that were immunologically indistinct from the same fragments obtained from plasma fibrinogen. The recombinant fibrinogen was shown to be biologically active in that it could form a thrombin-induced clot, which, in the presence of factor XIIIa, could undergo gamma chain dimerization and A alpha chain polymer formation.

Animals↗