Biomedical subjects

Jeffrey M Becker

Publications and source records attributed to Jeffrey M Becker.

35 records · Page 2Linked to original sources

PubMed2004

Wangiella (Exophiala) dermatitidis WdChs5p, a class V chitin synthase, is essential for sustained cell growth at temperature of infection.

The chitin synthase structural gene WdCHS5 was isolated from the black fungal pathogen of humans Wangiella dermatitidis. Sequence analysis revealed that the gene has a myosin motor-like-encoding region at its 5' end and a chitin synthase (class V)-encoding region at its 3' end. Northern blotting showed that WdCHS5 is expressed at high levels under conditions of stress. Analysis of the 5' upstream region of WdCHS5 fused to a reporter gene indicated that one or more of the potential regulatory elements present may have contributed to the high expression levels. Disruption of WdCHS5 produced mutants that grow normally at 25 degrees C but have severe growth and cellular abnormalities at 37 degrees C. Osmotic stabilizers, such as sorbitol and sucrose, rescued the wild-type phenotype, which indicated that the loss of WdChs5p causes cell wall integrity defects. Animal survival tests with a mouse model of acute infection showed that all wdchs5Delta mutants are less virulent than the parental strain. Reintroduction of the WdCHS5 gene into the wdchs5Delta mutants abolished the temperature-sensitive phenotype and reestablished their virulence. We conclude that the product of WdCHS5 is required for the sustained growth of W. dermatitidis at 37 degrees C and is of critical importance to its virulence.

Alkaline Phosphatase↗

PubMed2003

Synthesis and biophysical characterization of a multidomain peptide from a Saccharomyces cerevisiae G protein-coupled receptor.

We attached peptides corresponding to the seventh transmembrane domain (TMD7) of the alpha-mating factor receptor (Ste2p) of Saccharomyces cerevisiae to a hydrophilic, 40-residue fragment of the carboxyl terminus of this G protein-coupled receptor. Peptides corresponding to (a) the 40-residue portion of the carboxyl tail (T-40), (b) the tail plus a part of TMD7 (M7-12-T40), and (c) to the tail plus the full TMD7 (M7-24-T40) were chemically synthesized and purified. The molecular mass and primary sequence of these peptides were confirmed by mass spectrometry and tandem mass spectrometry procedures. Circular dichroism (CD) revealed that T-40 was disordered in phosphate buffer and in the presence of 1,2-dimyristoyl-sn-glycero-3-phosphocholine/1,2-dimyristoyl-sn-glycero-3-[phospho-racemic-(1-glycerol)] bilayers. In contrast, M7-12-T40 and M7-24-T40 peptides were partially helical in the presence of vesicles, and difference CD spectroscopy showed that the transmembrane regions of these peptides were 42 and 94% helical, respectively. CD analysis also demonstrated that M7-24-T40 retained its secondary structure in the presence of 1-palmitoyl-2-hydroxy-sn-glycero-3-[phospho-racemic-(1-glycerol)] micelles at 0.5 mm concentration. Thus, the tail and the transmembrane domain of the multidomain 64-amino acid residue peptide manifest individual conformational preferences. Measurement of tryptophan fluorescence indicated that the transmembrane domain integrated into bilayers in a manner similar to that expected for this region in the native state of the receptor. This study demonstrated that the tail of Ste2p can be used as a hydrophilic template to study transmembrane domain structure using techniques such as CD and NMR spectroscopy.

Amino Acid Sequence↗

PubMed2003

Sequences in the intracellular loops of the yeast pheromone receptor Ste2p required for G protein activation.

The alpha-factor receptor of the yeast Saccharomyces cerevisiae encoded by the STE2 gene is a member of the large family of G protein-coupled receptors (GPCRs) that mediate multiple signal transduction pathways. The third intracellular loop of GPCRs has been identified as a likely site of interaction with G proteins. To determine the extent of allowed substitutions within this loop, we subjected a stretch of 21 amino acids (Leu228-Leu248) to intensive random mutagenesis and screened multiply substituted alleles for receptor function. The 91 partially functional mutant alleles that were recovered contained 96 unique amino acid substitutions. Every position in this region can be replaced with at least two other types of amino acids without a significant effect on function. The tolerance for nonconservative substitutions indicates that activation of the G protein by ligand-bound receptors involves multiple intramolecular interactions that do not strongly depend on particular sequence elements. Many of the functional mutant alleles exhibit greater than normal levels of signaling, consistent with an inhibitory role for the third intracellular loop. Removal of increasing numbers of positively charged residues from the loop by site-directed mutagenesis causes a progressive loss of signaling function, indicating that the overall net charge of the loop is important for receptor function. Introduction of negatively charged residues also leads to a reduced level of signaling. The defects in signaling caused by substitution of charged amino acids are not caused by changes in the abundance of receptors at the cell surface.

Amino Acid Sequence↗

PubMed2003

Protein surface mapping by chemical oxidation: structural analysis by mass spectrometry.

The solvent-accessible surface area of proteins is important in biological function for many reasons, including protein-protein interactions, protein folding, and catalytic sites. Here we present a chemical technique to oxidize amino acid side chains in a model protein, apomyoglobin, and subsequent elucidation of the effect of solvent accessibility on the sites of oxidation. Under conditions of low protein oxidation (zero to three oxygen atoms added per apomyoglobin molecule), we have positively identified five oxidation sites by liquid chromatography-tandem mass spectrometry and high-resolution Fourier transform mass spectrometry. Our results indicate that all oxidized amino acids, with the exception of methionine, have highly solvent-accessible side chains, but the rate of oxidation may not be dictated solely by solvent accessibility and amino acid identity.

Amino Acid Sequence↗

PubMed2003

Biosynthesis and biophysical analysis of domains of a yeast G protein-coupled receptor.

The alpha-factor receptor(Ste2p) is required for the sexual conjugation of the yeast Saccharomyces cerevisiae. Ste2p belongs to the G protein-coupled receptor (GPCR) family sharing a common heptahelical transmembrane structure. Biological synthesis of regions of Ste2p fused to a leader protein in Escherichia coli yielded milligram quantities of polypeptides that corresponded to one or two transmembrane domains. Fusion proteins were characterized by polyacrylamide gel electrophoresis, high performance liquid chromatography, and mass spectrometry. CD studies on the fusion proteins in trifluoroethanol:water mixtures indicated the existence of alpha-helical structures in the single- and the double-transmembrane domains. NMR experiments were performed in CDCl(3):CD(3)OH:H(2)O (4:4:1) on the (15)N-labeled single-transmembrane peptide showing a clear dispersion of the nitrogen-amide proton correlation cross peaks indicative of a high-purity, uniformly labeled molecule. The results indicate that single- and double-transmembrane domains of a GPCR can be produced by biosynthetic methods in quantities and purity sufficient for biophysical studies.

Amino Acid Sequence↗

PubMed2003

Candida albicans septin mutants are defective for invasive growth and virulence.

Hyphal growth of Candida albicans is implicated as an important virulence factor for this opportunistic human pathogen. Septin proteins, a family of cytoskeletal elements that regulate membrane events and are important for proper morphogenesis of C. albicans, were examined for their role in tissue invasion and virulence in the mouse model of systemic infection. In vitro, septin mutants are only mildly defective for hyphal growth in liquid culture but display pronounced defects for invasive growth into agar. In vivo, the septin mutants were found to exhibit attenuated virulence. However, mice infected with the mutants displayed high fungal burdens in their kidneys without obvious symptoms of disease. Histological examination of infected kidneys revealed defects in organ invasion for the cdc10 Delta and cdc11 Delta deletion mutants, which displayed both reduced tissue penetration and noninvasive fungal masses. Thus, the septin proteins are necessary for invasive growth, which appears to be more important to the successful pathogenesis of C. albicans than hyphal growth alone.

Agar↗

PubMed2002

Tyr266 in the sixth transmembrane domain of the yeast alpha-factor receptor plays key roles in receptor activation and ligand specificity.

To identify interactions between Ste2p, a G protein-coupled receptor of the yeast Saccharomyces cerevisiae, and its tridecapeptide ligand, alpha-factor (WHWLQLKPGQPMY), a variety of alpha-factor analogues were used in conjunction with site-directed mutagenesis of a targeted portion of Ste2p transmembrane domain six. Alanine substitution of residues in the 262-270 region of Ste2p did not affect pheromone binding or signal transduction, except for the Y266A mutant, which did not transduce signal yet exhibited only a small decrease in alpha-factor binding affinity. Substitutions with Ser, Leu, or Lys at Y266 also generated signaling-defective receptors. In contrast, Phe or Trp substitution at Y266 retained receptor function, suggesting that aromaticity at this position was critical. When coexpressed with WT receptor, the Y266A receptor exhibited a strong dominant-negative phenotype, indicating that this mutant bound G protein. A partial tryptic digest revealed that, in the presence of agonist, a different digestion profile for Y266A receptor was generated in comparison to that for WT receptor. The difference in trypsin-sensitive sites and their negative dominance indicated that the Y266A receptor was not able to switch into an "activated" conformation upon ligand binding. In comparison to WT Ste2p, the mutantY266A receptor showed increased binding affinity for N-terminal, alanine-substituted alpha-factor analogues (residues 1-4) and the antagonist [desW(1),desH(2)]alpha-factor. A substantial decrease in affinity was observed for alpha-factor analogues with Ala substitutions from residues 5-13. The results suggest that Y266 is part of the binding pocket that recognizes the N-terminal portion of alpha-factor and is involved in the transformation of Ste2p into an activated state upon agonist binding.

Binding Sites↗

PubMed2002

High resolution NMR analysis of the seven transmembrane domains of a heptahelical receptor in organic-aqueous medium.

The NMR properties of seven peptides representing the transmembrane domains of the alpha-factor receptor from Saccharomyces cerevisiae were examined in trifluoroethanol/water (4:1) at 10 to 55 degrees C. The parameters extracted indicated all peptides were helical in this membrane mimetic solvent. Using chemical shift indices as the criterion, helicity varied from 64 to 83%. The helical residues in the peptides corresponded to the region predicted to cross the hydrocarbon interior of the bilayer. A study of a truncated 25-residue peptide corresponding to domain 2 gave evidence that the helix extended all the way to the N-terminus of this peptide, indicating that sequence and not chain end effects are very important in helix termination for our model peptides. Both nuclear Overhauser effect spectroscopy (NOESY) connectivities and chemical shift indices revealed significant perturbations around prolyl residues in the helices formed by transmembrane domains 6 and 7. Molecular models of the transmembrane domains indicate that helices for domains 6 and 7 are severely kinked at these prolyl residues. The helix perturbation around proline 258 in transmembrane domain 6 correlates with mutations that cause phenotypic changes in this receptor.

Cell Membrane↗

PubMed2002

Residues in the first extracellular loop of a G protein-coupled receptor play a role in signal transduction.

The Saccharomyces cerevisiae pheromone, alpha-factor (WHWLQLKPGQPMY), and Ste2p, its G protein-coupled receptor, were used as a model system to study ligand-receptor interaction. Cys-scanning mutagenesis on each residue of EL1, the first extracellular loop of Ste2p, was used to generate a library of 36 mutants with a single Cys residue substitution. Mutation of most residues of EL1 had only negligible effects on ligand affinity and biological activity of the mutant receptors. However, five mutants were identified that were either partially (L102C and T114C) or severely (N105C, S108C, and Y111C) compromised in signaling but retained binding affinities similar to those of wild-type receptor. Three-dimensional modeling, secondary structure predictions, and subsequent circular dichroism studies on a synthetic peptide with amino acid sequence corresponding to EL1 suggested the presence of a helix corresponding to EL1 residues 106 to 114 followed by two short beta-strands (residues 126 to 135). The distinctive periodicity of the five residues with a signal-deficient phenotype combined with biophysical studies suggested a functional involvement in receptor activation of a face on a 3(10) helix in this region of EL1. These studies indicate that EL1 plays an important role in the conformational switch that activates the Ste2p receptor to initiate the mating pheromone signal transduction pathway.

Amino Acid Sequence↗

PubMed2002

Identification of a contact region between the tridecapeptide alpha-factor mating pheromone of Saccharomyces cerevisiae and its G protein-coupled receptor by photoaffinity labeling.

Saccharomyces cerevisiae haploid cells communicate with their opposite mating type through peptide pheromones (alpha-factor and a-factor) that activate G protein-coupled receptors (GPCRs). S. cerevisiaewas used as a model system for the study of peptide-responsive GPCRs. Here, we detail the synthesis and characterization of a number of alpha-factor (Trp-His-Trp-Leu-Gln-Leu-Lys-Pro-Gly-Gln-Pro-Met-Tyr) pheromone analogues containing the photo-cross-linkable group 4-benzoyl-L-phenylalanine (Bpa). Following characterization, one analogue, [Bpa(1), Tyr(3), Arg(7), Phe(13)]alpha-factor, was radioiodinated and used as a probe for Ste2p, the GPCR for alpha-factor. Binding of the di-iodinated probe was saturable (K(d) = 200 nM) and competable by alpha-factor. Cross-linking into Ste2p was specific for this receptor and reversed by the wild-type pheromone. Chemical and enzymatic cleavage of the receptor/radioprobe complex indicated that cross-linking occurred on a portion of Ste2p spanning residues 251-294 which encompasses transmembrane domain 6, the extracellular loop between transmembrane domains 6 and 7, and transmembrane domain 7. This fragment was verified using T7-epitope-tagged Ste2p and a biotinylated, photoactivatable alpha-factor. After cross-linking with the biotinylated photoprobe and trypsin cleavage, the cross-linked receptor fragment was revealed by both an anti T7-epitope antibody and a biotin probe. This is the first determination of a specific contact region between a Class IV GPCR and its ligand. The results demonstrate that Bpa alpha-factor probes are useful in determining contacts between alpha-factor and Ste2p and initiate mapping of the ligand binding site of this GPCR.

Affinity Labels↗

PubMed2002

The chain length dependence of helix formation of the second transmembrane domain of a G protein-coupled receptor of Saccharomyces cerevisiae.

The chain length dependence of helix formation of transmembrane peptides in lipids was investigated using fragments corresponding to the second transmembrane domain of the alpha-factor receptor from Saccharomyces cerevisiae. Seven peptides with chain lengths of 10 (M2-10; FKYLLSNYSS), 14 (M2-14), 18 (M2-18), 22 (M2-22), 26 (M2-26), 30 (M2-30) and 35 (M2-35; RSRKTPIFIINQVSLFLIILHSALYFKYLLSNYSS) residues, respectively, were synthesized. CD spectra revealed that M2-10 was disordered, and all of the other peptides assumed partially alpha-helical secondary structures in 99% trifluoroethanol (TFE)/H(2)O. In 50% TFE/H(2)O, M2-30 assumed a beta-like structure. The other six peptides exhibited the same CD patterns as those found in 99% TFE/H(2)O. In 1,2-dimyristoyl-sn-glycero-3-phosphocholine/1,2-dimyristoyl-sn-glycero-3-phospho-rac-(1-glycerol) (4:1 ratio) vesicles, M2-22, M2-26, and M2-35 formed alpha-helical structures, whereas the other peptides formed beta-like structures. Fourier transform infrared spectroscopy in 1,2-dimyristoyl-sn-glycero-3-phosphocholine/1,2-dimyristoyl-sn-glycero-3-phospho-rac-(1-glycerol) (4:1) multilayers showed that M2-10, M2-14, M2-18, and M2-30 assumed beta-structures in this environment. Another homologous 30-residue peptide (M2-30B), missing residues SNYSS from the N terminus and extending to RSRKT on the C terminus, was helical in lipid bilayers, suggesting that residues at the termini of transmembrane domains influence their biophysical properties. Attenuated total reflection Fourier transform infrared spectroscopy revealed that M2-22, M2-26, M2-30B, and M2-35 were alpha-helical and oriented at angles of 12 degrees, 13 degrees, 36 degrees, and 34 degrees, respectively, with respect to the multilayer normal. This study showed that chain length must be taken into consideration when using peptides representing single transmembrane domains as surrogates for regions of an intact receptor. Furthermore, this work indicates that the tilt angle and conformation of transmembrane portions of G protein-coupled receptors may be estimated by detailed spectroscopic measurements of single transmembrane peptides.

Amino Acid Sequence↗

PubMed2002

Peptide transport in plants.

Recent completion of the Arabidopsis genome revealed that this organism has ten times more peptide transporters than any other sequenced organism (prokaryote or eukaryote). These transporters are found in three protein families: the ABC-type transporters; the di- and tripeptide transporters; and the newly described tetra- and pentapeptide oligopetide transporters. The abundance of these transporters suggests that they play diverse and important roles in plant growth and development. Possible substrates for these transporters include glutathione, gamma-glutamyl peptides, hormone-amino acid conjugates, phytosulfokine, peptide-like compounds and peptide phytotoxins. However, the exact role of peptide transport in plants is still undefined.

ATP-Binding Cassette Transporters↗

PubMed2002

An oligopeptide transporter gene family in Arabidopsis.

We have identified nine oligopeptide transporter (OPT) orthologs (AtOPT1 to AtOPT9) in Arabidopsis. These proteins show significant sequence similarity to OPTs of Candida albicans (CaOpt1p), Schizosaccharomyces pombe (Isp4p), and Saccharomyces cerevisiae (Opt1p and Opt2p). Hydrophilicity plots of the OPTs suggest that they are integral membrane proteins with 12 to 14 transmembrane domains. Sequence comparisons showed that the AtOPTs form a distinct subfamily when compared with the fungal OPTs. Two highly conserved motifs (NPG and KIPPR) were found among all OPT members. The identification of multiple OPTs in Arabidopsis suggests that they may play different functional roles. This idea is supported by the fact that AtOPTs have a distinct, tissue-specific expression pattern. The cDNAs encoding seven of the AtOPTs were cloned into a yeast vector under the control of a constitutive promoter. AtOPT4 expressed in S. cerevisiae mediated the uptake of KLG-[3H]L. Similarly, expression of five of the seven AtOPT proteins expressed in yeast conferred the ability to uptake tetra- and pentapeptides as measured by growth. This study provides new evidence for multiple peptide transporter systems in Arabidopsis, suggesting an important physiological role for small peptides in plants.

Amino Acid Sequence↗

PubMed1997

The topoisomerase I gene from Candida albicans.

We report here the cloning of the Candida albicans genomic topoisomerase I gene (TOP1) by use of PCR and subsequent hybridization. The predicted protein sequence shared 58.8% identity with the Saccharomyces cerevisiae topoisomerase I and 30-50% identity with other eukaryotic topoisomerase I proteins. A conditional gene disruption strain (CWJ477) was constructed so that one copy of TOP1 was deleted and the other copy of TOP1 was placed under a regulatable promoter. Under repressed conditions, cells grew slowly and cell morphology was abnormal. The virulence of CWJ477 was markedly reduced in a mouse model system, and that of the single gene knockout-strain was slightly attenuated, indicating that TOP1 might play a role in the infection of C. albicans in mice in a dose-dependent manner. Despite the reduced virulence of both the single and double knockout strains, viable cells of the pathogen were recovered from the kidneys as late as 22 d post-infection.

Amino Acid Sequence↗

PubMed1997

An oligopeptide transport gene from Candida albicans.

A Candida albicans oligopeptide transport gene, OPT1, was cloned from a C. albicans genomic library through heterologous expression in the Saccharomyces cerevisiae di-/tripeptide transport mutant PB1X-9B. When transformed with a plasmid harbouring OPT1, S. cerevisiae PB1X-9B, which did not express tetra-/pentapeptide transport activity under the conditions used, was conferred with an oligopeptide transport phenotype, as indicated by growth on the tetrapeptide Lys-Leu-Leu-Gly, sensitivity to toxic tetra- and pentapeptides, and an increase in the initial uptake rate of the radiolabelled tetrapeptide Lys-Leu-Gly-[3H]Leu. The level of oligopeptide transport was found to be influenced in the heterologous host by the source of nitrogen used for growth. The entire 3.8 kb fragment containing the oligopeptide transport activity was sequenced and an ORF of 2349 nucleotides containing a 58 nucleotide intron was identified. The deduced protein product of 783 amino acid residues contained 12 hydrophobic regions suggestive of a membrane transport protein. Sequence comparisons revealed that similar proteins are encoded by genes from S. cerevisiae and Schizosaccharomyces pombe and that OPT1 is not a member of the ABC or PTR membrane transport families.

Amino Acid Sequence↗

PubMed1995

Cloning of a Candida albicans peptide transport gene.

A Candida albicans peptide transport gene, CaPTR2, was cloned from a C. albicans genomic library by functional complementation of a peptide transport deficient mutant (strain ptr2-2) of Saccharomyces cerevisiae. CaPTR2 restored peptide transport to transformants as determined by uptake of radiolabelled dileucine, growth on dipeptides as sources of required amino acids, and restoration of growth inhibition by toxic peptides. Plasmid curing experiments demonstrated that the peptide transport phenotype was plasmid borne. CaPTR2 was localized to chromosome R of C. albicans by contour-clamped homologous electric field gel chromosome blots. Deletion subclones and frameshift mutagenesis were used to narrow the peptide transport complementing region to a 5.1 kb DNA fragment. DNA sequencing of the complementing region identified an ORF of 1869 bp containing an 84 nucleotide intron. The deduced amino acid sequence predicts a protein of 70 kDa consisting of 623 amino acids with 12 hydrophobic segments. A high level of identity was found between the predicted protein and peptide transport proteins of S. cerevisiae and Arabidopsis thaliana. This study represents the first steps in the genetic characterization of peptide transport in C. albicans and initiates a molecular approach for the study of drug delivery against this pathogen.

Amino Acid Sequence↗

PubMedDate not supplied

Substrate preference is altered by mutations in the fifth transmembrane domain of Ptr2p, the di/tri-peptide transporter of Saccharomyces cerevisiae.

The integral membrane protein Ptr2p transports di/tri-peptides into the yeast Saccharomyces cerevisiae. The sequence FYXXINXG (FYING motif) in the 5th transmembrane domain (TM5) is invariably conserved among the members of the PTR (Peptide TRansport) family ranging from yeast to human. To test the role of TM5 in Ptr2p function, Ala-scanning mutagenesis of the 22 residues comprising TM5 was completed. All mutated transporters, with the exception of the Y248A mutant, were expressed as determined by immunoblots. In peptide-dependent growth assays, ten mutants of the non-FYING residues grew as well as wild-type Ptr2p on all twelve different peptides tested. All of the FYING motif mutants, except the non-expressed Y248A, plus seven other mutants in TM5 exhibited differential growth on peptides including Leu-Leu and Met-Met-Met indicating that these mutations conferred substrate preference. In assays measuring direct uptake of the radioactive peptides (3)H-Leu-Leu or (14)C-Met-Met-Met, the F, I and G mutants of the FYING motif did not demonstrate accumulation of these peptides over a ten minute interval. The mutation N252A of the FYING motif, along with L240A, M250A, and L258A, exhibited differential substrate preference for Met-Met-Met over Leu-Leu. Other mutations (T239A, Q241A, N242A, M245A, and A260) resulted in preference for Leu-Leu over Met-Met-Met. These data demonstrate that TM5, in particular its conserved FYING motif, is involved in substrate preference of Ptr2p.

Alanine↗