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David M Bedwell

Publications and source records attributed to David M Bedwell.

16 recordsLinked to original sources
PubMed2006

Clinical doses of amikacin provide more effective suppression of the human CFTR-G542X stop mutation than gentamicin in a transgenic CF mouse model.

Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene cause the disease cystic fibrosis. We previously reported that gentamicin administration suppressed a CFTR premature stop mutation in a Cftr-/- mouse model carrying a human CFTR-G542X (hCFTR-G542X) transgene, resulting in the appearance of hCFTR protein and function. However, the high doses used in that study resulted in peak serum levels well beyond the levels typically administered to humans. To address this problem, we identified doses of both gentamicin and amikacin that resulted in peak serum levels within their accepted clinical ranges. We then asked whether these doses could suppress the hCFTR-G542X mutation in the Cftr-/- hCFTR-G542X mouse model. Our results indicate that low doses of each compound restored some hCFTR protein expression and function, as shown by immunofluorescence and short-circuit current measurements. However, we found that amikacin suppressed the hCFTR-G542X premature stop mutation more effectively than gentamicin when administered at these clinically relevant doses. Because amikacin is also less toxic than gentamicin, it may represent a superior choice for suppression therapy in patients that carry a premature stop mutation in the CFTR gene.

Amikacin↗
PubMed2006

Eukaryotic release factor 1 phosphorylation by CK2 protein kinase is dynamic but has little effect on the efficiency of translation termination in Saccharomyces cerevisiae.

Protein synthesis requires a large commitment of cellular resources and is highly regulated. Previous studies have shown that a number of factors that mediate the initiation and elongation steps of translation are regulated by phosphorylation. In this report, we show that a factor involved in the termination step of protein synthesis is also subject to phosphorylation. Our results indicate that eukaryotic release factor 1 (eRF1) is phosphorylated in vivo at serine 421 and serine 432 by the CK2 protein kinase (previously casein kinase II) in the budding yeast Saccharomyces cerevisiae. Phosphorylation of eRF1 has little effect on the efficiency of stop codon recognition or nonsense-mediated mRNA decay. Also, phosphorylation is not required for eRF1 binding to the other translation termination factor, eRF3. In addition, we provide evidence that the putative phosphatase Sal6p does not dephosphorylate eRF1 and that the state of eRF1 phosphorylation does not influence the allosuppressor phenotype associated with a sal6Delta mutation. Finally, we show that phosphorylation of eRF1 is a dynamic process that is dependent upon carbon source availability. Since many other proteins involved in protein synthesis have a CK2 protein kinase motif near their extreme C termini, we propose that this represents a common regulatory mechanism that is shared by factors involved in all three stages of protein synthesis.

Amino Acid Sequence↗
PubMed2006

Tpa1p is part of an mRNP complex that influences translation termination, mRNA deadenylation, and mRNA turnover in Saccharomyces cerevisiae.

In this report, we show that the Saccharomyces cerevisiae protein Tpa1p (for termination and polyadenylation) influences translation termination efficiency, mRNA poly(A) tail length, and mRNA stability. Tpa1p is encoded by the previously uncharacterized open reading frame YER049W. Yeast strains carrying a deletion of the TPA1 gene (tpa1Delta) exhibited increased readthrough of stop codons, and coimmunoprecipitation assays revealed that Tpa1p interacts with the translation termination factors eRF1 and eRF3. In addition, the tpa1Delta mutation led to a 1.5- to 2-fold increase in the half-lives of mRNAs degraded by the general 5'-->3' pathway or the 3'-->5' nonstop decay pathway. In contrast, this mutation did not have any affect on the nonsense-mediated mRNA decay pathway. Examination of mRNA poly(A) tail length revealed that poly(A) tails are longer than normal in a tpa1Delta strain. Consistent with a potential role in regulating poly(A) tail length, Tpa1p was also found to coimmunoprecipitate with the yeast poly(A) binding protein Pab1p. These results suggest that Tpa1p is a component of a messenger ribonucleoprotein complex bound to the 3' untranslated region of mRNAs that affects translation termination, deadenylation, and mRNA decay.

3' Untranslated Regions↗
PubMed2006

Distinct paths to stop codon reassignment by the variant-code organisms Tetrahymena and Euplotes.

The reassignment of stop codons is common among many ciliate species. For example, Tetrahymena species recognize only UGA as a stop codon, while Euplotes species recognize only UAA and UAG as stop codons. Recent studies have shown that domain 1 of the translation termination factor eRF1 mediates stop codon recognition. While it is commonly assumed that changes in domain 1 of ciliate eRF1s are responsible for altered stop codon recognition, this has never been demonstrated in vivo. To carry out such an analysis, we made hybrid proteins that contained eRF1 domain 1 from either Tetrahymena thermophila or Euplotes octocarinatus fused to eRF1 domains 2 and 3 from Saccharomyces cerevisiae. We found that the Tetrahymena hybrid eRF1 efficiently terminated at all three stop codons when expressed in yeast cells, indicating that domain 1 is not the sole determinant of stop codon recognition in Tetrahymena species. In contrast, the Euplotes hybrid facilitated efficient translation termination at UAA and UAG codons but not at the UGA codon. Together, these results indicate that while domain 1 facilitates stop codon recognition, other factors can influence this process. Our findings also indicate that these two ciliate species used distinct approaches to diverge from the universal genetic code.

Animals↗
PubMed2005

Discrimination between defects in elongation fidelity and termination efficiency provides mechanistic insights into translational readthrough.

The suppression of stop codons (termed translational readthrough) can be caused by a decreased accuracy of translation elongation or a reduced efficiency of translation termination. In previous studies, the inability to determine the extent to which each of these distinct processes contributes to a readthrough phenotype has limited our ability to evaluate how defects in the translational machinery influence the overall termination process. Here, we describe the combined use of misincorporation and readthrough reporter systems to determine which of these mechanisms contributes to translational readthrough in Saccharomyces cerevisiae. The misincorporation reporter system was generated by introducing a series of near-cognate mutations into functionally important residues in the firefly luciferase gene. These constructs allowed us to monitor the incidence of elongation errors by monitoring the level of firefly luciferase activity from a mutant allele inactivated by a single missense mutation. In this system, an increase in luciferase activity should reflect an increased level of misincorporation of the wild-type amino acid that provides an estimate of the overall fidelity of translation elongation. Surprisingly, we found that growth in the presence of paromomycin stimulated luciferase activity for only a small subset of the mutant proteins examined. This suggests that the ability of this aminoglycoside to induce elongation errors is limited to a subset of near-cognate mismatches. We also found that a similar bias in near-cognate misreading could be induced by the expression of a mutant form of ribosomal protein (r-protein) S9B or by depletion of r-protein L12. We used this misincorporation reporter in conjunction with a readthrough reporter system to show that alterations at different regions of the ribosome influence elongation fidelity and termination efficiency to different extents.

Aspartic Acid↗
PubMed2005

Inhibition of phosphoglucomutase activity by lithium alters cellular calcium homeostasis and signaling in Saccharomyces cerevisiae.

Phosphoglucomutase is a key enzyme of glucose metabolism that interconverts glucose-1-phosphate and glucose-6-phosphate. Loss of the major isoform of phosphoglucomutase in Saccharomyces cerevisiae results in a significant increase in the cellular glucose-1-phosphate-to-glucose-6-phosphate ratio when cells are grown in medium containing galactose as carbon source. This imbalance in glucose metabolites was recently shown to also cause a six- to ninefold increase in cellular Ca2+ accumulation. We found that Li+ inhibition of phosphoglucomutase causes a similar elevation of total cellular Ca2+ and an increase in 45Ca2+ uptake in a wild-type yeast strain grown in medium containing galactose, but not glucose, as sole carbon source. Li+ treatment also reduced the transient elevation of cytosolic Ca2+ response that is triggered by exposure to external CaCl2 or by the addition of galactose to yeast cells starved of a carbon source. Finally, we found that the Ca2+ over-accumulation induced by Li+ exposure was significantly reduced in a strain lacking the vacuolar Ca2+-ATPase Pmc1p. These observations suggest that Li+ inhibition of phosphoglucomutase results in an increased glucose-1-phosphate-to-glucose-6-phosphate ratio, which results in an accelerated rate of vacuolar Ca2+ uptake via the Ca2+-ATPase Pmc1p.

Calcium↗
PubMed2004

The Ca2+ homeostasis defects in a pgm2Delta strain of Saccharomyces cerevisiae are caused by excessive vacuolar Ca2+ uptake mediated by the Ca2+-ATPase Pmc1p.

Loss of the major isoform of phosphoglucomutase (PGM) causes an accumulation of glucose 1-phosphate when yeast cells are grown with galactose as the carbon and energy source. Remarkably, the pgm2Delta strain also exhibits a severe imbalance in intracellular Ca(2+) homeostasis when grown under these conditions. In the present study, we examined how the pgm2Delta mutation alters yeast Ca(2+) homeostasis in greater detail. We found that a shift from glucose to galactose as the carbon source resulted in a 2-fold increase in the rate of cellular Ca(2+) uptake in wild-type cells, whereas Ca(2+) uptake increased 8-fold in the pgm2Delta mutant. Disruption of the PMC1 gene, which encodes the vacuolar Ca(2+)-ATPase Pmc1p, suppressed the Ca(2+)-related phenotypes observed in the pgm2Delta strain. This suggests that excessive vacuolar Ca(2+) uptake is tightly coupled to these defects in Ca(2+) homeostasis. An in vitro assay designed to measure Ca(2+) sequestration into intracellular compartments confirmed that the pgm2Delta mutant contained a higher level of Pmc1p-dependent Ca(2+) transport activity than the wild-type strain. We found that this increased rate of vacuolar Ca(2+) uptake also coincided with a large induction of the unfolded protein response in the pgm2Delta mutant, suggesting that Ca(2+) uptake into the endoplasmic reticulum compartment was reduced. These results indicate that the excessive Ca(2+) uptake and accumulation previously shown to be associated with the pgm2Delta mutation are due to a severe imbalance in the distribution of cellular Ca(2+) into different intracellular compartments.

Calcium↗
PubMed2004

GTP hydrolysis by eRF3 facilitates stop codon decoding during eukaryotic translation termination.

Translation termination in eukaryotes is mediated by two release factors, eRF1 and eRF3. eRF1 recognizes each of the three stop codons (UAG, UAA, and UGA) and facilitates release of the nascent polypeptide chain. eRF3 is a GTPase that stimulates the translation termination process by a poorly characterized mechanism. In this study, we examined the functional importance of GTP hydrolysis by eRF3 in Saccharomyces cerevisiae. We found that mutations that reduced the rate of GTP hydrolysis also reduced the efficiency of translation termination at some termination signals but not others. As much as a 17-fold decrease in the termination efficiency was observed at some tetranucleotide termination signals (characterized by the stop codon and the first following nucleotide), while no effect was observed at other termination signals. To determine whether this stop signal-dependent decrease in the efficiency of translation termination was due to a defect in either eRF1 or eRF3 recycling, we reduced the level of eRF1 or eRF3 in cells by expressing them individually from the CUP1 promoter. We found that the limitation of either factor resulted in a general decrease in the efficiency of translation termination rather than a decrease at a subset of termination signals as observed with the eRF3 GTPase mutants. We also found that overproduction of eRF1 was unable to increase the efficiency of translation termination at any termination signals. Together, these results suggest that the GTPase activity of eRF3 is required to couple the recognition of translation termination signals by eRF1 to efficient polypeptide chain release.

Base Sequence↗
PubMed2004

Leaky termination at premature stop codons antagonizes nonsense-mediated mRNA decay in S. cerevisiae.

The Nonsense-Mediated mRNA Decay (NMD) pathway mediates the rapid degradation of mRNAs that contain premature stop mutations in eukaryotic organisms. It was recently shown that mutations in three yeast genes that encode proteins involved in the NMD process, UPF1, UPF2, and UPF3, also reduce the efficiency of translation termination. In the current study, we compared the efficiency of translation termination in a upf1Delta strain and a [PSI(+)] strain using a collection of translation termination reporter constructs. The [PSI(+)] state is caused by a prion form of the polypeptide chain release factor eRF3 that limits its availability to participate in translation termination. In contrast, the mechanism by which Upf1p influences translation termination is poorly understood. The efficiency of translation termination is primarily determined by a tetranucleotide termination signal consisting of the stop codon and the first nucleotide immediately 3' of the stop codon. We found that the upf1Delta mutation, like the [PSI(+)] state, decreases the efficiency of translation termination over a broad range of tetranucleotide termination signals in a unique, context-dependent manner. These results suggest that Upf1p may associate with the termination complex prior to polypeptide chain release. We also found that the increase in readthrough observed in a [PSI(+)]/upf1Delta strain was larger than the readthrough observed in strains carrying either defect alone, indicating that the upf1Delta mutation and the [PSI(+)] state influence the termination process in distinct ways. Finally, our analysis revealed that the mRNA destabilization associated with NMD could be separated into two distinct forms that correlated with the extent the premature stop codon was suppressed. The minor component of NMD was a 25% decrease in mRNA levels observed when readthrough was >/=0.5%, while the major component was represented by a larger decrease in mRNA abundance that was observed only when readthrough was </=0.5%. This low threshold for the onset of the major component of NMD indicates that mRNA surveillance is an ongoing process that occurs throughout the lifetime of an mRNA.

Codon, Nonsense↗
PubMed2003

Extracellular Ca(2+) sensing contributes to excess Ca(2+) accumulation and vacuolar fragmentation in a pmr1Delta mutant of S. cerevisiae.

Previous studies have suggested that yeast strains lacking the Ca(2+)-ATPase Pmr1p are unable to maintain an adequate level of Ca(2+) within the Golgi apparatus. It is thought that this compartmental store depletion induces a signal that causes an increased rate of Ca(2+) uptake and accumulation in a manner similar to the capacitative Ca(2+) entry (CCE) response in non-excitable mammalian cells. To explore this model further, we examined cellular Ca(2+) uptake and accumulation in a pmr1Delta strain grown in the presence of a reduced level of divalent cations. We found that the level of Ca(2+) uptake and accumulation in a pmr1Delta strain increased as the concentration of divalent cations in the growth medium decreased. These results are inconsistent with a model in which cellular Ca(2+) uptake and accumulation are determined solely by the depletion of Ca(2+) in an intracellular compartment. Instead, our results suggest that a second regulatory mechanism couples cellular Ca(2+) uptake to the availability of Ca(2+) in the extracellular environment. Furthermore, we found that various conditions that increase the level of cytosolic Ca(2+) correlate with vacuolar fragmentation in wild-type (WT), pmr1Delta and pmr1Delta/pmc1Delta yeast strains. This suggests that vacuolar fragmentation might function as a normal physiological response to Ca(2+) stress that increases the vacuolar surface/volume ratio, thereby maximizing the sequestration of this important signaling molecule.

Blotting, Northern↗
PubMed2003

A Saccharomyces cerevisiae mutant unable to convert glucose to glucose-6-phosphate accumulates excessive glucose in the endoplasmic reticulum due to core oligosaccharide trimming.

D-Glucose is the preferred carbon and energy source for most eukaryotic cells. Immediately following its uptake, glucose is rapidly phosphorylated to glucose-6-phosphate (Glc-6-P). The yeast Saccharomyces cerevisiae has three enzymes (Hxk1p, Hxk2p, and Glk1p) that convert glucose to Glc-6-P. In the present study, we found that yeast mutants lacking any two of these enzymes retain the ability to efficiently convert glucose to Glc-6-P and thus maintain a low level of cellular glucose. However, a mutant strain lacking all three glucose-phosphorylating enzymes contained up to 225-fold more intracellular glucose than normal. Drugs that inhibit the synthesis or the trimming of the lipid-linked core oligosaccharide Glu(3)Man(9)GlcNac(2) effectively reduced the accumulation of glucose. Similarly, mutations that block the addition of glucose residues to the core oligosaccharide moiety, such as alg5Delta or alg6Delta, also diminished glucose accumulation. These results indicate that the intracellular glucose accumulation observed in the glucose phosphorylation mutant results primarily from the trimming of glucose residues from core oligosaccharide chains within the endoplasmic reticulum (ER). Consistent with this conclusion, both [(14)C]glucose exchange and subcellular fractionation experiments indicate that much of the accumulated glucose is retained within an intracellular compartment, suggesting that the efficient transport of glucose from the ER to the cytosol in yeast may be coupled to its rephosphorylation to Glc-6-P. The high level of cellular glucose was associated with an increased level of protein glycation and the release of glucose into the culture medium via its transit through the secretory pathway. Finally, we also found that the accumulation of glucose may lead to a subtle alteration in ion homeostasis, particularly Ca(2+) uptake. This suggests that this mutant strain may serve as a useful model to study the consequences of excessive glucose accumulation and protein glycation.

Culture Media↗
PubMed2002

Intracellular glucose 1-phosphate and glucose 6-phosphate levels modulate Ca2+ homeostasis in Saccharomyces cerevisiae.

The enzyme phosphoglucomutase plays a key role in cellular metabolism by virtue of its ability to interconvert Glc-1-P and Glc-6-P. It was recently shown that a yeast strain lacking the major isoform of phosphoglucomutase (pgm2Delta) accumulates a high level of Glc-1-P and exhibits several phenotypes related to altered Ca(2+) homeostasis when d-galactose is utilized as the carbon source (Fu, L., Miseta, A., Hunton, D., Marchase, R. B., and Bedwell, D. M. (2000) J. Biol. Chem. 275, 5431-5440). These phenotypes include increased Ca(2+) uptake and accumulation and sensitivity to high environmental Ca(2+) levels. In the present study, we overproduced the enzyme UDP-Glc pyrophosphorylase to test whether the overproduction of a downstream metabolite produced from Glc-1-P can also mediate changes in Ca(2+) homeostasis. We found that overproduction of UDP-Glc did not cause any alterations in Ca(2+) uptake or accumulation. We also examined whether Glc-6-P can influence cellular Ca(2+) homeostasis. A yeast strain lacking the beta-subunit of phosphofructokinase (pfk2Delta) accumulates a high level of Glc-6-P (Huang, D., Wilson, W. A., and Roach, P. J. (1997) J. Biol. Chem. 272, 22495-22501). We found that this increase in Glc-6-P led to a 1.5-2-fold increase in total cellular Ca(2+). We also found that the pgm2Delta/pfk2Delta strain, which accumulated high levels of both Glc-6-P and Glc-1-P, no longer exhibited the Ca(2+)-related phenotypes associated with high Glc-1-P levels in the pgm2Delta mutant. These results provide strong evidence that cellular Ca(2+) homeostasis is coupled to the relative levels of Glc-6-P and Glc-1-P in yeast.

Base Sequence↗
PubMed2002

Aminoglycoside suppression of a premature stop mutation in a Cftr-/- mouse carrying a human CFTR-G542X transgene.

Cystic fibrosis (CF) is caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) protein. Since approximately 5% of all mutant CF alleles are stop mutations, it can be calculated that approximately 10% of CF patients carry a premature stop mutation in at least one copy of the CFTR gene. Certain ethnic groups, such as the Ashkenazi Jewish population, carry a much higher percentage of CF stop mutations. Consequently, a therapeutic strategy aimed at suppressing this class of mutation would be highly desirable for the treatment of this common genetic disease. We have shown previously that aminoglycoside antibiotics can suppress premature stop mutations in the CFTR gene in a bronchial epithelial cell line [Nat Med (1997) 3:1280]. To address whether aminoglycosides can suppress a CFTR premature stop mutation in an animal model, we constructed a transgenic mouse with a null mutation in the endogenous CFTR locus (Cftr-/-) that also expressed a human CFTR-G542X cDNA under control of the intestinal fatty acid binding protein promoter. We then investigated whether the daily administration of the aminoglycoside antibiotics gentamicin or tobramycin could restore the expression of a detectable level of CFTR protein. Immunofluorescence staining of intestinal tissues from Cftr-/- hCFTR-G542X mice revealed that gentamicin treatment resulted in the appearance of hCFTR protein at the apical surface of the glands of treated mice. Weaker staining was also observed in the intestinal glands following tobramycin treatment. Short-circuit current measurements made on intestinal tissues from these mice demonstrated that a significant number of positive cAMP-stimulated transepithelial chloride current measurements could be observed following gentamicin treatment (P=0.008) and a near significant number following tobramycin treatment (P=0.052). When taken together, these results indicate that gentamicin, and to a lesser extent tobramycin, can restore the synthesis of functional hCFTR protein by suppressing the hCFTR-G542X premature stop mutation in vivo.

Action Potentials↗
PubMed2002

Clinically relevant aminoglycosides can suppress disease-associated premature stop mutations in the IDUA and P53 cDNAs in a mammalian translation system.

Recent studies have suggested that the use of aminoglycosides to suppress disease-causing nonsense mutations may be a promising new therapy for a large number of genetic diseases. However, gentamicin is currently the only clinically relevant aminoglycoside shown to suppress premature stop mutations in a mammalian system. We compared the ability of the clinically approved aminoglycosides gentamicin, tobramycin, and amikacin to suppress premature stop mutations. Using readthrough reporter constructs as well as mammalian cDNAs containing naturally occurring premature stop mutations, we found that each of these aminoglycosides can suppress many premature stop mutations in a context-dependent manner in a mammalian translation system. Our results indicate that the tetranucleotide termination signal (the stop codon and the nucleotide 3' of the stop codon) is the primary determinant for aminoglycoside-mediated suppression. The levels of termination suppression achieved by tobramycin were substantially lower than those observed with gentamicin. In contrast, amikacin stimulated suppression in a manner that was generally similar to gentamicin. Amikacin produced higher levels of readthrough than gentamicin at some contexts, demonstrating a unique pattern of context dependence. Experiments with mammalian cDNAs confirmed these results and demonstrated that these aminoglycosides can also suppress disease-associated premature stop mutations previously identified in the IDUA gene (responsible for the lysosomal storage disease mucopolysaccharidosis I) and the P53 gene (associated with many forms of cancer). Taken together, these results suggest that amikacin represents an alternative to gentamicin for suppression therapy in certain contexts, thus providing a means of optimizing the efficacy of aminoglycoside-mediated suppression of premature stop mutations.

Amikacin↗
PubMed2002

Hexose phosphorylation and the putative calcium channel component Mid1p are required for the hexose-induced transient elevation of cytosolic calcium response in Saccharomyces cerevisiae.

Saccharomyces cerevisiae responds to environ-mental stimuli such as an exposure to pheromone or to hexoses after carbon source limitation with a transient elevation of cytosolic calcium (TECC) response. In this study, we examined whether hexose transport and phosphorylation are necessary for the TECC response. We found that a mutant strain lacking most of the known hexose transporters was unable to carry out the TECC response when exposed to glucose. A mutant strain that lacked the ability to phosphorylate glucose was unable to respond to glucose addition, but displayed a normal TECC response after the addition of galactose. These results indicate that hexose uptake and phosphorylation are required to trigger the hexose-induced TECC response. We also found that the TECC response was significantly smaller than normal when the level of environmental calcium was reduced, and was abolished in a mid1 mutant that lacked a subunit of the high-affinity calcium channel of the yeast plasma membrane. These results indicate that most or all of the TECC response is mediated by an influx of calcium from the extracellular space. Our results indicate that this transient increase in plasma membrane calcium permeability may be linked to the accumulation of Glc-1-P (or a related glucose metabolite) in yeast.

Aequorin↗