Biomedical subjects

Judith L Fridovich-Keil

Publications and source records attributed to Judith L Fridovich-Keil.

12 recordsLinked to original sources

PubMed2006

Relationship between UDP-galactose 4'-epimerase activity and galactose sensitivity in yeast.

UDP-galactose 4'-epimerase (GALE) catalyzes the final step of the highly conserved Leloir pathway of galactose metabolism. Loss of GALE in humans results in a variant form of the metabolic disorder, galactosemia. Loss of GALE in yeast results in galactose-dependent growth arrest. Although the role of GALE in galactose metabolism has been recognized for decades, the precise relationship between GALE activity and galactose sensitivity has remained unclear. Here we have explored this relationship by asking the following. 1) Is GALE rate-limiting for galactose metabolism in yeast? 2) What is the relationship between GALE activity and galactose-dependent growth arrest in yeast? 3) What is the relationship between GALE activity and the abnormal accumulation of galactose metabolites in yeast? To answer these questions we engineered a strain of yeast in which GALE was doxycycline-repressible and studied these cells under conditions of intermediate GALE expression. Our results demonstrated a smooth linear relationship between galactose metabolism and GALE activity over a range from 0 to approximately 5% but a steep threshold relationship between growth rate in galactose and GALE activity over the same range. The relationship between abnormal accumulation of metabolites and GALE activity was also linear over the range from 0 to approximately 5%, suggesting that if the abnormal accumulation of metabolites underlies galactose-dependent growth-arrest in GALE-impaired yeast, either the impact of individual metabolites must be synergistic and/or the threshold of sensitivity must be very steep. Together these data reveal important points of similarity and contrast between the roles of GALE and galactose-1-phosphate uridylyltransferase in galactose metabolism in yeast and provide a framework for future studies in mammalian systems.

Cell Extracts↗

PubMed2006

Galactosemia: the good, the bad, and the unknown.

Alpha-D-galactose is metabolized in species ranging from E. coli to mammals predominantly via a series of sequential reactions collectively known as the Leloir pathway. Deficiency of any one of these enzymes in humans results in a form of the inherited metabolic disorder, galactosemia, although the symptoms and severity depend upon the enzyme impaired, and the degree of functional deficiency (Tyfield and Walter, 2002, The Metabolic and Molecular Bases of Inherited Disease. New York: McGraw Hill.). Studies of these enzymes, and the disorders associated with their loss, have led to a much deeper appreciation of the intricate and interwoven levels of regulation that govern their normal function. These insights have further identified likely mediators of outcome severity in patients, and have enabled a rational approach to the development of novel strategies of intervention.

Animals↗

PubMed2005

Epimerase-deficiency galactosemia is not a binary condition.

Epimerase-deficiency galactosemia results from the impairment of UDP-galactose 4'-epimerase (GALE), the third enzyme in the Leloir pathway of galactose metabolism. Originally identified as a clinically benign "peripheral" condition with enzyme impairment restricted to circulating blood cells, GALE deficiency was later demonstrated also to exist in a rare but clinically severe "generalized" form, with enzyme impairment affecting a range of tissues. Isolated cases of clinically and/or biochemically intermediate cases of epimerase deficiency have also been reported. We report here studies of 10 patients who, in the neonatal period, received the diagnosis of hemolysate epimerase deficiency. We have characterized these patients with regard to three parameters: (1) GALE activity in transformed lymphoblasts, representing a "nonperipheral" tissue, (2) metabolic sensitivity of those lymphoblasts to galactose challenge in culture, and (3) evidence of normal versus abnormal galactose metabolism in the patients themselves. Our results demonstrate two important points. First, whereas some of the patients studied exhibited near-normal levels of GALE activity in lymphoblasts, consistent with a diagnosis of peripheral epimerase deficiency, many did not. We detected a spectrum of GALE activity levels ranging from 15%-64% of control levels, demonstrating that epimerase deficiency is not a binary condition; it is a continuum disorder. Second, lymphoblasts demonstrating the most severe reduction in GALE activity also demonstrated abnormal metabolite levels in the presence of external galactose and, in some cases, also in the absence of galactose. These abnormalities included elevated galactose-1P, elevated UDP-galactose, and deficient UDP-glucose. Moreover, some of the patients themselves also demonstrated metabolic abnormalities, both on and off galactose-restricted diet. Long-term follow-up studies of these and other patients will be required to elucidate the clinical significance of these biochemical abnormalities and the potential impact of dietary intervention on outcome.

Base Sequence↗

PubMed2005

Mediators of galactose sensitivity in UDP-galactose 4'-epimerase-impaired mammalian cells.

UDP-galactose 4'-epimerase (GALE) catalyzes the final step in the Leloir pathway of galactose metabolism, interconverting UDP-galactose and UDP-glucose. Unlike its Escherichia coli counterpart, mammalian GALE also interconverts UDP-N-acetylgalactosamine and UDP-N-acetylglucosamine. Considering the key roles played by all four of these UDP-sugars in glycosylation, human GALE therefore not only contributes to the Leloir pathway, but also functions as a gatekeeper overseeing the ratios of important substrate pools required for the synthesis of glycosylated macromolecules. Defects in human GALE result in the disorder epimerase-deficiency galactosemia. To explore the relationship among GALE activity, substrate specificity, metabolic balance, and galactose sensitivity in mammalian cells, we employed a previously described GALE-null line of Chinese hamster ovary cells, ldlD. Using a transfection protocol, we generated ldlD derivative cell lines that expressed different levels of wild-type human GALE or E. coli GALE and compared the phenotypes and metabolic profiles of these lines cultured in the presence versus absence of galactose. We found that GALE-null cells accumulated abnormally high levels of Gal-1-P and UDP-Gal and abnormally low levels of UDP-Glc and UDP-GlcNAc in the presence of galactose and that human GALE expression corrected each of these defects. Comparing the human GALE- and E. coli GALE-expressing cells, we found that although GALE activity toward both substrates was required to restore metabolic balance, UDP-GalNAc activity was not required for cell proliferation in the presence of otherwise cytostatic concentrations of galactose. Finally, we found that uridine supplementation, which essentially corrected UDP-Glc and, to a lesser extent UDP-GlcNAc depletion, enabled ldlD cells to proliferate in the presence of galactose despite the continued accumulation of Gal-1-P and UDP-Gal. These data offer important insights into the mechanism of galactose sensitivity in epimerase-impaired cells and suggest a potential novel therapy for patients with epimerase-deficiency galactosemia.

Animals↗

PubMed2005

Functional characterization of the K257R and G319E-hGALE alleles found in patients with ostensibly peripheral epimerase deficiency galactosemia.

Epimerase deficiency galactosemia is an autosomal recessive condition resulting from the impairment of UDP-galactose 4'-epimerase (hGALE). Although a small number of clinically severe patients have been reported who exhibit "generalized" GALE deficiency, the vast majority exhibit an apparently benign "peripheral" form of the disorder in which enzyme impairment is restricted to the circulating red and white blood cells. Previously, preliminary data were reported suggesting that GALE deficiency is 10-fold more common among African-Americans than among non-African-Americans, and that two missense mutations, K257R and G319E, are found in at least some of these patients. We report here functional studies of these alleles involving expression of the substituted human enzymes in a null-background strain of yeast. Although under normal assay conditions both substituted proteins demonstrate enzyme activities indistinguishable from the wild-type, one (G319E) demonstrates mild impairment under conditions of substrate limitation. No impairments are evident under conditions of cofactor (NAD) limitation. These results are consistent with the apparently benign status of peripheral epimerase deficiency galactosemia, but leave open the question of why patients with these substitutions demonstrate GALE deficiency in their red blood cells. While the possibility remains that K257R and G319E may cause tissue-specific impairments not recapitulated in vitro or in yeast, an equally if not more plausible explanation suggested by interspecies sequence alignments is that both substitutions may be polymorphisms that exist in linkage disequilibrium with other, as yet unidentified causal mutations.

Alleles↗

PubMed2004

Both KH and non-KH domain sequences are required for polyribosome association of Scp160p in yeast.

Scp160p is a 160 kDa RNA-binding protein in yeast previously demonstrated to associate with specific messages as an mRNP component of both soluble and membrane-bound polyribosomes. Although the vast majority of Scp160p sequence consists of 14 closely spaced KH domains, comparative sequence analyses also demonstrate the presence of a potential nuclear localization sequence located between KH domains 3 and 4, as well as a 110 amino acid non-KH N-terminal region that includes a potential nuclear export sequence (NES). As a step toward investigating the structure/function relationships of Scp160p, we generated two truncated alleles, FLAG.SCP160DeltaN1, encoding a protein product that lacks the first 74 amino acids, including the potential NES, and FLAG.SCP160DeltaC1, encoding a protein product that lacks the final KH domain (KH14). We report here that the N-truncated protein, expressed as a green fluorescent protein fusion in yeast, remains cytoplasmic, with no apparent nuclear accumulation. Biochemical studies further demonstrate that although the N-truncated protein remains competent to form RNPs, the C-truncated protein does not. Furthermore, polyribosome association is severely compromised for both truncated proteins. Perhaps most important, both truncated alleles appear only marginally functional in vivo, as demonstrated by the inability of each to complement scp160/eap1 synthetic lethality in a tester strain. Together, these data challenge the notion that Scp160p normally shuttles between the nucleus and cytoplasm, and further implicate polyribosome association as an essential component of Scp160p function in vivo. Finally, these data underscore the vital roles of both KH and non-KH domain sequences in Scp160p.

Alleles↗

PubMed2004

Determinants of function and substrate specificity in human UDP-galactose 4'-epimerase.

UDP-galactose 4'-epimerase (GALE) interconverts UDP-galactose and UDP-glucose in the final step of the Leloir pathway. Unlike the Escherichia coli enzyme, human GALE (hGALE) also efficiently interconverts a larger pair of substrates: UDP-N-acetylgalactosamine and UDP-N-acetylglucosamine. The basis of this differential substrate specificity has remained obscure. Recently, however, x-ray crystallographic data have both predicted essential active site residues and suggested that differential active site cleft volume may be a key factor in determining GALE substrate selectivity. We report here a direct test of this hypothesis. In brief, we have created four substituted alleles: S132A, Y157F, S132A/Y157F, and C307Y-hGALE. While the first three substitutions were predicted to disrupt catalytic activity, the fourth was predicted to reduce active site cleft volume, thereby limiting entry or rotation of the larger but not the smaller substrate. All four alleles were expressed in a null-background strain of Saccharomyces cerevisiae and characterized in terms of activity with regard to both UDP-galactose and UDP-N-acetylgalactosamine. The S132A/Y157F and C307Y-hGALE proteins were also overexpressed in Pichia pastoris and purified for analysis. In all forms tested, the Y157F, S132A, and Y157F/S132A-hGALE proteins each demonstrated a complete loss of activity with respect to both substrates. In contrast, the C307Y-hGALE demonstrated normal activity with respect to UDP-galactose but complete loss of activity with respect to UDP-N-acetylgalactosamine. Together, these results serve to validate the wild-type hGALE crystal structure and fully support the hypothesis that residue 307 acts as a gatekeeper mediating substrate access to the hGALE active site.

Alleles↗

PubMed2003

Genetic and biochemical interactions between SCP160 and EAP1 in yeast.

Scp160p is a multiple KH-domain RNA-binding protein in yeast known to associate with polyribosomes as an mRNP component, although its biological role remains unclear. As a genetic approach to examine Scp160p function, we applied an ethyl methanesulfonate (EMS) screen for loci synthetically lethal with scp160 loss, and identified a single candidate gene, EAP1, whose protein product functions in translation as an eIF4E-binding protein, with additional uncharacterized spindle pole body functions. To reconfirm scp160/eap1 synthetic lethality, we constructed a strain null for both genes, supported by an SCP160 maintenance plasmid. We used this strain to establish a quantitative assay for both Scp160p and Eap1p functions in vivo, and applied this assay to demonstrate that Y109A EAP1, a previously described allele of EAP1 that cannot bind eIF4E, is markedly impaired with regard to its SCP160-related activity. In addition, we explored the possibility of physical interaction between Eap1p and Scp160p, and discovered that Eap1p associates with Scp160p-containing complexes in an RNA-dependent manner. Finally, we probed the impact of EAP1 loss on Scp160p, and vice versa, and found that loss of each gene resulted in a significant change in either the complex associations or subcellular distribution of the other protein. These results clearly support the hypothesis that Scp160p plays a role in translation, demonstrate that the interaction of SCP160 and EAP1 is biologically significant, and provide important tools for future studies of the in vivo functions of both genes.

Alleles↗

PubMed2003

Scp160p associates with specific mRNAs in yeast.

Scp160p is a multiple KH-domain RNA-binding protein in yeast that has been demonstrated previously to associate with both soluble and membrane-bound polyribosomes as an mRNP component. One key question that has remained unanswered, however, is whether the mRNAs in these mRNP complexes are random or specific. We have addressed this question using microarray analyses of RNAs released from affinity isolated Scp160p-containing complexes, compared with total RNA controls from the same lysates. Our results, confirmed by quantitative RT-PCR analysis, clearly demonstrate that Scp160p associates with specific rather than with random messages, and that among the enriched targets are DHH1, YOR338W, BIK1, YOL155C and NAM8. Furthermore, loss of Scp160p resulted in a significant change in both the abundance and distribution between soluble and membrane-associated fractions for at least one of these messages (YOR338W), and in a subtle yet significant shift from soluble polyribosomes to soluble mRNPs for at least two of these target messages (DHH1 and YOR338W). Together, these data not only identify specific mRNA targets associated with Scp160p in vivo, they demonstrate that the association of Scp160p with these messages is biologically relevant.

DEAD-box RNA Helicases↗

PubMed2002

Structural analysis of the Y299C mutant of Escherichia coli UDP-galactose 4-epimerase. Teaching an old dog new tricks.

UDP-galactose 4-epimerase catalyzes the interconversion of UDP-Gal and UDP-Glc during normal galactose metabolism. The mammalian form of the enzyme, unlike its Escherichia coli counterpart, can also interconvert UDP-GalNAc and UDP-GlcNAc. One key feature of the epimerase reaction mechanism is the rotation of a 4-ketopyranose intermediate in the active site. By comparing the high resolution x-ray structures of both the bacterial and human forms of the enzyme, it was previously postulated that the additional activity in the human epimerase was due to replacement of the structural equivalent of Tyr-299 in the E. coli enzyme with a cysteine residue, thereby leading to a larger active site volume. To test this hypothesis, the Y299C mutant form of the E. coli enzyme was prepared and its three-dimensional structure solved as described here. Additionally, the Y299C mutant protein was assayed for activity against both UDP-Gal and UDP-GalNAc. These studies have revealed that, indeed, this simple mutation did confer UDP-GalNAc/UDP-GlcNAc converting activity to the bacterial enzyme with minimal changes in its three-dimensional structure. Specifically, although the Y299C mutation in the bacterial enzyme resulted in a loss of epimerase activity with regard to UDP-Gal by almost 5-fold, it resulted in a gain of activity against UDP-GalNAc by more than 230-fold.

Crystallography, X-Ray↗

PubMed2002

Impact of patient mutations on heterodimer formation and function in human galactose-1-P uridylyltransferase.

Impairment of the human enzyme galactose-1-P uridylyltransferase (hGALT) results in the potentially lethal disorder, galactosemia. One of the fundamental questions with regard to this dimeric enzyme involves the possible influence of patient mutations on heterodimer formation and activity. Indeed, considering that many if not most galactosemia patients are compound heterozygotes, this is an issue of clinical as well as basic science interest. We have utilized a yeast expression system for the human enzyme to test whether each of a small number of mutations in hGALT (S135L, F171S, F171W, Q188R, N314D, and R333W) impact either heterodimer formation or function. Our results clearly demonstrate that while a majority of the alleles tested show precisely random patterns of subunit assortment, two deviate slightly but significantly from this pattern. Similarly, while some heterodimers exhibit apparent independence of subunit activity, others do not. These data not only demonstrate that common patient mutations in hGALT can influence both heterodimer formation and function in heterozygotes, they further raise the question of whether such interactions may also occur between different mutant alleles in compound heterozygotes (i.e., patients). Indeed, such influences may underlie some of the biochemical and clinical heterogeneity observed in the galactosemia patient population.

Dimerization↗

PubMedDate not supplied

Differential roles of the Leloir pathway enzymes and metabolites in defining galactose sensitivity in yeast.

The metabolism of galactose via enzymes of the Leloir pathway: galactokinase, galactose-1-P uridylyltransferase, and UDP galactose-4'-epimerase, is a process that has been conserved from Escherichia coli through humans. Impairment of this pathway in patients results in the disease galactosemia. Despite decades of study, the underlying pathophysiology in galactosemia remains unknown. Here we have defined the functional and metabolic implications of impaired galactose metabolism in yeast, by asking two questions: (1) What is the impact of loss of each of the three Leloir enzymes on the ability of cells to metabolize galactose, and on their sensitivity to galactose, and (2) what is the relationship between gal-1P and galactose-sensitivity in yeast? Our results demonstrate that only transferase-null cells are able to deplete their medium of galactose; deletion of kinase or epimerase halts this process. In contrast, only kinase-null cultures grow well in glycerol/ethanol medium despite the addition of galactose; both transferase and epimerase-null yeast arrest growth under these conditions. Indeed, epimerase-null yeast arrest growth at galactose concentrations 10-fold lower than do their transferase-null counterparts. Secondary deletion of kinase relieves growth arrest in both strains. Finally, rather than a continuous relationship between gal-1P and growth arrest, we observed a threshold level of gal-1P (approximately 10 nmol/mg cell DM) above which both transferase-null and epimerase-null cultures could not grow. These results both confirm and significantly extend prior knowledge of galactose metabolism in yeast, and set the stage for future studies into the mediators and mechanism of Leloir-impaired galactose sensitivity in eukaryotes.

Cell Proliferation↗