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941.
Steffen Kutter Manfred S. Weiss Georg Wille Ralph Golbik Michael Spinka Stephan K?nig 《The Journal of biological chemistry》2009,284(18):12136-12144
The mechanism by which the enzyme pyruvate decarboxylase from two yeast
species is activated allosterically has been elucidated. A total of seven
three-dimensional structures of the enzyme, of enzyme variants, or of enzyme
complexes from two yeast species, three of them reported here for the first
time, provide detailed atomic resolution snapshots along the activation
coordinate. The prime event is the covalent binding of the substrate pyruvate
to the side chain of cysteine 221, thus forming a thiohemiketal. This reaction
causes the shift of a neighboring amino acid, which eventually leads to the
rigidification of two otherwise flexible loops, one of which provides two
histidine residues necessary to complete the enzymatically competent active
site architecture. The structural data are complemented and supported by
kinetic investigations and binding studies, providing a consistent picture of
the structural changes occurring upon enzyme activation.Pyruvate decarboxylases (EC 4.1.1.1) catalyze the non-oxidative
decarboxylation of pyruvate, yielding acetaldehyde and carbon dioxide.
Together with the enzyme alcohol dehydrogenase (EC 1.1.1.1), which reduces the
acetaldehyde to ethanol with the help of the co-substrate NADH, it represents
the metabolic pathway of alcoholic fermentation.
PDC3 is localized in
the cytosol of cells from yeasts, plant seeds, and a few bacteria. The
catalytic activity of PDC depends on the presence of the cofactor thiamine
diphosphate (ThDP), which is bound mainly via a divalent metal ion (magnesium
in most cases) to the protein moiety. Many detailed kinetic studies have been
published on yeast PDC wild types
(1–9).
A number of ScPDC variants were analyzed, too
(1–9).
Some active site variants (E51A, D28A, E477Q) proved to be almost
catalytically inactive. PDCs are multisubunit enzymes. The typical molecular
mass of one subunit is 59–61 kDa. The tetramer is the catalytically
active state of most PDCs. Higher oligomers (octamers) have been described for
PDCs from plant seeds (10,
11) or some fungi
(12). However, studies on
structure function relationships of yeast PDCs showed that the dimer is the
minimum functional unit of the enzyme displaying considerable catalytic
activity (13,
14). The two closely related
pyruvate decarboxylases from Saccharomyces cerevisiae
(ScPDC) and Kluyveromyces lactis (KlPDC) are well
characterized ThDP-dependent enzymes, which share 86.3% identical amino acid
residues. They have been studied in great detail by means of kinetic
investigations and spectroscopic studies. Both enzymes are allosterically
regulated as reflected by sigmoid steady state kinetics and lag phases in
their progress curves. The substrate PYR activates the initially inactive
yeast PDCs in a time-dependent manner. Kinetic studies reveal a slow
isomerization as triggered by substrate binding to a separate regulatory site
(15). A number of substrate
surrogates have been identified, which are able to activate PDC as well. The
effects of pyruvamide (PA; for the chemical structure, see
Scheme 1) on the activation
kinetics have been studied in detail for ScPDC
(15) and for KlPDC
(16). Phosphonate analogues
(among them methyl acetylphosphonate, MAP,
Scheme 1) of pyruvate have been
applied to elucidate the catalytic cycle
(17–21)
or to trap reaction intermediates in crystal structures
(22–24).
Chemical modification of PDCs with group-specific reagents pointed to an
important role of cysteine residues
(25). Site-directed
mutagenesis of cysteine residues to alanine or serine demonstrated that
residue Cys-221 might be the decisive one for enzyme activation
(1,
4,
26,
27). Consequently, it was
postulated that the region around Cys-221 is the regulatory site of PDC, and
formation of a thiohemiketal at this side chain was proposed. However, a
number of questions remained elusive. (i) How is the activator fixed at the
regulatory site? (ii) What are the prime structural properties of the active
state as compared with the inactive state? (iii) How is the signal transmitted
from the regulatory to the active site? (iv) Which are the decisive features
of the active site in the activated state that render efficient catalysis
possible? To answer these questions, we present here the crystal structures of
KlPDC with the bound substrate surrogate MAP and of the
ScPDC variants D28E and E477Q with bound substrate PYR along with
kinetic studies on the activating effect of both activators and binding
studies using the small angle x-ray solution scattering (SAXS) method.Open in a separate windowSCHEME 1.Chemical structures of the substrate pyruvate, the activators pyruvamide
and methyl acetylphosphonate, and the thiohemiketal from pyruvate and
cysteine, respectively. 相似文献
942.
Benjamin A Raby Kristel Van Steen Jessica Lasky-Su Kelan Tantisira Feige Kaplan Scott T Weiss 《Respiratory research》2009,10(1):67
Background
Glucocorticoid function is dependent on efficient translocation of the glucocorticoid receptor (GR) from the cytoplasm to the nucleus of cells. Importin-13 (IPO13) is a nuclear transport receptor that mediates nuclear entry of GR. In airway epithelial cells, inhibition of IPO13 expression prevents nuclear entry of GR and abrogates anti-inflammatory effects of glucocorticoids. Impaired nuclear entry of GR has been documented in steroid-non-responsive asthmatics. We hypothesize that common IPO13 genetic variation influences the anti-inflammatory effects of inhaled corticosteroids for the treatment of asthma, as measured by change in methacholine airway hyperresponsiveness (AHR-PC20).Methods
10 polymorphisms were evaluated in 654 children with mild-to-moderate asthma participating in the Childhood Asthma Management Program (CAMP), a clinical trial of inhaled anti-inflammatory medications (budesonide and nedocromil). Population-based association tests with repeated measures of PC20 were performed using mixed models and confirmed using family-based tests of association.Results
Among participants randomized to placebo or nedocromil, IPO13 polymorphisms were associated with improved PC20 (i.e. less AHR), with subjects harboring minor alleles demonstrating an average 1.51–2.17 fold increase in mean PC20 at 8-months post-randomization that persisted over four years of observation (p = 0.01–0.005). This improvement was similar to that among children treated with long-term inhaled corticosteroids. There was no additional improvement in PC20 by IPO13 variants among children treated with inhaled corticosteroids.Conclusion
IPO13 variation is associated with improved AHR in asthmatic children. The degree of this improvement is similar to that observed with long-term inhaled corticosteroid treatment, suggesting that IPO13 variation may improve nuclear bioavailability of endogenous glucocorticoids. 相似文献943.
944.
Kevin T. Gobeske Sunit Das Michael A. Bonaguidi Craig Weiss Jelena Radulovic John F. Disterhoft John A. Kessler 《PloS one》2009,4(10)
Exposure to exercise or to environmental enrichment increases the generation of new neurons in the adult hippocampus and promotes certain kinds of learning and memory. While the precise role of neurogenesis in cognition has been debated intensely, comparatively few studies have addressed the mechanisms linking environmental exposures to cellular and behavioral outcomes. Here we show that bone morphogenetic protein (BMP) signaling mediates the effects of exercise on neurogenesis and cognition in the adult hippocampus. Elective exercise reduces levels of hippocampal BMP signaling before and during its promotion of neurogenesis and learning. Transgenic mice with decreased BMP signaling or wild type mice infused with a BMP inhibitor both exhibit remarkable gains in hippocampal cognitive performance and neurogenesis, mirroring the effects of exercise. Conversely, transgenic mice with increased BMP signaling have diminished hippocampal neurogenesis and impaired cognition. Exercise exposure does not rescue these deficits, suggesting that reduced BMP signaling is required for environmental effects on neurogenesis and learning. Together, these observations show that BMP signaling is a fundamental mechanism linking environmental exposure with changes in cognitive function and cellular properties in the hippocampus. 相似文献
945.
The human Y chromosome carries four human Y-chromosomal euchromatin/heterochromatin transition regions, all of which are characterized by the presence of interchromosomal segmental duplications. The Yq11.1/Yq11.21 transition region harbours a peculiar segment composed of an imperfectly organized tandem-repeat structure encoding four members of the double homeobox (DUX) gene family. By comparative fluorescence in situ hybridization (FISH) analysis we have documented the primary appearance of Y-chromosomal DUX genes (DUXY) on the gibbon Y chromosome. The major amplification and dispersal of DUXY paralogs occurred after the gibbon and hominid lineages had diverged. Orthologous DUXY loci of human and chimpanzee show a highly similar structural organization. Sequence alignment survey, phylogenetic reconstruction and recombination detection analyses of human and chimpanzee DUXY genes revealed the existence of all copies in a common ancestor. Comparative analysis of the circumjacent beta-satellites indicated that DUXY genes and beta-satellites evolved in concert. However, evolutionary forces acting on DUXY genes may have induced amino acid sequence differences in the orthologous chimpanzee and human DUXY open reading frames (ORFs). The acquisition of complete ORFs in human copies might relate to evolutionary advantageous functions indicating neo-functionalization. We propose an evolutionary scenario in which an ancestral tandem array DUX gene cassette transposed to the hominoid Y chromosome followed by lineage-specific chromosomal rearrangements paved the way for a species-specific evolution of the Y-chromosomal members of a large highly diverged homeobox gene family. 相似文献
946.
Carola W. Meyer Asja Wagener Nadine Rink Claudia Hantschel Gerhard Heldmaier Martin Klingenspor Gudrun A. Brockmann 《Obesity (Silver Spring, Md.)》2009,17(11):1988-1993
To constitute a valuable resource to identify individual genes involved in the development of obesity, a novel mouse model, the Berlin Fat Mouse Inbred line 860 (BFMI860), was established. In order to characterize energy intake and energy expenditure in obese BFMI860 mice, we performed two independent sets of experiments in male BFMI860 and B6 control mice (10 per line). In experiment 1, we analyzed body fat content noninvasively by dual‐energy X‐ray absorptiometry and measured resting metabolic rate at thermoneutrality (RMRt) and respiratory quotient (RQ) in week 6, 10, and 18. In a second experiment, energy digested (energy intake minus fecal energy loss) was determined by bomb calorimetry from week 6 through week 12. BFMI860 mice were heavier and had higher fat mass (final body fat content was 24.7% compared with 14.6% in B6). They also showed fatty liver syndrome. High body fat accumulation in BFMI860 mice was restricted to weeks 6–10 and was accompanied by hyperphagia, higher energy digestion, higher RQs, and abnormally high blood triglyceride levels. Lean mass–adjusted RMRt was not altered between lines. These results indicate that in BFMI860 mice, the excessive accumulation of body fat is associated with altered lipid metabolism, high energy intake, and energy digestion. Assuming that BFMI860 mice and their obese phenotypes are of polygenic nature, this line is an excellent model for the study of obesity in humans, especially for juvenile obesity and hyperlipidemia. 相似文献
947.
Wolfgang Sturm Anton Sandhofer Julia Engl Markus Laimer Clemens Molnar Susanne Kaser Helmut Weiss Herbert Tilg Christoph F. Ebenbichler Josef R. Patsch 《Obesity (Silver Spring, Md.)》2009,17(9):1783-1788
Endothelial dysfunction and increased intima–media thickness (IMT) have been found in obese patients. Both regional fat distribution and liver steatosis may influence these markers of subclinical atherosclerosis. We sought to determine the interrelationships of endothelial function, carotid IMT, visceral and subcutaneous adipose tissue accumulation, and liver steatosis in severely obese subjects. In 64 severely obese patients (BMI 42.3 ± 4.3 kg/m²), we determined (i) endothelial function as flow‐mediated dilation (FMD) of the brachial artery, (ii) carotid IMT, (iii) visceral fat diameter, and (iv) degree of liver steatosis using ultrasound. FMD was associated inversely with visceral fat diameter and degree of steatosis (r = ?0.577, P < 0.0001 and r = ?0.523, P < 0.0001, respectively). Carotid IMT correlated with visceral fat mass (r = 0.343, P = 0.007) but not with liver steatosis. After adjustment for conventional cardiovascular risk factors, FMD was predicted independently by the visceral fat diameter, age, and sex (r2 = 0.48, P < 0.0001), but not by the degree of liver steatosis or plasma adiponectin levels. In contrast, age and sex were the only predictors of IMT (r2 = 0.33, P < 0.001). In obese patients, visceral fat diameter is a major determinant of endothelial dysfunction, independent of traditional risk factors or the degree of liver steatosis and plasma adiponectin. Measurement of visceral fat diameter by ultrasound is a novel and simple method to identify subjects with an increased risk for atherosclerosis within an obese population. 相似文献
948.
Dana B. Hancock Isabelle Romieu Min Shi Juan-Jose Sienra-Monge Hao Wu Grace Y. Chiu Huiling Li Blanca Estela del Rio-Navarro Saffron A. G. Willis-Owens Scott T. Weiss Benjamin A. Raby Hong Gao Celeste Eng Rocio Chapela Esteban G. Burchard Hua Tang Patrick F. Sullivan Stephanie J. London 《PLoS genetics》2009,5(8)
Many candidate genes have been studied for asthma, but replication has varied. Novel candidate genes have been identified for various complex diseases using genome-wide association studies (GWASs). We conducted a GWAS in 492 Mexican children with asthma, predominantly atopic by skin prick test, and their parents using the Illumina HumanHap 550 K BeadChip to identify novel genetic variation for childhood asthma. The 520,767 autosomal single nucleotide polymorphisms (SNPs) passing quality control were tested for association with childhood asthma using log-linear regression with a log-additive risk model. Eleven of the most significantly associated GWAS SNPs were tested for replication in an independent study of 177 Mexican case–parent trios with childhood-onset asthma and atopy using log-linear analysis. The chromosome 9q21.31 SNP rs2378383 (p = 7.10×10−6 in the GWAS), located upstream of transducin-like enhancer of split 4 (TLE4), gave a p-value of 0.03 and the same direction and magnitude of association in the replication study (combined p = 6.79×10−7). Ancestry analysis on chromosome 9q supported an inverse association between the rs2378383 minor allele (G) and childhood asthma. This work identifies chromosome 9q21.31 as a novel susceptibility locus for childhood asthma in Mexicans. Further, analysis of genome-wide expression data in 51 human tissues from the Novartis Research Foundation showed that median GWAS significance levels for SNPs in genes expressed in the lung differed most significantly from genes not expressed in the lung when compared to 50 other tissues, supporting the biological plausibility of our overall GWAS findings and the multigenic etiology of childhood asthma. 相似文献
949.
We developed eight polymorphic microsatellite markers for the endangered salmonid Parahucho perryi from genomic libraries enriched for (GACA)n and (GATA)n repeat sequences. Emphasis was placed on developing highly polymorphic,
perfect repeats that could be scored with confidence. Six tetra- and two di-nucleotide loci were screened in 49 individuals
from two populations on Sakhalin Island, Russia. Allelic variation was high with eight to 23 alleles per locus and expected
heterozygosity ranging from 0.48 to 0.93. These highly variable markers should prove useful in evaluating inbreeding, gene
flow and population structure in Sakhalin taimen throughout its range. 相似文献
950.
Michal Simovitch Hagit Sason Shulamit Cohen Eitan Erez Zahavi Naomi Melamed‐Book Aryeh Weiss Benjamin Aroeti Ilan Rosenshine 《Cellular microbiology》2010,12(4):489-505
Enterohaemorrhagic Escherichia coli and enteropathogenic E. coli are enteropathogens characterized by their ability to induce the host cell to form actin‐rich structures, termed pedestals. A type III secretion system, through which the pathogens deliver effector proteins into infected host cells, is essential for their virulence and pedestal formation. Enterohaemorrhagic E. coli encodes two similar effectors, EspM1 and EspM2, which activate the RhoA signalling pathway and induce the formation of stress fibres upon infection of host cells. We confirm these observations and in addition show that EspM inhibits the formation of actin pedestals. Moreover, we show that translocation of EspM into polarized epithelial cells induces dramatic changes in the tight junction localization and in the morphology and architecture of infected polarized monolayers. These changes are manifested by altered localization of the tight junctions and ‘bulging out’ morphology of the cells. Surprisingly, despite the dramatic changes in their architecture, the cells remain alive and the epithelial monolayer maintains a normal barrier function. Taken together, our results show that the EspM effectors inhibit pedestal formation and induce tight junction mislocalization as well as dramatic changes in the architecture of the polarized monolayer. 相似文献