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991.
992.
993.
994.
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
995.
Jingxin Wang Naduparambil K Jacob Katherine J Ladner Amer Beg James D Perko Stephan M Tanner Sandya Liyanarachchi Richard Fishel Denis C Guttridge 《EMBO reports》2009,10(11):1272-1278
Nuclear factor (NF)-κB is a positive regulator of tumour development and progression, but how it functions in normal cells leading to oncogenesis is not clear. As cellular senescence has proven to be an intrinsic tumour suppressor mechanism that cells must overcome to establish deregulated growth, we used primary fibroblasts to follow NF-κB function in cells transitioning from senescence to subsequent immortalization. Our findings show that RelA/p65−/− murine fibroblasts immortalize at considerably faster rates than RelA/p65+/+ cells. The ability of RelA/p65−/− fibroblasts to escape senescence earlier is due to their genomic instability, characterized by high frequencies of DNA mutations, gene deletions and gross chromosomal translocations. This increase in genomic instability is closely related to a compromised DNA repair that occurs in both murine RelA/p65−/− fibroblasts and tissues. Significantly, these results can also be duplicated in human fibroblasts lacking NF-κB. Altogether, our findings present a fresh perspective on the role of NF-κB as a tumour suppressor, which acts in pre-neoplastic cells to maintain cellular senescence by promoting DNA repair and genomic stability. 相似文献
996.
997.
Lisa E. Creary Pinar Ulug Stephan Menzel Colin A. McKenzie Neil A. Hanchard Veronica Taylor Martin Farrall Terrence E. Forrester Swee Lay Thein 《PloS one》2009,4(1)
Fetal haemoglobin (HbF) is a major ameliorating factor in sickle cell disease. We investigated if a quantitative trait locus on chromosome 6q23 was significantly associated with HbF and F cell levels in individuals of African descent. Single nucleotide polymorphisms (SNPs) in a 24-kb intergenic region, 33-kb upstream of the HBS1L gene and 80-kb upstream of the MYB gene, were typed in 177 healthy Afro-Caribbean subjects (AC) of approximately 7% European admixture, 631 healthy Afro-Germans (AG, a group of African and German descendents located in rural Jamaica with about 20% European admixture), 87 West African and Afro-Caribbean individuals with sickle cell anaemia (HbSS), as well as 75 Northern Europeans, which served as a contrasting population. Association with a tag SNP for the locus was detected in all four groups (AC, P = 0.005, AG, P = 0.002, HbSS patients, P = 0.019, Europeans, P = 1.5×10−7). The association signal varied across the interval in the African-descended groups, while it is more uniform in Europeans. The 6q QTL for HbF traits is present in populations of African origin and is also acting in sickle cell anaemia patients. We have started to distinguish effects originating from European and African ancestral populations in our admixed study populations. 相似文献
998.
Bridge ES Schoech SJ Bowman R Wingfield JC 《Journal of experimental zoology. Part A, Ecological genetics and physiology》2009,311(1):35-44
Florida Scrub-Jays (Aphelocoma coerulescens) in a suburban environment with year-round access to multiple sources of abundant, human-source foods consistently breed earlier each year and have lower baseline levels of circulating corticosterone (CORT) than jays in a nearby wildland setting. These findings suggest that food supplies influence CORT levels, which in turn may partially determine the timing of reproduction. However, wildland birds with access to high-quality supplemental foods did not advance breeding or lower CORT levels to the degree observed in the suburbs. Therefore, rather than quality or quantity of food consumed, the perception of a reliable and predictable food supply may be an important factor in determining laying dates. If a predictable food supply accelerates the reproductive process, it follows that food provided on an unpredictable schedule may slow reproduction. We subjected captive Western Scrub-Jays (A. californica) to a 30-day photoperiod transition from short- to long-days and tested whether birds with access to food on an unpredictable schedule exhibited delayed or reduced production of reproductive hormones compared with birds given food on a predictable schedule. Baseline CORT concentrations increased slightly during the experiment, but did not differ between treatment groups. Birds with unpredictable food had slightly lower testosterone levels relative to controls, but there was no effect on estradiol or luteinizing hormone. Our findings offer weak support for the hypothesis that an unpredictable food supply will delay the onset of reproduction; however, the artificial lab environment may limit the application of these findings to free-living populations. 相似文献
999.
Ruth Halaban Michael Krauthammer Mattia Pelizzola Elaine Cheng Daniela Kovacs Mario Sznol Stephan Ariyan Deepak Narayan Antonella Bacchiocchi Annette Molinaro Yuval Kluger Min Deng Nam Tran Wengeng Zhang Mauro Picardo Jan J. Enghild 《PloS one》2009,4(2)
Decitabine, an epigenetic modifier that reactivates genes otherwise suppressed by DNA promoter methylation, is effective for some, but not all cancer patients, especially those with solid tumors. It is commonly recognized that to overcome resistance and improve outcome, treatment should be guided by tumor biology, which includes genotype, epigenotype, and gene expression profile. We therefore took an integrative approach to better understand melanoma cell response to clinically relevant dose of decitabine and identify complementary targets for combined therapy. We employed eight different melanoma cell strains, determined their growth, apoptotic and DNA damage responses to increasing doses of decitabine, and chose a low, clinically relevant drug dose to perform whole-genome differential gene expression, bioinformatic analysis, and protein validation studies. The data ruled out the DNA damage response, demonstrated the involvement of p21Cip1 in a p53-independent manner, identified the TGFβ pathway genes CLU and TGFBI as markers of sensitivity to decitabine and revealed an effect on histone modification as part of decitabine-induced gene expression. Mutation analysis and knockdown by siRNA implicated activated β-catenin/MITF, but not BRAF, NRAS or PTEN mutations as a source for resistance. The importance of protein stability predicted from the results was validated by the synergistic effect of Bortezomib, a proteasome inhibitor, in enhancing the growth arrest of decitabine in otherwise resistant melanoma cells. Our integrative analysis show that improved therapy can be achieved by comprehensive analysis of cancer cells, identified biomarkers for patient''s selection and monitoring response, as well as targets for improved combination therapy. 相似文献
1000.
Stephan Fricke Manuela Ackermann Alexandra Stolzing Christoph Schimmelpfennig Nadja Hilger Jutta Jahns Guido Hildebrandt Frank Emmrich Peter Ruschpler Claudia P?sel Manja Kamprad Ulrich Sack 《PloS one》2009,4(7)