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71.
Despoina Lazarou Raphael Grougnet Athanasios Papadopoulos 《Mutation Research - Genetic Toxicology and Environmental Mutagenesis》2007,634(1-2):163-171
A polyphenolic mixture derived from sesame-seed perisperm (SSP) strongly reduced the mutagenicity of hydrogen peroxide (H2O2), sodium azide (NaN3), and benzo[a]pyrene (BaP) in strains TA100 and/or TA98 of Salmonella typhimurium. It exhibited desmutagenic activity against H2O2, BaP in TA98 and/or TA100 and biomutagenic activity (apparently by affecting the DNA-repair system) against NaN3 in strain TA100. According to in vitro experiments the polyphenolic mixture inhibited the activity of the CYP1A1 (EROD) enzyme responsible for the activation of BaP in the Ames’ test, as well as that of the cytosolic enzyme GST.A cytosolic fraction from liver of male Wistar rats treated with either 20% SSP in the food, or 3 mg or 6 mg of polyphenolic mixture/20 g food/day for a time period of 8 weeks reduced the mutagenic potential of BaP in strains TA100 and TA98, with the cytosolic fraction from rats treated with SSP causing the strongest reduction. Furthermore, a microsomal fraction from the 20% SSP-treated rats inhibited the mutagenicity of BaP in strains TA100 (26.3%) and TA98 (23%). In contrast, a microsomal fraction from rats treated with 3 mg of polyphenolic mixture stimulated the mutagenicity of BaP in TA100 but reduced it in TA98, while for the microsomal fraction from rats treated with 6 mg of polyphenolic mixture, these effects on TA100 and TA98 were reversed. 相似文献
72.
73.
Control of Periplasmic Interdomain Thiol:Disulfide Exchange in the
Transmembrane Oxidoreductase
DsbD
Despoina A. I. Mavridou Julie M. Stevens Alan D. Goddard Antony C. Willis Stuart J. Ferguson Christina Redfield 《The Journal of biological chemistry》2009,284(5):3219-3226
The bacterial protein DsbD transfers reductant from the cytoplasm to the
otherwise oxidizing environment of the periplasm. This reducing power is
required for several essential pathways, including disulfide bond formation
and cytochrome c maturation. DsbD includes a transmembrane domain
(tmDsbD) flanked by two globular periplasmic domains (nDsbD/cDsbD); each
contains a cysteine pair involved in electron transfer via a disulfide
exchange cascade. The final step in the cascade involves reduction of the
Cys103-Cys109 disulfide of nDsbD by Cys461 of
cDsbD. Here we show that a complex between the globular periplasmic domains is
trapped in vivo only when both are linked by tmDsbD. We have found
previously (Mavridou, D. A., Stevens, J. M., Ferguson, S. J.,
& Redfield, C. (2007) J. Mol. Biol. 370
,643
-658) that the attacking
cysteine (Cys461) in isolated cDsbD has a high
pKa value (10.5) that makes this thiol relatively
unreactive toward the target disulfide in nDsbD. Here we show using NMR that
active-site pKa values change significantly when cDsbD
forms a complex with nDsbD. This modulation of pKa values
is critical for the specificity and function of cDsbD. Uncomplexed cDsbD is a
poor nucleophile, allowing it to avoid nonspecific reoxidation; however, in
complex with nDsbD, the nucleophilicity of cDsbD increases permitting
reductant transfer. The observation of significant changes in active-site
pKa values upon complex formation has wider implications
for understanding reactivity in thiol:disulfide oxidoreductases.DsbD is a unique protein that transfers reductant across the cytoplasmic
membrane to the periplasm in many Gram-negative bacteria
(1,
2). Provision of reductant to
the periplasm is required because this compartment is otherwise considered to
be an oxidizing environment
(2). DsbD includes three
domains, each containing a pair of cysteine residues that perform a series of
disulfide exchange reactions (Fig.
1A). In the first step, the transmembrane domain (tmDsbD)
accepts electrons from thioredoxin in the cytoplasm; these are then
transferred to the periplasmic C-terminal domain (cDsbD) and finally to the
N-terminal domain (nDsbD), which is also located in the periplasm
(3-5).
nDsbD acts as a junction point for several pathways that require reductant,
including the general disulfide isomerase system and the pathway that is
thought to reduce the cysteine thiols of apocytochromes in the cytochrome
c biogenesis pathway
(6). In Gram-positive bacteria,
CcdA, an integral membrane protein, and ResA, which has a thioredoxin fold,
provide the reductant required for cytochrome c maturation
(7).Open in a separate windowFIGURE 1.Schematic representation of DsbD.
A, proposed pathway of
electron flow from thioredoxin (TrxA) in the cytoplasm, via the three
domains of DsbD, to the cytochrome c maturation (Ccm) and
disulfide bond isomerization pathways in the periplasm is shown. The crystal
structure of nDsbD is from Protein Data Bank code 1L6P
(8), cDsbD from Protein Data
Bank code 1UC7 (11), and the
nDsbD-cDsbD complex from Protein Data Bank code 1VRS
(12). The cyan boxes
indicate the thrombin cleavage sites introduced into full-length DsbD to allow
detection of the nDsbD-cDsbD complex following its formation in vivo.
The cysteine residues are shown in yellow. B, schematic
representation of the active site of cDsbD in the covalent complex with nDsbD
(12). Some active-site
residues of cDsbD are indicated in stick representation and the
inter-domain disulfide (Cys461-SS-Cys109) is shown in
yellow.Structural studies have sought to explain how DsbD functions and interacts
with its various partners. The structures of the two soluble periplasmic
domains have been determined (Fig.
1A, left). nDsbD has an immunoglobulin-like
structure (8,
9) and is the only known
thiol:disulfide oxidoreductase with this fold. cDsbD has the more typical
thioredoxin fold found in many oxidoreductases; this has the characteristic
active-site CXXC motif
(10,
11). A covalent complex
between single-cysteine variants of each of these two domains was produced
in vitro and its x-ray structure solved
(12), revealing the interface
between the two domains (Fig.
1A, right). Although this mixed disulfide is
accepted as a physiological intermediate in the function of DsbD, an in
vivo complex between the two soluble domains has not been reported
previously (3). Further
complexes between nDsbD and its other physiological partners have also been
trapped and their structures examined
(9,
13). Interestingly, all of the
interaction partners of nDsbD are thioredoxin-like proteins; similarities in
their folds are congruous with common interaction interfaces
(14). However, only cDsbD will
reduce nDsbD, whereas nDsbD will reduce several partners. This raises
questions about how the direction of reductant flow is maintained and
controlled within the series of disulfide-exchange reactions.As part of our structural and mechanistic characterization of DsbD and its
domains in solution, we have previously measured by NMR the
pKa values of the active-site cysteine pair,
Cys461 and Cys464, of cDsbD (numbered according to the
full-length Escherichia coli DsbD sequence)
(15). An unusually high
pKa value of 10.5 was measured for the N-terminal cysteine
of the CXXC motif, Cys461, and the pKa
value of the second cysteine, Cys464, was significantly higher than
the maximum pH value that was studied (pH 12.2). The pKa
value of 10.5 is the highest reported for the N-terminal cysteine of the
CXXC motif in a thioredoxin fold. The striking consequence of the
elevated pKa value is that the active-site cysteine of
cDsbD, Cys461, is not strongly nucleophilic, raising critical
questions about how this cysteine reacts with the disulfide in nDsbD. It was
demonstrated using site-directed mutagenesis that the negatively charged side
chains of Asp455 and Glu468, which are located close to
the CXXC motif (Fig.
1B), are responsible for the unusually high
pKa value of Cys461; mutation of one or both of
these residues to Asn and Gln, respectively, resulted in decreases in the
pKa value of Cys461 from 10.5 to 9.9 (E468Q),
to 9.3 (D455N), and to 8.6 (D455N/E468Q). The pKa values
for Asp455 were found to be 5.9 and 6.6 in oxidized and reduced
cDsbD; these values are significantly higher than the value of ∼4 for an
unperturbed aspartic acid. We postulated that the properties of the amino acid
side chains in the immediate environment of the cysteines in cDsbD would
change upon complex formation with nDsbD, changing the reactivity of the
cysteines and explaining how the reaction between the two domains is initiated
(15). Specifically, we
proposed that an increase in the pKa value of
Asp455 upon complex formation would lead to a decrease in the
pKa value of Cys461, thereby making it a better
nucleophile. Stirnimann et al.
(10) previously presented
pKa calculations suggesting an increase in the
Asp455 pKa value upon complex formation.The aim of this work has been to determine the molecular basis of the
control of the reactivity of the active-site cysteine residues in cDsbD, using
NMR to compare the active-site properties of cDsbD alone and in its
physiological complex with nDsbD. We demonstrate that the
pKa value of Asp455 is elevated by at least 1.1
pH units when cDsbD forms a complex with nDsbD. This modulation of the
pKa value is critical for the specificity and function of
cDsbD. These in vitro studies are complemented by in vivo
studies on complex formation, in which we have trapped the nDsbD-cDsbD complex
for the first time. The results of our experiments explain how the
intramolecular disulfide cascade within the soluble domains of DsbD functions,
and demonstrate the importance of the transmembrane domain in controlling and
facilitating complex formation between the soluble domains. 相似文献
74.
Active-site properties of the oxidized and reduced C-terminal domain of DsbD obtained by NMR spectroscopy 总被引:3,自引:0,他引:3
The periplasmic C-terminal domain of the Escherichia coli DsbD protein (cDsbD) has a thioredoxin fold. The two cysteine residues in the CXXC motif serve as the reductant for the disulfide bond of the N-terminal domain which can in turn act as a reductant for various periplasmic partners. The resulting disulfide bond in cDsbD is reduced via an unknown mechanism by the transmembrane helical domain of the protein. We show by NMR analysis of (13)C, (15)N-labelled cDsbD that the protein is rigid, is stable to extremes of pH and undergoes only localized conformational changes in the vicinity of the CXXC motif, and in adjacent regions of secondary structure, upon undergoing the reduced/oxidized transition. pK(a) values have been determined, using 2D NMR, for the N-terminal cysteine of the CXXC motif, Cys461, as well as for other active-site residues. It is demonstrated using site-directed mutagenesis that the negative charges of the side-chains of Asp455 and Glu468 in the active site contribute to the unusually high pK(a) value, 10.5, of Cys461. This value is higher than expected from knowledge of the reduction potential of cDsbD. In a double mutant of cDsbD, D455N/E468Q, the pK(a) value of Cys461 is lowered to 8.6, a value close to that expected for an unperturbed cysteine residue. The pK(a) value of the second cysteine in wild-type cDsbD, Cys464, is significantly higher than the maximum pH value that was studied (pH 12.2). 相似文献
75.
76.
77.
In many ecological communities, extinctions following habitat loss do not happen immediately. Understanding this delay is a major challenge, with conservation implications. In this issue, Otsu et al. show how landscape and management features affect the time lag. With this research as a starting point, we highlight the gaps and challenges still remaining in the study of extinction debt, especially in plant communities. 相似文献
78.
Canella Radea Aristeidis Parmakelis Vassilis Papadogiannis Despoina Charou Kostas A. Triantis 《ZooKeys》2013,(350):1-20
Hydrobioid freshwater gastropods were collected from mainland and insular Greece. Several threatened taxa, such as Graecoanatolica vegorriticola, Pseudamnicola negropontina, Pseudamnicola pieperi, Pseudobithynia eubooensis and Pseudoislamia balcanica, were recorded from new localities. Trichonia trichonica, which has been considered extinct from its type locality for the last twenty eight years, was re-discovered, whereas the presence of Daphniola exigua, G. vegorriticola, Marstoniopsis graeca, P. pieperi and Pseudobithynia trichonis in their type localities was verified. The taxonomic status of P. negropontina and the newly discovered populations of G. vegorriticola was elucidated using COI sequence data. The new data recorded during this survey indicate that the IUCN status of some Greek endemic hydrobioids needs to be updated. 相似文献
79.
Stuart D. Woodcock Karl Syson Richard H. Little Danny Ward Despoina Sifouna James K. M. Brown Stephen Bornemann Jacob G. Malone 《PLoS genetics》2021,17(4)
An important prelude to bacterial infection is the ability of a pathogen to survive independently of the host and to withstand environmental stress. The compatible solute trehalose has previously been connected with diverse abiotic stress tolerances, particularly osmotic shock. In this study, we combine molecular biology and biochemistry to dissect the trehalose metabolic network in the opportunistic human pathogen Pseudomonas aeruginosa PAO1 and define its role in abiotic stress protection. We show that trehalose metabolism in PAO1 is integrated with the biosynthesis of branched α-glucan (glycogen), with mutants in either biosynthetic pathway significantly compromised for survival on abiotic surfaces. While both trehalose and α-glucan are important for abiotic stress tolerance, we show they counter distinct stresses. Trehalose is important for the PAO1 osmotic stress response, with trehalose synthesis mutants displaying severely compromised growth in elevated salt conditions. However, trehalose does not contribute directly to the PAO1 desiccation response. Rather, desiccation tolerance is mediated directly by GlgE-derived α-glucan, with deletion of the glgE synthase gene compromising PAO1 survival in low humidity but having little effect on osmotic sensitivity. Desiccation tolerance is independent of trehalose concentration, marking a clear distinction between the roles of these two molecules in mediating responses to abiotic stress. 相似文献
80.
Antimutagenic properties of a polyphenol-enriched extract derived from sesame-seed perisperm 总被引:1,自引:0,他引:1
A polyphenolic mixture derived from sesame-seed perisperm (SSP) strongly reduced the mutagenicity of hydrogen peroxide (H(2)O(2)), sodium azide (NaN(3)), and benzo[a]pyrene (BaP) in strains TA100 and/or TA98 of Salmonella typhimurium. It exhibited desmutagenic activity against H(2)O(2), BaP in TA98 and/or TA100 and biomutagenic activity (apparently by affecting the DNA-repair system) against NaN(3) in strain TA100. According to in vitro experiments the polyphenolic mixture inhibited the activity of the CYP1A1 (EROD) enzyme responsible for the activation of BaP in the Ames' test, as well as that of the cytosolic enzyme GST. A cytosolic fraction from liver of male Wistar rats treated with either 20% SSP in the food, or 3mg or 6 mg of polyphenolic mixture/20 g food/day for a time period of 8 weeks reduced the mutagenic potential of BaP in strains TA100 and TA98, with the cytosolic fraction from rats treated with SSP causing the strongest reduction. Furthermore, a microsomal fraction from the 20% SSP-treated rats inhibited the mutagenicity of BaP in strains TA100 (26.3%) and TA98 (23%). In contrast, a microsomal fraction from rats treated with 3mg of polyphenolic mixture stimulated the mutagenicity of BaP in TA100 but reduced it in TA98, while for the microsomal fraction from rats treated with 6 mg of polyphenolic mixture, these effects on TA100 and TA98 were reversed. 相似文献