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1.
Johansson E Fanø M Bynck JH Neuhard J Larsen S Sigurskjold BW Christensen U Willemoës M 《The Journal of biological chemistry》2005,280(4):3051-3059
dCTP deaminase (EC 3.5.4.13) catalyzes the deamination of dCTP forming dUTP that via dUTPase is the main pathway providing substrate for thymidylate synthase in Escherichia coli and Salmonella typhimurium. dCTP deaminase is unique among nucleoside and nucleotide deaminases as it functions without aid from a catalytic metal ion that facilitates preparation of a water molecule for nucleophilic attack on the substrate. Two active site amino acid residues, Arg(115) and Glu(138), were identified by mutational analysis as important for activity in E. coli dCTP deaminase. None of the mutant enzymes R115A, E138A, or E138Q had any detectable activity but circular dichroism spectra for all mutant enzymes were similar to wild type suggesting that the overall structure was not changed. The crystal structures of wild-type E. coli dCTP deaminase and the E138A mutant enzyme have been determined in complex with dUTP and Mg(2+), and the mutant enzyme also with the substrate dCTP and Mg(2+). The enzyme is a third member of the family of the structurally related trimeric dUTPases and the bifunctional dCTP deaminase-dUTPase from Methanocaldococcus jannaschii. However, the C-terminal fold is completely different from dUTPases resulting in an active site built from residues from two of the trimer subunits, and not from three subunits as in dUTPases. The nucleotides are well defined as well as Mg(2+) that is tridentately coordinated to the nucleotide phosphate chains. We suggest a catalytic mechanism for the dCTP deaminase and identify structural differences to dUTPases that prevent hydrolysis of the dCTP triphosphate. 相似文献
2.
The bacteriophage T4 genome encodes most of its own enzymes for dNTP synthesis, which form a complex in infected Escherichia coli. The T4 thymidylate synthase (TS) and the T4 deoxycytidylate deaminase (CD) catalyze sequential reactions and are physically linked within this complex [McGaughey, K. M., Wheeler, L. J., Moore, J. T., Maley, G. F. , Maley, F., and Mathews, C. K. (1996) J. Biol. Chem. 271, 23037-23042]. From the crystal structure of T4TS [Finer-Moore, J. S., Maley, G. F., Maley, F., Montfort, W. R., and Stroud, R. M. (1994) Biochemistry 33, 15459-15468], it appears that three regions corresponding to insertions relative to E. coli TS lie on one side of the enzyme surface. We have investigated the residual activity of T4TS in response to complete deletion or substitution mutagenesis of these insertions. Two deletions generated in the small domain (residues 70-103) reduced the TS activity to 0.2% and 0.7% of the wild-type activity, with the latter able to complement growth of a thyA- E. coli strain in the absence of thymidine. By insertion of exogenous sequences variable in length and in sequence into these deletion mutants, enzyme activity increased to 44% that of the wild type. Restoration of the TS activity depended mostly on the hydrophobicity of the inserted residues. The sites of insertions also displayed distinct permissiveness for accommodating the exogenous insertions. Deletions and substitutions near the C-terminus resulted in complete inactivation of the T4TS. Proteolysis experiments revealed that the modified surface loops of the small domain were still accessible and flexible for protein-protein interactions. We have used ELISA to detect a physical association between T4TS and T4CD and compared the binding affinity of WT T4TS for two purified insertion mutants of T4CD. The results obtained showed that the native sequences of the small domain inserts are not required for T4TS/T4CD complex formation. 相似文献
3.
Complete amino acid sequence of an allosteric enzyme, T2 bacteriophage deoxycytidylate deaminase 总被引:4,自引:0,他引:4
The amino acid sequence of deoxycytidylate deaminase isolated from T2 phage-infected Escherichia coli has been determined. The enzyme is a hexamer, consisting of identical polypeptide subunits, each composed of 188 amino acids with a calculated Mr = 20,560. The primary structure was established by automatic Edman degradation of the intact carboxymethylated protein and of peptides derived from the protein by cleavage with cyanogen bromide, trypsin, chymotrypsin, the Staphylococcus aureus V8 protease, and 2-(2-nitrophenylsulfenyl)-3-methyl-3-bromoindolenine. Knowledge of the primary structure of deoxycytidylate deaminase should aid in determining the allosteric binding site of the negative effector, dTTP, recently reported (Maley, F., and Maley, G.F. (1982) J. Biol. Chem. 257, 11876-11878), and eventually that of the enzyme's positive regulator, dCTP, as well as its substrate. The deaminase has been crystallized through the use of polyethylene glycol; a scanning electron micrograph is presented. 相似文献
4.
Effect of Cleavage Mutants on Syncytium Formation Directed by the Wild-Type Fusion Protein of Newcastle Disease Virus 总被引:3,自引:0,他引:3
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The effects of Newcastle disease virus (NDV) fusion (F) glycoprotein cleavage mutants on the cleavage and syncytium-forming activity of the wild-type F protein were examined. F protein cleavage mutants were made by altering amino acids in the furin recognition region (amino acids 112 to 116) in the F protein of a virulent strain of NDV. Four mutants were made: Q114P replaced the glutamine residue with proline; K115G replaced lysine with glycine; double mutant K115G, R113G replaced both a lysine and an arginine with glycine residues; and a triple mutant, R112G, K115G, F117L, replaced three amino acids to mimic the sequence found in avirulent strains of NDV. All mutants except Q114P were cleavage negative and fusion negative. However, addition of exogenous trypsin cleaved all mutant F proteins and activated fusion. As expected for an oligomeric protein, the fusion-negative mutants had a dominant negative phenotype: cotransfection of wild-type and mutant F protein cDNAs resulted in an inhibition of syncytium formation. The presence of the mutant F protein did not inhibit cleavage of the wild-type protein. Furthermore, evidence is presented that suggests that the mutant protein and the wild-type protein formed heterooligomers. By measuring the syncytium-forming activity of the wild-type protein at various ratios of expression of mutant and wild-type protein, results were obtained that are most consistent with the notion that the size of the functionally active NDV F protein in these assays is a single oligomer, likely a trimer. That a larger oligomer, containing a mix of both wild-type and mutant F proteins, has partial activity cannot, however, be ruled out. 相似文献
5.
Thymark M Johansson E Larsen S Willemoës M 《Archives of biochemistry and biophysics》2008,470(1):20-26
In Escherichia coli and Salmonella typhimurium about 80% of the dUMP used for dTMP synthesis is derived from deamination of dCTP. The dCTP deaminase produces dUTP that subsequently is hydrolyzed by dUTPase to dUMP and diphosphate. The dCTP deaminase is regulated by dTTP that inhibits the enzyme by binding to the active site and induces an inactive conformation of the trimeric enzyme. We have analyzed the role of residues previously suggested to play a role in catalysis. The mutant enzymes R115Q, S111C, S111T and E138D were all purified and analyzed for activity. Only S111T and E138D displayed detectable activity with a 30- and 140-fold reduction in kcat, respectively. Furthermore, S111T and E138D both showed altered dTTP inhibition compared to wild-type enzyme. S111T was almost insensitive to the presence of dTTP. With the E138D enzyme the dTTP dependent increase in cooperativity of dCTP saturation was absent, although the dTTP inhibition itself was still cooperative. Modeling of the active site of the S111T enzyme indicated that this enzyme is restricted in forming the inactive dTTP binding conformer due to steric hindrance by the additional methyl group in threonine. The crystal structure of E138D in complex with dUTP showed a hydrogen bonding network in the active site similar to wild-type enzyme. However, changes in the hydrogen bond lengths between the carboxylate and a catalytic water molecule as well as a slightly different orientation of the pyrimidine ring of the bound nucleotide may provide an explanation for the reduced activity. 相似文献
6.
Three-dimensional structure of the R115E mutant of T4-bacteriophage 2'-deoxycytidylate deaminase 总被引:1,自引:0,他引:1
2'-Deoxycytidylate deaminase (dCD) converts deoxycytidine 5'-monophosphate (dCMP) to deoxyuridine 5'-monophosphate and is a major supplier of the substrate for thymidylate synthase, an important enzyme in DNA synthesis and a major target for cancer chemotherapy. Wild-type dCD is allosterically regulated by the end products of its metabolic pathway, deoxycytidine 5'-triphosphate and deoxythymidine 5'-triphosphate, which act as an activator and an inhibitor, respectively. The first crystal structure of a dCD, in the form of the R115E mutant of the T4-bacteriophage enzyme complexed with the active site inhibitor pyrimidin-2-one deoxyribotide, has been determined at 2.2 A resolution. This mutant of dCD is active, even in the absence of the allosteric regulators. The molecular topology of dCD is related to that of cytidine deaminase (CDA) but with modifications for formation of the binding site for the phosphate group of dCMP. The enzyme has a zinc ion-based mechanism that is similar to that of CDA. A second zinc ion that is present in bacteriophage dCD, but absent in mammalian dCD and CDA, is important for the structural integrity of the enzyme and for the binding of the phosphate group of the substrate or inhibitor. Although the R115E mutant of dCD is a dimer in solution, it crystallizes as a hexamer, mimicking the natural state of the wild-type enzyme. Residues 112 and 115, which are known to be important for the binding of the allosteric regulators, are found in a pocket that is at the intersubunit interfaces in the hexamer but distant from the substrate-binding site. The substrate-binding site is composed of residues from a single protein molecule and is sequestered in a deep groove. This groove is located at the outer surface of the hexamer but ends at the subunit interface that also includes residue 115. It is proposed that the absence of subunit interactions at this interface in the dimeric R115E mutant renders the substrate-binding site accessible. In contrast, for the wild-type enzyme, binding of dCTP induces an allosteric effect that affects the subunit interactions and results in an increase in the accessibility of the binding site. 相似文献
7.
8.
Active site mutants of human cyclophilin A separate peptidyl-prolyl isomerase activity from cyclosporin A binding and calcineurin inhibition. 总被引:12,自引:0,他引:12
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L. D. Zydowsky F. A. Etzkorn H. Y. Chang S. B. Ferguson L. A. Stolz S. I. Ho C. T. Walsh 《Protein science : a publication of the Protein Society》1992,1(9):1092-1099
Based on recent X-ray structural information, six site-directed mutants of human cyclophilin A (hCyPA) involving residues in the putative active site--H54, R55, F60, Q111, F113, and H126--have been constructed, overexpressed, and purified from Escherichia coli to homogeneity. The proteins W121A (Liu, J., Chen, C.-M., & Walsh, C.T., 1991a, Biochemistry 30, 2306-2310), H54Q, R55A, F60A, Q111A, F113A, and H126Q were assayed for cis-trans peptidyl-prolyl isomerase (PPIase) activity, their ability to bind the immunosuppressive drug cyclosporin A (CsA), and protein phosphatase 2B (calcineurin) inhibition in the presence of CsA. Results indicate that H54Q, Q111A, F113A, and W121A retain 3-15% of the catalytic efficiency (kcat/Km) of wild-type recombinant hCyPA. The remaining three mutants (R55A, F60A, and H126Q) each retain less than 1% of the wild-type catalytic efficiency, indicating participation by these residues in PPIase catalysis. Each of the mutants bound to a CsA affinity matrix. The mutants R55A, F60A, F113A, and H126Q inhibited calcineurin in the presence of CsA, whereas W121A did not. Although CsA is a competitive inhibitor of PPIase activity, it can complex with enzymatically inactive cyclophilins and inhibit the phosphatase activity of calcineurin. 相似文献
9.
De novo synthesis of thymidylate via deoxycytidine in dcd (dCTP deaminase) mutants of Escherichia coli.
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dcd (dCTP deaminase) mutants of Escherichia coli were reported not to require thymidine for growth even though most of the thymidylate that is synthesized de novo arises from cytosine nucleotides through a pathway involving dCTP deaminase. We found, however, that the fresh introduction of dcd mutations into many strains of E. coli produced a requirement for thymidine for optimum aerobic growth, but the mutants readily reverted to prototrophy via mutations in other genes. One such mutation was in deoA, the gene for deoxyuridine phosphorylase. However, a dcd deo mutant became thymidine dependent once again if a cdd mutation (affecting deoxycytidine deaminase) were introduced. The results indicate that dcd mutants utilize an alternative pathway of TMP synthesis in which deoxycytidine and deoxyuridine are intermediates. A cdd mutation blocks the pathway by preventing the conversion of deoxycytidine to deoxyuridine, whereas a deoA mutation enhances it by sparing deoxyuridine from catabolism. The deoxycytidine must arise from dCTP or dCDP via unknown steps. It is not known to what extent this pathway is utilized in wild-type cells, which, unlike the dcd mutants, do not accumulate dCTP. 相似文献
10.
At neutral pH, equine beta-lactoglobulin (ELG) is monomeric, whereas bovine beta-lactoglobulin (BLG) exists as a dimer. To understand the difference in the oligomerization properties between ELG and BLG, three mutants of ELG (LP, I, and LPI) were constructed by substituting amino acids responsible for important interactions at the dimer interface of BLG into ELG. The mutant LP has an AB loop mutation (S34A/E35Q), the mutant I has an I strand mutation (G145M/R146H/V147I/Q148R/I149L/V150S/P151F/D152N/L153P) and the mutant LPI includes both the LP and I mutations. The far- and near-UV CD spectra of the three mutants are similar to that of the wild-type ELG, indicating that the secondary and the tertiary structures of ELG are not significantly affected by the mutations. Ultracentrifuge analysis shows that all three mutants are monomeric at neutral pH, suggesting that the protein sequences in the AB loop and I strand of BLG alone cannot support dimerization of ELG. Thus, structural differences must exist between ELG and BLG that prevent the ELG mutants from forming the same interactions as BLG at the dimer interface. 相似文献
11.
Random mutagenesis of human cytochrome p450 2A6 and screening with indole oxidation products. 总被引:6,自引:0,他引:6
Cytochrome P450 (P450) 2A6 mutants from randomized libraries generated in the substrate recognition sequence (SRS) regions were screened in Escherichia coli on the basis of indole metabolism. SRS 3 and 4 libraries yielded colonies that produced indigo at least as well as wild-type (WT) P450 2A6, and some colonies were consistently more blue upon replating. One mutant, F209T, showed indole 3-hydroxylation WT. The double mutant L240C/N297Q consistently produced very blue colonies. Five mutants yielded mixtures of pigments from indole different than WT, as judged by visible spectra and HPLC of products. When bacteria expressing the mutants were grown in the presence of each of 26 substituted indoles, a variety of patterns of formation of different dyes was seen with several of the mutants. This approach has potential value in understanding P450 2A6 function and generating new dyestuffs and other products. 相似文献
12.
Effect of glycosylation on yeast invertase oligomer stability 总被引:5,自引:0,他引:5
Yeast external invertase is a glycoprotein that exists as a dimer that can associate to form tetramers, hexamers, and octamers (Chu, F., Watorek, W., and Maley, F. (1983) Arch. Biochem. Biophys. 223, 543-555; Esmon, P. C., Esmon, B. E., Schauer, I. E., Taylor, A., and Schekman, R. (1987) J. Biol. Chem., 262, 4395-4401), a process that is facilitated by the attached oligosaccharide chains. We have studied this association by high performance liquid chromatography on a gel filtration matrix, by which procedure wild-type bakers' yeast invertase gives two peaks, and invertase from a core mutant (mnn1 mnn9) of Saccharomyces cerevisiae X2180 gives three peaks. Concentration of an invertase solution by freezing drives the dimers into higher aggregates that, at 30 degrees C, re-equilibrate to a mixture of smaller forms, the composition of which depends on pH, concentration, and time. The invertase from a mutant, mnn1 mnn9 dpg1, which underglycosylates its glycoproteins and produces invertase with 4-7 oligosaccharide chains, forms oligomers of much lower stability than the mnn1 mnn9 invertase, which has 8-11 carbohydrate chains. Both of these mutants release external invertase from the periplasm into the medium during growth, but we conclude that defects in the cell wall structure may be more important in this release than an altered tendency of the invertases to aggregate. Investigation of aggregate formation by electron microscopy revealed that all invertases, including the internal nonglycosylated enzyme, form octamers under appropriate conditions. 相似文献
13.
Szczesna D Ghosh D Li Q Gomes AV Guzman G Arana C Zhi G Stull JT Potter JD 《The Journal of biological chemistry》2001,276(10):7086-7092
The effect of the familial hypertrophic cardiomyopathy mutations, A13T, F18L, E22K, R58Q, and P95A, found in the regulatory light chains of human cardiac myosin has been investigated. The results demonstrate that E22K and R58Q, located in the immediate extension of the helices flanking the regulatory light chain Ca(2+) binding site, had dramatically altered Ca(2+) binding properties. The K(Ca) value for E22K was decreased by approximately 17-fold compared with the wild-type light chain, and the R58Q mutant did not bind Ca(2+). Interestingly, Ca(2+) binding to the R58Q mutant was restored upon phosphorylation, whereas the E22K mutant could not be phosphorylated. In addition, the alpha-helical content of phosphorylated R58Q greatly increased with Ca(2+) binding. The A13T mutation, located near the phosphorylation site (Ser-15) of the human cardiac regulatory light chain, had 3-fold lower K(Ca) than wild-type light chain, whereas phosphorylation of this mutant increased the Ca(2+) affinity 6-fold. Whereas phosphorylation of wild-type light chain decreased its Ca(2+) affinity, the opposite was true for A13T. The alpha-helical content of the A13T mutant returned to the level of wild-type light chain upon phosphorylation. The phosphorylation and Ca(2+) binding properties of the regulatory light chain of human cardiac myosin are important for physiological function, and alteration any of these could contribute to the development of hypertrophic cardiomyopathy. 相似文献
14.
A Edixhoven-Bosdijk F W M de Rooij E de Baar-Heesakkers J H P Wilson 《Cellular and molecular biology, including cyto-enzymology》2002,48(8):861-866
To find an explanation for survival of homozygous or compound heterozygous variants of acute intermittent porphyria, we studied the three mutant forms of porphobilinogen deaminase (PBG-d) described in the four reported patients with homozygous acute intermittent porphyria. Wild-type human PBG-d and the PBG-d R167W, R167Q and R173Q mutants were expressed in Escherichia coli and the recombinant mutant human enzyme were examined for enzyme activity. Specific antibodies against human PBG-d detected the three human PBG-d mutants. All three had less than 2% of wild-type enzyme activity when examined under customary assay conditions (pH 8.0), but the R167W and R167Q mutants were found to have about 25% of normal activity when assayed at pH 7.0. This residual activity at a more physiological pH provides an explanation for survival when these mutations are inherited in a homozygous or compound heterozygous fashion. 相似文献
15.
Neuraminidase (NA) mutations conferring resistance to NA inhibitors were believed to compromise influenza virus fitness. Unexpectedly, an oseltamivir-resistant A/Brisbane/59/2007 (Bris07)-like H1N1 H275Y NA variant emerged in 2007 and completely replaced the wild-type (WT) strain in 2008-2009. The NA of such variant contained additional NA changes (R222Q, V234M and D344N) that potentially counteracted the detrimental effect of the H275Y mutation on viral fitness. Here, we rescued a recombinant Bris07-like WT virus and 4 NA mutants/revertants (H275Y, H275Y/Q222R, H275Y/M234V and H275Y/N344D) and characterized them in vitro and in ferrets. A fluorometric-based NA assay was used to determine Vmax and Km values. Replicative capacities were evaluated by yield assays in ST6Gal1-MDCK cells. Recombinant NA proteins were expressed in 293T cells and surface NA activity was determined. Infectivity and contact transmission experiments were evaluated for the WT, H275Y and H275Y/Q222R recombinants in ferrets. The H275Y mutation did not significantly alter Km and Vmax values compared to WT. The H275Y/N344D mutant had a reduced affinity (Km of 50 vs 12 μM) whereas the H275Y/M234V mutant had a reduced activity (22 vs 28 U/sec). In contrast, the H275Y/Q222R mutant showed a significant decrease of both affinity (40 μM) and activity (7 U/sec). The WT, H275Y, H275Y/M234V and H275Y/N344D recombinants had comparable replicative capacities contrasting with H275Y/Q222R mutant whose viral titers were significantly reduced. All studied mutations reduced the cell surface NA activity compared to WT with the maximum reduction being obtained for the H275Y/Q222R mutant. Comparable infectivity and transmissibility were seen between the WT and the H275Y mutant in ferrets whereas the H275Y/Q222R mutant was associated with significantly lower lung viral titers. In conclusion, the Q222R reversion mutation compromised Bris07-like H1N1 virus in vitro and in vivo. Thus, the R222Q NA mutation present in the WT virus may have facilitated the emergence of NAI-resistant Bris07 variants. 相似文献
16.
D. Xie S. Gulnik E. Gustchina B. Yu W. Shao W. Qoronfleh A. Nathan J. W. Erickson 《Protein science : a publication of the Protein Society》1999,8(8):1702-1707
The monomer-dimer equilibrium for the human immunodeficiency virus type 1 (HIV-1) protease has been investigated under physiological conditions. Dimer dissociation at pH 7.0 was correlated with a loss in beta-sheet structure and a lower degree of ANS binding. An autolysis-resistant mutant, Q7K/L33I/L63I, was used to facilitate sedimentation equilibrium studies at neutral pH where the wild-type enzyme is typically unstable in the absence of bound inhibitor. The dimer dissociation constant (KD) of the triple mutant was 5.8 microM at pH 7.0 and was below the limit of measurement (approximately 100 nM) at pH 4.5. Similar studies using the catalytically inactive D25N mutant yielded a KD value of 1.0 microM at pH 7.0. These values differ significantly from a previously reported value of 23 nM obtained indirectly from inhibitor binding measurements (Darke et al., 1994). We show that the discrepancy may result from the thermodynamic linkage between the monomer-dimer and inhibitor binding equilibria. Under conditions where a significant degree of monomer is present, both substrates and competitive inhibitors will shift the equilibrium toward the dimer, resulting in apparent increases in dimer stability and decreases in ligand binding affinity. Sedimentation equilibrium studies were also carried out on several drug-resistant HIV-1 protease mutants: V82F, V82F/I84V, V82T/I84V, and L90M. All four mutants exhibited reduced dimer stability relative to the autolysis-resistant mutant at pH 7.0. Our results indicate that reductions in drug affinity may be due to the combined effects of mutations on both dimer stability and inhibitor binding. 相似文献
17.
Farnesyl diphosphate synthase is a homodimer of subunits having typically two aspartate-rich motifs with two sets of substrate binding sites for an allylic diphosphate and isopentenyl diphosphate per molecule of a homodimeric enzyme. To determine whether each subunit contains an independent active site or whether the active sites are created by intersubunit interaction, we constructed several expression plasmids that overproduce hybrid-type heterodimers of Bacillus stearothermophilus FPP synthases constituting different types of mutated monomers, which exhibit little catalytic activity as homodimers, by combining two tandem fps genes for the manipulated monomer subunit with a highly efficient promoter trc within an overexpression pTrc99A plasmid. A heterodimer of a combination of subunits of the wild type and of R98E, a mutant subunit which exhibits little enzymatic activity as a dimer form (R98E)(2), exhibited 78% of the activity of the wild-type homodimer enzyme, (WT)(2). Moreover, when a hybrid-type heterodimeric dimer of FPP synthase mutant subunits (R98E/F220A) was prepared, the FPP synthase activity was 18- and 390-fold of that of each of the almost inactive mutants as a dimeric enzymes, (R98E)(2) and (F220A)(2) [Koyama, T., et al. (1995) Biochem. Biophys. Res. Commun. 212, 681-686], respectively. These results suggest that the subunits of the FPP synthase interact with each other to form a shared active site in the homodimer structure rather than an independent active site in each subunit. 相似文献
18.
Ano Bom AP Rangel LP Costa DC de Oliveira GA Sanches D Braga CA Gava LM Ramos CH Cepeda AO Stumbo AC De Moura Gallo CV Cordeiro Y Silva JL 《The Journal of biological chemistry》2012,287(33):28152-28162
Over 50% of all human cancers lose p53 function. To evaluate the role of aggregation in cancer, we asked whether wild-type (WT) p53 and the hot-spot mutant R248Q could aggregate as amyloids under physiological conditions and whether the mutant could seed aggregation of the wild-type form. The central domains (p53C) of both constructs aggregated into a mixture of oligomers and fibrils. R248Q had a greater tendency to aggregate than WT p53. Full-length p53 aggregated into amyloid-like species that bound thioflavin T. The amyloid nature of the aggregates was demonstrated using x-ray diffraction, electron microscopy, FTIR, dynamic light scattering, cell viabilility assay, and anti-amyloid immunoassay. The x-ray diffraction pattern of the fibrillar aggregates was consistent with the typical conformation of cross β-sheet amyloid fibers with reflexions of 4.7 Å and 10 Å. A seed of R248Q p53C amyloid oligomers and fibrils accelerated the aggregation of WT p53C, a behavior typical of a prion. The R248Q mutant co-localized with amyloid-like species in a breast cancer sample, which further supported its prion-like effect. A tumor cell line containing mutant p53 also revealed massive aggregation of p53 in the nucleus. We conclude that aggregation of p53 into a mixture of oligomers and fibrils sequestrates the native protein into an inactive conformation that is typical of a prionoid. This prion-like behavior of oncogenic p53 mutants provides an explanation for the negative dominance effect and may serve as a potential target for cancer therapy. 相似文献
19.
Kagawa T Watanabe N Mochizuki K Numari A Ikeno Y Itoh J Tanaka H Arias IM Mine T 《American journal of physiology. Gastrointestinal and liver physiology》2008,294(1):G58-G67
Progressive familial cholestasis (PFIC) 2 and benign recurrent intrahepatic cholestasis (BRIC) 2 are caused by mutations in the bile salt export pump (BSEP, ABCB11) gene; however, their prognosis differs. PFIC2 progresses to cirrhosis and requires liver transplantation, whereas BRIC2 is clinically benign. To identify the molecular mechanism(s) responsible for the phenotypic differences, eight PFIC2 and two BRIC2 mutations were introduced in rat Bsep, which was transfected in MDCK II cells. Taurocholate transport activity, protein expression, and subcellular distribution of these mutant proteins were studied in a polarized MDCK II monolayer. The taurocholate transport activity was approximately half of the wild-type (WT) in BRIC2 mutants (A570T and R1050C), was substantially less in two PFIC2 mutants (D482G and E297G), and was almost abolished in six other PFIC2 mutants (K461E, G982R, R1153C, R1268Q, 3767-3768insC, and R1057X). Bsep protein expression levels correlated closely with transport activity, except for R1057X. The half-life of the D482G mutant was shorter than that of the WT (1.35 h vs. 3.49 h in the mature form). BRIC2 mutants and three PFIC mutants (D482G, E297G, and R1057X) were predominantly distributed in the apical membrane. The other PFIC2 mutants remained intracellular. The R1057X mutant protein was stably expressed and trafficked to the apical membrane, suggesting that the COOH-terminal tail is required for transport activity but not for correct targeting. In conclusion, taurocholate transport function was impaired in proportion to rapid degradation of Bsep protein in the mutants, which were aligned in the following order: A570T and R1050C > D482G > E297G > K461E, G982R, R1153C, R1268Q, 3767-3768insC, and R1057X. These results may explain the phenotypic difference between BRIC2 and PFIC2. 相似文献
20.
McLaughlin LA Paine MJ Kemp CA Maréchal JD Flanagan JU Ward CJ Sutcliffe MJ Roberts GC Wolf CR 《The Journal of biological chemistry》2005,280(46):38617-38624
We have previously shown that Phe(120), Glu(216), and Asp(301) in the active site of cytochrome P450 2D6 (CYP2D6) play a key role in substrate recognition by this important drug-metabolizing enzyme (Paine, M. J., McLaughlin, L. A., Flanagan, J. U., Kemp, C. A., Sutcliffe, M. J., Roberts, G. C., and Wolf, C. R. (2003) J. Biol. Chem. 278, 4021-4027 and Flanagan, J. U., Maréchal, J.-D., Ward, R., Kemp, C. A., McLaughlin, L. A., Sutcliffe, M. J., Roberts, G. C., Paine, M. J., and Wolf, C. R. (2004) Biochem. J. 380, 353-360). We have now examined the effect of mutations of these residues on interactions of the enzyme with the prototypical CYP2D6 inhibitor, quinidine. Abolition of the negative charge at either or both residues 216 and 301 decreased quinidine inhibition of bufuralol 1'-hydroxylation and dextromethorphan O-demethylation by at least 100-fold. The apparent dissociation constants (K(d)) for quinidine binding to the wild-type enzyme and the E216D and D301E mutants were 0.25-0.50 microm. The amide substitution of Glu(216) or Asp(301) resulted in 30-64-fold increases in the K(d) for quinidine. The double mutant E216Q/D301Q showed the largest decrease in quinidine affinity, with a K(d) of 65 microm. Alanine substitution of Phe(120), Phe(481),or Phe(483) had only a minor effect on the inhibition of bufuralol 1'-hydroxylation and dextromethorphan O-demethylation and on binding. In contrast to the wild-type enzyme, a number of the mutants studied were found to be able to metabolize quinidine. E216F produced O-demethylated quinidine, and F120A and E216Q/D301Q produced both O-demethylated quinidine and 3-hydroxyquinidine metabolites. Homology modeling and molecular docking were used to predict the modes of quinidine binding to the wild-type and mutant enzymes; these were able to rationalize the experimental observations. 相似文献