| 1. |
|
|
| 2. |
- Coelho, Paulo G., et al.
(författare)
-
Osseointegration of metallic devices : Current trends based on implant hardware design
- 2014
-
Ingår i: Archives of Biochemistry and Biophysics. - : Elsevier. - 0003-9861 .- 1096-0384. ; 561, s. 99-108
-
Forskningsöversikt (refereegranskat)abstract
- Osseointegration of metallic devices has been one of the most successful treatments in rehabilitative dentistry and medicine over the past five decades. While highly successful, the quest for designing surgical instrumentation and associated implantable devices that hastens osseointegration has been perpetual and has often been approached as single variable preclinical investigations. The present manuscript presents how the interplay between surgical instrumentation and device macrogeometry not only plays a key role on both early and delayed stages of osseointegration, but may also be key in how efficient smaller length scale designing (at the micrometer and nanometer scale levels) may be in hastening early stages of osseointegration. (C) 2014 Elsevier Inc. All rights reserved.
|
|
| 3. |
- Fedulova, Natalia, 1980-, et al.
(författare)
-
Characterization of porcine Alpha-class glutathione transferase A1-1
- 2011
-
Ingår i: Archives of Biochemistry and Biophysics. - : Elsevier BV. - 0003-9861 .- 1096-0384. ; 507:2, s. 205-211
-
Tidskriftsartikel (refereegranskat)abstract
- An Alpha-class glutathione transferase (GST) has been cloned from pig gonads. In addition to two conservative point mutations our nucleotide sequence presents a frame shift resulting from a missing A as compared to a previously published porcine GST A1-1 sequence. The deduced C-terminal amino-acid segment of the protein differs between the two variants. Repeated sequencing of cDNA isolated from different tissuesand animals ruled out the possibility of a cloning artifact, and the deduced amino acid sequence ofour clone showed higher similarity to related mammalian GST sequences. Hereafter, we refer to ourcloned enzyme as GST A1-1 and to the previously published enzyme as GST A1-1*. The study of the tissue distribution of the GSTA1 mRNA revealed high expression levels in many organs, in particular adipose tissue, liver, and pituitary gland. Porcine GST A1-1 was expressed in Escherichia coli and its kinetic properties were determined using alternative substrates. The catalytic activity in steroid isomerization reactionswas at least 10-fold lower than the corresponding values for porcine GST A2-2, whereas the activity with 1-chloro-2,4-dinitrobenzene was approximately 8-fold higher. Differences in the H-site residues of mammalian Alpha-class GSTs may explain the catalytic divergence.
|
|
| 4. |
|
|
| 5. |
- Hoffmann, Inga, et al.
(författare)
-
7,8- and 5,8-linoleate diol synthases support the heterolytic scission of oxygen-oxygen bonds by different amide residues.
- 2013
-
Ingår i: Archives of Biochemistry and Biophysics. - : Elsevier BV. - 0003-9861 .- 1096-0384. ; 539:1, s. 87-91
-
Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract
- Linoleate diol synthases (LDS) are fungal dioxygenase-cytochrome P450 fusion enzymes. They oxidize 18:2n-6 sequentially to 8R-hydroperoxylinoleic acid (8R-HPODE) and 7S,8S- or 5S,8R-dihydroxylinoleic acids (DiHODE) by intramolecular oxygen transfer. The P450 domains contain a conserved sequence, Ala-Asn-Gln-Xaa-Gln, presumably located in the I-helices. The Asn938Leu replacement of 7,8-LDS of Gaeumannomyces graminis virtually abolished and the Asn938Asp and Asn938Gln replacements reduced the hydroperoxide isomerase activity. Gln941Leu and Gln941Glu substitutions had little effects. Replacements of the homologous Asn(887) and Gln(890) residues of 5,8-LDS of Aspergillus fumigatus yielded the opposite results. Asn887Leu and Asn887Gln of 5,8-LDS retained 5,8-DiHODE as the main metabolite with an increased formation of 6,8- and 8,11-DiHODE, whereas Gln890Leu almost abolished the 5,8-LDS activity. Replacement of Gln(890) with Glu also retained 5,8-DiHODE as the main product, but shifted oxygenation from C-5 to C-7 and C-11 and to formation of epoxyalcohols by homolytic scission of 8R-HPODE. P450 hydroxylases usually contain an "acid-alcohol" pair in the I-helices for the heterolytic scission of O-2 and formation of compound I (Por(+.) Fe(IV)=0) and water. The function of the acid-alcohol pair appears to be replaced by two different amide residues, Asn(938) of 7,8-LDS and Gln(890) of 5,8-LDS, for heterolysis of 8R-HPODE to generate compound I.
|
|
| 6. |
- Hoffmann, Inga, 1984-, et al.
(författare)
-
Expression of 5,8-LDS of Aspergillus fumigatus and its dioxygenase domain : a comparison with 7,8-LDS, 10-dioxygenase, and cyclooxygenase
- 2011
-
Ingår i: Archives of Biochemistry and Biophysics. - : Elsevier BV. - 0003-9861 .- 1096-0384. ; 506:2, s. 216-222
-
Tidskriftsartikel (refereegranskat)abstract
- 5,8-Linoleate diol synthase (5,8-LDS) of Aspergillus fumigatus was cloned, expressed, and compared with 7,8-LDS of the Take-all fungus. Replacements of Tyr and Cys in the conserved YRWH and FXXGPHXCLG sequences abolished 8R-dioxygenase (8-DOX) and hydroperoxide isomerase activities, respectively. The predicted α-helices of LDS were aligned with α-helices of cyclooxygenase-1 (COX-1) to identify the 8-DOX domains. N-terminal expression constructs of 5,8- and 7,8-LDS (674 of 1079, and 673 of 1165 residues), containing one additional α-helix compared to cyclooxygenase-1, yielded prominent 8R-DOX activities with apparently unchanged or slightly lower substrate affinities, respectively. Val-328 of 5,8-LDS did not influence the position of oxygenation in contrast to the homologous residues Val-349 of COX-1 and Leu-384 of 10R-dioxygenase. We conclude that ∼675 amino acids are sufficient to support 8-DOX activity.
|
|
| 7. |
- Horvath, Istvan, et al.
(författare)
-
Modulation of α-synuclein fibrillization by ring-fused 2-pyridones : templation and inhibition involve oligomers with different structure
- 2013
-
Ingår i: Archives of Biochemistry and Biophysics. - : Elsevier. - 0003-9861 .- 1096-0384. ; 532:2, s. 84-90
-
Tidskriftsartikel (refereegranskat)abstract
- In a recent study we discovered that a ring-fused 2-pyridone compound triggered fibrillization of a key protein in Parkinson's disease, α-synuclein. To reveal how variations in compound structure affect protein aggregation, we now prepared a number of strategic analogs and tested their effects on α-synuclein amyloid fiber formation in vitro. We find that, in contrast to the earlier templating effect, some analogs inhibit α-synuclein fibrillization. For both templating and inhibiting compounds, the key species formed in the reactions are α-synuclein oligomers that contain compound. Despite similar macroscopic appearance, the templating and inhibiting oligomers are distinctly different in secondary structure content. When the inhibitory oligomers are added in seed amounts, they inhibit fresh α-synuclein aggregation reactions. Our study demonstrates that small chemical changes to the same central fragment can result in opposite effects on protein aggregation.
|
|
| 8. |
- Jernerén, Fredrik, et al.
(författare)
-
Linoleate 9R-dioxygenase and allene oxide synthase activities of Aspergillus terreus
- 2010
-
Ingår i: Archives of Biochemistry and Biophysics. - : Elsevier BV. - 0003-9861 .- 1096-0384. ; 495:1, s. 67-73
-
Tidskriftsartikel (refereegranskat)abstract
- Oxygenation of linoleic acid by Aspergillus terreus was studied with LC-MS/MS. 9(R)-Hydroperoxy-10(E),12(Z)-octadecadienoic acid (9R-HpODE) was identified along with 10(R)-hydroxy-8(E),12(Z)-octadecadienoic acid and variable amounts of 8(R)-hydroxy-9(Z),12(Z)-octadecadienoic acid. 9R-HpODE was formed from [11S-2H]18:2n-6 with loss of the deuterium label, suggesting antarafacial hydrogen abstraction and oxygenation. Two polar metabolites were identified as 9-hydroxy-10-oxo-12(Z)-octadecenoic acid (alpha-ketol) and 13-hydroxy-10-oxo-11(E)-octadecenoic acid (gamma-ketol), likely formed by spontaneous hydrolysis of an unstable allene oxide, 9(R),10-epoxy-10,12(Z)-octadecadienoic acid. alpha-Linolenic acid and 20:2n-6 were oxidized to hydroperoxy fatty acids at C-9 and C-11, respectively, but alpha- and gamma-ketols of these fatty acids could not be detected. The genome of A. terreus lacks lipoxygenases, but contains genes homologous to 5,8-linoleate diol synthases and linoleate 10R-dioxygenases of aspergilli. Our results demonstrate that linoleate 9R-dioxygenase linked to allene oxide synthase activities can be expressed in fungi.
|
|
| 9. |
- Josephy, P. David, et al.
(författare)
-
Functional studies of single-nucleotide polymorphic variants of human glutathione transferase T1-1 involving residues in the dimer interface
- 2011
-
Ingår i: Archives of Biochemistry and Biophysics. - : Elsevier BV. - 0003-9861 .- 1096-0384. ; 513:2, s. 87-93
-
Tidskriftsartikel (refereegranskat)abstract
- Glutathione transferase T1-1 catalyses detoxication and bioactivation processes in which glutathione conjugates are formed from endogenous and xenobiotic substrates, including alkylating agents and halogenated alkanes. Although the common null polymorphism of the human GSTT1 gene has been studied extensively, little is known about the consequences of GSTT1 single-nucleotide polymorphisms (SNPs). Here, we have examined the effects of two SNPs that alter amino acid residues in the dimer interface of the GST T1-1 protein and one that causes a conservative substitution in the core of the subunit. Variant proteins were expressed in an Escherichia coli strain in which the metabolism of ethylene dibromide to a glutathione conjugate leads to lacZ reversion mutations. We measured the kinetic properties of the enzymes with the characteristic substrate 1,2-epoxy-3-(p-nitrophenoxy)propane (EPNP) and determined the specific activities with several other substrates. Circular dichroism spectroscopy was used to measure protein thermal denaturation profiles. Variant T104P, which has been reported as inactive, showed weak but detectable activity with each substrate. Variant R76S was expressed at lower levels and showed much-reduced thermal stability. The results are interpreted in the context of the three-dimensional structure of human GST T1-1.
|
|
| 10. |
- Larsson, Anna-Karin, et al.
(författare)
-
Molecular evolution of Theta-class glutathione transferase for enhanced activity with the anticancer drug 1,3-bis-(2-chloroethyl)-1-nitrosourea and other alkylating agents
- 2010
-
Ingår i: Archives of Biochemistry and Biophysics. - : Elsevier BV. - 0003-9861 .- 1096-0384. ; 497:1-2, s. 28-34
-
Tidskriftsartikel (refereegranskat)abstract
- Glutathione transferase (GST) displaying enhanced activity with the cytostatic drug 1,3-bis-(2-chloroethyl)-1-nitrosourea (BCNU) and structurally related alkylating agents was obtained by molecular evolution. Mutant libraries created by recursive recombination of cDNA coding for human and rodent Theta-class GSTs were heterologously expressed in Escherichia coli and screened with the surrogate substrate 4-nitrophenethyl bromide (NPB) for enhanced alkyltransferase activity. A mutant with a 70-fold increased catalytic efficiency with NPB, compared to human GST T1-1, was isolated. The efficiency in degrading BCNU had improved 170-fold, significantly more than with the model substrate NPB. The enhanced catalytic activity of the mutant GST was also 2-fold higher with BCNU than wild-type mouse GST T1-1, which is 80-fold more efficient than wild-type human GST T1-1. We propose that GSTs catalyzing inactivation of anticancer drugs may find clinical use in protecting sensitive normal tissues to toxic side-effects in treated patients, and as selectable markers in gene therapy.
|
|
| 11. |
- Lindberg, Diana, 1970-, et al.
(författare)
-
Mutations in salt-bridging residues at the interface of the core and lid domains of epoxide hydrolase StEH1 affect regioselectivity, protein stability and hysteresis
- 2010
-
Ingår i: Archives of Biochemistry and Biophysics. - Amsterdam : Elsevier. - 0003-9861 .- 1096-0384. ; 495:2, s. 165-173
-
Tidskriftsartikel (refereegranskat)abstract
- Epoxide hydrolase, StEH1, shows hysteretic behavior in the catalyzed hydrolysis of trans-2-methylstyrene oxide (2-MeSO)(1). Linkage between protein structure dynamics and catalytic function was probed in mutant enzymes in which surface-located salt-bridging residues were substituted. Salt-bridges at the interface of the alpha/beta-hydrolase fold core and lid domains, as well as between residues in the lid domain, between Lys(179)Asp(202), Glu(215)-Arg(41) and Arg(236)-Glu(136) were disrupted by mutations, K179Q E215Q, R236Q and R236Q. All mutants displayed enzyme activity with styrene oxide (SO) and 2-MeSO when assayed at 30 degrees C. Disruption of salt-bridges altered the rates for isomerization between distinct Michaelis complexes, with (1R,2R)-2-MeSO as substrate, presumably as a result of increased dynamics of involved protein segments. Another indication of increased flexibility was a lowered thermostability in all mutants. We propose that the alterations to regioselectivity in these mutants derive from an increased mobility in protein segments otherwise stabilized by salt bridging interactions.
|
|
| 12. |
- Lindqvist, Johan, et al.
(författare)
-
Sexually dimorphic myofilament function in a mouse model of nemaline myopathy
- 2014
-
Ingår i: Archives of Biochemistry and Biophysics. - : Elsevier BV. - 0003-9861 .- 1096-0384. ; 564, s. 37-42
-
Tidskriftsartikel (refereegranskat)abstract
- Nemaline myopathy, the most common congenital myopathy, is characterized by mutations in genes encoding myofilament proteins such as skeletal alpha-actin. These mutations are thought to ultimately lead to skeletal muscle weakness. Interestingly, some of the mutations appear to be more potent in males than in females. The underlying mechanisms remain obscure but may be related to sex-specific differences in the myofilament function of both limb and respiratory muscles. To verify this, in the present study, we used skeletal muscles (tibialis anterior and diaphragm) from a transgenic mouse model harbouring the His40Tyr amino acid substitution in skeletal alpha-actin. In this animal model, 60% of males die by 13weeks of age (the underlying causes of death are obscure but probably due to respiratory insufficiency) whereas females have a normal lifespan. By recording and analysing the mechanics of membrane-permeabilized myofibres, we only observed sex-related differences in the tibialis anterior muscles. Indeed, the concomitant deficits in maximal steady-state isometric force and stiffness of myofibres were less exacerbated in transgenic females than in males, potentially explaining the lower potency in limb muscles. However, the absence of sex-difference in the diaphragm muscles was rather unexpected and suggests that myofilament dysfunction does not solely underlie the sexually dimorphic phenotypes.
|
|
| 13. |
- Luo, Jinghui, et al.
(författare)
-
Ribonucleotide reductase inhibition by p-alkoxyphenols studied by molecular docking and molecular dynamics simulations.
- 2011
-
Ingår i: Archives of Biochemistry and Biophysics. - : Elsevier BV. - 0003-9861 .- 1096-0384. ; 516:1, s. 29-34
-
Tidskriftsartikel (refereegranskat)abstract
- Ribonucleotide reductase (RNR) is necessary for production of the precursor deoxyribonucleotides for DNA synthesis. Class Ia RNR functions via a stable free radical in one of the two components protein R2. The enzyme mechanism involves long range (proton coupled) electron transfer between protein R1 and the tyrosyl radical in protein R2. Earlier experimental studies showed that p-alkoxyphenols inhibit RNR. Here, molecular docking and molecular dynamics simulations involving protein R2 suggest an inhibition mechanism for p-alkoxyphenols . A low energy binding pocket is identified in protein R2. The preferred configuration provides a structural basis explaining their specific binding to the Escherichia coli and mouse R2 proteins. Trp48 (E. coli numbering), on the electron transfer pathway, is involved in the interactions with the inhibitors. The relative order of the binding energies calculated for the phenol derivatives to protein R2 is correlated with earlier experimental data on inhibition efficiency, in turn related to increasing size of the hydrophobic alkyl substituents. Using the configuration identified by molecular docking as a starting point for molecular dynamics simulations, we find that the p-allyloxyphenol interrupts the catalytic electron transfer pathway of the R2 protein by forming hydrogen bonds with Trp48 and Asp237, thus explaining the inhibitory activity of p-alkoxyphenols.
|
|
| 14. |
- Löfgren Söderberg, Kajsa, et al.
(författare)
-
Targeting prion propagation using peptide constructs with signal sequence motifs
- 2014
-
Ingår i: Archives of Biochemistry and Biophysics. - : Elsevier BV. - 0003-9861 .- 1096-0384. ; 564, s. 254-261
-
Tidskriftsartikel (refereegranskat)abstract
- Synthetic peptides with sequences derived from the cellular prion protein (PrPc) unprocessed N-terminus are able to counteract the propagation of proteinase K resistant prions (PrPRes, indicating the presence of the prion isoform of the prion protein) in cell cultures (Lofgren et al., 2008). The anti-prion peptides have characteristics like cell penetrating peptides (CPPs) and consist of the prion protein hydrophobic signal sequence followed by a polycationic motif (residues KKRPKP), in mouse PrPc corresponding to residues 1-28. Here we analyze the sequence elements required for the anti-prion effect of KKRPKP-conjugates. Neuronal GT1-1 cells were infected with either prion strain RML or 22L Variable peptide constructs originating from the mPrP(1-28) sequence were analyzed for anti-prion effects, measured as disappearance of proteinase K resistant prions (PrPRes) in the infected cell cultures. We find that even a 5 amino acid N-terminal shortening of the signal peptide abolishes the anti-prion effect. We show that the signal peptide from PrPc can be replaced with the signal peptide from the Neural cell adhesion molecule-1; NCAMl(1-19), with a retained capacity to reduce PrPRes levels. The anti-prion effect is lost if the polycationic N-terminal PrPc-motif is conjugated to any conventional CPP, such as TAT(48-60), transportan-10 or penetratin. We propose a mechanism by which a signal peptide from a secretory or cell surface protein acts to promote the transport of a prion-binding polycationic PrPc-motif to a subcellular location where prion conversion occurs (most likely the Endosome Recycling Compartment), thereby targeting prion propagation.
|
|
| 15. |
|
|
| 16. |
- Nahálková, Jarmila, 1969-, et al.
(författare)
-
TPPII, MYBBP1A and CDK2 form a protein–protein interaction network
- 2014
-
Ingår i: Archives of Biochemistry and Biophysics. - : Elsevier. - 0003-9861 .- 1096-0384. ; 564, s. 128-135
-
Tidskriftsartikel (refereegranskat)abstract
- Tripeptidyl-peptidase II (TPPII) is an aminopeptidase with suggested regulatory effects on cell cycle, apoptosis and senescence. A protein–protein interaction study revealed that TPPII physically interacts with the tumor suppressor MYBBP1A and the cell cycle regulator protein CDK2. Mutual protein–protein interaction was detected between MYBBP1A and CDK2 as well. In situ Proximity Ligation Assay (PLA) using HEK293 cells overexpressing TPPII forming highly enzymatically active oligomeric complexes showed that the cytoplasmic interaction frequency of TPPII with MYBBP1A increased with the protein expression of TPPII and using serum-free cell growth conditions. A specific reversible inhibitor of TPPII, butabindide, suppressed the cytoplasmic interactions of TPPII and MYBBP1A both in control HEK293 and the cells overexpressing murine TPPII. The interaction of MYBBP1A with CDK2 was confirmed by in situPLA in two different mammalian cell lines. Functional link between TPPII and MYBBP1A has been verified by gene expression study during anoikis, where overexpression of TPP II decreased mRNA expression level of MYBBP1A at the cell detachment conditions. All three interacting proteins TPPII, MYBBP1A and CDK2 have been previously implicated in the research for development of tumor-suppressing agents. This is the first report presenting mutual protein–protein interaction network of these proteins.
|
|
| 17. |
- Nilsson, Tomas, et al.
(författare)
-
LC-MS/MS analysis of epoxyalcohols and epoxides of arachidonic acid and their oxygenation by recombinant CYP4F8 and CYP4F22
- 2010
-
Ingår i: Archives of Biochemistry and Biophysics. - : Elsevier BV. - 0003-9861 .- 1096-0384. ; 494:1, s. 64-71
-
Tidskriftsartikel (refereegranskat)abstract
- CYP4F22 and CYP4F8 are expressed in epidermis, and mutations of CYP4F22 are associated with lamellar ichthyosis. Epoxyalcohols (HEETs) and epoxides (EETs) of 20:4n-6 appear to be important for the water permeability barrier of skin. Our aim was to study the MS/MS spectra and fragmentation of these compounds and to determine whether they were oxidized by CYP4F22 or CYP4F8 expressed in yeast. HEETs were prepared from 15-hydroperoxyeicosatetraenoic acid (15-HPETE), 12-HPETE, and their [(2)H(8)]labeled isotopomers, and separated by normal phase-HPLC with MS/MS analysis. CYP4F22 oxygenated 20:4n-6 at C-18, whereas metabolites of HEETs could not be identified. CYP4F8 formed omega3 hydroxy metabolites of HEETs derived from 12R-HPETE with 11,12-epoxy-10-hydroxy configuration, but not HEETs derived from 15S-HPETE. 8,9-EET and 11,12-EET were also subject to omega3 hydroxylation by CYP4F8. We conclude that CYP4F8 and CYP4F22 oxidize 20:4n-6 and that CYP4F8 selectively oxidizes 8,9-EET, 11,12-EET, and 10,11R,12R-HEET at the omega3 position.
|
|
| 18. |
- Norlin, Maria, et al.
(författare)
-
Androgen receptor-mediated regulation of the anti-atherogenic enzyme CYP27A1 involves the JNK/c-jun pathway
- 2011
-
Ingår i: Archives of Biochemistry and Biophysics. - : Elsevier BV. - 0003-9861 .- 1096-0384. ; 506:2, s. 236-241
-
Tidskriftsartikel (refereegranskat)abstract
- CYP27A1, an enzyme with several important roles in cholesterol homeostasis and vitamin D3 metabolism, has been ascribed anti-atherogenic properties. This study addresses an important problem regarding how this enzyme, involved in cholesterol metabolism in the liver and peripheral tissues, is regulated. Our results identify the human CYP27A1 gene as a new target for the JNK/c-jun pathway. Initial experiments showed that an inhibitor of c-Jun N-terminal kinase (JNK) downregulated basal CYP27A1 promoter activity whereas overexpression of JNK slightly enhanced promoter activity. Androgen receptor (AR)-mediated upregulation of mRNA levels and endogenous enzyme activity was recently reported. In the present study, the AR antagonist nilutamide blocked the androgen induction of CYP27A1. The present data revealed that inhibition of the JNK/c-jun pathway abolishes the AR-mediated effect on CYP27A1 transcription and enzyme activity, whereas overexpression of JNK markedly increased androgenic upregulation of CYP27A1. In conclusion, the current results indicate involvement of the JNK/c-jun pathway in AR-mediated upregulation of human CYP27A1. The link to JNK signaling is interesting since inflammatory processes may upregulate CYP27A1 to clear cholesterol from peripheral tissues.
|
|
| 19. |
|
|
| 20. |
- Palmieri-Thiers, Cynthia, et al.
(författare)
-
Identification of putative residues involved in the accessibility of the substrate-binding site of lipoxygenase by site-directed mutagenesis studies
- 2011
-
Ingår i: Archives of Biochemistry and Biophysics. - : Elsevier BV. - 0003-9861 .- 1096-0384. ; 509:1, s. 82-89
-
Tidskriftsartikel (refereegranskat)abstract
- Lipoxygenases (LOXs) are a class of widespread dioxygenases catalyzing the hydroperoxidation of polyunsaturated fatty acids (PUFA). Recently, we isolated a cDNA encoding a LOX, named olive LOX1, from olive fruit of which the deduced amino acid sequence shows more than 50% identity with plant LOXs. In the present study, a model of olive LOX1 based on the crystal structure of soybean LOX-1 as template has been generated and two bulky amino acid residues highly conserved in LOXs (Phe277) and in plant LOXs (Tyr280), located at the putative entrance of catalytic site were identified. These residues may perturb accessibility of the substrate-binding site and therefore were substituted by less space-filling residues. Kinetic studies using linoleic and linolenic acids as substrates were carried out on wild type and mutants. The results show that the removal of steric bulk at the entrance of the catalytic site induces an increase of substrate affinity and of catalytic efficiency, and demonstrate that penetration of substrates into active site of olive LOX1 requires the movement of the side chains of the Phe277 and Tyr280 residues. This study suggests the involvement of these residues in the accessibility of the substrate-binding site in the lipoxygenase family.
|
|
| 21. |
|
|
| 22. |
- Ryge, Marija Rakonjac, et al.
(författare)
-
A mutation interfering with 5-lipoxygenase domain interaction leads to increased enzyme activity
- 2014
-
Ingår i: Archives of Biochemistry and Biophysics. - : Elsevier BV. - 0003-9861 .- 1096-0384. ; 545, s. 179-185
-
Tidskriftsartikel (refereegranskat)abstract
- 5-Lipoxygenase (5-LOX) catalyzes two steps in conversion of arachidonic acid to proinflammatory leukotrienes. Lipoxygenases, including human 5-LOX, consist of an N-terminal C2-like beta-sandwich and a catalytic domain. We expressed the 5-LOX domains separately, these were found to interact in the yeast two-hybrid system. The 5-LOX structure suggested association between Arg(101) in the beta-sandwich and Asp(166) in the catalytic domain, due to electrostatic interaction as well as hydrogen bonds. Indeed, mutagenic replacements of these residues led to loss of two-hybrid interaction. Interestingly, when Arg(101) was mutated to Asp in intact 5-LOX, enzyme activity was increased. Thus, higher initial velocity of the reaction (v(init)) and increased final amount of products were monitored for 5-LOX-R101D, at several different assay conditions. In the 5-LOX crystal structure, helix alpha 2 and adjacent loops (including Asp(166)) of the 5-LOX catalytic domain has been proposed to form a flexible lid controlling access to the active site, and lid movement would be determined by bonding of lid residues to the C2-like beta-sandwich. The more efficient catalysis following disruption of the R101-D166 ionic association supports the concept of such a flexible lid in human 5-LOX. (C) 2014 Elsevier Inc. All rights reserved.
|
|
| 23. |
- Santacruz-Perez, Carolina, et al.
(författare)
-
A specific interdomain interaction preserves the structural and binding properties of the ModA protein from the phytopathogen Xanthomonas citri domain interaction and transport in ModA
- 2013
-
Ingår i: Archives of Biochemistry and Biophysics. - : Elsevier BV. - 0003-9861 .- 1096-0384. ; 539:1, s. 20-30
-
Tidskriftsartikel (refereegranskat)abstract
- The periplasmic-binding proteins in ATP-binding cassette systems (ABC Transporters) are responsible for the capture and delivery of ligands to their specific transporters, triggering a series of ATP-driven conformational changes that leads to the transport of the ligand. Structurally consisting of two lobes, the proteins change conformation after interaction with the ligand. The structure of the molybdate-binding protein (ModA) from Xanthomonas citri, bound to molybdate, was previously solved by our group and an interdomain interaction, mediated by a salt bridge between K127 and D59, apparently supports the binding properties and keeps the domains closed. To determinate the importance of this interaction, we built two ModA mutants, K127S and D59A, and analysed their functional and structural properties. Based on a set of spectroscopic experiments, crystallisation trials, structure determination and molecular dynamics (MD) simulations, we showed that the salt bridge is essential to maintain the structure and binding properties. Additionally, the MD simulations revealed that this mutant adopted a more compact structure that packed down the ligand-binding pocket. From the closed bound to open structure, the positioning of the helices forming the dipole and the salt bridge are essential to induce an intermediate state.
|
|
| 24. |
- Schedin-Weiss, Sophia, et al.
(författare)
-
Kinetic evidence that allosteric activation of antithrombin by heparin is mediated by two sequential conformational changes
- 2010
-
Ingår i: Archives of Biochemistry and Biophysics. - : Elsevier BV. - 0003-9861 .- 1096-0384. ; 504:2, s. 169-176
-
Tidskriftsartikel (refereegranskat)abstract
- The serpin, antithrombin, requires allosteric activation by a sequence-specific pentasaccharide unit of heparin or heparan sulfate glycosaminoglycans to function as an anticoagulant regulator of blood clotting proteases. Surprisingly, X-ray structures have shown that the pentasaccharide produces similar induced-fit changes in the heparin binding site of native and latent antithrombin despite large differences in the heparin affinity and global conformation of these two forms. Here we present kinetic evidence for similar induced-fit mechanisms of pentasaccharide binding to native and latent antithrombins and kinetic simulations which together support a three-step mechanism of allosteric activation of native antithrombin involving two successive conformational changes. Equilibrium binding studies of pentasaccharide interactions with native and latent antithrombins and the salt dependence of these interactions suggest that each conformational change is associated with distinct spectroscopic changes and is driven by a progressively better fit of the pentasaccharide in the binding site. The observation that variant antithrombins that cannot undergo the second conformational change bind the pentasaccharide like latent antithrombin and are partially activated suggests that both conformational changes contribute to allosteric activation, in agreement with a recently proposed model of allosteric activation.
|
|
| 25. |
- Wennman, Anneli, et al.
(författare)
-
Kinetic investigation of the rate-limiting step of manganese- and iron-lipoxygenases
- 2014
-
Ingår i: Archives of Biochemistry and Biophysics. - : Elsevier BV. - 0003-9861 .- 1096-0384. ; 555, s. 9-15
-
Tidskriftsartikel (refereegranskat)abstract
- Lipoxygenases (LOX) oxidize polyunsaturated fatty acids to hydroperoxides, which are generated by proton coupled electron transfer to the metal center with (FeOH-)-O-III or (MnOH-)-O-III. Hydrogen abstraction by (FeOH-)-O-III of soybean LOX-1 (sLOX-1) is associated with a large deuterium kinetic isotope effect (D-KIE). Our goal was to compare the D-KIE and other kinetic parameters at different temperatures of sLOX-1 with 13R-LOX with catalytic manganese (13R-MnLOX). The reaction rate and the D-KIE of sLOX-1 with unlabeled and [11-H-2(2)]18:2n-6 were almost temperature independent with an apparent D-KIE of similar to 56 at 30 degrees C, which is in agreement with previous studies. In contrast, the reaction rate of 13R-MnLOX increased 7-fold with temperature (8-50 degrees C), and the apparent D-KIE decreased linearly from similar to 38 at 8 degrees C to similar to 20 at 50 degrees C. The kinetic lag phase of 13R-MnLOX was consistently extended at low temperatures. The Phe337Ile mutant of 13R-MnLOX, which catalyzes antarafacial hydrogen abstraction and oxygenation in analogy with sLOX-1, retained the large D-KIE and its temperature-dependent reaction rate. The kinetic differences between 13R-MnLOX and sLOX-1 may be due to protein dynamics, hydrogen donor-acceptor distances, and to the metal ligands, which may not equalize the 0.7 V-gap between the redox potentials of the free metals.
|
|
| 26. |
|
|
| 27. |
- Nurmemmedov, Elmar, et al.
(författare)
-
Kinetic behaviour of WT 1's zinc finger domain in binding to the alpha-actinin-1 mRNA.
- 2010
-
Ingår i: Archives of Biochemistry and Biophysics. - : Elsevier BV. - 0003-9861. ; 497, s. 21-27
-
Tidskriftsartikel (refereegranskat)abstract
- The zinc finger transcription factor Wilms tumour protein (WT 1) is known for its essential involvement in the development of the genitourinary system as well as of other organs and tissues. WT 1 is capable of selectively binding either DNA or mRNA targets. A KTS insertion due to alternative splicing between the zinc fingers 3 and 4 and an unconventional zinc finger 1 are the unique features that distinguish WT 1 from classical DNA-binding C(2)H(2)-type zinc finger proteins. The DNA binding characteristics of WT 1 are well studied. Due to lack of information about its native RNA targets, no extensive research has been directed at how WT 1 binds RNA. Using surface plasmon resonance, this study attempts to understand the binding behaviour of WT 1 zinc fingers with its recently reported and first putative mRNA target, ACT 34, whose stem-loop structure is believed to be critical for the interactions with WT 1. We have analysed the interactions of five WT 1 zinc finger truncations with wild-type ACT 34 and four variants. Our results indicate that WT 1 zinc fingers bind ACT 34 in a specific manner, and that this occurs as interplay of all four zinc fingers. We also report that a sensitive kinetic balance, which is equilibrated by both zinc finger 1 and KTS, regulates the interaction with ACT 34. The stem-loop and the flanking nucleotides are important elements for specific recognition by WT 1 zinc fingers.
|
|
