1. |
- Abou-Hamdan, Abbas, et al.
(författare)
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Functional design of bacterial superoxide : quinone oxidoreductase
- 2022
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Ingår i: Biochimica et Biophysica Acta - Bioenergetics. - : Elsevier BV. - 0005-2728 .- 1879-2650. ; 1863:7
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Tidskriftsartikel (refereegranskat)abstract
- The superoxide anion - molecular oxygen reduced by a single electron - is produced in large amounts by enzymatic and adventitious reactions. It can perform a range of cellular functions, including bacterial warfare and iron uptake, signalling and host immune response in eukaryotes. However, it also serves as precursor for more deleterious species such as the hydroxyl anion or peroxynitrite and defense mechanisms to neutralize superoxide are important for cellular health. In addition to the soluble proteins superoxide dismutase and superoxide reductase, recently the membrane embedded diheme cytochrome b561 (CybB) from E. coli has been proposed to act as a superoxide:quinone oxidoreductase. Here, we confirm superoxide and cellular ubiquinones or menaquinones as natural substrates and show that quinone binding to the enzyme accelerates the reaction with superoxide. The reactivity of the substrates is in accordance with the here determined midpoint potentials of the two b hemes (+48 and -23 mV / NHE). Our data suggest that the enzyme can work near the diffusion limit in the forward direction and can also catalyse the reverse reaction efficiently under physiological conditions. The data is discussed in the context of described cytochrome b561 proteins and potential physiological roles of CybB.
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2. |
- Behboudi, Afrouz, 1967, et al.
(författare)
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Detailed chromosomal and radiation hybrid mapping in the proximal part of rat Chromosome 10 and gene order comparison with mouse and human.
- 2002
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Ingår i: Mammalian genome : official journal of the International Mammalian Genome Society. - : Springer Science and Business Media LLC. - 0938-8990 .- 1432-1777. ; 13:6, s. 302-9
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Tidskriftsartikel (refereegranskat)abstract
- The rat provides valuable and sometimes unique models of human complex diseases. To fully exploit the rat models in biomedical research, it is important to have access to detailed knowledge of the rat genome organization as well as its relation to the human genome. Rat Chromosome 10 (RNO10) harbors several important cancer-related genes. Deletions in the proximal part of RNO10 were repeatedly found in a rat model for endometrial cancer. To identify functional and positional candidate genes in the affected region, we used radiation hybrid (RH) mapping and single- and dual-color fluorescence in situ hybridization (FISH) techniques to construct a detailed chromosomal map of the proximal part of RNO10. The regional localization of 14 genes, most of them cancer-related ( Grin2a, Gspt1, Crebbp, Gfer, Tsc2, Tpsb1, Il9r, Il4, Irf1, Csf2, Sparc, Tp53, Thra1, Gh1), and of five microsatellite markers ( D10Mit10, D10Rat42, D10Rat50, D10Rat72, and D10Rat165) was determined on RNO10. For a fifteenth gene, Ppm1b, which had previously been assigned to RNO10, the map position was corrected to RNO6q12-q13.
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5. |
- Drobin, Dan, et al.
(författare)
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Tranexamic acid does not prevent rebleeding in an uncontrolled hemorrhage porcine model
- 2005
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Ingår i: Journal of Trauma. - : Lippincott Williams & Wilkins. - 0022-5282 .- 1529-8809. ; 59:4, s. 976-983
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND: Fluid resuscitation after uncontrolled hemorrhage might promote rebleeding and irreversible shock. Tranexamic acid is a procoagulant drug that limits blood loss after surgery of the hip, knee, and heart. We hypothesized that pretreatment with tranexamic acid reduces the rebleeding in uncontrolled hemorrhage and thereby allows safe administration of crystalloid fluid resuscitation.METHODS: A 120-minute intravenous infusion of 100 mL/kg of Ringer's solution was given to 24 pigs (mean weight, 20 kg) 10 minutes after lacerating the infrarenal aorta. The animals were randomized to receive an intravenous injection of 15 mg/kg of tranexamic acid or placebo just before starting the resuscitation. Rebleeding events were monitored by two ultrasonic probes positioned proximal and distal to the laceration.RESULTS: Tranexamic acid had no effect on the number of rebleeding events, bled volume, or mortality. The initial bleeding stopped within 4 minutes after the injury. The five animals that died suffered from 4.4 rebleeding events on average, which tripled the total blood loss, whereas the survivors had only 1.3 such events during fluid resuscitation (p < 0.02). At autopsy, death was associated with a larger total hemorrhage; the blood recovered from the abdomen weighed 1.4 kg (median) in nonsurvivors and 0.6 kg in survivors (p < 0.001), with the difference being attributable to rebleeding.CONCLUSION: Rebleeding events increased the amount of blood lost and the mortality in uncontrolled aortic hemorrhage. Tranexamic acid offered no benefit.
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7. |
- Lundgren, Camilla A. K, et al.
(författare)
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Scavenging of superoxide by a membrane-bound superoxide oxidase
- 2018
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Ingår i: Nature Chemical Biology. - : Springer Science and Business Media LLC. - 1552-4450 .- 1552-4469. ; 14, s. 788-793
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Tidskriftsartikel (refereegranskat)abstract
- Superoxide is a reactive oxygen species produced during aerobic metabolism in mitochondria and prokaryotes. It causes damage to lipids, proteins and DNA and is implicated in cancer, cardiovascular disease, neurodegenerative disorders and aging. As protection, cells express soluble superoxide dismutases, disproportionating superoxide to oxygen and hydrogen peroxide. Here, we describe a membrane-bound enzyme that directly oxidizes superoxide and funnels the sequestered electrons to ubiquinone in a diffusion-limited reaction. Experiments in proteoliposomes and inverted membranes show that the protein is capable of efficiently quenching superoxide generated at the membrane in vitro. The 2.0 Å crystal structure shows an integral membrane di-heme cytochrome b poised for electron transfer from the P-side and proton uptake from the N-side. This suggests that the reaction is electrogenic and contributes to the membrane potential while also conserving energy by reducing the quinone pool. Based on this enzymatic activity, we propose that the enzyme family be denoted superoxide oxidase (SOO).
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8. |
- Moe, Agnes, et al.
(författare)
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The respiratory supercomplex from C. glutamicum
- 2022
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Ingår i: Structure. - : Elsevier BV. - 0969-2126 .- 1878-4186. ; 30:3, s. 338-349
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Tidskriftsartikel (refereegranskat)abstract
- Corynebacterium glutamicum is a preferentially aerobic gram-positive bacterium belonging to the phylum Actinobacteria, which also includes the pathogen Mycobacterium tuberculosis. In these bacteria, respiratory complexes III and IV form a CIII2CIV2 supercomplex that catalyzes oxidation of menaquinol and reduction of dioxygen to water. We isolated the C. glutamicum supercomplex and used cryo-EM to determine its structure at 2.9 Å resolution. The structure shows a central CIII2 dimer flanked by a CIV on two sides. A menaquinone is bound in each of the QN and QP sites in each CIII and an additional menaquinone is positioned ∼14 Å from heme bL. A di-heme cyt. cc subunit electronically connects each CIII with an adjacent CIV, with the Rieske iron-sulfur protein positioned with the iron near heme bL. Multiple subunits interact to form a convoluted sub-structure at the cytoplasmic side of the supercomplex, which defines a path for proton transfer into CIV.
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10. |
- Sjöstrand, Dan, et al.
(författare)
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A rapid expression and purification condition screening protocol for membrane protein structural biology
- 2017
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Ingår i: Protein Science. - : Wiley. - 0961-8368 .- 1469-896X. ; 26:8, s. 1653-1666
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Tidskriftsartikel (refereegranskat)abstract
- Membrane proteins control a large number of vital biological processes and are often medically important-not least as drug targets. However, membrane proteins are generally more difficult to work with than their globular counterparts, and as a consequence comparatively few high-resolution structures are available. In any membrane protein structure project, a lot of effort is usually spent on obtaining a pure and stable protein preparation. The process commonly involves the expression of several constructs and homologs, followed by extraction in various detergents. This is normally a time-consuming and highly iterative process since only one or a few conditions can be tested at a time. In this article, we describe a rapid screening protocol in a 96-well format that largely mimics standard membrane protein purification procedures, but eliminates the ultracentrifugation and membrane preparation steps. Moreover, we show that the results are robustly translatable to large-scale production of detergent-solubilized protein for structural studies. We have applied this protocol to 60 proteins from an E. coli membrane protein library, in order to find the optimal expression, solubilization and purification conditions for each protein. With guidance from the obtained screening data, we have also performed successful large-scale purifications of several of the proteins. The protocol provides a rapid, low cost solution to one of the major bottlenecks in structural biology, making membrane protein structures attainable even for the small laboratory.
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11. |
- Sjöstrand, Dan, et al.
(författare)
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Disruption of the GDNF Binding Site in NCAM DissociatesLigand Binding and Homophilic Cell Adhesion
- 2007
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Ingår i: Journal of Biological Chemistry. - Bethesda, MD : American Society for Biochemistry and Molecular Biology. - 0021-9258 .- 1083-351X. ; 282:17, s. 12734-12740
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Tidskriftsartikel (refereegranskat)abstract
- Most plasma membrane proteins are capable of sensing multiple cell-cell and cell-ligand interactions, but the extent towhich this functional versatility is founded on their modular design is less clear. We have identified the third immunoglobulin domain of the Neural Cell Adhesion Molecule (NCAM) as the necessary and sufficient determinant for its interaction with Glial Cell Line-derived Neurotrophic Factor (GDNF). Four charged contacts were identified by molecular modeling as the main contributors to binding energy. Their mutation abolished GDNF binding to NCAM but left intact the ability of NCAM tomediate cell adhesion, indicating that the two functions are genetically separable. The GDNF-NCAM interface allows complex formation with the GDNF family receptor α1, shedding light on the molecular architecture of a multicomponent GDNF receptor.
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12. |
- Sjöstrand, Dan
(författare)
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GDNF and its receptors : a structure-function analysis of the NCAM-GDNF-GFRalpha1 complex and an examination of potential receptor candidates
- 2008
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Neurotrophic factors are small, secreted proteins that are important players in the development and maintenance of the nervous system. Their functions span from neuronal survival and migration to neurite outgrowth and axon guidance, and in some cases even programmed cell death. Glial cell line-derived neurotrophic factor (GDNF) is the founding member of a family of four neurotrophic factors; the GDNF family of ligands. GDNF and its fellow family members Neurturin, Artemin and Persephin signal by forming a tripartite complex with one GPI-anchored receptor (GFRalpha1, -2, -3 or -4, respectively) and one transmembrane receptor, the latter being responsible for transmitting the signal through the plasma membrane into the cell. The transmembrane receptor can be either one of two molecules: The receptor tyrosine kinase Ret or the Neural Cell Adhesion Molecule (NCAM). The interaction determinants of the Ret-GDNF-GFRalpha1 complex have been thoroughly analyzed, as well as the functional importance of its structural features. The more recently discovered NCAM-GDNF-GFRalpha1 complex, however, is much less studied. In paper I we mapped the GDNF binding domain in NCAM to the third immunoglobulin-like domain by analysing the ligand-binding properties of a large set of deletion mutants. Molecular modeling of the interaction interface revealed four charged contacts, and mutation of the NCAM residues involved in these contacts created a receptor unable to bind GDNF while keeping its cell adhesion properties, thus separating these two functions of NCAM. In paper II we used a similar approach to map the NCAM-GFRalpha1 binding site. The major GFRalpha1-binding site in NCAM was mapped to the fourth Ig-like domain, and we found that the primary NCAM-binding site resides in the N-terminal domain of GFRalpha1. Deletion of the latter domain did not affect the ability of GFRalpha1 to potentiate GDNF-NCAM binding, implying that GFRalpha1 facilitates signaling by a ligand presentation mechanism rather than by allosterically changing the GDNF-binding properties of NCAM. We also showed that deletion of the NCAM-binding N-terminal domain severely impaired the ability of GFRalpha1 to interfere with NCAM-mediated cell adhesion. This suggests that the strong GFRalpha1-NCAM interactions mediated by the N-terminal domain of GFRalpha1 are required for efficient modulation of NCAMmediated cell adhesion. Although the GDNF family of ligands appears to be a vertebrate-specific invention, orthologs of the receptors are present in other phyla. In paper III we examined the Drosophila Ret ortholog dRet, and showed that it did not bind GDNF, nor did it display any cell adhesion promoting properties despite its cadherin-like domain structural features. Interestingly, we found that a chimeric mammalian Ret molecule that included the dRet kinase domain was capable of inducing PC12 cell differentiation upon stimulation with GDNF and soluble GFRalpha1, suggesting that this functional feature of the intracellular domain is evolutionarily conserved. GDNF and GFRalpha1 promote the migration and differentiation of cortical interneurons from the medial ganglionic eminence (MGE), a process in which neither RET nor NCAM appear to be involved, suggesting the possible existence of a third transmembrane receptor. In paper IV we examined two potential receptor candidates: The HGF receptor Met and the Neuregulin-1 receptor ErbB4. None of the two receptors were phosphorylated by GDNF in our biochemical assays, and neither HGF nor Neuregulin had any significant effects of their own on neurite outgrowth in our cultures. Intriguingly, two different Met inhibitors displayed outgrowthpromoting effects on their own. However, neither Met nor ErbB4 inhibitors impaired GDNFinduced neurite outgrowth, showing that these receptors are not involved in GDNF signaling in MGE neurons.
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13. |
- Smirnova, Irina A., et al.
(författare)
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Isolation of yeast complex IV in native lipid nanodiscs
- 2016
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Ingår i: Biochimica et Biophysica Acta - Biomembranes. - : Elsevier BV. - 0005-2736 .- 1879-2642. ; 1858:12, s. 2984-2992
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Tidskriftsartikel (refereegranskat)abstract
- We used the amphipathic styrene maleic acid (SMA) co-polymer to extract cytochrome c oxidase (CytcO) in its native lipid environment from S. cerevisiae mitochondria. Native nanodiscs containing one CytcO per disc were purified using affinity chromatography. The longest cross-sections of the native nanodiscs were 11 nm x 14 nm. Based on this size we estimated that each CytcO was surrounded by similar to 100 phospholipids. The native nanodiscs contained the same major phospholipids as those found in the mitochondrial inner membrane. Even though CytcO forms a supercomplex with cytochrome bc(1) in the mitochondria! membrane, cyt.bc(1) was not found in the native nanodiscs. Yet, the loosely-bound Respiratory SuperComplex factors were found to associate with the isolated CytcO. The native nanodiscs displayed an O-2-reduction activity of similar to 130 electrons CytcO(-1) s(-1) and the kinetics of the reaction of the fully reduced CytcO with 02 was essentially the same as that observed with CytcO in mitochondrial membranes. The kinetics of CO-ligand binding to the CytcO catalytic site was similar in the native nanodiscs and the mitochondrial membranes. We also found that excess SMA reversibly inhibited the catalytic activity of the mitochondrial CytcO, presumably by interfering with cyt. c binding. These data point to the importance of removing excess SMA after extraction of the membrane protein. Taken together, our data shows the high potential of using SMA-extracted CytcO for functional and structural studies.
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14. |
- Wiseman, Benjamin, et al.
(författare)
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Structure of a functional obligate complex III2IV2 respiratory supercomplex from Mycobacterium smegmatis
- 2018
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Ingår i: Nature Structural & Molecular Biology. - : Springer Science and Business Media LLC. - 1545-9993 .- 1545-9985. ; 25:12, s. 1128-1136
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Tidskriftsartikel (refereegranskat)abstract
- In the mycobacterial electron-transport chain, respiratory complex III passes electrons from menaquinol to complex IV, which in turn reduces oxygen, the terminal acceptor. Electron transfer is coupled to transmembrane proton translocation, thus establishing the electrochemical proton gradient that drives ATP synthesis. We isolated, biochemically characterized, and determined the structure of the obligate III2IV2 supercomplex from Mycobacterium smegmatis, a model for Mycobacterium tuberculosis. The supercomplex has quinol:O-2 oxidoreductase activity without exogenous cytochrome c and includes a superoxide dismutase subunit that may detoxify reactive oxygen species produced during respiration. We found menaquinone bound in both the Q(o) and Q(i) sites of complex III. The complex III-intrinsic diheme cytochrome cc subunit, which functionally replaces both cytochrome c(1) and soluble cytochrome c in canonical electron-transport chains, displays two conformations: one in which it provides a direct electronic link to complex IV and another in which it serves as an electrical switch interrupting the connection.
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15. |
- Zhou, Shu, et al.
(författare)
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Solution NMR structure of yeast Rcf1, a protein involved in respiratory supercomplex formation
- 2018
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Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 115:12, s. 3048-3053
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Tidskriftsartikel (refereegranskat)abstract
- The Saccharomyces cerevisiae respiratory supercomplex factor 1 (Rcf1) protein is located in the mitochondrial inner membrane where it is involved in formation of supercomplexes composed of respiratory complexes III and IV. We report the solution structure of Rcf1, which forms a dimer in dodecylphosphocholine (DPC) micelles, where each monomer consists of a bundle of five transmembrane (TM) helices and a short flexible soluble helix (SH). Three TM helices are unusually charged and provide the dimerization interface consisting of 10 putative salt bridges, defining a charge zipper motif. The dimer structure is supported by molecular dynamics (MD) simulations in DPC, although the simulations show a more dynamic dimer interface than the NMR data. Furthermore, CD and NMR data indicate that Rcf1 undergoes a structural change when reconstituted in liposomes, which is supported by MD data, suggesting that the dimer structure is unstable in a planar membrane environment. Collectively, these data indicate a dynamic monomer-dimer equilibrium. Furthermore, the Rcf1 dimer interacts with cytochrome c, suggesting a role as an electron-transfer bridge between complexes III and IV. The Rcf1 structure will help in understanding its functional roles at a molecular level.
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