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| 2. |
- Niegowski, Damian, 1978-, et al.
(author)
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Structural basis for synthesis of inflammatory mediators by human leukotriene C4 synthase
- 2007
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In: Nature. - : Springer Science and Business Media LLC. - 0028-0836 .- 1476-4687. ; 448:7153, s. 613-616
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Journal article (peer-reviewed)abstract
- Cysteinyl leukotrienes are key mediators in inflammation and have an important role in acute and chronic inflammatory diseases of the cardiovascular and respiratory systems, in particular bronchial asthma. In the biosynthesis of cysteinyl leukotrienes, conversion of arachidonic acid forms the unstable epoxide leukotriene A4 (LTA4). This intermediate is conjugated with glutathione (GSH) to produce leukotriene C4 (LTC4) in a reaction catalysed by LTC4 synthase1: this eaction is the key step in cysteinyl leukotriene formation. Here we present the rystal structure of the human LTC4 synthase in its apo and GSH-complexed forms to 2.00 and 2.15 A ̊resolution, respectively. The structure reveals a homotrimer, here each monomer is composed of four transmembrane segments. The structure of the enzyme in complex with substrate reveals that the active site enforces a orseshoe-shaped conformation on GSH, and effectively positions the thiol group or activation by a nearby arginine at the membrane–enzyme interface. In addition, the structure provides a model for how the v-end of the lipophilic co-substrate is pinned at one end of a hydrophobic cleft, providing a molecular ‘ruler’ to align the eactive epoxide at the thiol of glutathione. This provides new structural insights nto the mechanism of LTC4 formation, and also suggests that the observed inding and activation of GSH might be common for a family of homologous proteins mportant for inflammatory and detoxification responses.
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| 3. |
- Rinaldo-Matthis, Agnes, et al.
(author)
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Pre-Steady-State Kinetic Characterization of Thiolate Anion Formation in Human Leukotriene C-4 Synthase
- 2012
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In: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 51:4, s. 848-856
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Journal article (peer-reviewed)abstract
- Human leukotriene C-4 synthase (hLTC4S) is an integral membrane protein that catalyzes the committed step in the biosynthesis of cysteinyl-leukotrienes, i.e., formation of leukotriene C-4 (LTC4). This molecule, together with its metabolites LTD4 and LTE4, induces inflammatory responses, particularly in asthma, and thus, the enzyme is an attractive drug target. During the catalytic cycle, glutathione (GSH) is activated by hLTC4S that forms a nucleophilic thiolate anion that will attack LTA(4), presumably according to an S(N)2 reaction to form LTC4. We observed that GSH thiolate anion formation is rapid and occurs at all three monomers of the homotrimer and is concomitant with stoichiometric release of protons to the medium. The pK(a) (5.9) for enzyme-bound GSH thiol and the rate of thiolate formation were determined (k(obs) = 200 s(-1)). Taking advantage of a strong competitive inhibitor, glutathionesulfonic acid, shown here by crystallography to bind in the same location as GSH, we determined the overall dissociation constant (K-d(GS) = 14.3 mu M). The release of the thiolate was assessed using a GSH release experiment (1.3 s(-1)). Taken together, these data establish that thiolate anion formation in hLTC4S is not the rate-limiting step for the overall reaction of LTC4 production (k(cat) = 26 s(-1)), and compared to the related microsomal glutathione transferase 1, which displays very slow GSH thiolate anion formation and one-third of the sites reactivity, hLTC4S has evolved a different catalytic mechanism.
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- Wetterholm, Anders, et al.
(author)
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High-level expression, purification, and crystallization of recombinant rat leukotriene C4 synthase from the yeast Pichia pastoris
- 2008
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In: Protein Expression and Purification. - : Elsevier BV. - 1046-5928 .- 1096-0279. ; 60:1, s. 1-6
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Journal article (peer-reviewed)abstract
- Leukotriene C(4) synthase (LTC4S) is a member of the MAPEG family of integral membrane proteins and catalyzes the conjugation of leukotriene A(4) with glutathione to form leukotriene C(4), a powerful mediator of allergic inflammation and anaphylaxis. Structural information on this class of proteins would be highly useful for rational drug design. Here, we report the expression, purification, and crystallization of recombinant LTC4S from rat. The enzyme was expressed as an N-terminal hexa-histidine-tagged fusion protein in Pichia pastoris and purified with two steps of affinity chromatography on Ni-Sepharose and S-hexyl-glutathione agarose, followed by gel filtration. From 1l culture, we obtained 0.5-1 mg of apparently homogeneous protein with a specific LTC4S activity ranging between 36 and 49 micromol/mg/min. A small-scale screen identified dodecyl maltoside as a useful detergent for protein extraction and yielded a highly active protein. When tested separately in crystallization trials of the purified LTC4S, six out of seven detergents from all the maltoside family yielded diffracting crystals with the highest resolution at approximately 6 A. Hence, our approach holds promise for solving the structure of rat LTC4S and other members of the MAPEG family of integral membrane proteins.
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| 5. |
- Winblad, Bengt, et al.
(author)
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Donepezil in patients with severe Alzheimer's disease : double-blind, parallel-group, placebo-controlled study
- 2006
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In: The Lancet. - New York : Elsevier. - 0140-6736 .- 1474-547X. ; 367:9516, s. 1057-1065
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Journal article (peer-reviewed)abstract
- Background The cholinesterase inhibitor donepezil is used to treat mild-to-moderate Alzheimer's disease. Its efficacy in severe dementia has not been assessed and is controversial. Our aim was to ascertain the effectiveness of donepezil in patients with severe Alzheimer's disease, by focusing primarily on cognition and activities of daily living.Methods We did a 6-month, double-blind, parallel-group, placebo-controlled study in 248 patients with severe Alzheimer's disease (mini mental state examination score 1-10) who were living in assisted care nursing homes ran by trained staff in Sweden. We assigned patients oral donepezil (5 mg per day for 30 days then up to 10 mg per day thereafter, n=128) or matched placebo (n=120). Our primary endpoints were change from baseline to month 6 in the severe impairment battery (SIB) and modified Alzheimer's Disease Cooperative Study activities of daily living inventory for severe Alzheimer's disease (ADCS-ADL-severe). We analysed outcomes for patients with data at baseline and at one or more other timepoints (modified intent-to-treat population) with last observation carried forward used to replace missing data.Findings 95 patients assigned donepezil and 99 patients assigned placebo completed the study. Patients treated with donepezil improved more in SIB scores and declined less in ADCS-ADL-severe scores at 6 months after initiation of treatment compared with baseline than did controls (least squares [LS] mean difference, 5.7,95% Cl 1.5-9.8; p=0.008, and 1.7, 0.2-3.2; p=0.03, respectively). The incidence of adverse events was comparable between groups (donepezil 82% [n=105] vs placebo 76% [n=91]), with most being transient and mild or moderate in severity. More patients discontinued treatment because of adverse events in the donepezil group (n=20) than in the placebo group (n=8).Interpretation Donepezil improves cognition and preserves function in individuals with severe Alzheimer's disease who live in nursing homes.
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