| 1. |
- Claesson, Magnus, et al.
(författare)
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Crystal structure of the glycosyltransferase SnogD from the biosynthetic pathway of nogalamycin in Streptomyces nogalater
- 2012
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Ingår i: The FEBS journal. - Stockholm : Karolinska Institutet, Dept of Medical Biochemistry and Biophysics. - 1742-464X .- 1742-4658.
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Tidskriftsartikel (refereegranskat)abstract
- The glycosyltransferase SnogD from Streptomyces nogalater transfers a nogalamine moiety to the metabolic intermediate 3′,4′-demethoxynogalose-1-hydroxynogalamycinone during the final steps of biosynthesis of the aromatic polyketide nogalamycin. The crystal structure of recombinant SnogD, as an apo-enzyme and with a bound nucleotide, 2-deoxyuridine-5′-diphosphate, was determined to 2.6 Å resolution. Reductive methylation of SnogD was crucial for reproducible preparation of diffraction quality crystals due to creation of an additional intermolecular salt bridge between methylated lysine residue Lys384 and Glu374* from an adjacent molecule in the crystal lattice. SnogD is a dimer both in solution and in the crystal, and the enzyme subunit displays a fold characteristic of the GT-B family of glycosyltransferases. Binding of the nucleotide is associated with rearrangement of two active-site loops. Site-directed mutagenesis shows that two active-site histidine residues, His25 and His301, are critical for the glycosyltransferase activities of SnogD both in vivo and in vitro. The crystal structures and the functional data are consistent with a role for His301 in binding of the diphosphate group of the sugar donor substrate, and a function of His25 as a catalytic base in the glycosyl transfer reaction.
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| 2. |
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| 3. |
- Andersson, Mattias K., et al.
(författare)
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Arg143 and Lys192 of the human mast cell chymase mediate the preference for acidic amino acids in position P2′ of substrates
- 2010
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Ingår i: The FEBS Journal. - : Wiley. - 1742-464X .- 1742-4658. ; 277:10, s. 2255-2267
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Tidskriftsartikel (refereegranskat)abstract
- Chymases are chymotrypsin-like serine proteases that are found in large amounts in mast cell granules. So far, the extended cleavage specificities of eight such chymases have been determined, and four of these were shown to have a strong preference for acidic amino acids at position P2'. These enzymes have basic amino acids in positions 143 and 192 (Arg and Lys, respectively). We therefore hypothesized that Arg143 and Lys192 of human chymase mediate the preference for acidic amino acids at position P2' of substrates. In order to address this question, we performed site-directed mutagenesis of these two positions in human chymase. Analysis of the extended cleavage specificities of two single mutants (Arg143 -> Gln and Lys192 -> Met) and the combined double mutant revealed an altered specificity for P2' amino acids, whereas all other positions were essentially unaffected. A weakened preference for acidic amino acids at position P2' was observed for the two single mutants, whereas the double mutant lacked this preference. Therefore, we conclude that positions 143 and 192 in human chymase contribute to the strong preference for negatively charged amino acids at position P2'. This is the first time that a similar combined effect has been shown to influence the cleavage specificity, apart from position P1, among the chymases. Furthermore, the conservation of the preference for acidic P2' amino acids for several mast cell chymases clearly indicates that other substrates than angiotensin I may be major in vivo targets for these enzymes.
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| 4. |
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| 5. |
- Azinas, Stavros, et al.
(författare)
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D-strand perturbation and amyloid propensity in beta-2 microglobulin
- 2011
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Ingår i: The FEBS Journal. - : John Wiley & Sons. - 1742-464X .- 1742-4658. ; 278:13, s. 2349-58
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Tidskriftsartikel (refereegranskat)abstract
- Proteins hosting main β-sheets adopt specific strategies to avoid intermolecular interactions leading to aggregation and amyloid deposition. Human beta-2 microglobulin (β2m) displays a typical immunoglobulin fold and is known to be amyloidogenic in vivo. Upon severe kidney deficiency, β2m accumulates in the bloodstream, triggering, over the years, pathological deposition of large amyloid aggregates in joints and bones. A β-bulge observed on the edge D β-strand of some β2m crystal structures has been suggested to be crucial in protecting the protein from amyloid aggregation. Conversely, a straight D-strand, observed in different crystal structures of monomeric β2m, could promote amyloid aggregation. More recently, the different conformations observed for the β2m D-strand have been interpreted as the result of intrinsic flexibility, rather than being assigned to a functional protective role against aggregation. To shed light on such contrasting picture, the mutation Asp53→Pro was engineered in β2m, aiming to impair the formation of a regular/straight D-strand. Such a mutant was characterized structurally and biophysically by CD, X-ray crystallography and MS, in addition to an assessment of its amyloid aggregation trends in vitro. The results reported in the present study highlight the conformational plasticity of the edge D-strand, and show that even perturbing the D-strand structure through a Pro residue has only marginal effects on protecting β2m from amyloid aggregation in vitro.
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| 6. |
- Belotserkovsky, Jaroslav M., 1980-, et al.
(författare)
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Mutations in 16S rRNA that suppress cold-sensitive initiation factor 1 affect ribosomal subunit association
- 2011
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Ingår i: The FEBS Journal. - : Wiley. - 1742-464X .- 1742-4658. ; 278:18, s. 3508-3517
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Tidskriftsartikel (refereegranskat)abstract
- A mutation in the infA gene encoding initiation factor 1 (IF1) gives rise to a cold-sensitive phenotype. An Escherichia coli strain with this mutation was used as a tool to select for second-site suppressors that compensate for the cold sensitivity and map specifically to rRNA. Several suppressor mutants with altered 16S rRNA that partially restore growth of an IF1 mutant strain in the cold were isolated and characterized. Suppressor mutations were found in helix (h) 18, h32, h34 and h41 in 16S rRNA. These mutations are not clustered to any particular region in 16S rRNA and none overlap previously reported sites of interaction with IF1. While the isolated suppressors are structurally diverse, they are functionally related because all affect ribosomal subunit association in vivo. Furthermore, in vitro subunit-association experiments indicate that most of the suppressor mutations directly influence ribosomal subunit association even though none of these are confined to any of the known intersubunit bridges. These results are consistent with the model that IF1 is an rRNA chaperone that induces large-scale conformational changes in the small ribosomal subunit, and as a consequence modulates initiation of translation by affecting subunit association.
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| 7. |
- Belotserkovsky, Jaroslav M., 1980-, et al.
(författare)
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Suppression of a cold-sensitive mutant initiation factor 1 by alterations in the 23S rRNA maturation region
- 2011
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Ingår i: The FEBS Journal. - : Wiley. - 1742-464X .- 1742-4658. ; 278:10, s. 1745-1756
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Tidskriftsartikel (refereegranskat)abstract
- Genetic selection has been used to isolate second-site suppressors of a defective cold-sensitive initiation factor I (IF1) R69L mutant of Escherichia coli. The suppressor mutants specifically map to a single rRNA operon on a plasmid in a strain with all chromosomal rRNA operons deleted. Here, we describe a set of suppressor mutations that are located in the processing stem of precursor 23S rRNA. These mutations interfere with processing of the 23S rRNA termini. A lesion of RNase III also suppresses the cold sensitivity. Our results suggest that the mutant IF1 strain is perturbed at the level of ribosomal subunit association, and the suppressor mutations partially compensate for this defect by disrupting rRNA maturation. These results support the notion that IF1 is an RNA chaperone and that translation initiation is coupled to ribosomal maturation.
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| 8. |
- Björnerås, Johannes, et al.
(författare)
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Direct detection of neuropeptide dynorphin A binding to the second extracellular loop of the kappa opioid receptor using a soluble protein scaffold
- 2014
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Ingår i: The FEBS Journal. - : Wiley. - 1742-464X .- 1742-4658. ; 281:3, s. 814-824
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Tidskriftsartikel (refereegranskat)abstract
- The molecular determinants for selectivity of ligand binding to membrane receptors are of key importance for the understanding of cellular signalling, as well as for rational therapeutic intervention. In the present study, we target the interaction between the kappa opioid receptor (KOR) and its native peptide ligand dynorphin A (DynA) using solution state NMR spectroscopy, which is generally made difficult by the sheer size of membrane bound receptors. Our method is based on 'transplantation' of an extracellular loop of KOR into a 'surrogate' scaffold; in this case, a soluble beta-barrel. Our results corroborate the general feasibility of the method, showing that the inserted receptor segment has negligible effects on the properties of the scaffold protein, at the same time as maintaining an ability to bind its native DynA ligand. Upon DynA binding, only small induced chemical shift changes of the KOR loop were observed, whereas chemical shift changes of DynA and NMR paramagnetic relaxation data show conclusively that the peptide interacts with the inserted loop. The binding interface is composed of a disordered part of the KOR loop and involves both electrostatic and hydrophobic interactions. Even so, simultaneous effects along the DynA sequence upon binding show that control of the recognition is a concerted event.
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| 9. |
- Blikstad, Cecilia, et al.
(författare)
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Substrate scope and selectivity in offspring to an enzyme subjected to directed evolution
- 2014
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Ingår i: The FEBS Journal. - : Wiley. - 1742-464X .- 1742-4658. ; 281:10, s. 2387-2398
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Tidskriftsartikel (refereegranskat)abstract
- We have analyzed the effects of mutations inserted during directed evolution of a specialized enzyme, Escherichia coli S-1,2-propanediol oxidoreductase (FucO). The kinetic properties of evolved variants have been determined and the observed differences have been rationalized by modeling the tertiary structures of isolated variants and the wild-type enzyme. The native substrate, S-1,2-propanediol, as well as phenylacetaldehyde and 2S-3-phenylpropane-1,2-diol, which are new substrates accepted by isolated variants, were docked into the active sites. The study provides a comprehensive picture of how acquired catalytic properties have arisen via an intermediate generalist enzyme, which had acquired a single mutation (L259V) in the active site. Further mutagenesis of this generalist resulted in a new specialist catalyst. We have also been able to relate the native enzyme activities to the evolved ones and linked the differences to individual amino acid residues important for activity and selectivity. F254 plays a dual role in the enzyme function. First, mutation of F254 into an isoleucine weakens the interactions with the coenzyme thereby increasing its dissociation rate from the active site and resulting in a four-fold increase in turnover number with S-1,2-propanediol. Second, F254 is directly involved in binding of aryl-substituted substrates via π–π interactions. On the other hand, N151 is critical in determining the substrate scope since the side chain amide group stabilizes binding of 1,2-substituted diols and is apparently necessary for enzymatic activity with these substrates. Moreover, the side chain of N151 introduces steric hindrance, which prevents high activity with phenylacetaldehyde. Additionally, the hydroxyl group of T149 is required to maintain the catalytically important hydrogen bonding network.
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| 10. |
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| 11. |
- Bolognesi, B, et al.
(författare)
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The N-terminus of amyloid-beta plays a crucial role in its aggregation and toxicity
- 2010
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Ingår i: The FEBS Journal. - : Wiley-Blackwell. - 1742-464X .- 1742-4658. ; 277:Suppl. 1, s. 79-80
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract
- The aggregation of Amyloid Beta (Aß) peptide into insolubleamyloid fibrils that deposit in the brain is one of the primarypathogenic events in Alzheimer’s disease. We have previouslyshown, using a Drosophila model of Aß toxicity, that the N terminus of the Aß peptide, despite being unstructured in themature Aß fibril, nonetheless affects Aß induced neurodegeneration in vivo. In order to understand the contribution of the N terminusof Aß to its aggregation behaviour, we have investigated anumber of rationally designed N-terminal mutants in vitro. We find that single amino acid mutations in this region affect significantlythe kinetics of Aß aggregation in vitro as measured by arange of spectroscopic techniques. Furthermore, we observe striking differences in the morphology of the aggregated speciesformed by these different Aß mutants when imaged with TEM or AFM and also in the ß-sheet content of their mature fibrils. Interestingly, mutants with an increased net charge or lower hydrophobicity tend to show slower aggregation kinetics, and to form more ordered aggregates whereas mutations that reduce net charge or increase hydrophobicity favour faster aggregation kinetics and poorly structured aggregates. In addition, the exposed hydrophobicity of aggregates formed in the early stages of aggregation is correlated to their toxicity. These findings demonstrate not only that the N-terminus of the Aß peptide plays a crucial role in its aggregation and toxicity but also suggest that this region of Aß may modulate in vivo toxicity by altering the conformations of aggregates that it forms.
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| 12. |
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| 13. |
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| 14. |
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| 15. |
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| 16. |
- Cedersund, Gunnar
(författare)
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Conclusions via unique predictions obtained despite unidentifiability - new definitions and a general method
- 2012
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Ingår i: The FEBS Journal. - : Wiley-Blackwell. - 1742-464X .- 1742-4658. ; 279:18, s. 3513-3527
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Tidskriftsartikel (refereegranskat)abstract
- It is often predicted that model-based data analysis will revolutionize biology, just as it has physics and engineering. A widely used tool within such analysis is hypothesis testing, which focuses on model rejections. However, the fact that a systems biology model is non-rejected is often a relatively weak statement, as such models usually are highly over-parametrized with respect to the available data, and both parameters and predictions may therefore be arbitrarily uncertain. For this reason, we formally define and analyse the concept of a core prediction. A core prediction is a uniquely identified property that must be fulfilled if the given model structure is to explain the data, even if the individual parameters are non-uniquely identified. It is shown that such a prediction is as strong a conclusion as a rejection. Furthermore, a new method for core prediction analysis is introduced, which is beneficial for the uncertainty of specific model properties, as the method only characterizes the space of acceptable parameters in the relevant directions. This avoids the curse of dimensionality associated with the generic characterizations used by previously proposed methods. Analysis on examples shows that the new method is comparable to profile likelihood with regard to practical identifiability, and thus generalizes profile likelihood to the more general problem of observability. If used, the concepts and methods presented herein make it possible to distinguish between a conclusion and a mere suggestion, which hopefully will contribute to a more justified confidence in systems biology analyses.
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| 17. |
- Chen, Yang, et al.
(författare)
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Staphylococcus aureus elongation factor G - structure and analysis of a target for fusidic acid
- 2010
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Ingår i: The FEBS Journal. - : Wiley. - 1742-464X .- 1742-4658. ; 277:18, s. 3789-3803
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Tidskriftsartikel (refereegranskat)abstract
- Fusidic acid (FA) is a bacteriostatic antibiotic that locks elongation factor G (EF-G) on the ribosome in a post-translocational state. It is used clinically against Gram-positive bacteria such as pathogenic strains of Staphylococcus aureus, but no structural information has been available for EF-G from these species. We have solved the apo crystal structure of EF-G from S. aureus to 1.9 A resolution. This structure shows a dramatically different overall conformation from previous structures of EF-G, although the individual domains are highly similar. Between the different structures of free or ribosome-bound EF-G, domains III-V move relative to domains I-II, resulting in a displacement of the tip of domain IV relative to domain G. In S. aureus EF-G, this displacement is about 25 A relative to structures of Thermus thermophilus EF-G in a direction perpendicular to that in previous observations. Part of the switch I region (residues 46-56) is ordered in a helix, and has a distinct conformation as compared with structures of EF-Tu in the GDP and GTP states. Also, the switch II region shows a new conformation, which, as in other structures of free EF-G, is incompatible with FA binding. We have analysed and discussed all known fusA-based fusidic acid resistance mutations in the light of the new structure of EF-G from S. aureus, and a recent structure of T. thermophilus EF-G in complex with the 70S ribosome with fusidic acid [Gao YG et al. (2009) Science326, 694-699]. The mutations can be classified as affecting FA binding, EF-G-ribosome interactions, EF-G conformation, and EF-G stability.
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| 18. |
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| 19. |
- Egeblad, Louise, et al.
(författare)
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Structural and functional studies of the human phosphoribosyltransferase domain containing protein 1
- 2010
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Ingår i: The FEBS Journal. - : Wiley. - 1742-464X .- 1742-4658. ; 277:23, s. 4920-30
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Tidskriftsartikel (refereegranskat)abstract
- Human hypoxanthine-guanine phosphoribosyltransferase (HPRT) (EC 2.4.2.8) catalyzes the conversion of hypoxanthine and guanine to their respective nucleoside monophosphates. Human HPRT deficiency as a result of genetic mutations is linked to both Lesch-Nyhan disease and gout. In the present study, we have characterized phosphoribosyltransferase domain containing protein 1 (PRTFDC1), a human HPRT homolog of unknown function. The PRTFDC1 structure has been determined at 1.7 Å resolution with bound GMP. The overall structure and GMP binding mode are very similar to that observed for HPRT. Using a thermal-melt assay, a nucleotide metabolome library was screened against PRTFDC1 and revealed that hypoxanthine and guanine specifically interacted with the enzyme. It was subsequently confirmed that PRTFDC1 could convert these two bases into their corresponding nucleoside monophosphate. However, the catalytic efficiency (k(cat)/K(m)) of PRTFDC1 towards hypoxanthine and guanine was only 0.26% and 0.09%, respectively, of that of HPRT. This low activity could be explained by the fact that PRTFDC1 has a Gly in the position of the proposed catalytic Asp of HPRT. In PRTFDC1, a water molecule at the position of the aspartic acid side chain position in HPRT might be responsible for the low activity observed by acting as a weak base. The data obtained in the present study indicate that PRTFDC1 does not have a direct catalytic role in the nucleotide salvage pathway.
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| 20. |
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| 21. |
- Fontana, Jacopo M., et al.
(författare)
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Calcium oscillations triggered by cardiotonic steroids
- 2013
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Ingår i: The FEBS Journal. - : Wiley. - 1742-464X .- 1742-4658. ; 280:21, s. 5450-5455
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Forskningsöversikt (refereegranskat)abstract
- Na+, K+-ATPase (NKA) is well known for its function as an ion pump. Studies during the last decade have revealed an additional role for NKA as a signal transducer. In this brief review, we describe how cardiotonic steroids, which are highly specific NKA ligands, trigger slow Ca2+ oscillations by promoting the interaction between NKA and the inositol trisphosphate receptor, and how this Ca2+ signal activates the NF-B subunit p65 and increases the expression of the antiapoptotic factor Bcl-xL. The potential tissue-protective effects of this signal are discussed.
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| 22. |
- Fowler, Christopher J.
(författare)
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Transport of endocannabinoids across the plasma membrane and within the cell
- 2013
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Ingår i: The FEBS Journal. - : Wiley. - 1742-464X .- 1742-4658. ; 280:9, s. 1895-1904
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Forskningsöversikt (refereegranskat)abstract
- Endocannabinoids are readily accumulated from the extracellular space by cells. Although their uptake properties have the appearance of a process of facilitated diffusion, it is by no means clear as to whether there is a plasma membrane transporter dedicated to this task. Intracellular carrier proteins that shuttle the endocannabinoid anandamide from the plasma membrane to its intracellular targets such as the metabolic enzyme, fatty acid amide hydrolase, have been identified. These include proteins with other primary functions, such as fatty-acid-binding proteins and heat shock protein70, and possibly a fatty acid amide hydrolase-like anandamide transporter protein. Thus, anandamide uptake can be adequately described as a diffusion process across the plasma membrane followed by intracellular carrier-mediated transport to effector molecules, catabolic enzymes and sequestration sites, although it is recognized that different cells are likely to utilize different mechanisms of endocannabinoid transport depending upon the utility of the endocannabinoid for the cell in question.
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| 23. |
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| 24. |
- Fritz, Günter, et al.
(författare)
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Natural and amyloid self-assembly of S100 proteins : structural basis of functional diversity
- 2010
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Ingår i: The FEBS Journal. - : Wiley. - 1742-464X .- 1742-4658. ; 277:22, s. 4578-4590
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Tidskriftsartikel (refereegranskat)abstract
- The S100 proteins are 10-12 kDa EF-hand proteins that act as central regulators in a multitude of cellular processes including cell survival, proliferation, differentiation and motility. Consequently, many S100 proteins are implicated and display marked changes in their expression levels in many types of cancer, neurodegenerative disorders, inflammatory and autoimmune diseases. The structure and function of S100 proteins are modulated by metal ions via Ca(2+) binding through EF-hand motifs and binding of Zn(2+) and Cu(2+) at additional sites, usually at the homodimer interfaces. Ca(2+) binding modulates S100 conformational opening and thus promotes and affects the interaction with p53, the receptor for advanced glycation endproducts and Toll-like receptor 4, among many others. Structural plasticity also occurs at the quaternary level, where several S100 proteins self-assemble into multiple oligomeric states, many being functionally relevant. Recently, we have found that the S100A8/A9 proteins are involved in amyloidogenic processes in corpora amylacea of prostate cancer patients, and undergo metal-mediated amyloid oligomerization and fibrillation in vitro. Here we review the unique chemical and structural properties of S100 proteins that underlie the conformational changes resulting in their oligomerization upon metal ion binding and ultimately in functional control. The possibility that S100 proteins have intrinsic amyloid-forming capacity is also addressed, as well as the hypothesis that amyloid self-assemblies may, under particular physiological conditions, affect the S100 functions within the cellular milieu.
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| 25. |
- Furukawa, Kentaro, et al.
(författare)
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Genetic screening for suppressor mutations of the hyper- or non-filamentous growth of the yeast osmotic signalling mutants
- 2010
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Ingår i: FEBS JOURNAL. - 1742-464X .- 1742-4658. ; 277:Suppl.1, s. 142-143
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- In certain yeast Saccharomyces cerevisiae strains, diploid cells develop pseudohyphae under nitrogen starvation, while haploid cells produce invasive filaments that penetrate the agar in rich medium. We have recently reported that these morphological developments are strongly inhibited under hyper-osmotic condition through the high-osmolarity glycerol (HOG) response MAPK pathway (Furukawa et al., 2009, Mol Microbiol). Deletion of the HOG1 MAPK gene enhances morphological developments, while expression of active Hog1 inhibits those even in the absence of hyper-osmotic stress. Moreover, it has been reported that HOG1 affects morphology and pathogenicity also in other fungal species. Candida albicans hog1 mutant cells form (pseudo)hyphae and show related transcriptome changes even in the absence of morphogenetic signals. Cryptococcus neoformans hog1 mutant cells enhance production of capsule and melanin, which are crucial virulence factors. Hence, fungal Hog1 orthologues are thought to be central negative regulators of many aspects of morphological developments and virulence, and studies on the underlying mechanisms are relevant for the identification of novel targets for antifungal therapy. In order to more deeply understand the inhibitory role of Hog1 in morphological developments, the proteins or signalling pathways affected by Hog1 need to be identified. In this study, we attempted to screen suppressor mutations of the hyper- or non-filamentous growth which are caused by HOG1 deletion or active Hog1, respectively. On the basis of the screening results, the mechanism by which Hog1 negatively regulates morphological developments will be discussed.
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| 26. |
- Garcia-Salcedo, Raúl, et al.
(författare)
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Glucose de-repression by yeast AMP-activated protein kinase SNF1 is controlled via at least two independent steps
- 2014
-
Ingår i: Febs Journal. - : Wiley. - 1742-464X .- 1742-4658. ; 281:7, s. 1901-1917
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Tidskriftsartikel (refereegranskat)abstract
- The AMP-activated protein kinase, AMPK, controls energy homeostasis in eukaryotic cells but little is known about the mechanisms governing the dynamics of its activation/deactivation. The yeast AMPK, SNF1, is activated in response to glucose depletion and mediates glucose de-repression by inactivating the transcriptional repressor Mig1. Here we show that overexpression of the Snf1-activating kinase Sak1 results, in the presence of glucose, in constitutive Snf1 activation without alleviating glucose repression. Co-overexpression of the regulatory subunit Reg1 of the Glc-Reg1 phosphatase complex partly restores glucose regulation of Snf1. We generated a set of 24 kinetic mathematical models based on dynamic data of Snf1 pathway activation and deactivation. The models that reproduced our experimental observations best featured (a) glucose regulation of both Snf1 phosphorylation and dephosphorylation, (b) determination of the Mig1 phosphorylation status in the absence of glucose by Snf1 activity only and (c) a regulatory step directing active Snf1 to Mig1 under glucose limitation. Hence it appears that glucose de-repression via Snf1-Mig1 is regulated by glucose via at least two independent steps: the control of activation of the Snf1 kinase and directing active Snf1 to inactivating its target Mig1.
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| 27. |
- Ge, Changrong, et al.
(författare)
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Membrane remodeling capacity of a vesicle-inducing glycosyltransferase
- 2014
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Ingår i: The FEBS Journal. - : Wiley. - 1742-464X .- 1742-4658. ; 281:16, s. 3667-3684
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Tidskriftsartikel (refereegranskat)abstract
- Intracellular vesicles are abundant in eukaryotic cells but absent in the Gram-negative bacterium Escherichia coli. However, strong overexpression of a monotopic glycolipid-synthesizing enzyme, monoglucosyldiacylglycerol synthase from Acholeplasma laidlawii (alMGS), leads to massive formation of vesicles in the cytoplasm of E. coli. More importantly, alMGS provides a model system for the regulation of membrane properties by membrane-bound enzymes, which is critical for maintaining cellular integrity. Both phenomena depend on how alMGS binds to cell membranes, which is not well understood. Here, we carry out a comprehensive investigation of the membrane binding of alMGS by combining bioinformatics methods with extensive biochemical studies, structural modeling and molecular dynamics simulations. We find that alMGS binds to the membrane in a fairly upright manner, mainly by residues in the N-terminal domain, and in a way that induces local enrichment of anionic lipids and a local curvature deformation. Furthermore, several alMGS variants resulting from substitution of residues in the membrane anchoring segment are still able to generate vesicles, regardless of enzymatic activity. These results clarify earlier theories about the driving forces for vesicle formation, and shed new light on the membrane binding properties and enzymatic mechanism of alMGS and related monotopic GT-B fold glycosyltransferases.
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| 28. |
- Geijer, Cecilia, 1980, et al.
(författare)
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Functional analysis of the Ashbya gossypii Fps1 homolog
- 2010
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Ingår i: FEBS Journal. - 1742-464X .- 1742-4658. ; 277:Supplement 1
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- Saccharomyces cerevisiae aquaglyceroporin Fps1 plays a central role in yeast osmoregulation, controlling the intracellular level of the compatible solute glycerol. When a cell encounter hyperosmotic conditions, Fps1 closes rapidly to ensure retention and accumulation of glycerol. In adaptation to lower external osmolarity, Fps1 opens again to release glycerol and hence turgor pressure. Mutants lacking Fps1 can not withstand a hypo-osmotic shock as well as wild type cells. Fps1 has unusually long N- and C-terminal extensions, and regulatory domains that are crucial for the gating mechanism have been identified on both termini. Fps1 also facilitates passive uptake of other small molecules such as arsenite and acidic acid. The filamentous fungi Ashbya gossypii Fps1 homolog (AgFps1) has shorter termini than Fps1. The aim of this study is to determine the function of AgFps1 by heterologous expression in S. cerevisiae fps1Δ mutants, and to study the physiological role of AgFps1 by deletion analysis in Ashbya gossypii. We can show that heterologous expression of AgFps1 in S. cerevisiae can substitute for Fps1 by releasing excessive glycerol upon a hypo-osmotic shock. AgFps1 expressed in S. cerevisiae appears to be hyperactive under hyperosmotic conditions, and exchanging the N- and C-terminal extensions for the corresponding termini of Fps1 is not sufficient to generate a regulated channel. Further, successful deletion of AgFPS1 renders an Ashbya gossypii mutant more resistant to arsenite than wild type fungi, indicating that AgFps1 transports arsenite.
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| 29. |
- Geijer, Cecilia, 1980, et al.
(författare)
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Initiation of the transcriptional response to hyperosmotic shock correlates with the potential for volume recovery.
- 2013
-
Ingår i: The FEBS journal. - : Wiley. - 1742-4658 .- 1742-464X. ; 280:16, s. 3854-67
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Tidskriftsartikel (refereegranskat)abstract
- The control of activity and localization of transcription factors is critical for appropriate transcriptional responses. In eukaryotes, signal transduction components such as mitogen-activated protein kinase (MAPK) shuttle into the nucleus to activate transcription. It is not known in detail how different amounts of nuclear MAPK over time affect the transcriptional response. In the present study, we aimed to address this issue by studying the high osmolarity glycerol (HOG) system in Saccharomyces cerevisiae. We employed a conditional osmotic system, which changes the period of the MAPK Hog1 signal independent of the initial stress level. We determined the dynamics of the Hog1 nuclear localization and cell volume by single-cell analysis in well-controlled microfluidics systems and compared the responses with the global transcriptional output of cell populations. We discovered that the onset of the initial transcriptional response correlates with the potential of cells for rapid adaptation; cells that are capable of recovering quickly initiate the transcriptional responses immediately, whereas cells that require longer time to adapt also respond later. This is reflected by Hog1 nuclear localization, Hog1 promoter association and the transcriptional response, but not Hog1 phosphorylation, suggesting that a presently uncharacterized rapid adaptive mechanism precedes the Hog1 nuclear response. Furthermore, we found that the period of Hog1 nuclear residence affects the amplitude of the transcriptional response rather than the spectrum of responsive genes.
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| 30. |
- Gossani, Cristiani, et al.
(författare)
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Evolutionary analysis of multidrug resistance genes in fungi – impact of gene duplication and family conservation
- 2014
-
Ingår i: The FEBS Journal. - : John Wiley & Sons. - 1742-464X .- 1742-4658. ; 281:22, s. 4967-4977
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Tidskriftsartikel (refereegranskat)abstract
- Although the emergence of bacterial drug resistance is of great concern to the scientific community, few studies have evaluated this phenomenon systematically in fungi by using genome‐wide datasets. In the present study, we assembled a large compendium of Saccharomyces cerevisiae chemical genetic data to study the evolution of multidrug resistance genes (MDRs) in the fungal lineage. We found that MDRs typically emerge in widely conserved families, most of which containing homologs from pathogenic fungi, such as Candida albicans and Coccidioides immitis, which could favor the evolution of drug resistance in those species. By integrating data from chemical genetics with protein family conservation, genetic and protein interactions, we found that gene families rarely have more than one MDR, indicating that paralogs evolve asymmetrically with regard to multidrug resistance roles. Furthermore, MDRs have more genetic and protein interaction partners than non‐MDRs, supporting their participation in complex biochemical systems underlying the tolerance to multiple bioactive molecules. MDRs share more chemical genetic interactions with other MDRs than with non‐MDRs, regardless of their evolutionary affinity. These results suggest the existence of an intricate system involved in the global drug tolerance phenotypes. Finally, MDRs are more likely to be hit repeatedly by mutations in laboratory evolution experiments, indicating that they have great adaptive potential. The results presented here not only reveal the main genomic features underlying the evolution of MDRs, but also shed light on the gene families from which drug resistance is more likely to emerge in fungi.
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| 31. |
- Guerra, Lina, et al.
(författare)
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Do bacterial genotoxins contribute to chronic inflammation, genomic instability and tumor progression?
- 2011
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Ingår i: The FEBS Journal. - : John Wiley & Sons. - 1742-464X .- 1742-4658. ; 278:23, s. 4577-4588
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Forskningsöversikt (refereegranskat)abstract
- Cytolethal distending toxin, produced by several Gram-negative bacteria, and colibactin, secreted by several commensal and extraintestinal pathogenic Escherichia coli strains, are the first bacterial genotoxins to be described to date. Exposure to cytolethal distending toxin and colibactin induces DNA damage, and consequently activates the DNA damage response, resulting in cell cycle arrest of the intoxicated cells and DNA repair. Irreversible DNA damage will lead to cell death by apoptosis or to senescence. It is well established that chronic exposure to DNA damaging agents, either endogenous (reactive oxygen species) or exogenous (ionizing radiation), may cause genomic instability as a result of the alteration of genes coordinating the DNA damage response, thus favoring tumor initiation and progression. In this review, we summarize the state of the art of the biology of cytolethal distending toxin and colibactin, focusing on the activation of the DNA damage response and repair pathways, and discuss the cellular responses induced in intoxicated cells, as well as how prolonged intoxication may lead to chronic inflammation, the accumulation of genomic instability, and tumor progression in both in vitro and in vivo models.
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| 32. |
- Gustavsson, Anna-Karin, 1986, et al.
(författare)
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Allosteric regulation of phosphofructokinase controls the emergence of glycolytic oscillations in isolated yeast cells
- 2014
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Ingår i: The FEBS Journal. - : Wiley. - 1742-464X .- 1742-4658. ; 281:12, s. 2784-2793
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Tidskriftsartikel (refereegranskat)abstract
- Oscillations are widely distributed in nature and synchronization of oscillators has been described at the cellular level (e.g. heart cells) and at the population level (e.g. fireflies). Yeast glycolysis is the best known oscillatory system, although it has been studied almost exclusively at the population level (i.e. limited to observations of average behaviour in synchronized cultures). We studied individual yeast cells that were positioned with optical tweezers in a microfluidic chamber to determine the precise conditions for autonomous glycolytic oscillations. Hopf bifurcation points were determined experimentally in individual cells as a function of glucose and cyanide concentrations. The experiments were analyzed in a detailed mathematical model and could be interpreted in terms of an oscillatory manifold in a three-dimensional state-space; crossing the boundaries of the manifold coincides with the onset of oscillations and positioning along the longitudinal axis of the volume sets the period. The oscillatory manifold could be approximated by allosteric control values of phosphofructokinase for ATP and AMP.
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| 33. |
- Gustavsson, Anna-Karin, 1986, et al.
(författare)
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FEBS Journal Prize Lecture: Sustained glycolytic oscillations in individual isolated yeast cells
- 2013
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Ingår i: FEBS Journal. - : Wiley. - 1742-4658 .- 1742-464X. ; 280:Suppl. S1
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- Yeast glycolytic oscillations have been extensively studied since the 1950s in dense populations of cells and in cell-free extracts. Until recently, sustained oscillations had only been observed at the population level, i.e. for synchronized cultures at high biomass concentrations. One question that had not been satisfactorily addressed was whether individual cells display qualitatively different behaviour from the mean behaviour of a population of cells. We were able to observe sustained oscillations in individual isolated cells using a sophisticated experimental setup in which the concentration of metabolites in glycolysis was quantified by measuring the autofluorescence intensity from NADH molecules in the individual cells, the extracellular environment was controlled both spatially and temporally using microfluidics, and the cell density and position of the cell array within the microfluidic flow chamber was varied using optical tweezers. We thus showed that a high cell density is not a requirement for induction of oscillatory behaviour. A detailed kinetic model for the cellular reactions was adjusted to describe isolated cells in a microfluidic flow chamber. It was successfully used to simulate the heterogeneity in the oscillatory response of the individual cells, assuming small differences in a single internal parameter. In further studies we have investigated the precise conditions for autonomous oscillations at the single cell level. We have also investigated how the extracellular environment affects the characteristics of the oscillations and the heterogeneity between cells. This setup also enables studies of cell-to-cell distance and flowrate dependence on cell communication and synchronization.
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| 34. |
- Hagman, Arne, et al.
(författare)
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Analysis on yeast short-term Crabtree effect and its origin.
- 2014
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Ingår i: The FEBS Journal. - : Wiley. - 1742-464X .- 1742-4658. ; 281:21, s. 4805-4814
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Tidskriftsartikel (refereegranskat)abstract
- The short-term Crabtree effect is defined as the immediate appearance of aerobic alcoholic fermentation upon a pulse of excess sugar to sugar-limited yeast cultures. In this paper we characterized ten different yeast species, having a clearly defined phylogenetic relationship. Yeast species were cultivated under glucose-limited conditions, and upon a glucose pulse we studied their general carbon metabolism. We generated an extensive collection of data on glucose and oxygen consumption, and ethanol and carbon dioxide generation. We conclude that Pichia, Debaryomyces, Eremothecium and Kluyveromyces marxianus yeasts did not exhibit any significant ethanol formation, while Kluyveromyces lactis behaved as an intermediate yeast, and Lachancea, Torulaspora, Vanderwaltozyma and Saccharomyces yeasts exhibited rapid ethanol accumulation. Based on our previous data set covering over forty yeast species for the presence of the long-term Crabtree effect and our present data, we can speculate that the origin of the short-term effect may coincide with the origin of the long-term Crabtree effect in the Saccharomycetales lineage, taking place approximately 150 million years ago. This article is protected by copyright. All rights reserved.
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| 35. |
- Hammarström, Per
(författare)
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The dynamic amyloid landscape
- 2010
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Ingår i: The FEBS Journal. - : Wiley-Blackwell. - 1742-464X .- 1742-4658. ; 277:Suppl. 1, s. 14-14
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract
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| 36. |
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| 37. |
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| 38. |
- Härdin, Hanna M., et al.
(författare)
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Clusters of reaction rates and concentrations in protein networks such as the phosphotransferase system
- 2014
-
Ingår i: The FEBS Journal. - : Wiley. - 1742-464X .- 1742-4658. ; 281:2, s. 531-548
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Forskningsöversikt (refereegranskat)abstract
- To understand the functioning of living cells, it is often helpful or even necessary to exploit inherent timescale disparities and focus on long-term dynamic behaviour. In the present study, we explore this type of behaviour for the biochemical network of the phosphotransferase system. We show that, during the slow phase that follows a fast initial transient, the network reaction rates are partitioned into clusters corresponding to connected parts of the reaction network. Rates within any of these clusters assume essentially the same value: differences within each cluster are vastly smaller than that from one cluster to another. This rate clustering induces an analogous clustering of the reactive compounds: only the molecular concentrations on the interface between these clusters are produced and consumed at substantially different rates and hence change considerably during the slow phase. The remaining concentrations essentially assume their steady-state values already by the end of the transient phase. Further, we find that this clustering phenomenon occurs for a large number of parameter values and also for models with different topologies; to each of these models, there corresponds a particular network partitioning. Our results show that, in spite of its complexity, the phosphotransferase system tends to behave in a rather simple (yet versatile) way. The persistence of clustering for the perturbed models we examined suggests that it is likely to be encountered in various environmental conditions, as well as in other signal transduction pathways with network structures similar to that of the phosphotransferase system.
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| 39. |
- Ignatovica, Vita, et al.
(författare)
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Identification and analysis of functionally important amino acids in human purinergic 12 receptor using a Saccharomyces cerevisiae expression system
- 2012
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Ingår i: The FEBS Journal. - : Wiley. - 1742-464X .- 1742-4658. ; 279:1, s. 180-191
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Tidskriftsartikel (refereegranskat)abstract
- The purinergic 12 receptor (P2Y12) is a major drug target for anticoagulant therapies, but little is known about the regions involved in ligand binding and activation of this receptor. We generated four randomized P2Y12 libraries and investigated their ligand binding characteristics. P2Y12 was expressed in a Saccharomyces cerevisiae model system. Four libraries were generated with randomized amino acids at positions 181, 256, 265 and 280. Mutant variants were screened for functional activity in yeast using the natural P2Y12 ligand ADP. Activation results were investigated using quantitative structure-activity relationship (QSAR) models and ligand-receptor docking. We screened four positions in P2Y12 for functional activity by substitution with amino acids with diverse physiochemical properties. This analysis revealed that positions E181, R256 and R265 alter the functional activity of P2Y12 in a specific manner. QSAR models for E181 and R256 mutant libraries strongly supported the experimental data. All substitutions of amino acid K280 were completely inactive, highlighting the crucial role of this residue in P2Y12 function. Ligand-receptor docking revealed that K280 is likely to be a key element in the ligand-binding pocket of P2Y12. The results of this study demonstrate that positions 181, 256, 265 and 280 of P2Y12 are important for the functional integrity of the receptor. Moreover, K280 appears to be a crucial feature of the P2Y12 ligand-binding pocket. These results are important for rational design of novel antiplatelet agents.
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| 40. |
- Ingvarsson, Henrik, 1975-, et al.
(författare)
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Flexibility and communication within the structure of the Mycobacterium smegmatis methionyl-tRNA synthetase
- 2010
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Ingår i: The FEBS Journal. - : Wiley. - 1742-464X .- 1742-4658. ; 277:19, s. 3947-3962
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Tidskriftsartikel (refereegranskat)abstract
- Two structures of monomeric methionyl-tRNA synthetase, from Mycobacterium smegmatis, in complex with the ligands methionine/adenosine and methionine, were analyzed by X-ray crystallography at 2.3 Å and at 2.8 Å, respectively. The structures demonstrated the flexibility of the multidomain enzyme. A new conformation of the structure was identified in which the connective peptide domain bound more closely to the catalytic domain than described previously. The KMSKS(301-305) loop in our structures was in an open and inactive conformation that differed from previous structures by a rotation of the loop of about 90° around hinges located at Asn297 and Val310. The binding of adenosine to the methionyl-tRNA synthetase methionine complex caused a shift in the KMSKS domain that brought it closer to the catalytic domain. The potential use of the adenosine-binding site for inhibitor binding was evaluated and a potential binding site for a specific allosteric inhibitor was identified.
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| 41. |
- Johansson, Renzo, et al.
(författare)
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High-resolution crystal structures of the flavoprotein NrdI in oxidized and reduced states – an unusual flavodoxin
- 2010
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Ingår i: The FEBS Journal. - : Wiley. - 1742-464X .- 1742-4658. ; 277:20, s. 4265-4277
-
Tidskriftsartikel (refereegranskat)abstract
- The small flavoprotein NrdI is an essential component of the class Ib ribonucleotide reductase system in many bacteria. NrdI interacts with the class Ib radical generating protein NrdF. It is suggested to be involved in the rescue of inactivated diferric centres or generation of active dimanganese centres in NrdF. Although NrdI bears a superficial resemblance to flavodoxin, its redox properties have been demonstrated to be strikingly different. In particular, NrdI is capable of two-electron reduction, whereas flavodoxins are exclusively one-electron reductants. This has been suggested to depend on a lesser destabilization of the negatively-charged hydroquinone state than in flavodoxins. We have determined the crystal structures of NrdI from Bacillus anthracis, the causative agent of anthrax, in the oxidized and semiquinone forms, at resolutions of 0.96 and 1.4 Å, respectively. These structures, coupled with analysis of all curated NrdI sequences, suggest that NrdI defines a new structural family within the flavodoxin superfamily. The conformational behaviour of NrdI in response to FMN reduction is very similar to that of flavodoxins, involving a peptide flip in a loop near the N5 atom of the flavin ring. However, NrdI is much less negatively charged than flavodoxins, which is expected to affect its redox properties significantly. Indeed, sequence analysis shows a remarkable spread in the predicted isoelectric points of NrdIs, from approximately pH 4–10. The implications of these observations for class Ib ribonucleotide reductase function are discussed.
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| 42. |
- Kallberg, Yvonne, et al.
(författare)
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Classification of the short-chain dehydrogenase/reductase superfamily using hidden Markov models
- 2010
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Ingår i: FEBS JOURNAL. - : Blackwell Publishing Ltd. - 1742-464X .- 1742-4658. ; 277:10, s. 2375-2386
-
Tidskriftsartikel (refereegranskat)abstract
- The short-chain dehydrogenase/reductase (SDR) superfamily now has over 47 000 members, most of which are distantly related, with typically 20-30% residue identity in pairwise comparisons, making it difficult to obtain an overview of this superfamily. We have therefore developed a family classification system, based upon hidden Markov models (HMMs). To this end, we have identified 314 SDR families, encompassing about 31 900 members. In addition, about 9700 SDR forms belong to families with too few members at present to establish valid HMMs. In the human genome, we find 47 SDR families, corresponding to 82 genes. Thirteen families are present in all three domains (Eukaryota, Bacteria, and Archaea), and are hence expected to catalyze fundamental metabolic processes. The majority of these enzymes are of the extended type, in agreement with earlier findings. About half of the SDR families are only found among bacteria, where the classical SDR type is most prominent. The HMM-based classification is used as a basis for a sustainable and expandable nomenclature system.
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| 43. |
- Korkmaz, Brice, et al.
(författare)
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Discriminating between the activities of human cathepsin G and chymase using fluorogenic substrates
- 2011
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Ingår i: The FEBS Journal. - : Wiley. - 1742-464X .- 1742-4658. ; 278:15, s. 2635-2646
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Tidskriftsartikel (refereegranskat)abstract
- Cathepsin G (CG) (EC 3.4.21.20) and chymase (EC 3.4.21.39) are two closely-related chymotrypsin-like proteases that are released from cytoplasmic granules of activated mast cells and/or neutrophils. We investigated the potential for their substrate-binding subsites to discriminate between their substrate specificities, aiming to better understand their respective role during the progression of inflammatory diseases. In addition to their preference for large aromatic residues at P1, both preferentially accommodate small hydrophilic residues at the S1' subsite. Despite significant structural differences in the S2' subsite, both prefer an acidic residue at that position. The Ala226/Glu substitution at the bottom of the CG S1 pocket, which allows CG but not chymase to accommodate a Lys residue at P1, is the main structural difference, allowing discrimination between the activities of these two proteases. However, a Lys at P1 is accommodated much less efficiently than a Phe, and the corresponding substrate is cleaved by beta 2-tryptase (EC 3.4.21.59). We optimized a P1 Lys-containing substrate to enhance sensitivity towards CG and prevent cleavage by chymase and beta 2-tryptase. The resulting substrate (ABZ-GIEPKSDPMPEQ-EDDnp) [ where ABZ is O-aminobenzoic acid and EDDnp is N-(2,4-dinitrophenyl)-ethylenediamine] was cleaved by CG but not by chymase and tryptase, with a specificity constant of 190 mM(-1).s(-1). This allows the quantification of active CG in cells or tissue extracts where it may be present together with chymase and tryptase, as we have shown using a HMC-1 cell homogenate and a sputum sample from a patient with severe asthma.
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| 44. |
- Kumar, Saroj, et al.
(författare)
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Conformational changes of recombinant Ca2+-ATPase studied by reaction-induced infrared difference spectroscopy
- 2013
-
Ingår i: The FEBS Journal. - : Wiley. - 1742-464X .- 1742-4658. ; 280:21, s. 5398-5407
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Tidskriftsartikel (refereegranskat)abstract
- Recombinant Ca2+-ATPase was expressed in Saccharomycescerevisiae with a biotin-acceptor domain linked to its C-terminus by a thrombin cleavage site. We obtained 200g of similar to 70% pure recombinant sarcoendoplasmic reticulum Ca2+-ATPase isoform1a (SERCA1a) from a 6-L yeast culture. The catalytic cycle of SERCA1a was followed in real time using rapid scan FTIR spectroscopy. Different intermediate states (Ca(2)E1P and Ca(2)E2P) of the recombinant protein were accumulated using different buffer compositions. The difference spectra of their formation from Ca(2)E1 had the same spectral features as those from the native rabbit SERCA1a. The enzyme-specific activity for the active enzyme fraction in both samples was also similar. The results show that the recombinant protein obtained from the yeast-based expression system has similar structural and dynamic properties as native rabbit SERCA1a. It is now possible to apply this expression system together with IR spectroscopy to the investigation of the role of individual amino acids.
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| 45. |
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| 46. |
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| 47. |
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| 48. |
- Lind, Jesper, et al.
(författare)
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Structural features of proinsulin C-peptide oligomeric and amyloid states
- 2010
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Ingår i: The FEBS Journal. - : Wiley. - 1742-464X .- 1742-4658. ; 277:18, s. 3759-68
-
Tidskriftsartikel (refereegranskat)abstract
- The formation and structure of proinsulin C-peptide oligomers has been investigated by PAGE, NMR spectroscopy and dynamic light scattering. The results obtained show that C-peptide forms oligomers of different sizes, and that their formation and size distribution is altered by salt and divalent metal ions, which indicates that the aggregation process is mediated by electrostatic interactions. It is further demonstrated that the size distribution of the C-peptide oligomers, in agreement with previous studies, is altered by insulin, which supports a physiologically relevant interaction between these two peptides. A small fraction of oligomers has previously been suggested to be in equilibrium with a dominant fraction of soluble monomers, and this pattern also is observed in the present study. The addition of modest amounts of sodium dodecyl sulphate at low pH increases the relative amount of oligomers, and this effect was used to investigate the details of both oligomer formation and structure by a combination of biophysical techniques. The structural properties of the SDS-induced oligomers, as obtained by thioflavin T fluorescence, CD spectroscopy and IR spectroscopy, demonstrate that soluble aggregates are predominantly in β-sheet conformation, and that the oligomerization process shows characteristic features of amyloid formation. The formation of large, insoluble, β-sheet amyloid-like structures will alter the equilibrium between monomeric C-peptide and oligomers. This leads to the conclusion that the oligomerization of C-peptide may be relevant also at low concentrations.
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| 49. |
- Lindgren, Mikael, et al.
(författare)
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Amyloid oligomers: spectroscopic characterization of amyloidogenic protein states
- 2010
-
Ingår i: The FEBS Journal. - : Wiley. - 1742-464X .- 1742-4658. ; 277:6, s. 1380-1388
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Forskningsöversikt (refereegranskat)abstract
- It is assumed that protein fibrils manifested in amyloidosis result from an aggregation reaction involving small misfolded protein sequences being in an oligomeric or prefibrillar state. This review covers recent optical spectroscopic studies of amyloid protein misfolding, oligomerization and amyloid fibril growth. Although amyloid fibrils have been studied using established protein-characterization techniques throughout the years, their oligomeric precursor states require sensitive detection in real-time. Here, fluorescent staining is commonly performed using thioflavin T and other small fluorescent molecules such as 4-(dicyanovinyl)- julolidine and 1-amino-8-naphtalene sulphonate that have high affinity to hydrophobic patches. Thus, populated oligomeric intermediates and related prefibrillar structures have been reported for several human amyloidogenic systems, including amyloid-beta protein, prion protein, transthyretin, alpha-synuclein, apolipoprotein C-II and insulin. To obtain information on the progression of the intermediate states, these were monitored in terms of fluorescence parameters, such as anisotropy, and quantum efficiency changes upon protein binding. Recently, new antibody stains have allowed precise monitoring of the oligomer size and distributions using multicolor labelling and single molecule detection. Moreover, a pentameric thiophene derivative (p-FTAA) was reported to indicate early precursors during A-beta(1-40) fibrillation, and was also demonstrated in real-time visualization of cerebral protein aggregates in transgenic AD mouse models by multiphoton microscopy. Conclusively, molecular probes and optical spectroscopy are now entering a phase enabling the in vivo interrogation of the role of oligomers in amyloidosis. Such techniques used in parallel with in vitro experiments, of increasing detail, will probably couple structure to pathogenesis in the near future.
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| 50. |
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