| 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. |
- Ahmadpour, Doryaneh, 1973, et al.
(författare)
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Hitchhiking on vesicles: a way to harness age-related proteopathies?
- 2020
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Ingår i: FEBS Journal. - : Wiley. - 1742-464X .- 1742-4658. ; 287:23, s. 5068-5079
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Tidskriftsartikel (refereegranskat)abstract
- Central to proteopathies and leading to most age-related neurodegenerative disorders is a failure in protein quality control (PQC). To harness the toxicity of misfolded and damaged disease proteins, such proteins are either refolded, degraded by temporal PQC, or sequestered by spatial PQC into specific, organelle-associated, compartments within the cell. Here, we discuss the impact of vesicle trafficking pathways in general, and syntaxin 5 in particular, as key players in spatial PQC directing misfolded proteins to the surface of vacuole and mitochondria, which facilitates their clearance and detoxification. Since boosting vesicle trafficking genetically can positively impact on spatial PQC and make cells less sensitive to misfolded disease proteins, we speculate that regulators of such trafficking might serve as therapeutic targets for age-related neurological disorders.
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| 4. |
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| 5. |
- Aliashkevich, Alena, et al.
(författare)
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LD-transpeptidases : the great unknown among the peptidoglycan cross-linkers
- 2022
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Ingår i: The FEBS Journal. - : John Wiley & Sons. - 1742-464X .- 1742-4658. ; 289:16, s. 4718-4730
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Forskningsöversikt (refereegranskat)abstract
- The peptidoglycan (PG) cell wall is an essential polymer for the shape and viability of bacteria. Its protective role is in great part provided by its mesh-like character. Therefore, PG-cross-linking enzymes like the penicillin-binding proteins (PBPs) are among the best targets for antibiotics. However, while PBPs have been in the spotlight for more than 50 years, another class of PG-cross-linking enzymes called LD-transpeptidases (LDTs) seemed to contribute less to PG synthesis and, thus, has kept an aura of mystery. In the last years, a number of studies have associated LDTs with cell wall adaptation to stress including β-lactam antibiotics, outer membrane stability, and toxin delivery, which has shed light onto the biological meaning of these proteins. Furthermore, as some species display a great abundance of LD-cross-links in their cell wall, it has been hypothesized that LDTs could also be the main synthetic PG-transpeptidases in some bacteria. In this review, we introduce these enzymes and their role in PG biosynthesis and we highlight the most recent advances in understanding their biological role in diverse species.
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| 6. |
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| 7. |
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| 8. |
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| 9. |
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| 10. |
- Andersen, Gorm, et al.
(författare)
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A gene duplication led to specialized gamma-aminobutyrate and beta-alanine aminotransferase in yeast
- 2007
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Ingår i: The FEBS Journal. - : Wiley. - 1742-464X .- 1742-4658. ; 274:7, s. 1804-1817
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Tidskriftsartikel (refereegranskat)abstract
- In humans, beta-alanine (BAL) and the neurotransmitter gamma-aminobutyrate (GABA) are transaminated by a single aminotransferase enzyme. Apparently, yeast originally also had a single enzyme, but the corresponding gene was duplicated in the Saccharomyces kluyveri lineage. SkUGA1 encodes a homologue of Saccharomyces cerevisiae GABA aminotransferase, and SkPYD4 encodes an enzyme involved in both BAL and GABA transamination. SkPYD4 and SkUGA1 as well as S. cerevisiae UGA1 and Schizosaccharomyces pombe UGA1 were subcloned, over-expressed and purified. One discontinuous and two continuous coupled assays were used to characterize the substrate specificity and kinetic parameters of the four enzymes. It was found that the cofactor pyridoxal 5'-phosphate is needed for enzymatic activity and alpha-ketoglutarate, and not pyruvate, as the amino group acceptor. SkPyd4p preferentially uses BAL as the amino group donor (V-max/K-m = 0.78 U.mg(-1).mM(-1)), but can also use GABA (V-max/K-m = 0.42 U.mg(-1).mM(-1)), while SkUga1p only uses GABA (V-max/K-m = 4.01 U.mg(-1).mM(-1)). SpUga1p and ScUga1p transaminate only GABA and not BAL. While mammals degrade BAL and GABA with only one enzyme, but in different tissues, S. kluyveri and related yeasts have two different genes/enzymes to apparently 'distinguish' between the two reactions in a single cell. It is likely that upon duplication similar to 200 million years ago, a specialized Uga1p evolved into a 'novel' transaminase enzyme with broader substrate specificity.
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| 11. |
- Andersson, Annika, et al.
(författare)
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Membrane integration and topology of RIFIN and STEVOR proteins of the Plasmodium falciparum parasite
- 2020
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Ingår i: The FEBS Journal. - : Wiley. - 1742-464X .- 1742-4658. ; 287:13, s. 2744-2762
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Tidskriftsartikel (refereegranskat)abstract
- The malarial parasite Plasmodium exports its own proteins to the cell surfaces of red blood cells (RBCs) during infection. Examples of exported proteins include members of the repetitive interspersed family (RIFIN) and subtelomeric variable open reading frame (STEVOR) family of proteins from Plasmodium falciparum. The presence of these parasite-derived proteins on surfaces of infected RBCs triggers the adhesion of infected cells to uninfected cells (rosetting) and to the vascular endothelium potentially obstructing blood flow. While there is a fair amount of information on the localization of these proteins on the cell surfaces of RBCs, less is known about how they can be exported to the membrane and the topologies they can adopt during the process. The first step of export is plausibly the cotranslational insertion of proteins into the endoplasmic reticulum (ER) of the parasite, and here, we investigate the insertion of three RIFIN and two STEVOR proteins into the ER membrane. We employ a well-established experimental system that uses N-linked glycosylation of sites within the protein as a measure to assess the extent of membrane insertion and the topology it assumes when inserted into the ER membrane. Our results indicate that for all the proteins tested, transmembranes (TMs) 1 and 3 integrate into the membrane, so that the protein assumes an overall topology of Ncyt-Ccyt. We also show that the segment predicted to be TM2 for each of the proteins likely does not reside in the membrane, but is translocated to the lumen.
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| 12. |
- 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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| 13. |
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| 14. |
- 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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| 15. |
- Bajusz, Csaba, et al.
(författare)
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The nuclear activity of the actin-binding Moesin protein is necessary for gene expression in Drosophila
- 2021
-
Ingår i: The FEBS Journal. - : Wiley. - 1742-464X .- 1742-4658. ; 288:16, s. 4812-4832
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Tidskriftsartikel (refereegranskat)abstract
- Ezrin-Radixin-Moesin (ERM) proteins play an essential role in the cytoplasm by cross-linking actin filaments with plasma membrane proteins. Research has identified the nuclear localization of ERMs, as well as the involvement of a single Drosophila ERM protein, Moesin, in nuclear mRNA exports. However, the question of how important the nuclear activity of ERM proteins are for the life of an organism has so far not been explored. Here, we present the first attempt to reveal the in vivo relevance of nuclear localization of Moesin in Drosophila. With the help of a nuclear export signal, we decreased the amount of Moesin in the nuclei of the animals. Furthermore, we observed various developmental defects, demonstrating the importance of ERM function in the nucleus for the first time. Transcriptome analysis of the mutant flies revealed that the lack of nuclear Moesin function leads to expression changes in nearly 700 genes, among them heat-shock genes. This result together with additional findings revealed that in Drosophila the expression of protein chaperones requires the nuclear functions of Moesin.
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| 16. |
- Bardales, José R., et al.
(författare)
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Identification of multiple isoforms of the cAMP-dependent protein kinase catalytic subunit in the bivalve mollusc Mytilus galloprovincialis
- 2008
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Ingår i: The FEBS Journal. - : Wiley. - 1742-464X .- 1742-4658. ; 275:18, s. 4479-4489
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Tidskriftsartikel (refereegranskat)abstract
- Several isoforms of the cAMP-dependent protein kinase catalytic subunit (C-subunit) were separated from the posterior adductor muscle and the mantle tissues of the sea mussel Mytilus galloprovincialis by cation exchange chromatography, and identified by: (a) protein kinase activity; (b) antibody recognition; and (c) peptide mass fingerprinting. Some of the isozymes seemed to be tissue-specific, and all them were phosphorylated at serine and threonine residues and showed slight but significant differences in their apparent molecular mass values, which ranged from 41.3 to 44.5 kDa. The results from the MS analysis suggest that at least some of the mussel C-subunit isoforms arise as a result of alternative splicing events. Furthermore, several peptide sequences from mussel C-subunits, determined by de novo sequencing, showed a high degree of homology with the mammalian Calpha-isoform, and contained some structural motifs that are essential for catalytic function. On the other hand, no significant differences were observed in the kinetic parameters of C-subunit isoforms, determined by using synthetic peptides as substrate and inhibitor. However, the C-subunit isoforms separated from the mantle tissue differed in their ability to phosphorylate in vitro some proteins present in a mantle extract.
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| 17. |
- Bartish, Galyna, et al.
(författare)
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Amino acids Thr56 and Thr58 are not essential for elongation factor 2 function in yeast
- 2007
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Ingår i: The FEBS Journal. - : Wiley. - 1742-464X .- 1742-4658. ; 274:20, s. 5285-5297
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Tidskriftsartikel (refereegranskat)abstract
- Yeast elongation factor 2 is an essential protein that contains two highly conserved threonine residues, T56 and T58, that could potentially be phosphorylated by the Rck2 kinase in response to environmental stress. The importance of residues T56 and T58 for elongation factor 2 function in yeast was studied using site directed mutagenesis and functional complementation. Mutations T56D, T56G, T56K, T56N and T56V resulted in nonfunctional elongation factor 2 whereas mutated factor carrying point mutations T56M, T56C, T56S, T58S and T58V was functional. Expression of mutants T56C, T56S and T58S was associated with reduced growth rate. The double mutants T56M/T58W and T56M/T58V were also functional but the latter mutant caused increased cell death and considerably reduced growth rate. The results suggest that the physiological role of T56 and T58 as phosphorylation targets is of little importance in yeast under standard growth conditions. Yeast cells expressing mutants T56C and T56S were less able to cope with environmental stress induced by increased growth temperatures. Similarly, cells expressing mutants T56M and T56M/T58W were less capable of adapting to increased osmolarity whereas cells expressing mutant T58V behaved normally. All mutants tested were retained their ability to bind to ribosomes in vivo. However, mutants T56D, T56G and T56K were under-represented on the ribosome, suggesting that these nonfunctional forms of elongation factor 2 were less capable of competing with wild-type elongation factor 2 in ribosome binding. The presence of nonfunctional but ribosome binding forms of elongation factor 2 did not affect the growth rate of yeast cells also expressing wild-type elongation factor 2.
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| 18. |
- Beatriz Badia, Mariana, et al.
(författare)
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Specific Arabidopsis thaliana malic enzyme isoforms can provide anaplerotic pyruvate carboxylation function in Saccharomyces cerevisiae
- 2017
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Ingår i: The FEBS Journal. - : WILEY. - 1742-464X .- 1742-4658. ; 284:4, s. 654-665
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Tidskriftsartikel (refereegranskat)abstract
- NAD(P)-malic enzyme (NAD(P)-ME) catalyzes the reversible oxidative decarboxylation of malate to pyruvate, CO2, and NAD(P)H and is present as a multigene family in Arabidopsis thaliana. The carboxylation reaction catalyzed by purified recombinant Arabidopsis NADP-ME proteins is faster than those reported for other animal or plant isoforms. In contrast, no carboxylation activity could be detected in vitro for the NAD-dependent counterparts. In order to further investigate their putative carboxylating role in vivo, Arabidopsis NAD(P)-ME isoforms, as well as the NADP-ME2del2 (with a decreased ability to carboxylate pyruvate) and NADP-ME2R115A (lacking fumarate activation) versions, were functionally expressed in the cytosol of pyruvate carboxylase-negative (Pyc(-)) Saccharomyces cerevisiae strains. The heterologous expression of NADP-ME1, NADP-ME2 (and its mutant proteins), and NADP-ME3 restored the growth of Pyc(-) S. cerevisiae on glucose, and this capacity was dependent on the availability of CO2. On the other hand, NADP-ME4, NAD-ME1, and NAD-ME2 could not rescue the Pyc(-) strains from C-4 auxotrophy. NADP-ME carboxylation activity could be measured in leaf crude extracts of knockout and over-expressing Arabidopsis lines with modified levels of NADP-ME, where this activity was correlated with the amount of NADP-ME2 transcript. These results indicate that specific A. thaliana NADP-ME isoforms are able to play an anaplerotic role in vivo and provide a basis for the study on the carboxylating activity of NADP-ME, which may contribute to the synthesis of C-4 compounds and redox shuttling in plant cells.
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| 19. |
- Beckman, Marie, et al.
(författare)
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Activation of cathepsin D by glycosaminoglycans
- 2009
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Ingår i: The FEBS Journal. - : Wiley. - 1742-464X .- 1742-4658. ; 276:24, s. 7343-7352
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Tidskriftsartikel (refereegranskat)abstract
- We have previously shown that heparin can increase the activity of the proenzyme form of Alzheimer's beta-site amyloid precursor protein cleaving enzyme 1 (BACE1). Cathepsin D (CD) is a member of the aspartic protease family and has sequence similarity to BACE1. Therefore, we examined whether heparin and other glycosaminoglycans (GAGs) can influence the activity of CD. Heparin and other GAGs were found to stimulate the activity of recombinant proCD. Desulfation of heparin almost abolished the stimulation, indicating that sulfate groups were important for the stimulatory effect. In addition, the stimulation was dependent on the length of the GAG chain, as larger GAGs were more potent in their ability to stimulate proCD than shorter fragments. In the presence of heparin, limited autocatalytic proteolysis of the proenzyme was increased, suggesting that heparin increases the activity of proCD by accelerating the conversion of proCD, which has little activity, to pseudoCD, an active form lacking residues 1-26 of the prodomain. Furthermore, the activity of spleen-derived mature CD, which lacks the entire 44 amino acid residue prodomain, was also increased by heparin, indicating that the catalytic domain of CD contains at least one region to which GAGs bind and stimulate enzyme activity. Because heparin also stimulated the activity of pseudoCD, proenzyme activation was probably accelerated by the interaction of heparin with the catalytic domain of pseudoCD. However, it is possible that heparin may also activate the proenzyme directly. On the basis of this study, we propose that GAGs may regulate CD activity in vivo.
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| 20. |
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| 21. |
- 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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| 22. |
- 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
-
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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| 23. |
- Bhalerao, Rishikesh P.
(författare)
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When to branch: seasonal control of shoot architecture in trees
- 2021
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Ingår i: The Febs Journal. - : Wiley. - 1742-464X .- 1742-4658. ; 289, s. 8062-8070
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Tidskriftsartikel (refereegranskat)abstract
- Long-lived perennial plants optimize their shoot architecture by responding to seasonal cues. The main strategy used by plants of temperate and boreal regions with respect to surviving the extremely unfavourable conditions of winter comprises the protection of their apical and lateral meristematic tissues. This involves myriads of transcriptional, translational and metabolic changes in the plants because shoot architecture is controlled by multiple pathways that regulate processes such as bud formation and flowering, small RNAs, environmental factors (especially light quality, photoperiod and temperature), hormones, and sugars. Recent studies have begun to reveal how these pathways are recruited for the seasonal adaptation and regulation of shoot architecture in perennial plants, including the role of a regulatory module consisting of antagonistic players terminal flower 1 (TFL1) and like-ap1 (LAP1) in the hybrid aspen. Here, we review recent progress in our understanding of the genetic control of shoot architecture in perennials compared to in annuals.
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| 24. |
- 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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| 25. |
- 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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| 26. |
- Blomberg, Jeanette, 1977-, et al.
(författare)
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Reduced FAS transcription in clones of U937 cells that have acquired resistance to Fas-induced apoptosis
- 2009
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Ingår i: The FEBS Journal. - : Wiley. - 1742-464X .- 1742-4658. ; 276:2, s. 497-508
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Tidskriftsartikel (refereegranskat)abstract
- Susceptibility to cell death is a prerequisite for the elimination of tumour cells by cytotoxic immune cells, chemotherapy or irradiation. Activation of the death receptor Fas is critical for the regulation of immune cell homeostasis and efficient killing of tumour cells by apoptosis. To define the molecular changes that occur during selection for insensitivity to Fas-induced apoptosis, a resistant variant of the U937 cell line was established. Individual resistant clones were isolated and characterized. The most frequently observed defect in the resistant cells was reduced Fas expression, which correlated with decreased FAS transcription. Clones with such reduced Fas expression also displayed partial cross-resistance to tumour necrosis factor-alpha stimulation, but the mRNA expression of tumour necrosis factor receptors was not decreased. Reintroduction of Fas conferred susceptibility to Fas but not to tumour necrosis factor-alpha stimulation, suggesting that several alterations could be present in the clones. The reduced Fas expression could not be explained by mutations in the FAS coding sequence or promoter region, or by silencing through methylations. Protein kinase B and extracellular signal-regulated kinase, components of signalling pathways downstream of Ras, were shown to be activated in some of the resistant clones, but none of the three RAS genes was mutated, and experiments using chemical inhibitors could not establish that the activation of these proteins was the cause of Fas resistance as described in other systems. Taken together, the data illustrate that Fas resistance can be caused by reduced Fas expression, which is a result of an unidentified mode of regulation.
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| 27. |
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| 28. |
- 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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| 29. |
- Borisova, Anna, et al.
(författare)
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Sequencing, biochemical characterization, crystal structure and molecular dynamics of cellobiohydrolase Cel7A from Geotrichum candidum 3C
- 2015
-
Ingår i: FEBS Journal. - : Wiley. - 1742-464X .- 1742-4658. ; 282, s. 4515-4537
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Tidskriftsartikel (refereegranskat)abstract
- The ascomycete Geotrichum candidum is a versatile and efficient decay fungus that is involved, for example, in biodeterioration of compact discs; notably, the 3C strain was previously shown to degrade filter paper and cotton more efficiently than several industrial enzyme preparations. Glycoside hydrolase (GH) family 7 cellobiohydrolases (CBHs) are the primary constituents of industrial cellulase cocktails employed in biomass conversion, and feature tunnel-enclosed active sites that enable processive hydrolytic cleavage of cellulose chains. Understanding the structure-function relationships defining the activity and stability of GH7 CBHs is thus of keen interest. Accordingly, we report the comprehensive characterization of the GH7 CBH secreted by G. candidum (GcaCel7A). The bimodular cellulase consists of a family 1 cellulose-binding module (CBM) and linker connected to a GH7 catalytic domain that shares 64% sequence identity with the archetypal industrial GH7 CBH of Hypocrea jecorina (HjeCel7A). GcaCel7A shows activity on Avicel cellulose similar to HjeCel7A, with less product inhibition, but has a lower temperature optimum (50 degrees C versus 60-65 degrees C, respectively). Five crystal structures, with and without bound thio-oligosaccharides, show conformational diversity of tunnel-enclosing loops, including a form with partial tunnel collapse at subsite -4 not reported previously in GH7. Also, the first O-glycosylation site in a GH7 crystal structure is reported - on a loop where the glycan probably influences loop contacts across the active site and interactions with the cellulose surface. The GcaCel7A structures indicate higher loop flexibility than HjeCel7A, in accordance with sequence modifications. However, GcaCel7A retains small fluctuations in molecular simulations, suggesting high processivity and low endo-initiation probability, similar to HjeCel7A.
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| 30. |
- Boukhelifa, Malika, et al.
(författare)
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The proline-rich protein palladin is a binding partner for profilin
- 2006
-
Ingår i: The FEBS Journal. - : Wiley. - 1742-464X .- 1742-4658. ; 273:1, s. 26-33
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Tidskriftsartikel (refereegranskat)abstract
- Palladin is an actin-associated protein that has been suggested to play critical roles in establishing cell morphology and maintaining cytoskeletal organization in a wide variety of cell types. Palladin has been shown previously to bind directly to three different actin-binding proteins vasodilator-stimulated phosphoprotein (VASP), α-actinin and ezrin, suggesting that it functions as an organizing unit that recruits actin-regulatory proteins to specific subcellular sites. Palladin contains sequences resembling a motif known to bind profilin. Here, we demonstrate that palladin is a binding partner for profilin, interacting with profilin via a poly proline-containing sequence in the amino-terminal half of palladin. Double-label immunofluorescence staining shows that palladin and profilin partially colocalize in actin-rich structures in cultured astrocytes. Our results suggest that palladin may play an important role in recruiting profilin to sites of actin dynamics.
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| 31. |
- Bresell, Anders, et al.
(författare)
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Bioinformatic and enzymatic characterization of the MAPEG superfamily
- 2005
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Ingår i: The FEBS Journal. - : Wiley. - 1742-464X .- 1742-4658. ; 272:7, s. 1688-1703
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Tidskriftsartikel (refereegranskat)abstract
- The membrane associated proteins in eicosanoid and glutathione metabolism (MAPEG) superfamily includes structurally related membrane proteins with diverse functions of widespread origin. A total of 136 proteins belonging to the MAPEG superfamily were found in database and genome screenings. The members were found in prokaryotes and eukaryotes, but not in any archaeal organism. Multiple sequence alignments and calculations of evolutionary trees revealed a clear subdivision of the eukaryotic MAPEG members, corresponding to the six families of microsomal glutathione transferases (MGST) 1, 2 and 3, leukotriene C4 synthase (LTC4), 5-lipoxygenase activating protein (FLAP), and prostaglandin E synthase. Prokaryotes contain at least two distinct potential ancestral subfamilies, of which one is unique, whereas the other most closely resembles enzymes that belong to the MGST2/FLAP/LTC4 synthase families. The insect members are most similar to MGST1/prostaglandin E synthase. With the new data available, we observe that fish enzymes are present in all six families, showing an early origin for MAPEG family differentiation. Thus, the evolutionary origins and relationships of the MAPEG superfamily can be defined, including distinct sequence patterns characteristic for each of the subfamilies. We have further investigated and functionally characterized representative gene products from Escherichia coli, Synechocystis sp., Arabidopsis thaliana and Drosophila melanogaster, and the fish liver enzyme, purified from pike (Esox lucius). Protein overexpression and enzyme activity analysis demonstrated that all proteins catalyzed the conjugation of 1-chloro-2,4-dinitrobenzene with reduced glutathione. The E. coli protein displayed glutathione transferase activity of 0.11 µmol·min−1·mg−1 in the membrane fraction from bacteria overexpressing the protein. Partial purification of the Synechocystis sp. protein yielded an enzyme of the expected molecular mass and an N-terminal amino acid sequence that was at least 50% pure, with a specific activity towards 1-chloro-2,4-dinitrobenzene of 11 µmol·min−1·mg−1. Yeast microsomes expressing the Arabidopsis enzyme showed an activity of 0.02 µmol·min−1·mg−1, whereas the Drosophila enzyme expressed in E. coli was highly active at 3.6 µmol·min−1·mg−1. The purified pike enzyme is the most active MGST described so far with a specific activity of 285 µmol·min−1·mg−1. Drosophila and pike enzymes also displayed glutathione peroxidase activity towards cumene hydroperoxide (0.4 and 2.2 µmol·min−1·mg−1, respectively). Glutathione transferase activity can thus be regarded as a common denominator for a majority of MAPEG members throughout the kingdoms of life whereas glutathione peroxidase activity occurs in representatives from the MGST1, 2 and 3 and PGES subfamilies.
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| 32. |
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| 33. |
- Bromée, Torun, et al.
(författare)
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Neuropeptide Y-family receptors Y6 and Y7 in chicken : Cloning, pharmacological characterization, tissue distribution and conserved synteny with human chromosome region
- 2006
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Ingår i: The FEBS Journal. - : Federation of European Biochemical Societies. - 1742-464X .- 1742-4658. ; 273:9, s. 2048-2063
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Tidskriftsartikel (refereegranskat)abstract
- The peptides of the neuropeptide Y (NPY) family exert their functions, including regulation of appetite and circadian rhythm, by binding to G-protein coupled receptors. Mammals have five subtypes, named Y1, Y2, Y4, Y5 and Y6, and recently Y7 has been discovered in fish and amphibians. In chicken we have previously characterized the first four subtypes and here we describe Y6 and Y7. The genes for Y6 and Y7 are located 1 megabase apart on chromosome 13, which displays conserved synteny with human chromosome 5 that harbours the Y6 gene. The porcine PYY radioligand bound the chicken Y6 receptor with a Kd of 0.80 ± 0.36 nm. No functional coupling was demonstrated. The Y6 mRNA is expressed in hypothalamus, gastrointestinal tract and adipose tissue. Porcine PYY bound chicken Y7 with a Kd of 0.14 ± 0.01 nm (mean ± SEM), whereas chicken PYY surprisingly had a much lower affinity, with a Ki of 41 nm, perhaps as a result of its additional amino acid at the N terminus. Truncated peptide fragments had greatly reduced affinity for Y7, in agreement with its closest relative, Y2, in chicken and fish, but in contrast to Y2 in mammals. This suggests that in mammals Y2 has only recently acquired the ability to bind truncated PYY. Chicken Y7 has a much more restricted tissue distribution than other subtypes and was only detected in adrenal gland. Y7 seems to have been lost in mammals. The physiological roles of Y6 and Y7 remain to be identified, but our phylogenetic and chromosomal analyses support the ancient origin of these Y receptor genes by chromosome duplications in an early (pregnathostome) vertebrate ancestor.
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| 34. |
- Brown, Christian, et al.
(författare)
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Structural and functional characterization of the microtubule interacting and trafficking domains of two oomycete chitin synthases
- 2016
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Ingår i: The FEBS Journal. - : Wiley. - 1742-464X .- 1742-4658. ; 283:16, s. 3072-3088
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Tidskriftsartikel (refereegranskat)abstract
- Chitin synthases (Chs) are responsible for the synthesis of chitin, a key structural cell wall polysaccharide in many organisms. They are essential for growth in certain oomycete species, some of which are pathogenic to diverse higher organisms. Recently, a Microtubule Interacting and Trafficking (MIT) domain, which is not found in any fungal Chs, has been identified in some oomycete Chs proteins. Based on experimental data relating to the binding specificity of other eukaryotic MIT domains, there was speculation that this domain may be involved in the intracellular trafficking of Chs proteins. However, there is currently no evidence for this or any other function for the MIT domain in these enzymes. To attempt to elucidate their function, MIT domains from two Chs enzymes from the oomycete Saprolegnia monoica were cloned, expressed and characterized. Both were shown to interact strongly with the plasma membrane component phosphatidic acid, and to have additional putative interactions with proteins thought to be involved in protein transport and localization. Aiding our understanding of these data, the structure of the first MIT domain from a carbohydrate-active enzyme (MIT1) was solved by NMR, and a model structure of a second MIT domain (MIT2) was built by homology modelling. Our results suggest a potential function for these MIT domains in the intracellular transport and/or regulation of Chs enzymes in the oomycetes.
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| 35. |
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| 36. |
- Bushnev, S., et al.
(författare)
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Chemical biosupercapacitors for biomedical application
- 2017
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Ingår i: The FEBS Journal. - : John Wiley & Sons. - 1742-464X .- 1742-4658. ; 284:S1, s. 207-207
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract
- The development of miniature autonomous bioelectronic devices that function in the human or animal internal environments is one of the most popular areas of bioelectronics. In recent works, a concept was developed for the creation of charge-storing fuel cells, or in other words self-charging supercapacitors based on (bio)electrodes with a dual function of generation and accumulation of electric charge and operating in both continuous and pulse modes. The main purpose of this work is to create a potentially implantable biodevice with a dual function of generation and accumulation of electrical charge on the basis of a membraneless nanobiocomposite biocathode with CNT/PANI/MvBOx composite material and a bioanode with GOx/AuNPs composite material, as well as investigation of their stability and efficiency in solutions close to the human blood. Nanobiocomposite materials are widely used as components of electronic devices for biomedical applications (biosensors, bio-fuel cells, biobataries, etc.) Modern bioelectronic devices based on nanocomposite materials can be used to influence organs and tissues, as well as for point delivery of drugs. Electrically conductive polymers are usually synthesized by chemical methods in an acid medium by oxidative polymerization of the monomer. This approach has a number of disadvantages, in particular, contamination of the final product with residual monomers and oxidant degradation products. Therefore, in this paper, electrochemical and enzymatic methods for the synthesis of electrically conducting polymers have been tested, which may be an alternative to chemical polymerization.
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| 37. |
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| 38. |
- Carlsson, Jonas, et al.
(författare)
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Investigation and prediction of the severity of p53 mutants using parameters from structural calculations
- 2009
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Ingår i: The FEBS Journal. - : Wiley. - 1742-464X .- 1742-4658. ; 276:15, s. 4142-4155
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Tidskriftsartikel (refereegranskat)abstract
- A method has been developed to predict the effects of mutations in the p53 cancer suppressor gene. The new method uses novel parameters combined with previously established parameters. The most important parameter is the stability measure of the mutated structure calculated using molecular modelling. For each mutant, a severity score is reported, which can be used for classification into deleterious and nondeleterious. Both structural features and sequence properties are taken into account. The method has a prediction accuracy of 77% on all mutants and 88% on breast cancer mutations affecting WAF1 promoter binding. When compared with earlier methods, using the same dataset, our method clearly performs better. As a result of the severity score calculated for every mutant, valuable knowledge can be gained regarding p53, a protein that is believed to be involved in over 50% of all human cancers.
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| 39. |
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| 40. |
- Cavinato, Maria, et al.
(författare)
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Targeting cellular senescence based on interorganelle communication, multilevel proteostasis, and metabolic control
- 2021
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Ingår i: The FEBS Journal. - : Wiley. - 1742-464X .- 1742-4658. ; 228:12, s. 3834-3854
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Forskningsöversikt (refereegranskat)abstract
- Cellular senescence, a stable cell division arrest caused by severe damage and stress, is a hallmark of aging in vertebrates including humans. With progressing age, senescent cells accumulate in a variety of mammalian tissues, where they contribute to tissue aging, identifying cellular senescence as a major target to delay or prevent aging. There is an increasing demand for the discovery of new classes of small molecules that would either avoid or postpone cellular senescence by selectively eliminating senescent cells from the body (i.e., 'senolytics') or inactivating/switching damage-inducing properties of senescent cells (i.e., 'senostatics/senomorphics'), such as the senescence-associated secretory phenotype. Whereas compounds with senolytic or senostatic activity have already been described, their efficacy and specificity has not been fully established for clinical use yet. Here, we review mechanisms of senescence that are related to mitochondria and their interorganelle communication, and the involvement of proteostasis networks and metabolic control in the senescent phenotype. These cellular functions are associated with cellular senescence in in vitro and in vivo models but have not been fully exploited for the search of new compounds to counteract senescence yet. Therefore, we explore possibilities to target these mechanisms as new opportunities to selectively eliminate and/or disable senescent cells with the aim of tissue rejuvenation. We assume that this research will provide new compounds from the chemical space which act as mimetics of caloric restriction, modulators of calcium signaling and mitochondrial physiology, or as proteostasis optimizers, bearing the potential to counteract cellular senescence, thereby allowing healthy aging.
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| 41. |
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| 42. |
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| 43. |
- 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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| 44. |
- Cedersund, Gunnar, et al.
(författare)
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Systems biology : Model Based Evaluation and Comparison of Potential Explanations for Given Biological Data
- 2009
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Ingår i: The FEBS Journal. - : Wiley-Blackwell. - 1742-464X .- 1742-4658. ; 276:4, s. 903-922
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Forskningsöversikt (refereegranskat)abstract
- Systems biology and its usage of mathematical modeling to analyse biological data is rapidly becoming an established approach to biology. A crucial advantage of this approach is that more information can be extracted from observations of intricate dynamics, which allows nontrivial complex explanations to be evaluated and compared. In this minireview we explain this process, and review some of the most central available analysis tools. The focus is on the evaluation and comparison of given explanations for a given set of experimental data and prior knowledge. Three types of methods are discussed: (a) for evaluation of whether a given model is sufficiently able to describe the given data to be nonrejectable; (b) for evaluation of whether a slightly superior model is significantly better; and (c) for a general evaluation and comparison of the biologically interesting features in a model. The most central methods are reviewed, both in terms of underlying assumptions, including references to more advanced literature for the theoretically oriented reader, and in terms of practical guidelines and examples, for the practically oriented reader. Many of the methods are based upon analysis tools from statistics and engineering, and we emphasize that the systems biology focus on acceptable explanations puts these methods in a nonstandard setting. We highlight some associated future improvements that will be essential for future developments of model based data analysis in biology.
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| 45. |
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| 46. |
- 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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| 47. |
- Czerwiec, E, et al.
(författare)
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Novel gamma-carboxyglutamic acid-containing peptides from the venom of Conus textile
- 2006
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Ingår i: The FEBS Journal. - : Wiley. - 1742-464X .- 1742-4658. ; 273:12, s. 2779-2788
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Tidskriftsartikel (refereegranskat)abstract
- The cone snail is the only invertebrate system in which the vitamin K-dependent carboxylase (or gamma-carboxylase) and its product gamma-carboxyglutamic acid (Gla) have been identified. It remains the sole source of structural information of invertebrate gamma-carboxylase substrates. Four novel Gla-containing peptides were purified from the venom of Conus textile and characterized using biochemical methods and mass spectrometry. The peptides Gla(1)-TxVI, Gla(2)-TxVI/A, Gla(2)-TxVI/B and Gla(3)-TxVI each have six Cys residues and belong to the O-superfamily of conotoxins. All four conopeptides contain 4-trans-hydroxyproline and the unusual amino acid 6-L >-bromotryptophan. Gla(2)-TxVI/A and Gla(2)-TxVI/B are isoforms with an amidated C-terminus that differ at positions +1 and +13. Three isoforms of Gla(3)-TxVI were observed that differ at position +7: Gla(3)-TxVI, Glu7-Gla(3)-TxVI and Asp7-Gla(3)-TxVI. The cDNAs encoding the precursors of the four peptides were cloned. The predicted signal sequences (amino acids -46 to -27) were nearly identical and highly hydrophobic. The predicted propeptide region (-20 to -1) that contains the gamma-carboxylation recognition site (gamma-CRS) is very similar in Gla(2)-TxVI/A, Gla(2)-TxVI/B and Gla(3)-TxVI, but is more divergent for Gla(1)-TxVI. Kinetic studies utilizing the Conus gamma-carboxylase and synthetic peptide substrates localized the gamma-CRS of Gla(1)-TxVI to the region -14 to -1 of the polypeptide precursor: the K-m was reduced from 1.8 mM for Gla (1)-TxVI lacking a propeptide to 24 mu M when a 14-residue propeptide was attached to the substrate. Similarly, addition of an 18-residue propeptide to Gla(2)-TxVI/B reduced the K-m value tenfold.
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| 48. |
- Dahl, Göran, et al.
(författare)
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Effects on protease inhibition by modifying of helicase residues in hepatitis C virus nonstructural protein 3
- 2007
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Ingår i: The FEBS Journal. - : Wiley. - 1742-464X .- 1742-4658. ; 274:22, s. 5979-5986
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Tidskriftsartikel (refereegranskat)abstract
- This study of the full-length bifunctional nonstructural protein 3 from hepatitis C virus (HCV) has revealed that residues in the helicase domain affect the inhibition of the protease. Two residues (Q526 and H528), apparently located in the interface between the S2 and S4 binding pockets of the substrate binding site of the protease, were selected for modification, and three enzyme variants (Q526A, H528A and H528S) were expressed, purified and characterized. The substitutions resulted in indistinguishable Km values and slightly lower kcat values compared to the wild-type. The Ki values for a series of structurally diverse protease inhibitors were affected by the substitutions, with increases or decreases up to 10-fold. The inhibition profiles for H528A and H528S were different, confirming that not only did the removal of the imidazole side chain have an effect, but also that minor differences in the nature of the introduced side chain influenced the characteristics of the enzyme. These results indicate that residues in the helicase domain of nonstructural protein 3 can influence the protease, supporting our hypothesis that full-length hepatitis C virus nonstructural protein 3 should be used for protease inhibitor optimization and characterization. Furthermore, the data suggest that inhibitors can be designed to interact with residues in the helicase domain, potentially leading to more potent and selective compounds.
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| 49. |
- Dahlroth, Sue-Li, et al.
(författare)
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Crystal structure of the shutoff and exonuclease protein from the oncogenic Kaposi's sarcoma-associated herpesvirus
- 2009
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Ingår i: The FEBS Journal. - : Wiley. - 1742-464X .- 1742-4658. ; 276:22, s. 6636-6645
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Tidskriftsartikel (refereegranskat)abstract
- The Kaposi's sarcoma-associated herpesvirus protein SOX (shut off and exonuclease) and its Epstein–Barr virus homolog, BGLF5, are active during the early lytic phase and belong to the alkaline nuclease family. Both proteins have been shown to be bifunctional, being responsible for DNA maturation as well as host shutoff at the mRNA level. We present the crystal structure of SOX determined at 1.85 Å resolution. By modeling DNA binding, we have identified catalytic residues that explain the preferred 5'-exonuclease activity of the alkaline nucleases. The presence of a crevice suitable for binding duplex DNA supports a role for herpes alkaline nucleases in recombination events preceding packaging of viral DNA. Direct interaction with dsDNA is supported by oligonucleotide binding data. Mutations specifically affecting host shutoff map to a surface region of the N-terminal domain, implying an essential role in protein–protein interactions, and link the RNase activity of the enzyme to mRNA degradation pathways.
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| 50. |
- Danielsson, Anna, et al.
(författare)
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Insulin resistance in human adipocytes occurs downstream of IRS1 after surgical cell isolation but at the level of phosphorylation of IRS1 in type 2 diabetes
- 2005
-
Ingår i: The FEBS Journal. - : Wiley. - 1742-464X .- 1742-4658. ; 272:1, s. 141-151
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Tidskriftsartikel (refereegranskat)abstract
- Insulin resistance is a cardinal feature of type 2 diabetes and also a consequence of trauma such as surgery. Directly after surgery and cell isolation, adipocytes were insulin resistant, but this was reversed after overnight incubation in 10% CO2 at 37 °C. Tyrosine phosphorylation of the insulin receptor and insulin receptor substrate (IRS)1 was insulin sensitive, but protein kinase B (PKB) and downstream metabolic effects exhibited insulin resistance that was reversed by overnight incubation. MAP-kinases ERK1/2 and p38 were strongly phosphorylated after surgery, but was dephosphorylated during reversal of insulin resistance. Phosphorylation of MAP-kinase was not caused by collagenase treatment during cell isolation and was present also in tissue pieces that were not subjected to cell isolation procedures. The insulin resistance directly after surgery and cell isolation was different from insulin resistance of type 2 diabetes; adipocytes from patients with type 2 diabetes remained insulin resistant after overnight incubation. IRS1, PKB, and downstream metabolic effects, but not insulin-stimulated tyrosine phosphorylation of insulin receptor, exhibited insulin resistance. These findings suggest a new approach in the study of surgery-induced insulin resistance and indicate that human adipocytes should recover after surgical procedures for analysis of insulin signalling. Moreover, we pinpoint the signalling dysregulation in type 2 diabetes to be the insulin-stimulated phosphorylation of IRS1 in human adipocytes.
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