| 1. |
- Blombach, Fabian, et al.
(författare)
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Archaeal MBF1 binds to 30S and 70S ribosomes via its helix-turn-helix domain
- 2014
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Ingår i: Biochemical Journal. - 0264-6021 .- 1470-8728. ; 462, s. 373-384
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Tidskriftsartikel (refereegranskat)abstract
- MBF1 (multi-protein bridging factor 1) is a protein containing a conserved HTH (helix-turn-helix) domain in both eukaryotes and archaea. Eukaryotic MBF1 has been reported to function as a transcriptional co-activator that physically bridges transcription regulators with the core transcription initiation machinery of RNA polymerase II. In addition, MBF1 has been found to be associated with polyadenylated mRNA in yeast as well as in mammalian cells. aMBF1 (archaeal MBF1) is very well conserved among most archaeal lineages; however, its function has so far remained elusive. To address this, we have conducted a molecular characterization of this aMBF1. Affinity purification of interacting proteins indicates that aMBF1 binds to ribosomal subunits. On sucrose density gradients, aMBF1 co-fractionates with free 30S ribosomal subunits as well as with 70S ribosomes engaged in translation. Binding of aMBF1 to ribosomes does not inhibit translation. Using NMR spectroscopy, we show that aMBF1 contains a long intrinsically disordered linker connecting the predicted N-terminal zinc-ribbon domain with the C-terminal HTH domain. The HTH domain, which is conserved in all archaeal and eukaryotic MBF1 homologues, is directly involved in the association of aMBF1 with ribosomes. The disordered linker of the ribosome-bound aMBF1 provides the N-terminal domain with high flexibility in the aMBF1 ribosome complex. Overall, our findings suggest a role for aMBF1 in the archaeal translation process.
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| 2. |
- Dobritzsch, Doreen, 1972-, et al.
(författare)
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Structural and functional characterization of ochratoxinase, a novel mycotoxin-degrading enzyme
- 2014
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Ingår i: Biochemical Journal. - 0264-6021 .- 1470-8728. ; 462:3, s. 441-452
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Tidskriftsartikel (refereegranskat)abstract
- Ochratoxin, with ochratoxin A as the dominant form, is one of the five major mycotoxins most harmful to humans and animals. It is produced by Aspergillus and Penicillium species and occurs in a wide range of agricultural products. Detoxification of contaminated food is a challenging health issue. In the present paper we report the identification, characterization and crystal structure (at 2.2 angstrom) of a novel microbial ochratoxinase from Aspergillus niger. A putative amidase gene encoding a 480 amino acid polypeptide was cloned and homologously expressed in A. niger. The recombinant protein is N-terminally truncated, thermostable, has optimal activity at pH similar to 6 and 66 degrees C, and is more efficient in ochratoxin A hydrolysis than carboxypeptidase A and Y, the two previously known enzymes capable of degrading this mycotoxin. The subunit of the homo-octameric enzyme folds into a two-domain structure characteristic of a metal dependent amidohydrolase, with a twisted TIM (triosephosphateisomerase)-barrel and a smaller beta-sandwich domain. The active site contains an aspartate residue for acid base catalysis, and a carboxylated lysine and four histidine residues for binding of a binuclear metal centre.
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| 3. |
- Doyle, Siamsa, et al.
(författare)
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Trafficking modulator TENin1 inhibits endocytosis, causes endomembrane protein accumulation at the pre-vacuolar compartment and impairs gravitropic response in Arabidopsis thaliana
- 2014
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Ingår i: Biochemical Journal. - 0264-6021 .- 1470-8728. ; 460, s. 177-185
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Tidskriftsartikel (refereegranskat)abstract
- Auxin gradients are established and maintained by polarized distribution of auxin transporters that undergo constitutive endocytic recycling from the PM (plasma membrane) and are essential for the gravitropic response in plants. The present study characterizes an inhibitor of endomembrane protein trafficking, TE1 (trafficking and endocytosis inhibitor 1/TENin1) that reduces gravitropic root bending in Arabidopsis thaliana seedlings. Short-term TE1 treatment causes accumulation of PM proteins, including the BR (brassinosteroid) receptor BRI1 (BR insensitive 1), PIP2a (PM intrinsic protein 2a) and the auxin transporter PIN2 (PIN-FORMED 2) in a PVC (pre-vacuolar related compartment), which is sensitive to BFA (Brefeldin A). This compound inhibits endocytosis from the PM and promotes trafficking to the vacuole, consistent with inhibition of retrieval of proteins to the TGN (trans-Golgi network) from the PVC and the PM. However, trafficking of newly synthesized proteins to the PM is unaffected. The short-term protein trafficking inhibition and long-term effect on plant growth and survival caused by TE1 were fully reversible upon drug washout. Structure activity relationship studies revealed that only minor modifications were possible without loss of biological activity. Diversity in Arabidopsis ecotypes was also exploited to identify two Arabidopsis accessions that display reduced sensitivity to TE1. This compound and the resistant Arabidopsis accessions may be used as a resource in future studies to better understand endomembrane trafficking in plants.
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| 4. |
- Marino, Giada, et al.
(författare)
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Family-wide characterization of Matrix Metallo-proteinases from Arabidopsis thaliana reveals their distinct proteolytic activity and cleavage site specificity.
- 2014
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Ingår i: Biochemical Journal. - : Biochemical Journal. - 0264-6021 .- 1470-8728. ; 457:2, s. 335-346
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Tidskriftsartikel (refereegranskat)abstract
- Matrix metalloproteases (MMPs) are a family of zinc-dependent endopeptidases widely distributed throughout all kingdoms of life. In mammals, MMPs play key roles in many physiological and pathological processes including remodeling of the extracellular matrix. In the genome of the annual plant Arabidopsis thaliana five MMP-like proteins (At-MMPs) are encoded, but their function is unknown. Previous work on these enzymes was limited to gene expression analysis, and so far proteolytic activity has been shown only for At1-MMP. We expressed and purified the catalytic domains of all five At-MMPs as His-tagged proteins in E.coli to delineate the biochemical differences and similarities among the Arabidopsis MMP family members. We demonstrate that all five recombinant At-MMPs are active proteases with distinct preferences for different protease substrates. Furthermore, we performed a family-wide characterization of their biochemical properties and highlight similarities and differences in their cleavage site specificities as well as pH- and temperature dependent activities. Detailed analysis of their sequence specificity using Proteomic Identification of protease Cleavage Sites (PICS) revealed profiles similar to human MMPs with the exception of At5-MMP; homology models of the At-MMP catalytic domains supported these results. Our results suggest that each At-MMP may be involved in different proteolytic processes during plant growth and development.
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| 5. |
- O'Malley, Tiernan T., et al.
(författare)
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A beta dimers differ from monomers in structural propensity, aggregation paths and population of synaptotoxic assemblies
- 2014
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Ingår i: Biochemical Journal. - 0264-6021. ; 461, s. 413-426
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Tidskriftsartikel (refereegranskat)abstract
- Dimers of A beta (amyloid beta-protein) are believed to play an important role in Alzheimer's disease. In the absence of sufficient brain-derived dimers, we studied one of the only possible dimers that could be produced in vivo, [A beta](DiY) (dityrosine cross-linked A beta). For comparison, we used the A beta monomer and a design dimer cross-linked by replacement of Ser(26) with cystine [A beta S26C](2). We showed that similar to monomers, unaggregated dimers lack appreciable structure and fail to alter long-term potentiation. Importantly, dimers exhibit subtly different structural propensities from monomers and each other, and can self-associate to form larger assemblies. Although [A beta](DiY) and [A beta S26C](2) have distinct aggregation pathways, they both populate bioactive soluble assemblies for longer durations than A beta monomers. Our results indicate that the link between A beta dimers and Alzheimer's disease results from the ability of dimers to further assemble and form synaptotoxic assemblies that persist for long periods of time.
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| 6. |
- Reyhani, Vahid, et al.
(författare)
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Fibrin binds to collagen and provides a bridge for alpha V beta 3 integrin-dependent contraction of collagen gels
- 2014
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Ingår i: Biochemical Journal. - 0264-6021 .- 1470-8728. ; 462, s. 113-123
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Tidskriftsartikel (refereegranskat)abstract
- The functional significance of fibrin deposits typically seen in inflammatory lesions, carcinomas and in healing wounds is not fully understood. In the present study, we demonstrate that fibrinogen/fibrin specifically bound to native Col I (collagen type I) and used the Col I fibre network as a base to provide a functional interface matrix that connects cells to the Col I fibres through alpha V beta 3 integrins. This allowed murine myoblast C2C12 cells to contract the collagenous composite gel via alpha V beta 3 integrin. We show that fibrinogen specifically bound to immobilized native Col I at the site known to bind matrix metalloproteinase-1, discoidin domain receptor-2 and fibronectin, and that binding had no effect on Col I fibrillation. A specific competitive inhibitor blocking the Col-I-binding site for fibrinogen abolished the organization of fibrin into discernable fibrils, as well as the C2C12-mediated contraction of Col I gels. Our data show that fibrin can function as a linkage protein between Col I fibres and cells, and suggest that fibrin at inflammatory sites indirectly connects alpha V beta 3 integrins to Col I fibres and thereby promotes cell-mediated contraction of collagenous tissue structures.
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| 7. |
- van Dijk, Jesper R., et al.
(författare)
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Tumour-associated mutations of PA-TM-RING ubiquitin ligases RNF167/RNF13 identify the PA domain as a determinant for endosomal localization
- 2014
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Ingår i: Biochemical Journal. - 0264-6021 .- 1470-8728. ; 459:1, s. 27-36
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Tidskriftsartikel (refereegranskat)abstract
- Diverse cellular processes depend on endocytosis, intracellular vesicle trafficking, sorting and exocytosis, and processes that are regulated post-transcriptionally by modifications such as phosphorylation and ubiquitylation. The PA (protease-associated) domain E3 ligases, such as Godzilla(CG10277) in Drosophila melanogaster and RNF167 (RING finger protein 167) in humans, have been implicated in the regulation of cellular endosome trafficking. In the present study, we have characterized point mutations in the RING (really interesting new gene) domain of human RNF13 and RNF167, which have been identified in human tumour samples, that abrogate ubiquitin ligase activity as well as function. In the present study, we have also identified a functional role for the PA domain, which is required for endosomal localization of these proteins. Although the PA domain point mutations of RNF13 and RNF167 identified in human tumours are ligase active, the resultant mutant proteins are mislocalized within the cell. Thus the PA domain E3 ligases examined in the present study appear to require both E3 ligase activity as well as an intact PA domain to efficiently target and ubiquitylate their cellular substrates.
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| 8. |
- von Mach, Tobias, et al.
(författare)
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Ligand binding and complex formation of galectin-3 is modulated by pH variations
- 2014
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Ingår i: Biochemical Journal. - 0264-6021. ; 457:1, s. 107-115
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Tidskriftsartikel (refereegranskat)abstract
- Galectin-3-dependent clusters or lattices are formed at the surface as well as in distinct organelles of eukaryotic cells. Incorporation into membrane proximal networks can fix glycoproteins within subcellular domains or sort them into distinct transport pathways. In the present paper we analysed the effect of acidification on the sugar binding and self-oligomerization of galectin-3. Using a fluorescence anisotropy assay we measured decreasing galectin-3 affinities to the blood group antigen GalNAcα1- 3(Fucα1-2) Galβ1-4Glc under low pH conditions. Binding to the strong interaction partner N-acetyl-D-lactosamine was also lost at pH 5.0, whereas the less efficient ligand lactose was still able to bind. This indicates that variations in the binding specificity to distinct glycans can be observed by altering the pH. The formation of galectin-3-based complexes by interaction with the multivalent glycoproteins asialofetuin or transferrin was also obliterated at acidic pH and the ligand-binding affinity itself was modulated by oligomerization of the lectin.When galectin-3 was added to giant plasma membrane vesicles from the apical surface of epithelial cells, pH modulation could generate or eliminate the formation of membrane domains enriched with p75 NTR (neurotrophin receptor p75). In conclusion, the results of the present study suggest that the formation and composition of galectin-3 networks can be finetuned by changes in the environmental pH.
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