| 1. |
- Edman, Maria, et al.
(författare)
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Structural features of glycosyltransferases synthesizing major bilayer and nonbilayer-prone membrane lipids in Acholeplasma laidlawii and Streptococcus pneumoniae
- 2003
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Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 278:10, s. 8420-8428
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Tidskriftsartikel (refereegranskat)abstract
- In membranes of Acholeplasma laidlawii two consecutively acting glucosyltransferases, the (i) alpha-monoglucosyl-diacylglycerol. (MGlcDAG) synthase (aIMGS) (EC 2.4.1.157) and the (ii) alpha-diglucosyl-DAG (DGlcDAG) synthase (alDGS) (EC 2.4.1.208), are involved in maintaining (i) a certain anionic lipid surface charge density and (ii) constant nonbilayer/bilayer conditions (curvature packing stress), respectively. Cloning of the aIDGS gene revealed related uncharacterized sequence analogs especially in several Gram-positive pathogens, thermophiles and archaea, where the encoded enzyme function of a potential Streptococcus pneumoniae DGS gene (cpoA) was verified. A strong stimulation of aIDGS by phosphatidylglycerol (PG), cardiolipin, or nonbilayer-prone 1,3-DAG was observed, while only PG stimulated CpoA. Several secondary structure prediction and fold recognition methods were used together with SWISS-MODEL to build three-dimensional model structures for three MGS and two DGS lipid glycosyltransferases. Two Escherichia coli proteins with known structures were identified as the best templates, the membrane surface-associated two-domain glycosyltransferase MurG and the soluble GlcNAc epimerase. Differences in electrostatic surface potential between the different models and their individual domains suggest that electrostatic interactions play a role for the association to membranes. Further support for this was obtained when hybrids of the N- and C-domain, and full size alMGS with green fluorescent protein were localized to different regions of the E. coli inner membrane and cytoplasm in vivo. In conclusion, it is proposed that the varying abilities to bind, and sense lipid charge and curvature stress, are governed by typical differences in charge (pI values), amphiphilicity, and hydrophobicity for the N- and (catalytic) C-domains of these structurally similar membrane-associated enzymes.
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| 2. |
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| 3. |
- Granlund, Irene, et al.
(författare)
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The TL29 protein is lumen located, associated with PSII and not an ascorbate peroxidase
- 2009
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Ingår i: Plant and Cell Physiology. - : Oxford Journals. - 0032-0781 .- 1471-9053. ; 50:11, s. 1898-1910
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Tidskriftsartikel (refereegranskat)abstract
- The TL29 protein is one of the more abundant proteins in the thylakoid lumen of plant chloroplasts. Based on its sequence homology to ascorbate peroxidases, but without any supporting biochemical evidence, TL29 was suggested to be involved in the plant defense system against reactive oxygen species and consequently renamed to APX4. Our in vivo and in vitro analyses failed to show any peroxidase activity associated with TL29; it bound neither heme nor ascorbate. Recombinant overexpressed TL29 had no ascorbate-dependent peroxidase activity, and various mutational analyses aiming to convert TL29 into an ascorbate peroxidase failed. Furthermore, in the thylakoid lumen no such activity could be associated with TL29 and, additionally, TL29 knock-out mutants did not show any decreased peroxidase activity or increased content of radical oxygen species when grown under light stress. Instead we could show that TL29 is a lumen-located component associated with PSII.
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| 4. |
- Gustafsson, Lotta, et al.
(författare)
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Changes in proteasome structure and function caused by HAMLET in tumor cells.
- 2009
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Ingår i: PLoS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 4:4
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND: Proteasomes control the level of endogenous unfolded proteins by degrading them in the proteolytic core. Insufficient degradation due to altered protein structure or proteasome inhibition may trigger cell death. This study examined the proteasome response to HAMLET, a partially unfolded protein-lipid complex, which is internalized by tumor cells and triggers cell death. METHODOLOGY/PRINCIPAL FINDINGS: HAMLET bound directly to isolated 20S proteasomes in vitro and in tumor cells significant co-localization of HAMLET and 20S proteasomes was detected by confocal microscopy. This interaction was confirmed by co-immunoprecipitation from extracts of HAMLET-treated tumor cells. HAMLET resisted in vitro degradation by proteasomal enzymes and degradation by intact 20S proteasomes was slow compared to fatty acid-free, partially unfolded alpha-lactalbumin. After a brief activation, HAMLET inhibited proteasome activity in vitro and in parallel a change in proteasome structure occurred, with modifications of catalytic (beta1 and beta5) and structural subunits (alpha2, alpha3, alpha6 and beta3). Proteasome inhibition was confirmed in extracts from HAMLET-treated cells and there were indications of proteasome fragmentation in HAMLET-treated cells. CONCLUSIONS/SIGNIFICANCE: The results suggest that internalized HAMLET is targeted to 20S proteasomes, that the complex resists degradation, inhibits proteasome activity and perturbs proteasome structure. We speculate that perturbations of proteasome structure might contribute to the cytotoxic effects of unfolded protein complexes that invade host cells.
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| 5. |
- Hall, Michael, 1980-, et al.
(författare)
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The lumenal pentapeptide repeat proteins TL15 and TL20.3 are novel chaperone-like proteins in the chloroplast lumen of higher plants
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- In the thylakoid lumen of Arabidopsis thaliana, three pentapeptide repeat family proteins of unknown function are localized. Pentapeptide repeat proteins (PRP) are comprised of at least eight tandem repeats of five amino acids of the consensus sequence A(D/N)LXX, which fold into a quadrilateral beta helix structure. Here we have solved the crystal structure of the mature form of the lumenal PRP protein TL15 to 1.3 Å resolution. TL15 is comprised of a main pentapeptide domain, consisting of a total of 19 pentapeptide repeats which form five turns of a beta helix, and a C-terminal alpha helix domain consisting of two alpha helices. The alpha helices form a ‘cap’ at the C-terminal end of the beta helix and are connected by a disulphide bond between the conserved cysteine residues C122 and C142. Furthermore we show that the lumenal PRPs TL15 and TL20.3 can assist in refolding of a chemically denatured substrate in vitro, indicating foldase chaperone activity. The three lumenal PRPs have been previously identified as targets of thioredoxin, and interestingly we observed a greatly increased chaperone activity of TL15 and TL20.3 after reduction of their disulphide bonds. Our results provide the high resolution crystal structure of the TL15 protein and our analysis of chaperone activity suggests that TL15 and TL20.3 may constitute a novel type of redox-regulated molecular chaperones in the chloroplast lumen of higher plants.
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| 6. |
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| 7. |
- Lundberg, Erik, et al.
(författare)
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Crystal structure of the TL29 protein from Arabidopsis thaliana : An APX homolog without peroxidase activity
- 2011
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Ingår i: Journal of Structural Biology. - : Elsevier. - 1047-8477 .- 1095-8657. ; 176:1, s. 24-31
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Tidskriftsartikel (refereegranskat)abstract
- TL29 is a plant-specific protein found in the thylakoid lumen of chloroplasts. Despite the putative requirement in plants for a peroxidase close to the site of photosynthetic oxygen production, and the sequence homology of TL29 to ascorbate peroxidases, so far biochemical methods have not shown this enzyme to possess peroxidase activity. Here we report the three-dimensional X-ray crystal structure of recombinant TL29 from Arabidopsis thaliana at a resolution of 2.5 Å. The overall structure of TL29 is mainly alpha helical with six longer and six shorter helical segments. The TL29 structure resembles that of typical ascorbate peroxidases, however, crucial differences were found in regions that would be important for heme and ascorbate binding. Such differences suggest it to be highly unlikely that TL29 functions as a peroxidase.
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| 8. |
- Mishra, Yogesh, et al.
(författare)
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Active-site plasticity revealed in the asymmetric dimer of AnPrx6 the 1-Cys peroxiredoxin and molecular chaperone from Anabaena sp. PCC 7120
- 2017
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Ingår i: Scientific Reports. - : Nature Publishing Group. - 2045-2322. ; 7
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Tidskriftsartikel (refereegranskat)abstract
- Peroxiredoxins (Prxs) are vital regulators of intracellular reactive oxygen species levels in all living organisms. Their activity depends on one or two catalytically active cysteine residues, the peroxidatic Cys (C-P) and, if present, the resolving Cys (C-R). A detailed catalytic cycle has been derived for typical 2-Cys Prxs, however, little is known about the catalytic cycle of 1-Cys Prxs. We have characterized Prx6 from the cyanobacterium Anabaena sp. strain PCC7120 (AnPrx6) and found that in addition to the expected peroxidase activity, AnPrx6 can act as a molecular chaperone in its dimeric state, contrary to other Prxs. The AnPrx6 crystal structure at 2.3 angstrom resolution reveals different active site conformations in each monomer of the asymmetric obligate homo-dimer. Molecular dynamic simulations support the observed structural plasticity. A FSH motif, conserved in 1-Cys Prxs, precedes the active site PxxxTxxCp signature and might contribute to the 1-Cys Prx reaction cycle.
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| 9. |
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| 10. |
- Storm, Patrik, 1966-, et al.
(författare)
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Refolding and enzyme kinetic studies on the ferrochelatase of the cyanobacterium synechocystis sp. PCC 6803
- 2013
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Ingår i: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 8:2
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Tidskriftsartikel (refereegranskat)abstract
- Heme is a cofactor for proteins participating in many important cellular processes, including respiration, oxygen metabolism and oxygen binding. The key enzyme in the heme biosynthesis pathway is ferrochelatase (protohaem ferrolyase, EC 4.99.1.1), which catalyzes the insertion of ferrous iron into protoporphyrin IX. In higher plants, the ferrochelatase enzyme is localized not only in mitochondria, but also in chloroplasts. The plastidic type II ferrochelatase contains a C-terminal chlorophyll a/b (CAB) motif, a conserved hydrophobic stretch homologous to the CAB domain of plant light harvesting proteins and light-harvesting like proteins. This type II ferrochelatase, found in all photosynthetic organisms, is presumed to have evolved from the cyanobacterial ferrochelatase. Here we describe a detailed enzymological study on recombinant, refolded and functionally active type II ferrochelatase (FeCh) from the cyanobacterium Synechocystis sp. PCC 6803. A protocol was developed for the functional refolding and purification of the recombinant enzyme from inclusion bodies, without truncation products or soluble aggregates. The refolded FeCh is active in its monomeric form, however, addition of an N-terminal His6-tag has significant effects on its enzyme kinetics. Strikingly, removal of the C-terminal CAB-domain led to a greatly increased turnover number, kcat, compared to the full length protein. While pigments isolated from photosynthetic membranes decrease the activity of FeCh, direct pigment binding to the CAB domain of FeCh was not evident.
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