| 1. |
- Elbing, Karin, 1974, et al.
(författare)
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Transcriptional responses to glucose at different glycolytic rates in Saccharomyces cerevisiae
- 2004
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Ingår i: European Journal of Biochemistry. - : Wiley. - 0014-2956 .- 1432-1033. ; 271:23-24, s. 4855-4864
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Tidskriftsartikel (refereegranskat)abstract
- The addition of glucose to Saccharomyces cerevisiae cells causes reprogramming of gene expression. Glucose is sensed by membrane receptors as well as (so far elusive) intracellular sensing mechanisms. The availability of four yeast strains that display different hexose uptake capacities allowed us to study glucose-induced effects at different glycolytic rates. Rapid glucose responses were observed in all strains able to take up glucose, consistent with intracellular sensing. The degree of long-term responses, however, clearly correlated with the glycolytic rate: glucose-stimulated expression of genes encoding enzymes of the lower part of glycolysis showed an almost linear correlation with the glycolytic rate, while expression levels of genes encoding gluconeogenic enzymes and invertase (SUC2) showed an inverse correlation. Glucose control of SUC2 expression is mediated by the Snf1-Mig1 pathway. Mig1 dephosphorylation upon glucose addition is known to lead to repression of target genes. Mig1 was initially dephosphorylated upon glucose addition in all strains able to take up glucose, but remained dephosphorylated only at high glycolytic rates. Remarkably, transient Mig1-dephosphorylation was accompanied by the repression of SUC2 expression at high glycolytic rates, but stimulated SUC2 expression at low glycolytic rates. This suggests that Mig1-mediated repression can be overruled by factors mediating induction via a low glucose signal. At low and moderate glycolytic rates, Mig1 was partly dephosphorylated both in the presence of phosphorylated, active Snf1, and unphosphorylated, inactive Snf1, indicating that Mig1 was actively phosphorylated and dephosphorylated simultaneously, suggesting independent control of both processes. Taken together, it appears that glucose addition affects the expression of SUC2 as well as Mig1 activity by both Snf1-dependent and -independent mechanisms that can now be dissected and resolved as early and late/sustained responses.
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| 2. |
- Eriksson, Torny, et al.
(författare)
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Heterogeneity of homologously expressed Hypocrea jecorina (Trichoderma reesei) Cel7B catalytic module
- 2004
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Ingår i: European Journal of Biochemistry. - : Wiley. - 0014-2956 .- 1432-1033. ; 271:7, s. 1266-1276
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Tidskriftsartikel (refereegranskat)abstract
- The catalytic module of Hypocrea jecorina (previously Trichoderma reesei) Cel7B was homologously expressed by transformation of strain QM9414. Post-translational modifications in purified Cel7B preparations were analysed by enzymatic digestions, high performance chromatography, mass spectrometry and site-directed mutagenesis. Of the five potential sites found in the wild-type enzyme, only Asn56 and Asn182 were found to be N-glycosylated. GlcNAc(2)Man(5) was identified as the predominant N-glycan, although lesser amounts of GlcNAc(2)Man(7) and glycans carrying a mannophosphodiester bond were also detected. Repartition of neutral and charged glycan structures over the two glycosylation sites mainly accounts for the observed microheterogeneity of the protein. However, partial deamidation of Asn259 and a partially occupied O-glycosylation site give rise to further complexity in enzyme preparations.
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| 3. |
- Kang, J, et al.
(författare)
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Total chemical synthesis and NMR characterization of the glycopeptide tx5a, a heavily post-translationally modified conotoxin, reveals that the glycan structure is alpha-D-Gal-(1 -> 3)-alpha-D-GalNAc
- 2004
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Ingår i: European Journal of Biochemistry. - : Wiley. - 0014-2956 .- 1432-1033. ; 271:23-24, s. 4939-4949
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Tidskriftsartikel (refereegranskat)abstract
- The 13-amino acid glycopeptide tx5a (Gla-Cys-Cys-Gla-Asp-Gly-Trp*-Cys-Cys-Thr*-Ala-Ala-Hyp-OH, where Trp* = 6-bromotryptophan and Thr* = Gal-GalNAc-threonine), isolated from Conus textile, causes hyperactivity and spasticity when injected intracerebral ventricularly into mice. It contains nine post-translationally modified residues: four cysteine residues, two gamma-carboxyglutamic acid residues, and one residue each of 6-bromotryptophan, 4-trans-hydroxyproline and glycosylated threonine. The chemical nature of each of these has been determined with the exception of the glycan linkage pattern on threonine and the stereochemistry of the 6-bromotryptophan residue. Previous investigations have demonstrated that tx5a contains a disaccharide composed of N-acetylgalactosamine (GalNAc) and galactose (Gal), but the interresidue linkage was not characterized. We hypothesized that tx5a contained the T-antigen, beta-D-Gal-(1-->3)-alpha-D-GalNAc, one of the most common O-linked glycan structures, identified previously in another Conus glycopeptide, contalukin-G. We therefore utilized the peracetylated form of this glycan attached to Fmoc-threonine in an attempted synthesis. While the result-ing synthetic peptide (Gla-Cys-Cys-Gla-Asp-Gly-Trp*-Cys-Cys-Thr*-Ala-Ala-Hyp-OH, where Trp* =6-bromotryptophan and Thr* = beta-D-Gal-(1-->3)-alpha-D-GalNAc-threonine) and the native peptide had almost identical mass spectra, a comparison of their RP-HPLC chromatograms suggested that the two forms were not identical. Two-dimensional H-1 homonuclear and C-13-H-1 heteronuclear NMR spectroscopy of native tx5a isolated from Conus textile was then used to determine that the glycan present on tx5a indeed is not the aforementioned T-antigen, but rather alpha-D-Gal-(1-->3)-alpha-D-GalNAc.
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| 4. |
- Nicolaes, GAF, et al.
(författare)
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Altered inactivation pathway of factor Va by activated protein C in the presence of heparin
- 2004
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Ingår i: European Journal of Biochemistry. - : Wiley. - 0014-2956 .- 1432-1033. ; 271:13, s. 2724-2736
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Tidskriftsartikel (refereegranskat)abstract
- Inactivation of factor Va (FVa) by activated protein C (APC) is a predominant mechanism in the down-regulation of thrombin generation. In normal FVa, APC-mediated inactivation occurs after cleavage at Arg306 (with corresponding rate constant k'(306)) or after cleavage at Arg506 (k(506)) and subsequent cleavage at Arg306 (k(306)). We have studied the influence of heparin on APC-catalyzed FVa inactivation by kinetic analysis of the time courses of inactivation. Peptide bond cleavage was identified by Western blotting using FV-specific antibodies. In normal FVa, unfractionated heparin (UFH) was found to inhibit cleavage at Arg506 in a dose-dependent manner. Maximal inhibition of k(506) by UFH was 12-fold, with the secondary cleavage at Arg306 (k(306)) being virtually unaffected. In contrast, UFH stimulated the initial cleavage at Arg306 (k'(306)) two- to threefold. Low molecular weight heparin (Fragmin(R)) had the same effects on the rate constants of FVa inactivation as UFH, but pentasaccharide did not inhibit FVa inactivation. Analysis of these data in the context of the 3D structures of APC and FVa and of simulated APC-heparin and FVa-APC complexes suggests that the heparin-binding loops 37 and 70 in APC complement electronegative areas surrounding the Arg506 site, with additional contributions from APC loop 148. Fewer contacts are observed between APC and the region around the Arg306 site in FVa. The modeling and experimental data suggest that heparin, when bound to APC, prevents optimal docking of APC at Arg506 and promotes association between FVa and APC at position Arg306.
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| 5. |
- Tossavainen, H, et al.
(författare)
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NMR solution structure of calerythrin, an EF-hand calcium-binding protein from Saccharopolyspora erythraea
- 2003
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Ingår i: European Journal of Biochemistry. - : Wiley. - 0014-2956 .- 1432-1033. ; 270:11, s. 2505-2512
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Tidskriftsartikel (refereegranskat)abstract
- The structure of calerythrin, a prokaryotic 20 kDa calcium-binding protein has been determined by solution NMR spectroscopy. Distance, dihedral angle, J coupling, secondary chemical shift, residual dipolar coupling and radius of gyration restraints reveal four EF-hand motifs arranged in a compact globular structure. A tight turn in the middle of the amino acid sequence brings the two halves, each comprising a pair of EF-hands, close together. The structural similarity between calerythrin and the eukaryotic sarcoplasmic calcium-binding proteins is notable.
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| 6. |
- Henriksson, Maria, et al.
(författare)
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A nonphosphorylated 14-3-3 binding motif on exoenzyme S that is functional in vivo
- 2002
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Ingår i: European Journal of Biochemistry. - : Wiley. - 0014-2956 .- 1432-1033. ; 269:20, s. 4921-4929
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Tidskriftsartikel (refereegranskat)abstract
- 14-3-3 proteins play an important role in a multitude of signalling pathways. The interactions between 14-3-3 and other signalling proteins, such as Raf and KSR (kinase suppressor of Ras), occur in a phospho-specific manner. Recently, a phosphorylation-independent interaction has been reported to occur between 14-3-3 and several proteins, for example 5-phosphatase, p75NTR-associated cell death executor (NADE) and the bacterial toxin Exoenzyme S (ExoS), an ADP-ribosyltransferase from Pseudomonas aeruginosa. In this study we have identified the amino acid residues on ExoS, which are responsible for its specific interaction with 14-3-3. Furthermore, we show that a peptide derived from ExoS, containing the 14-3-3 interaction site, effectively competes out the interaction between ExoS and 14-3-3. In addition, competition with this peptide blocks ExoS modification of Ras in our Ras modification assay. We show that the ExoS protein interacts with all isoforms of the 14-3-3 family tested. Moreover, in vivo an ExoS protein lacking the 14-3-3 binding site has a reduced capacity to ADP ribosylate cytoplasmic proteins, e.g. Ras, and shows a reduced capacity to change the morphology of infected cells.
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| 7. |
- Tengel, Tobias, et al.
(författare)
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Conformational analysis by CD and NMR spectroscopy of a peptide encompassing the amphipathic domain of YopD from Yersinia
- 2002
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Ingår i: European Journal of Biochemistry. - : Wiley. - 0014-2956 .- 1432-1033. ; 269:15, s. 3659-3668
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Tidskriftsartikel (refereegranskat)abstract
- To establish an infection, Yersinia pseudotuberculosis utilizes a plasmid-encoded type III secretion machine that permits the translocation of several anti-host factors into the cytosol of target eukaryotic cells. Secreted YopD is essential for this process. Pre-secretory stabilization of YopD is mediated by an interaction with its cognate chaperone, LcrH. YopD possesses LcrH binding domains located in the N-terminus and in a predicted amphipathic domain located near the C-terminus. This latter domain is also critical for Yersinia virulence. In this study, we designed synthetic peptides encompassing the C-terminal amphipathic domain of YopD. A solution structure of YopD278−300, a peptide that strongly interacted with LcrH, was obtained by NMR methods. The structure is composed of a well-defined amphipathic α helix ranging from Phe280 to Tyr291, followed by a type I β turn between residues Val292 and His295. The C-terminal truncated peptides, YopD278−292 and YopD271−292, lacked helical structure, implicating the β turn in helix stability. An interaction between YopD278−300 and its cognate chaperone, LcrH, was observed by NMR through line-broadening effects and chemical shift differences between the free peptide and the peptide–LcrH complex. These effects were not observed for the unstructured peptide, YopD278−292, which confirms that the α helical structure of the YopD amphipathic domain is a critical binding region of LcrH.
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| 8. |
- Lake, Vanessa, et al.
(författare)
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ATPase activity of magnesium chelatase subunit I is required to maintain subunit D in vivo
- 2004
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Ingår i: European Journal of Biochemistry. - : Wiley. - 0014-2956 .- 1432-1033. ; 271:11, s. 2182-2188
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Tidskriftsartikel (refereegranskat)abstract
- During biosynthesis of chlorophyll, Mg2+ is inserted into protoporphyrin IX by magnesium chelatase. This enzyme consists of three different subunits of approximate to 40, 70 and 140 kDa. Seven barley mutants deficient in the 40 kDa magnesium chelatase subunit were analysed and it was found that this subunit is essential for the maintenance of the 70 kDa subunit, but not the 140 kDa subunit. The 40 kDa subunit has been shown to belong to the family of proteins called 'ATPases associated with various cellular activities', known to form ring-shaped oligomeric complexes working as molecular chaperones. Three of the seven barley mutants are semidominant mis-sense mutations leading to changes of conserved amino acid residues in the 40 kDa protein. Using the Rhodobacter capsulatus 40 and 70 kDa magnesium chelatase subunits we have analysed the effect of these mutations. Although having no ATPase activity, the deficient 40 kDa subunit could still associate with the 70 kDa protein. The binding was dependent on Mg2+ and ATP or ADP. Our study demonstrates that the 40 kDa subunit functions as a chaperon that is essential for the survival of the 70 kDa subunit in vivo. We conclude that the ATPase activity of the 40 kDa subunit is essential for this function and that binding between the two subunits is not sufficient to maintain the 70 kDa subunit in the cell. The ATPase deficient 40 kDa proteins fail to participate in chelation in a step after the association of the 40 and 70 kDa subunits. This step presumably involves a conformational change of the complex in response to ATP hydrolysis.
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| 9. |
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| 10. |
- Ramström, Margareta, et al.
(författare)
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A novel mass spectrometric approach to analysis of hormonal peptides in extracts of mouse pancreatic islets
- 2003
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Ingår i: European Journal of Biochemistry. - : Wiley. - 0014-2956 .- 1432-1033. ; 270:15, s. 3146-3152
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Tidskriftsartikel (refereegranskat)abstract
- Liquid chromatography mass spectrometry (LC-MS) is a valuable tool in the analysis of proteins and peptides. The combination of LC-MS with different fragmentation methods provides sequence information on components in complex mixtures. In this work, on-line packed capillary LC electrospray ionization Fourier transform ion cyclotron resonance MS was combined with two complementary fragmentation techniques, i.e. nozzle-skimmer fragmentation and electron capture dissociation, for the determination of hormonal peptides in an acid ethanol extract of mouse pancreatic islets. The most abundant peptides, those derived from proinsulin and proglucagon, were identified by their masses and additional sequence-tag information established their identities. Interestingly, the experiments demonstrated the presence of truncated C-peptides, des-(25-29)-C-peptide and des-(27-31)-C-peptide. These novel findings clearly illustrate the potential usefulness of the described technique for on-line sequencing and characterization of peptides in tissue extracts.
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