| 331. |
- Wiktelius, Eric, et al.
(författare)
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Novel class of glutathione transferases from cyanobacteria exhibit high catalytic activities towards naturally occurring isothiocyanates
- 2007
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Ingår i: Biochemical Journal. - 0264-6021 .- 1470-8728. ; 406, s. 115-123
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Tidskriftsartikel (refereegranskat)abstract
- In the present paper, we report a novel class of GSTs (glutathione transferases), called the Chi class, originating from cyanobacteria and with properties not observed previously in prokaryotic enzymes. GSTs constitute a widespread multifunctional group of proteins, of which mammalian enzymes are the best characterized. Although GSTs have their origin in prokaryotes, few bacterial representatives have been characterized in detail, and the catalytic activities and substrate specificities observed have generally been very modest. The few well-studied bacterial GSTs have largely unknown physiological functions. Genome databases reveal that cyanobacteria have an extensive arsenal of glutathione-associated proteins. We have studied two cyanobacterial GSTs which are the first examples of bacterial enzymes that are as catalytically efficient as the best mammalian enzymes. GSTs from the thermophile Thermosynechococcus elongatus BP-1 and from Synechococcus elongatus PCC 6301 were found to catalyse the conjugation of naturally occurring plant-derived isothiocyanates to glutathione at high rates. The cyanobacterial GSTs studied are smaller than previously described members of this enzyme family, but display many of the typical structural features that are characteristics of GSTs. They are also active towards several classical substrates, but at the same moderate rates that have been observed for other GSTs derived from prokaryotes. The cloning, expression and characterization of two cyanobacterial GSTs are described. The possible significance of the observed catalytic properties is discussed in the context of physiological relevance and GST evolution.
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| 332. |
- Wollberg, Patrik, et al.
(författare)
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The adapter protein APS associates with the multifunctional docking sites Tyr-568 and Tyr-936 in c-Kit
- 2003
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Ingår i: Biochemical Journal. - 0264-6021 .- 1470-8728. ; 370:Pt 3, s. 1033-1038
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Tidskriftsartikel (refereegranskat)abstract
- The adapter protein APS has previously been shown to be involved in recruiting the ubiquitin E3 ligase c-Cbl to the insulin receptor, the platelet-derived growth factor beta-receptor and the erythropoietin receptor, leading to increased degradation of the receptors and inhibition of mitogenesis. Here we demonstrate, by use of immobilized synthetic phosphopeptides corresponding to various autophosphorylated tyrosine residues in the receptor for stem-cell factor (c-Kit), that APS preferentially associates with phosphorylated Tyr-568 and Tyr-936. Tyr-568 has previously been identified as the binding site of the Src family of tyrosine kinases, the Csk-homologous kinase CHK, and the protein tyrosine phosphatase SHP-2. We have recently demonstrated that Tyr-936 is an autophosphorylation site involved in binding the adapter proteins Grb2 and Grb7. We could further demonstrate that the critical determinant for binding of APS is the presence of either a leucine or an isoleucine residue in the position +3 to the phosphorylated tyrosine. This allowed us to design mutants that selectively failed to associate with APS, while still associating with Src family members, SHP-2 and Grb2, respectively.
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| 333. |
- Wu, Jun, et al.
(författare)
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Functional studies of human intestinal alkaline sphingomyelinase by deglycosylation and mutagenesis
- 2005
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Ingår i: Biochemical Journal. - 0264-6021. ; 386, s. 153-160
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Tidskriftsartikel (refereegranskat)abstract
- Intestinal alk-SMase (alkaline sphingomyelinase) is an ectoenzyme related to the NPP (nucleotide phosphodiesterase) family. It has five potential N-glycosylation sites and predicated transmembrane domains at both the N- and C-termini. The amino acid residues forming the two metal-binding sites in NPP are conserved, and those of the active core are modified. We examined the functional changes of the enzyme induced by deglycosylation and mutagenesis. Treating alk-SMase cDNA-transfected COS-7 cells with tunicamycin rendered the expressed enzyme completely inactive. Mutations of the five potential N-glycosylation sites individually and in combination showed that these sites were all glycosylated and deficient glycosylation decreased the enzyme activity. Immunogold labelling showed that the wild-type enzyme was mainly located in the plasma membrane, whereas the C-terminal domain-truncated enzyme was released into the medium. Deglycosylation blocked the release of the enzyme that accumulated in endosome-like structures. The enzyme activity was also decreased by mutations of the residues forming the putative metalbinding sites and the active core. Substitution of the active core sequence with that of NPP or mutation of T75 in the core abolished the enzyme activity against sphingomyel in but failed to render the enzyme NPP active. Our results indicate that alk-SMase activity is severely affected by defective N-glycosylation and structural alterations of the putative metal-binding sites and the predicted active core.
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| 334. |
- Wu, Jun, et al.
(författare)
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Intestinal alkaline sphingomyelinase hydrolyses and inactivates platelet-activating factor by a phospholipase C activity.
- 2006
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Ingår i: The Biochemical journal. - 1470-8728. ; 394, s. 299-308
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Tidskriftsartikel (refereegranskat)abstract
- Alkaline sphingomyelinase (alk-SMase) is a new member of the NPP (nucleotide pyrophosphatase/phosphodiesterase) family that hydrolyses SM (sphingomyelin) to generate ceramide in the intestinal tract. The enzyme may protect the intestinal mucosa from inflammation and tumorigenesis. PAF (platelet-activating factor) is a pro-inflammatory phospholipid involved in pathogenesis of inflammatory bowel diseases. We examined whether alk-SMase can hydrolyse and inactivate PAF. [3H]Octadecyl-labelled PAF was incubated with purified rat intestinal alk-SMase or recombinant human alk-SMase expressed in COS-7 cells. The hydrolytic products were assayed with TLC and MS. We found that alkSMase cleaved the phosphocholine head group from PAF and generated 1-O-alkyl-2-acetyl-sn-glycerol. Differing from the activity against SM, the activity against PAF was optimal at pH 7.5, inhibited by EDTA and stimulated by 0.1-0.25 mM Zn2+. The activity was abolished by site mutation of the predicted metal-binding sites that are conserved in all NPP members. Similar to the activity against SM, the activity against PAF was dependent on bile salt, particularly taurocholate and taurochenodeoxycholate. The V(max) for PAF hydrolysis was 374 mumol x h(-1) x (mg of protein)(-1). The hydrolysis of PAF and SM could be inhibited by the presence of SM and PAF respectively, the inhibition of PAF hydrolysis by SM being stronger. The PAF-induced MAPK (mitogen-activated protein kinase) activation and IL-8 (interleukin 8) release in HT-29 cells, and chemotaxis in leucocytes were abolished by alk-SMase treatment. In conclusion, alk-SMase hydrolyses and inactivates PAF by a phospholipase C activity. The finding reveals a novel function, by which alk-SMase may counteract the development of intestinal inflammation and colon cancer.
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| 335. |
- Xu, Shengyuan, et al.
(författare)
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Purification of a 75 kDa protein from the organelle matrix of human neutrophils and identification as N-acetylglucosamine-6-sulphatase
- 2005
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Ingår i: Biochemical Journal. - 0264-6021 .- 1470-8728. ; 387:Pt 3, s. 841-7
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Tidskriftsartikel (refereegranskat)abstract
- A 75 kDa protein was purified to homogeneity from granule extracts of normal human granulocytes using Sephadex G-75 chromatography, Mono-S cation exchange chromatography and chromatofocusing. The protein consisted of one chain with a molecular mass of 75 kDa, as determined by SDS/PAGE. Tryptic peptide analysis by MALDI-TOF (matrix-assisted laser-desorption ionization-time-of-flight) MS and sequence analysis by MS/MS identified the protein to be N-acetylglucosamine-6-sulphatase (EC 3.1.6.14). The identity of the protein was confirmed by demostrating enzymatic activity towards the substrate N-acetylglucosamine 6-sulphate. The enzyme was active over a broad pH range with an optimum of pH 7.0, and showed a Km value of 13.0 mM and a Vmax value of ~1.8 µM/min per mg. The enzyme also showed O-desulphation activity towards heparan sulphate-derived saccharides. Subcellular fractionation of neutrophil organelles showed the presence of enzymatic activity mainly in the same fractions as primary granules. Furthermore, PMA treatment of the neutrophils induced release of the enzyme, indicating its matrix protein nature. The presence of N-acetylglucosamine-6-sulphatase in human neutrophils implies that neutrophils may play a role in the modulation of cell surface molecules and extracellular matrix by O-desulphation.
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| 336. |
- Yasmin, Lubna, et al.
(författare)
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Electrostatic interactions play a minor role in the binding of ExoS to 14-3-3 proteins
- 2010
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Ingår i: Biochemical Journal. - : Portland Press. - 0264-6021 .- 1470-8728. ; 427:2, s. 217-224
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Tidskriftsartikel (refereegranskat)abstract
- 14-3-3 proteins belong to a family of conserved molecules expressed in all eukaryotic cells that play an important role in a multitude of signalling pathways. 14-3-3 proteins bind either to phosphoserine/phosphothreonine residues or to sequence-specific non-phosphorylated motifs in more than 200 interaction partners [Pozuelo Rubio, Geraghty, Wong, Wood, Campbell, Morrice and Mackintosh (2004) Biochem. J. 379, 395-408]. These interactions result in cell-cycle regulation, apoptosis, stress responses, cell metabolism and malignant transformation. One example of a phosphorylation-independent interaction is the binding of 14-3-3 to ExoS (exoenzyme S), a bacterial ADP-ribosyltransferase toxin of Pseudomonas aeruginosa. In the present study, we have utilized additional biochemical and infection analyses to define further the structural basis of the interaction between ExoS and 14-3-3. An ExoS leucine-substitution mutant dramatically reduced the interaction potential with 14-3-3 suggesting that Leu422, Leu423, Leu426 and Leu428 of ExoS are important for its interaction with 14-3-3, its enzymatic activity and cytotoxicity. However, ExoS substitution mutants of residues that interact with 14-3-3 through an electrostatic interaction, such as Ser416, His418, Asp424 and Asp427, showed no reduction in their interaction potential with 14-3-3. These ExoS substitution mutants were also as aggressive as wild-type ExoS at inducing cell death and to modify endogenous ExoS target within the cell. In conclusion, electrostatic interaction between ExoS and 14-3-3 via polar residues (Ser416, His418, Asp424 and Asp427) appears to be of secondary importance. Thus the interaction between the 'roof' of the groove of 14-3-3 and ExoS relies more on hydrophobic interaction forces, which probably contributes to induce cell death after ExoS infection and activation.
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| 337. |
- Yu, Hao, et al.
(författare)
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beta 1 Integrin and alpha-dystroglycan binding sites are localized to different laminin-G-domain-like (LG) modules within the laminin alpha 5 chain G domain
- 2003
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Ingår i: Biochemical Journal. - 0264-6021. ; 371, s. 289-299
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Tidskriftsartikel (refereegranskat)abstract
- Laminins are a group of extracellular-matrix proteins important in development and disease. They are heterotrimers, and specific domains in the different chains have specialized functions. The G domain of the alpha5 chain has now been produced in transfected mammalian cells as single modules and two tandem arrays, alpha5LG1-3 and alpha5LG4-5 (LG is laminin G domain-like.). Using these fragments we produced specific polyclonal antibodies functional in immunoblotting and immunofluorescence studies and in solid-phase assays. Both alpha5LG tandem arrays had physiologically relevant affinities for sulphated ligands such as heparin and sulphatides. Cells adhered to these fragments and acquired a spread morphology when plated on a5LG1-3. Binding of integrins alpha3beta1 and alpha6beta1 was localized to the alpha5LG1-3 modules, and a-dystroglycan binding was localized to the a5LG4-5 modules, thus locating these activities to different LG modules within the laminin a5 G domain. However, both these activities were of relatively low affinity, indicating that integrin-mediated cell adhesion to the laminin 10/11 alpha5G domain depends on contributions from the other chains of the heterotrimer and that high-affinity a-dystroglycan binding could be dependent on specific Ca2+-ion-co-ordinating amino acids absent from alpha5LG4-5.
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| 338. |
- Zavialov, Anton
(författare)
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Caf1A usher possesses a Caf1 subunit-like domain that is crucial for Caf1 fibre secretion
- 2009
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Ingår i: Biochemical Journal. - 0264-6021 .- 1470-8728. ; 418, s. 541-551
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Tidskriftsartikel (refereegranskat)abstract
- The chaperone/usher pathway controls assembly of fibres of adhesive organelles of Gram-negative bacteria. The final steps of fibre assembly and fibre translocation to the cell surface are co-ordinated by the outer membrane proteins, ushers. Ushers consist of several soluble periplasmic domains and a single transmembrane beta-barrel. Here we report isolation and structural/functional characterization of a novel middle domain of the Caf1A usher from Yersinia pestis. The isolated UMD (usher middle domain) is a highly soluble monomeric protein capable of autonomous folding. A 2.8 angstrom (1 angstrom = 0.1 nm) resolution crystal structure of UMD revealed that this domain has an immunoglobulin-like fold similar to that of donor-strand-complemented Caf1 fibre subunit. Moreover, these proteins displayed significant structural similarity. Although UMD is in the middle of the predicted amphipathic beta-barrel of Caf1A, the usher still assembled in the membrane in the absence of this domain. UMD did not bind Caf1M-Caf1 complexes, but its presence was shown to be essential for Caf1 fibre secretion. The study suggests that UMD may play the role of a subunit-substituting protein (dummy subunit), plugging or priming secretion through the channel in the Caf1A usher. Comparison of isolated UMD with the recent strcture of the corresponding domain of PapC usher revealed high similarity of the core structures, suggesting a universal structural adaptation of FGL (F(1)G(1) long) and FGS (F(1)G(1) short) chaperone/usher pathways for the secretion of different types of fibres. The functional role of two topologically different states of this plug domain suggested by structural and biochemical results is discussed.
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| 339. |
- Zavialov, Andrey V, et al.
(författare)
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Human ADA2 belongs to a new family of growth factors with adenosine deaminase activity
- 2005
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Ingår i: Biochemical Journal. - 0264-6021 .- 1470-8728. ; 391:Pt 1, s. 51-57
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Tidskriftsartikel (refereegranskat)abstract
- Two distinct isoenzymes of ADA (adenosine deaminase), ADA1 and ADA2, have been found in humans. Inherited mutations in ADA1 result in SCID (severe combined immunodeficiency). This observation has led to extensive studies of the structure and function of this enzyme that have revealed an important role for it in lymphocyte activation. In contrast, the physiological role of ADA2 is unknown. ADA2 is found in negligible quantities in serum and may be produced by monocytes/macrophages. ADA2 activity in the serum is increased in various diseases in which monocyte/macrophage cells are activated. In the present study, we report that ADA2 is a heparin-binding protein. This allowed us to obtain a highly purified enzyme and to study its biochemistry. ADA2 was identified as a member of a new class of ADGFs (ADA-related growth factors), which is present in almost all organisms from flies to humans. Our results suggest that ADA2 may be active in sites of inflammation during hypoxia and in areas of turnout growth where the adenosine concentration is significantly elevated and the extracellular pH is acidic. Our finding that ADA2 co-purified and concentrated together with IgG in commercially available preparations offers an intriguing explanation for the observation that treatment with such preparations leads to non-specific immune-system stimulation.
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| 340. |
- Zavialov, Anton V, et al.
(författare)
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Resolving the energy paradox of chaperone/usher-mediated fibre assembly
- 2005
-
Ingår i: Biochemical Journal. - 0264-6021 .- 1470-8728. ; 389:Pt 3, s. 685-694
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Tidskriftsartikel (refereegranskat)abstract
- Periplasmic chaperone/usher machineries are used for assembly of filamentous adhesion organelles of Gram-negative pathogens in a process that has been suggested to be driven by folding energy. Structures of mutant chaperone–subunit complexes revealed a final folding transition (condensation of the subunit hydrophobic core) on the release of organelle subunit from the chaperone–subunit pre-assembly complex and incorporation into the final fibre structure. However, in view of the large interface between chaperone and subunit in the pre-assembly complex and the reported stability of this complex, it is difficult to understand how final folding could release sufficient energy to drive assembly. In the present paper, we show the X-ray structure for a native chaperone–fibre complex that, together with thermodynamic data, shows that the final folding step is indeed an essential component of the assembly process. We show that completion of the hydrophobic core and incorporation into the fibre results in an exceptionally stable module, whereas the chaperone–subunit pre-assembly complex is greatly destabilized by the high-energy conformation of the bound subunit. This difference in stabilities creates a free energy potential that drives fibre formation.
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