| 401. |
- Wells, T, et al.
(författare)
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Age-related changes in the composition, the molecular stoichiometry and the stability of proteoglycan aggregates extracted from human articular cartilage
- 2003
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Ingår i: Biochemical Journal. - 0264-6021. ; 370:1, s. 69-79
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Tidskriftsartikel (refereegranskat)abstract
- The heterogeneity of the components of proteoglycan aggregates, their stoichiometry within the aggregate and the aggregates' stability was investigated in normal human articular cartilage specimens (age-range newborn to 63 years). Proteoglycans were extracted from tissue by sequentially extracting them with PBS alone, PBS containing oligosaccharides of hyaluronan, and PBS containing solutions of increasing guanidinium chloride concentration (I M, 2 M, 3 M and 4 M). A high proportion of each of the components of the proteoglycan aggregate, i.e. uronic acid, sulphated glycosaminoglycan, hyaluronan binding domain of aggrecan (G1-domain), link protein (LP) and hyaluronan, was extracted from immature cartilage by PBS alone and PBS containing oligosaccharides of hyaluronan. This was in marked contrast to adult cartilage, which required high concentrations of guanidinium chloride for the efficient extraction of these components. The molar ratios of total G1-domain: LP and the G1-domain associated with aggrecan: LP also differed markedly between immature and mature cartilage and between each of the sequential extracts. The concentration of LP was less than that of the G1-domain in all extracts of cartilage from individuals over 13 years, but this was particularly noticeable in the I M guanidinium chloride extracts, and it was surmised that a deficiency in LP produces unstable aggregates in situ. The fragmentation of LP, which is known to occur with advancing age, did not influence the extractability of LP, and fragments were present in each of the sequential extracts. Therefore the generally accepted model of proteoglycan aggregation presented in the literature, which is mostly derived from analysis of immature animal cartilage, cannot be used to describe the structure and organization of aggregates in adult human articular cartilage, where a heterogeneous population of complexes exist that have varying degrees of stability.
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| 402. |
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| 403. |
- Wikström, Katarina, et al.
(författare)
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The anti-apoptotic effect of leukotriene D-4 involves the prevention of caspase 8 activation and Bid cleavage
- 2003
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Ingår i: Biochemical Journal. - 0264-6021. ; 371, s. 115-124
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Tidskriftsartikel (refereegranskat)abstract
- We have shown in a previous study that leukotriene D-4 (LTD4) signalling increases cell survival and proliferation in intestinal epithelial cells [ohd, Wikstrom and Sjolander (2000) Gastroenterology 119, 1007-1018]. This is highly interesting since inflammatory conditions of the bowel are associated with an increased risk of developing colon cancer. The enzyme cyclooxygenase 2 (COX-2) is important in this context since it is upregulated in colon cancer tissues and in tumour cell lines. Treatment with the COX-2-specific inhibitor N-(2-cyclohexyloxy-4-nitrophenyl)methane sulphonamide has been shown previously to cause apoptosis in intestinal epithelial cells. In the present study, we attempted to elucidate the underlying mechanisms and we can now show that a mitochondrial pathway is employed. Inhibition of COX-2 causes release of cytochrome c, as shown by both Western-blot and microscopy studies, and as with apoptosis, this is significantly decreased by LTD4. Since previous studies showed increased Bcl-2 levels on LTD4 stimulation, we further studied apoptotic regulation at the mitochondrial level. From this we could exclude the involvement of the anti-apoptotic protein Bcl-X-L as well as its pro-apoptotic counterpart Bax, since they are not expressed. Furthermore, the activity of the proapoptotic protein Bad (Bcl-2/Bcl-X-L-antagonist, causing cell death) was completely unaffected. However, inhibition of COX-2 caused cleavage of caspase 8 into a 41 kDa fragment associated with activation and caused the appearance of an activated 15 kDa fragment of Bid. This indicates that N-(2-cyclohexyloxy4-nitrophenyl)methane sulphonamide-induced apoptosis is mediated by the activation of caspase 8, via generation of truncated Bid, and thereafter release of cytochrome c. Interestingly, LTD4 not only reverses the effects induced by inhibition of COX-2 but also reduces the apoptotic potential by lowering the basal level of caspase 8 activation and truncated Bid generation.
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| 404. |
- 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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| 405. |
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| 406. |
- 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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| 407. |
- Zalem, Dani, et al.
(författare)
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Biochemical and structural characterization of the novel sialic acid-binding site of Escherichia coli heat-labile enterotoxin LT-IIb.
- 2016
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Ingår i: The Biochemical journal. - 1470-8728. ; 473:21, s. 3923-3936
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Tidskriftsartikel (refereegranskat)abstract
- The structurally related AB5-type heat-labile enterotoxins of Escherichia coli and Vibrio cholerae are classified into two major types. The type I group includes cholera toxin (CT) and E. coli LT-I, whereas the type II subfamily comprises LT-IIa, LT-IIb and LT-IIc. The carbohydrate-binding specificities of LT-IIa, LT-IIb and LT-IIc are distinctive from those of cholera toxin and E. coli LT-I. Whereas CT and LT-I bind primarily to the GM1 ganglioside, LT-IIa binds to gangliosides GD1a, GD1b and GM1, LT-IIb binds to the GD1a and GT1b gangliosides, and LT-IIc binds to GM1, GM2, GM3 and GD1a. These previous studies of the binding properties of type II B-subunits have been focused on ganglio core chain gangliosides. To further define the carbohydrate binding specificity of LT-IIb B-subunits, we have investigated its binding to a collection of gangliosides and non-acid glycosphingolipids with different core chains. A high-affinity binding of LT-IIb B-subunits to gangliosides with a neolacto core chain, such as Neu5Gcα3- and Neu5Acα3-neolactohexaosylceramide, and Neu5Gcα3- and Neu5Acα3-neolactooctaosylceramide was detected. An LT-IIb-binding ganglioside was isolated from human small intestine and characterized as Neu5Acα3-neolactohexaosylceramide. The crystal structure of the B-subunit of LT-IIb with the pentasaccharide moiety of Neu5Acα3-neolactotetraosylceramide (Neu5Ac-nLT: Neu5Acα3Galβ4GlcNAcβ3Galβ4Glc) was determined providing the first information for a sialic-binding site in this subfamily, with clear differences from that of CT and LT-I.
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| 408. |
- Zibrova, Darya, et al.
(författare)
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GFAT1 phosphorylation by AMPK promotes VEGF-induced angiogenesis
- 2017
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Ingår i: Biochemical Journal. - 0264-6021. ; 474:6, s. 983-1001
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Tidskriftsartikel (refereegranskat)abstract
- Activation of AMP-activated protein kinase (AMPK) in endothelial cells regulates energy homeostasis, stress protection and angiogenesis, but the underlying mechanisms are incompletely understood. Using a label-free phosphoproteomic analysis, we identified glutamine:fructose-6-phosphate amidotransferase 1 (GFAT1) as an AMPK substrate. GFAT1 is the rate-limiting enzyme in the hexosamine biosynthesis pathway (HBP) and as such controls the modification of proteins by O-linked β-N-acetylglucosamine (O-GlcNAc). In the present study, we tested the hypothesis that AMPK controls O-GlcNAc levels and function of endothelial cells via GFAT1 phosphorylation using biochemical, pharmacological, genetic and in vitro angiogenesis approaches. Activation of AMPK in primary human endothelial cells by 5-aminoimidazole-4-carboxamide riboside (AICAR) or by vascular endothelial growth factor (VEGF) led to GFAT1 phosphorylation at serine 243. This effect was not seen when AMPK was down-regulated by siRNA. Upon AMPK activation, diminished GFAT activity and reduced O-GlcNAc levels were observed in endothelial cells containing wild-type (WT)-GFAT1 but not in cells expressing non-phosphorylatable S243A-GFAT1. Pharmacological inhibition or siRNA-mediated down-regulation of GFAT1 potentiated VEGF-induced sprouting, indicating that GFAT1 acts as a negative regulator of angiogenesis. In cells expressing S243A-GFAT1, VEGF-induced sprouting was reduced, suggesting that VEGF relieves the inhibitory action of GFAT1/HBP on angiogenesis via AMPK-mediated GFAT1 phosphorylation. Activation of GFAT1/HBP by high glucose led to impairment of vascular sprouting, whereas GFAT1 inhibition improved sprouting even if glucose level was high. Our findings provide novel mechanistic insights into the role of HBP in angiogenesis. They suggest that targeting AMPK in endothelium might help to ameliorate hyperglycaemia-induced vascular dysfunction associated with metabolic disorders.
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| 409. |
- Österlund, Torben, et al.
(författare)
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Domain-structure analysis of recombinant rat hormone-sensitive lipase
- 1996
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Ingår i: Biochemical Journal. - 0264-6021. ; 319:Pt 2, s. 411-420
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Tidskriftsartikel (refereegranskat)abstract
- Hormone-sensitive lipase (HSL) plays a key role in lipid metabolism and overall energy homoeostasis, by controlling the release of fatty acids from stored triglycerides in adipose tissue. Lipases and esterases form a protein superfamily with a common structural fold, called the alpha/beta-hydrolase fold, and a catalytic triad of serine, aspartic or glutamic acid and histidine. Previous alignments between HSL and lipase 2 of Moraxella TA144 have been extended to cover a much larger part of the HSL sequence. From these extended alignments, possible sites for the catalytic triad and alpha/beta-hydrolase fold are suggested. Furthermore, it is proposed that HSL contains a structural domain with catalytic capacity and a regulatory module attached, as well as a structural N-terminal domain unique to this enzyme. In order to test the proposed domain structure, rat HSL was overexpressed and purified to homogeneity using a baculovirus/insect-cell expression system. The purification, resulting in > 99% purity, involved detergent solubilization followed by anion-exchange chromatography and hydrophobic-interaction chromatography. The purified recombinant enzyme was identical to rat adipose-tissue HSL with regard to specific activity, substrate specificity and ability to serve as a substrate for cAMP-dependent protein kinase. The recombinant HSL was subjected to denaturation by guanidine hydrochloride and limited proteolysis. These treatments resulted in more extensive loss of activity against phospholipid-stabilized lipid substrates than against water-soluble substrates, suggesting that the hydrolytic activity can be separated from recognition of lipid substrates. These data support the concept that HSL has at least two major domains.
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