| 61. |
- Stamenkovic, Jelena, et al.
(författare)
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Inhibition of the malate-aspartate shuttle in mouse pancreatic islets abolishes glucagon secretion without affecting insulin secretion
- 2015
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Ingår i: Biochemical Journal. - 0264-6021. ; 468, s. 49-63
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Tidskriftsartikel (refereegranskat)abstract
- Altered secretion of insulin as well as glucagon has been implicated in the pathogenesis of Type 2 diabetes (T2D), but the mechanisms controlling glucagon secretion from alpha-cells largely remain unresolved. Therefore, we studied the regulation of glucagon secretion from alpha TC1-6 (alpha TC1 clone 6) cells and compared it with insulin release from INS-1 832/13 cells. We found that INS-1 832/13 and alpha TC1-6 cells respectively secreted insulin and glucagon concentration-dependently in response to glucose. In contrast, tight coupling of glycolytic and mitochondrial metabolism was observed only in INS-1 832/13 cells. Although glycolytic metabolism was similar in the two cell lines, TCA (tricarboxylic acid) cycle metabolism, respiration and ATP levels were less glucose-responsive in alpha TC1-6 cells. Inhibition of the malate-aspartate shuttle, using phenyl succinate (PhS), abolished glucose-provoked ATP production and hormone secretion from alpha TC1-6 but not INS-1 832/13 cells. Blocking the malate-aspartate shuttle increased levels of glycerol 3-phosphate only in INS-1 832/13 cells. Accordingly, relative expression of constituents in the glycerol phosphate shuttle compared with malate-aspartate shuttle was lower in alpha TC1-6 cells. Our data suggest that the glycerol phosphate shuttle augments the malate-aspartate shuttle in INS-1 832/13 but not alpha TC1-6 cells. These results were confirmed in mouse islets, where PhS abrogated secretion of glucagon but not insulin. Furthermore, expression of the rate-limiting enzyme of the glycerol phosphate shuttle was higher in sorted primary beta-than in alpha-cells. Thus, suppressed glycerol phosphate shuttle activity in the alpha-cell may prevent a high rate of glycolysis and consequently glucagon secretion in response to glucose. Accordingly, pyruvate-and lactate-elicited glucagon secretion remains unaffected since their signalling is independent of mitochondrial shuttles.
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| 62. |
- Struglics, André, et al.
(författare)
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Calpain is involved in C-terminal truncation of human aggrecan
- 2010
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Ingår i: Biochemical Journal. - 0264-6021. ; 18, s. 10-10
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Tidskriftsartikel (refereegranskat)abstract
- Mature aggrecan is generally C-terminally truncated at several sites in the CS (chondroitin sulfate) region. Aggrecanases and MMPs (matrix metalloproteinases) have been suggested to be responsible for this digestion. To identify whether calpain, a common intracellular protease, has a specific role in the proteolysis of aggrecan we developed neoepitope antibodies (anti-PGVA, anti-GDLS and anti-EDLS) against calpain cleavage sites and used Western blot analysis to identify alpain-generated fragments in normal and OA (osteoarthritis) knee cartilage and SF (synovial fluid) samples. Our results showed that human aggrecan contains six calpain cleavage sites: one in the IGD (interglobular domain), one in the KS (keratan sulfate) region, two in the CS1 and two in the CS2 region. Kinetic studies of calpain proteolysis against aggrecan showed that the aggrecan molecule was cleaved in a specific order where cuts in CS1 was the most preferred and cuts in KS region was the second most preferred cleavage. OA and normal cartilage contained low amounts of a calpain-generated G1-PGVA fragment (0.5-2%) compared with aggrecanase-generated G1-TEGE (71-76%) and MMP-generated G1-IPEN (23-29%) fragments. Significant amounts of calpain-generated GDLS and EDLS fragments were found in OA and normal cartilage, and a ARGS-EDLS fragment was detected in arthritic SF samples. The results of the present study indicate that calpains are involved in the C-terminal truncation of aggrecan and might have a minor role in arthritic diseases.
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| 63. |
- Struglics, André, et al.
(författare)
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MMP proteolysis of the human extracellular matrix protein aggrecan is mainly a process of normal turnover
- 2012
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Ingår i: Biochemical Journal. - 0264-6021. ; 446, s. 213-223
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Tidskriftsartikel (refereegranskat)abstract
- Although it has been shown that aggrecanases are involved in aggrecan degradation, the role of MMP (matrix metalloproteinase) aggrecanolysis is less well studied. To investigate MMP proteolysis of human aggrecan, in the present study we used neoepitope antibodies against MMP cleavage sites and Western blot analysis to identify MMP-generated fragments in normal and OA (osteoarthritis/osteoarthritic) cartilage, and in normal, knee injury and OA and SF (synovial fluid) samples. MMP-3 in vitro digestion showed that aggrecan contains six MMP cleavage sites, in the IGD (interglobular domain), the KS (keratan sulfate) region, the border between the KS region and CS (chondroitin sulfate) region 1, the CS I region, and the border between the CS2 and the G3 domain, and kinetic studies showed a specific order of digestion where the cleavage between CS2 and the G3 domain was the most preferred. In vivo studies showed that OA cartilage contained (per dry weight) 3.4-fold more MMP-generated FFGV fragments compared with normal cartilage, and although aggrecanase-generated SF-ARGS concentrations were increased 14-fold in OA and knee-injured patients compared with levels in knee-healthy reference subjects, the SF-FFGV concentrations did not notably change. The results of the present study suggest that MMPs are mainly involved in normal aggrecan turnover and might have a less-active role in aggrecan degradation during knee injury and OA.
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| 64. |
- Sörensson, Carolin, et al.
(författare)
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Determination of primary sequence specificity of Arabidopsis MAPKs MPK3 and MPK6 leads to identification of new substrates
- 2012
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Ingår i: Biochemical Journal. - 0264-6021 .- 1470-8728. ; 446, s. 271-278
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Tidskriftsartikel (refereegranskat)abstract
- MAPKs (mitogen-activated protein kinases) are signalling components highly conserved among eukaryotes. Their diverse biological functions include cellular differentiation and responses to different extracellular stress stimuli. Although some substrates of MAPKs have been identified in plants, no information is available about whether amino acids in the primary sequence other than proline-directed phosphorylation (pS-P) contribute to kinase specificity towards substrates. in the present study, we used a random positional peptide library to search for consensus phosphorylation sequences for Arabidopsis MAPKs MPK3 and MPK6. These experiments indicated a preference towards the sequence L/P-P/X-S-P-R/K for both kinases. After bioinformatic processing, a number of novel candidate MAPK substrates were predicted and subsequently confirmed by in vitro kinase assays using bacterially expressed native Arabidopsis proteins as substrates. MPK3 and MPK6 phosphorylated all proteins tested more efficiently than did another MAPK, MPK4. These results indicate that the amino acid residues in the primary sequence surrounding the phosphorylation site of Arabidopsis MAPK substrates can contribute to MAPK specificity. Further characterization of one of these new substrates confirmed that AtIg80180.1 was phosphorylated in planta in a MAPK-dependent manner. Phenotypic analyses of Arabidopsis expressing phosphorylation site mutant forms of AtIg80180.1 showed clustered stomata and higher stomatal index in cotyledons expressing the phosphomimetic form of AtIg80180.1, providing a link between this new MAPK substrate and the defined role for MPK3 and MPK6 in stomatal patterning.
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| 65. |
- Thackray, Alana M., et al.
(författare)
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Genetic human prion disease modelled in PrP transgenic Drosophila
- 2017
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Ingår i: Biochemical Journal. - : Portland Press. - 0264-6021 .- 1470-8728. ; 474:19, s. 3253-3267
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Tidskriftsartikel (refereegranskat)abstract
- Inherited human prion diseases, such as fatal familial insomnia (FFI) and familial Creutzfeldt-Jakob disease (fCJD), are associated with autosomal dominant mutations in the human prion protein gene PRNP and accumulation of PrPSc, an abnormal isomer of the normal host protein PrPC, in the brain of affected individuals. PrPSc is the principal component of the transmissible neurotoxic prion agent. It is important to identify molecular pathways and cellular processes that regulate prion formation and prion-induced neurotoxicity. This will allow identification of possible therapeutic interventions for individuals with, or at risk from, genetic human prion disease. Increasingly, Drosophila has been used to model human neurodegenerative disease. An important unanswered question is whether genetic prion disease with concomitant spontaneous prion formation can be modelled in Drosophila We have used pUAST/PhiC31-mediated site-directed mutagenesis to generate Drosophila transgenic for murine or hamster PrP (prion protein) that carry single-codon mutations associated with genetic human prion disease. Mouse or hamster PrP harbouring an FFI (D178N) or fCJD (E200K) mutation showed mild Proteinase K resistance when expressed in Drosophila Adult Drosophila transgenic for FFI or fCJD variants of mouse or hamster PrP displayed a spontaneous decline in locomotor ability that increased in severity as the flies aged. Significantly, this mutant PrP-mediated neurotoxic fly phenotype was transferable to recipient Drosophila that expressed the wild-type form of the transgene. Collectively, our novel data are indicative of the spontaneous formation of a PrP-dependent neurotoxic phenotype in FFI- or CJD-PrP transgenic Drosophila and show that inherited human prion disease can be modelled in this invertebrate host.
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| 66. |
- Tryggvesson, Anders, 1975, et al.
(författare)
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Interaction specificity between the chaperone and proteolytic components of the cyanobacterial Clp protease
- 2012
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Ingår i: Biochemical Journal. - : Portland Press Ltd.. - 0264-6021 .- 1470-8728. ; 446, s. 311-320
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Tidskriftsartikel (refereegranskat)abstract
- The Clp protease is conserved among eubacteria and most eukaryotes, and uses ATP to drive protein substrate unfolding and translocation into a chamber of sequestered proteolytic active sites. In plant chloroplasts and cyanobacteria, the essential constitutive Clp protease consists of the Hsp100/ClpC chaperone partnering a proteolytic core of catalytic ClpP and noncatalytic ClpR subunits. In the present study, we have examined putative determinants conferring the highly specific association between ClpC and the ClpP3/R core from the model cyanobacterium Synechococcus elongatus. Two conserved sequences in the N-terminus of ClpR (tyrosine and proline motifs) and one in the N-terminus of ClpP3 (MPIG motif) were identified as being crucial for the ClpC-ClpP3/R association. These N-terminal domains also influence the stability of the ClpP3/R core complex itself. A unique C-terminal sequence was also found in plant and cyanobacterial ClpC orthologues just downstream of the P-loop region previously shown in Escherichia coli to be important for Hsp100 association to ClpP. This R motif in Synechococcus ClpC confers specificity for the ClpP3/R core and prevents association with E. coli ClpP; its removal from ClpC reverses this core specificity.
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| 67. |
- Uhrik, Lukas, et al.
(författare)
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Allosteric changes in HDM2 by the ATM phosphomimetic S395D mutation : implications on HDM2 function
- 2019
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Ingår i: Biochemical Journal. - : PORTLAND PRESS LTD. - 0264-6021 .- 1470-8728. ; 476, s. 3401-3411
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Tidskriftsartikel (refereegranskat)abstract
- Allosteric changes imposed by post-translational modifications regulate and differentiate the functions of proteins with intrinsic disorder regions. HDM2 is a hub protein with a large interactome and with different cellular functions. It is best known for its regulation of the p53 tumour suppressor. Under normal cellular conditions, HDM2 ubiquitinates and degrades p53 by the 26S proteasome but after DNA damage, HDM2 switches from a negative to a positive regulator of p53 by binding to p53 mRNA to promote translation of the p53 mRNA. This change in activity is governed by the ataxia telangiectasia mutated kinase via phosphorylation on serine 395 and is mimicked by the S395D phosphomimetic mutant. Here we have used different approaches to show that this event is accompanied by a specific change in the HDM2 structure that affects the HDM2 interactome, such as the N-termini HDM2-p53 protein-protein interaction. These data will give a better understanding of how HDM2 switches from a negative to a positive regulator of p53 and gain new insights into the control of the HDM2 structure and its interactome under different cellular conditions and help identify interphases as potential targets for new drug developments.
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| 68. |
- 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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| 69. |
- 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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| 70. |
- 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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