Michale Bouskila | University of Dundee (original) (raw)

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Papers by Michale Bouskila

Research paper thumbnail of TTBK2 kinase substrate specificity and the impact of spinocerebellar-ataxia-causing mutations on expression, activity, localization and development

Biochemical Journal, 2011

Mutations that truncate the C-terminal, non-catalytic moiety of the Tau tubulin Kinase 2 (TTBK2) ... more Mutations that truncate the C-terminal, non-catalytic moiety of the Tau tubulin Kinase 2 (TTBK2) cause the inherited, autosomal dominant inherited spinocerebellar ataxia type 11 (SCA11) movement disorder. In this study we first assess the substrate specificity of TTBK2 and demonstrate that it has an unusual preference for a phosphotyrosine at the +2 position relative to the phosphorylation site. We elaborate a peptide substrate (TTBKtide, RRKDLHDDEEDEAMSIYpA) that can be employed to quantify TTBK2 kinase activity. Through modelling and mutagenesis we identify a putative phosphate priming-groove within the TTBK2 kinase domain. We demonstrate that SCA11 truncating mutations promote TTBK2 protein expression, suppress kinase activity and lead to enhanced nuclear localisation. We generate an SCA11-mutation-carrying knock-in mouse and show that this leads to inhibition of endogenous TTBK2 protein kinase activity. Finally, we find that in homozygosity, the SCA11 mutation causes embryonic lethality at E10. These findings provide the first insights into some of the intrinsic properties of TTBK2 and reveal how SCA11causing mutations impact on protein expression, catalytic activity, localisation and development. We hope these findings will be helpful for future investigation of the regulation and function of TTBK2 and its role in SCA11.

Research paper thumbnail of Investigation of the hepatotoxicity profile of chemical entities using Liverbeads® and WIF-B9 in vitro models

The cytotoxicity proWle of various chemical entities was evaluated using two in vitro hepatocyte ... more The cytotoxicity proWle of various chemical entities was evaluated using two in vitro hepatocyte models. Liverbeads ® is a cryopreserved model consisting of primary hepatocytes entrapped in alginate beads. WIF-B9 is a hybrid cell line obtained by fusion of rat hepatoma (Fao) and human Wbroblasts (WI38). Various reference hepatotoxicants were tested and ranked according to their equivalent concentration 50 (EC50) for various biochemical endpoints (lactate dehydrogenase (LDH) release, 3-(4,5 dimethylthiazol 2yl)-2,5-diphenyl-2H tetrazolium bromure (MTT) activity, adenosine triphosphate (ATP) and glutathione (GSH) levels). The ranking obtained was comparable in both models and consistent with previously published results on hepatocyte monolayers. Ketoconazole, erythromycin estolate, retinoic acid, telithromycin and -naphthyl-isothiocyanate were among the most toxic chemicals in both models, with an EC50 < 200 M. Troleandomycin, spiramycin, erythromycin, diclofenac, taurodeoxycholate, warfarin, galactosamine, valproic acid and isoniazid were found to be less toxic. Few marked diVerences, potentially linked to metabolism pathways, were observed between EC50s in the two models for compounds such as cyclosporine A (10 and >831 M) and warfarin (5904 and 1489 M) in WIF-B9 and Liverbeads ® , respectively. The results obtained indicate that Liverbeads ® and WIF-B9 cells are reliable in vitro models to evaluate the hepatotoxic potential of a wide range of chemicals, irrespective of structure and pharmaceutical class.

Research paper thumbnail of Adiponectin: a relevant player in PPARγ-agonist-mediated improvements in hepatic insulin sensitivity

International Journal of Obesity, 2005

The potent insulin-sensitizing effects of peroxisome proliferator-activated receptor γ (PPARγ) ag... more The potent insulin-sensitizing effects of peroxisome proliferator-activated receptor γ (PPARγ) agonists are well established. However, it is still a matter of intense debate as to which tissue(s) represent the most critical sites of action for PPARγ agonists, and what the relevant target genes are that ultimately mediate the improvements in insulin sensitivity. The cell type with the highest levels of

Research paper thumbnail of Investigation of the hepatotoxicity profile of chemical entities using Liverbeads ® and WIF-B9 in vitro models

Toxicology in Vitro, 2006

The cytotoxicity profile of various chemical entities was evaluated using two in vitro hepatocyte... more The cytotoxicity profile of various chemical entities was evaluated using two in vitro hepatocyte models. Liverbeads® is a cryopreserved model consisting of primary hepatocytes entrapped in alginate beads. WIF-B9 is a hybrid cell line obtained by fusion of rat hepatoma (Fao) and human fibroblasts (WI38). Various reference hepatotoxicants were tested and ranked according to their equivalent concentration 50 (EC50) for various biochemical endpoints (lactate dehydrogenase (LDH) release, 3-(4,5 dimethylthiazol 2yl)-2,5-diphenyl-2H tetrazolium bromure (MTT) activity, adenosine triphosphate (ATP) and glutathione (GSH) levels). The ranking obtained was comparable in both models and consistent with previously published results on hepatocyte monolayers. Ketoconazole, erythromycin estolate, retinoic acid, telithromycin and α-naphthyl-isothiocyanate were among the most toxic chemicals in both models, with an EC50 < 200 μM. Troleandomycin, spiramycin, erythromycin, diclofenac, taurodeoxycholate, warfarin, galactosamine, valproic acid and isoniazid were found to be less toxic. Few marked differences, potentially linked to metabolism pathways, were observed between EC50s in the two models for compounds such as cyclosporine A (10 and >831 μM) and warfarin (5904 and 1489 μM) in WIF-B9 and Liverbeads®, respectively. The results obtained indicate that Liverbeads® and WIF-B9 cells are reliable in vitro models to evaluate the hepatotoxic potential of a wide range of chemicals, irrespective of structure and pharmaceutical class.

Research paper thumbnail of Insulin promotes glycogen synthesis in the absence of GSK3 phosphorylation in skeletal muscle

American Journal of Physiology-endocrinology and Metabolism, 2007

Insulin promotes dephosphorylation and activation of glycogen synthase (GS) by inactivating GSK3 ... more Insulin promotes dephosphorylation and activation of glycogen synthase (GS) by inactivating GSK3 through phosphorylation. Insulin also promotes glucose uptake and glucose-6-phosphate (G6P) production, which allosterically activates GS. The relative importance of these two regulatory mechanisms in the activation of GS in vivo is unknown. The aim of this study was to investigate if dephosphoryltion of GS mediated via GSK3 is required for normal glycogen synthesis in skeletal muscle with insulin. We employed GSK3 knock-in mice in which wild-type GSK3 and genes are replaced with mutant forms (GSK3 / S21A/S21A/S9A/S9A ), which are non-responsive to insulin. Although insulin failed to promote dephosphorylation and activation of GS in GSK3 / S21A/S21A/S9A/S9A mice, glycogen content in different muscles from these mice was similar compared to wild-type mice. Basal and adrenaline-stimulated activity of muscle glycogen phosphorylase was comparable between wild-type and GSK3 knock-in mice. Incubation of isolated soleus muscle in Krebs buffer containing 5.5 mM glucose in the presence or absence of insulin revealed that the levels of G6P, the rate of [ 14 C]glucose incorporation into glycogen, as well as an increase in total glycogen content were similar between wild-type and GSK3 knock-in mice. Injection of glucose containing [ 3 H]2-deoxy-glucose and [ 14 C]glucose also resulted in similar rates of muscle glucose uptake and glycogen synthesis in vivo between wild-type and GSK3 knock-in mice. These results suggest that insulin-mediated inhibition of GSK3

Research paper thumbnail of Allosteric Regulation of Glycogen Synthase Controls Glycogen Synthesis in Muscle

Cell Metabolism, 2010

Glycogen synthase (GS), a key enzyme in glycogen synthesis, is activated by the allosteric stimul... more Glycogen synthase (GS), a key enzyme in glycogen synthesis, is activated by the allosteric stimulator glucose-6-phosphate (G6P) and by dephosphorylation through inactivation of GS kinase-3 with insulin. The relative importance of these two regulatory mechanisms in controlling GS is not established, mainly due to the complex interplay between multiple phosphorylation sites and allosteric effectors. Here we identify a residue that plays an important role in the allosteric activation of GS by G6P. We generated knockin mice in which wild-type muscle GS was replaced by a mutant that could not be activated by G6P but could still be activated normally by dephosphorylation. We demonstrate that knockin mice expressing the G6P-insensitive mutant display an ∼80% reduced muscle glycogen synthesis by insulin and markedly reduced glycogen levels. Our study provides genetic evidence that allosteric activation of GS is the primary mechanism by which insulin promotes muscle glycogen accumulation in vivo.► A critical glucose-6-phosphate (G6P) action site on glycogen synthase (GS) identified ► A knockin mouse expressing G6P-insensitive mutant GS generated ► GS knockin mice display dramatically reduced ability to accumulate muscle glycogen ► Insulin promotes glycogen synthesis mainly via allosteric activation of GS by G6P

Research paper thumbnail of Allosteric Regulation Plays a Key Role in Muscle Glycogen Synthesis: 1352

Medicine and Science in Sports and Exercise, 2008

Research paper thumbnail of TTBK2 kinase substrate specificity and the impact of spinocerebellar-ataxia-causing mutations on expression, activity, localization and development

Biochemical Journal, 2011

Mutations that truncate the C-terminal, non-catalytic moiety of the Tau tubulin Kinase 2 (TTBK2) ... more Mutations that truncate the C-terminal, non-catalytic moiety of the Tau tubulin Kinase 2 (TTBK2) cause the inherited, autosomal dominant inherited spinocerebellar ataxia type 11 (SCA11) movement disorder. In this study we first assess the substrate specificity of TTBK2 and demonstrate that it has an unusual preference for a phosphotyrosine at the +2 position relative to the phosphorylation site. We elaborate a peptide substrate (TTBKtide, RRKDLHDDEEDEAMSIYpA) that can be employed to quantify TTBK2 kinase activity. Through modelling and mutagenesis we identify a putative phosphate priming-groove within the TTBK2 kinase domain. We demonstrate that SCA11 truncating mutations promote TTBK2 protein expression, suppress kinase activity and lead to enhanced nuclear localisation. We generate an SCA11-mutation-carrying knock-in mouse and show that this leads to inhibition of endogenous TTBK2 protein kinase activity. Finally, we find that in homozygosity, the SCA11 mutation causes embryonic lethality at E10. These findings provide the first insights into some of the intrinsic properties of TTBK2 and reveal how SCA11causing mutations impact on protein expression, catalytic activity, localisation and development. We hope these findings will be helpful for future investigation of the regulation and function of TTBK2 and its role in SCA11.

Research paper thumbnail of Investigation of the hepatotoxicity profile of chemical entities using Liverbeads® and WIF-B9 in vitro models

The cytotoxicity proWle of various chemical entities was evaluated using two in vitro hepatocyte ... more The cytotoxicity proWle of various chemical entities was evaluated using two in vitro hepatocyte models. Liverbeads ® is a cryopreserved model consisting of primary hepatocytes entrapped in alginate beads. WIF-B9 is a hybrid cell line obtained by fusion of rat hepatoma (Fao) and human Wbroblasts (WI38). Various reference hepatotoxicants were tested and ranked according to their equivalent concentration 50 (EC50) for various biochemical endpoints (lactate dehydrogenase (LDH) release, 3-(4,5 dimethylthiazol 2yl)-2,5-diphenyl-2H tetrazolium bromure (MTT) activity, adenosine triphosphate (ATP) and glutathione (GSH) levels). The ranking obtained was comparable in both models and consistent with previously published results on hepatocyte monolayers. Ketoconazole, erythromycin estolate, retinoic acid, telithromycin and -naphthyl-isothiocyanate were among the most toxic chemicals in both models, with an EC50 < 200 M. Troleandomycin, spiramycin, erythromycin, diclofenac, taurodeoxycholate, warfarin, galactosamine, valproic acid and isoniazid were found to be less toxic. Few marked diVerences, potentially linked to metabolism pathways, were observed between EC50s in the two models for compounds such as cyclosporine A (10 and >831 M) and warfarin (5904 and 1489 M) in WIF-B9 and Liverbeads ® , respectively. The results obtained indicate that Liverbeads ® and WIF-B9 cells are reliable in vitro models to evaluate the hepatotoxic potential of a wide range of chemicals, irrespective of structure and pharmaceutical class.

Research paper thumbnail of Adiponectin: a relevant player in PPARγ-agonist-mediated improvements in hepatic insulin sensitivity

International Journal of Obesity, 2005

The potent insulin-sensitizing effects of peroxisome proliferator-activated receptor γ (PPARγ) ag... more The potent insulin-sensitizing effects of peroxisome proliferator-activated receptor γ (PPARγ) agonists are well established. However, it is still a matter of intense debate as to which tissue(s) represent the most critical sites of action for PPARγ agonists, and what the relevant target genes are that ultimately mediate the improvements in insulin sensitivity. The cell type with the highest levels of

Research paper thumbnail of Investigation of the hepatotoxicity profile of chemical entities using Liverbeads ® and WIF-B9 in vitro models

Toxicology in Vitro, 2006

The cytotoxicity profile of various chemical entities was evaluated using two in vitro hepatocyte... more The cytotoxicity profile of various chemical entities was evaluated using two in vitro hepatocyte models. Liverbeads® is a cryopreserved model consisting of primary hepatocytes entrapped in alginate beads. WIF-B9 is a hybrid cell line obtained by fusion of rat hepatoma (Fao) and human fibroblasts (WI38). Various reference hepatotoxicants were tested and ranked according to their equivalent concentration 50 (EC50) for various biochemical endpoints (lactate dehydrogenase (LDH) release, 3-(4,5 dimethylthiazol 2yl)-2,5-diphenyl-2H tetrazolium bromure (MTT) activity, adenosine triphosphate (ATP) and glutathione (GSH) levels). The ranking obtained was comparable in both models and consistent with previously published results on hepatocyte monolayers. Ketoconazole, erythromycin estolate, retinoic acid, telithromycin and α-naphthyl-isothiocyanate were among the most toxic chemicals in both models, with an EC50 < 200 μM. Troleandomycin, spiramycin, erythromycin, diclofenac, taurodeoxycholate, warfarin, galactosamine, valproic acid and isoniazid were found to be less toxic. Few marked differences, potentially linked to metabolism pathways, were observed between EC50s in the two models for compounds such as cyclosporine A (10 and >831 μM) and warfarin (5904 and 1489 μM) in WIF-B9 and Liverbeads®, respectively. The results obtained indicate that Liverbeads® and WIF-B9 cells are reliable in vitro models to evaluate the hepatotoxic potential of a wide range of chemicals, irrespective of structure and pharmaceutical class.

Research paper thumbnail of Insulin promotes glycogen synthesis in the absence of GSK3 phosphorylation in skeletal muscle

American Journal of Physiology-endocrinology and Metabolism, 2007

Insulin promotes dephosphorylation and activation of glycogen synthase (GS) by inactivating GSK3 ... more Insulin promotes dephosphorylation and activation of glycogen synthase (GS) by inactivating GSK3 through phosphorylation. Insulin also promotes glucose uptake and glucose-6-phosphate (G6P) production, which allosterically activates GS. The relative importance of these two regulatory mechanisms in the activation of GS in vivo is unknown. The aim of this study was to investigate if dephosphoryltion of GS mediated via GSK3 is required for normal glycogen synthesis in skeletal muscle with insulin. We employed GSK3 knock-in mice in which wild-type GSK3 and genes are replaced with mutant forms (GSK3 / S21A/S21A/S9A/S9A ), which are non-responsive to insulin. Although insulin failed to promote dephosphorylation and activation of GS in GSK3 / S21A/S21A/S9A/S9A mice, glycogen content in different muscles from these mice was similar compared to wild-type mice. Basal and adrenaline-stimulated activity of muscle glycogen phosphorylase was comparable between wild-type and GSK3 knock-in mice. Incubation of isolated soleus muscle in Krebs buffer containing 5.5 mM glucose in the presence or absence of insulin revealed that the levels of G6P, the rate of [ 14 C]glucose incorporation into glycogen, as well as an increase in total glycogen content were similar between wild-type and GSK3 knock-in mice. Injection of glucose containing [ 3 H]2-deoxy-glucose and [ 14 C]glucose also resulted in similar rates of muscle glucose uptake and glycogen synthesis in vivo between wild-type and GSK3 knock-in mice. These results suggest that insulin-mediated inhibition of GSK3

Research paper thumbnail of Allosteric Regulation of Glycogen Synthase Controls Glycogen Synthesis in Muscle

Cell Metabolism, 2010

Glycogen synthase (GS), a key enzyme in glycogen synthesis, is activated by the allosteric stimul... more Glycogen synthase (GS), a key enzyme in glycogen synthesis, is activated by the allosteric stimulator glucose-6-phosphate (G6P) and by dephosphorylation through inactivation of GS kinase-3 with insulin. The relative importance of these two regulatory mechanisms in controlling GS is not established, mainly due to the complex interplay between multiple phosphorylation sites and allosteric effectors. Here we identify a residue that plays an important role in the allosteric activation of GS by G6P. We generated knockin mice in which wild-type muscle GS was replaced by a mutant that could not be activated by G6P but could still be activated normally by dephosphorylation. We demonstrate that knockin mice expressing the G6P-insensitive mutant display an ∼80% reduced muscle glycogen synthesis by insulin and markedly reduced glycogen levels. Our study provides genetic evidence that allosteric activation of GS is the primary mechanism by which insulin promotes muscle glycogen accumulation in vivo.► A critical glucose-6-phosphate (G6P) action site on glycogen synthase (GS) identified ► A knockin mouse expressing G6P-insensitive mutant GS generated ► GS knockin mice display dramatically reduced ability to accumulate muscle glycogen ► Insulin promotes glycogen synthesis mainly via allosteric activation of GS by G6P

Research paper thumbnail of Allosteric Regulation Plays a Key Role in Muscle Glycogen Synthesis: 1352

Medicine and Science in Sports and Exercise, 2008