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Neuroprotective effects of calmodulin peptide 76-121aa: disruption of calmodulin binding to mutant huntingtin.

Dudek NL, Dai Y, Muma NA - Brain Pathol. (2009)

Bottom Line: Importantly, the effect of the CaM-peptide shows selectivity, such that total TG activity is not significantly altered by expression of CaM-peptide nor is the activity of another CaM-dependent enzyme, CaM kinase II.In vitro, recombinant exon 1 of huntingtin with 44 glutamines (htt-exon1-44Q) binds to CaM-agarose; the addition of 10 microM of CaM-peptide significantly decreases the interaction of htt-exon1-44Q and CaM but not the binding between CaM and calcineurin, another CaM-binding protein.These data support the hypothesis that CaM regulates TG-catalyzed modifications of mutant huntingtin and that specific and selective disruption of the CaM-huntingtin interaction is potentially a new target for therapeutic intervention in HD.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Experimental Therapeutics, Loyola University Chicago School of Medicine, Maywood, IL, USA.

ABSTRACT
Huntington's disease (HD) is a neurodegenerative disease caused by mutant huntingtin protein containing an expanded polyglutamine tract, which may cause abnormal protein-protein interactions such as increased association with calmodulin (CaM). We previously demonstrated in HEK293 cells that a peptide containing amino acids 76-121 of CaM (CaM-peptide) interrupted the interaction between CaM and mutant huntingtin, reduced mutant huntingtin-induced cytotoxicity and reduced transglutaminase (TG)-modified mutant huntingtin. We now report that adeno-associated virus (AAV)-mediated expression of CaM-peptide in differentiated neuroblastoma SH-SY5Y cells, stably expressing an N-terminal fragment of huntingtin containing 148 glutamine repeats, significantly decreases the amount of TG-modified huntingtin and attenuates cytotoxicity. Importantly, the effect of the CaM-peptide shows selectivity, such that total TG activity is not significantly altered by expression of CaM-peptide nor is the activity of another CaM-dependent enzyme, CaM kinase II. In vitro, recombinant exon 1 of huntingtin with 44 glutamines (htt-exon1-44Q) binds to CaM-agarose; the addition of 10 microM of CaM-peptide significantly decreases the interaction of htt-exon1-44Q and CaM but not the binding between CaM and calcineurin, another CaM-binding protein. These data support the hypothesis that CaM regulates TG-catalyzed modifications of mutant huntingtin and that specific and selective disruption of the CaM-huntingtin interaction is potentially a new target for therapeutic intervention in HD.

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Expression of CaM-peptide in N-terminal mutant huntingtin expressing cells does not significantly affect CaM kinase II activity or expression. A. SH-SY5Y cells were transfected with vector (V), htt-N63-148Q (H) + V, CaM-peptide + V, or H + CaM-peptide. Cells were harvested 48 h post-transfection and CaM kinase II activity was evaluated by the SigmaTECT CaM kinase II Assay System as per the manufacturer's directions. Data shown are the mean ± standard error of the mean (SEM; n = 3). One-way analysis of variance (ANOVA) (F(3,8) = 2.669, P = 0.1187) and Newman-Keuls Multiple comparison indicate no significant differences among groups. B. The CaM kinase II assay was also performed in the absence of exogenous calmodulin. Data shown are the mean ± SEM. Two-way ANOVA indicates a significant main effect of EGTA (F(1,16) = 143.37, P < 0.0001). However, there was no significant main effect of transfection on CaM kinase II activity (F(3,16) = 0.86, P = 0.48). The interaction between EGTA and transfection was also not significant (F(3,16) = 1.89, P = 0.17). Newman-Keuls multiple comparison test indicates no significant differences in CaM kinase II activity among the various transfections in either the presence or absence of EGTA. C.Top, protein expression of CaM kinase II remained at the same level after transfections of either htt-N-148Q (H), CaM-peptide or the combination of those two constructs. Middle, myc antibody was used to confirm the expression of htt-N-148Q. Bottom, equal loading was verified by reprobing the same membrane with an actin antibody. Abbreviations: CaM = calmodulin; EGTA = ethylene glycol tetraacetic acid; H = htt-N63-148Q.
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fig06: Expression of CaM-peptide in N-terminal mutant huntingtin expressing cells does not significantly affect CaM kinase II activity or expression. A. SH-SY5Y cells were transfected with vector (V), htt-N63-148Q (H) + V, CaM-peptide + V, or H + CaM-peptide. Cells were harvested 48 h post-transfection and CaM kinase II activity was evaluated by the SigmaTECT CaM kinase II Assay System as per the manufacturer's directions. Data shown are the mean ± standard error of the mean (SEM; n = 3). One-way analysis of variance (ANOVA) (F(3,8) = 2.669, P = 0.1187) and Newman-Keuls Multiple comparison indicate no significant differences among groups. B. The CaM kinase II assay was also performed in the absence of exogenous calmodulin. Data shown are the mean ± SEM. Two-way ANOVA indicates a significant main effect of EGTA (F(1,16) = 143.37, P < 0.0001). However, there was no significant main effect of transfection on CaM kinase II activity (F(3,16) = 0.86, P = 0.48). The interaction between EGTA and transfection was also not significant (F(3,16) = 1.89, P = 0.17). Newman-Keuls multiple comparison test indicates no significant differences in CaM kinase II activity among the various transfections in either the presence or absence of EGTA. C.Top, protein expression of CaM kinase II remained at the same level after transfections of either htt-N-148Q (H), CaM-peptide or the combination of those two constructs. Middle, myc antibody was used to confirm the expression of htt-N-148Q. Bottom, equal loading was verified by reprobing the same membrane with an actin antibody. Abbreviations: CaM = calmodulin; EGTA = ethylene glycol tetraacetic acid; H = htt-N63-148Q.

Mentions: To determine if expression of CaM-peptide has effects on the activity of other CaM-dependent enzymes, we examined whether expression of CaM-peptide affects CaM-dependent protein kinase II (CaM kinase II) activity. SH-SY5Y cells were transfected with vector, htt-N63-148Q, CaM-peptide or a combination of htt-N63-148Q and CaM-peptide. We found no significant differences in CaM kinase II activity among the various transfections (Figure 6). Interestingly, expression of CaM-peptide alone resulted in a small but insignificant increase in CaM kinase II activity compared with all other transfections. We also examined the activity of CaM kinase II without the addition of exogenous CaM (addition of exogenous CaM is recommended, as described in the manufacturer's protocol). There was no significant effect of CaM-peptide under these assay conditions either (Figure 6).


Neuroprotective effects of calmodulin peptide 76-121aa: disruption of calmodulin binding to mutant huntingtin.

Dudek NL, Dai Y, Muma NA - Brain Pathol. (2009)

Expression of CaM-peptide in N-terminal mutant huntingtin expressing cells does not significantly affect CaM kinase II activity or expression. A. SH-SY5Y cells were transfected with vector (V), htt-N63-148Q (H) + V, CaM-peptide + V, or H + CaM-peptide. Cells were harvested 48 h post-transfection and CaM kinase II activity was evaluated by the SigmaTECT CaM kinase II Assay System as per the manufacturer's directions. Data shown are the mean ± standard error of the mean (SEM; n = 3). One-way analysis of variance (ANOVA) (F(3,8) = 2.669, P = 0.1187) and Newman-Keuls Multiple comparison indicate no significant differences among groups. B. The CaM kinase II assay was also performed in the absence of exogenous calmodulin. Data shown are the mean ± SEM. Two-way ANOVA indicates a significant main effect of EGTA (F(1,16) = 143.37, P < 0.0001). However, there was no significant main effect of transfection on CaM kinase II activity (F(3,16) = 0.86, P = 0.48). The interaction between EGTA and transfection was also not significant (F(3,16) = 1.89, P = 0.17). Newman-Keuls multiple comparison test indicates no significant differences in CaM kinase II activity among the various transfections in either the presence or absence of EGTA. C.Top, protein expression of CaM kinase II remained at the same level after transfections of either htt-N-148Q (H), CaM-peptide or the combination of those two constructs. Middle, myc antibody was used to confirm the expression of htt-N-148Q. Bottom, equal loading was verified by reprobing the same membrane with an actin antibody. Abbreviations: CaM = calmodulin; EGTA = ethylene glycol tetraacetic acid; H = htt-N63-148Q.
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig06: Expression of CaM-peptide in N-terminal mutant huntingtin expressing cells does not significantly affect CaM kinase II activity or expression. A. SH-SY5Y cells were transfected with vector (V), htt-N63-148Q (H) + V, CaM-peptide + V, or H + CaM-peptide. Cells were harvested 48 h post-transfection and CaM kinase II activity was evaluated by the SigmaTECT CaM kinase II Assay System as per the manufacturer's directions. Data shown are the mean ± standard error of the mean (SEM; n = 3). One-way analysis of variance (ANOVA) (F(3,8) = 2.669, P = 0.1187) and Newman-Keuls Multiple comparison indicate no significant differences among groups. B. The CaM kinase II assay was also performed in the absence of exogenous calmodulin. Data shown are the mean ± SEM. Two-way ANOVA indicates a significant main effect of EGTA (F(1,16) = 143.37, P < 0.0001). However, there was no significant main effect of transfection on CaM kinase II activity (F(3,16) = 0.86, P = 0.48). The interaction between EGTA and transfection was also not significant (F(3,16) = 1.89, P = 0.17). Newman-Keuls multiple comparison test indicates no significant differences in CaM kinase II activity among the various transfections in either the presence or absence of EGTA. C.Top, protein expression of CaM kinase II remained at the same level after transfections of either htt-N-148Q (H), CaM-peptide or the combination of those two constructs. Middle, myc antibody was used to confirm the expression of htt-N-148Q. Bottom, equal loading was verified by reprobing the same membrane with an actin antibody. Abbreviations: CaM = calmodulin; EGTA = ethylene glycol tetraacetic acid; H = htt-N63-148Q.
Mentions: To determine if expression of CaM-peptide has effects on the activity of other CaM-dependent enzymes, we examined whether expression of CaM-peptide affects CaM-dependent protein kinase II (CaM kinase II) activity. SH-SY5Y cells were transfected with vector, htt-N63-148Q, CaM-peptide or a combination of htt-N63-148Q and CaM-peptide. We found no significant differences in CaM kinase II activity among the various transfections (Figure 6). Interestingly, expression of CaM-peptide alone resulted in a small but insignificant increase in CaM kinase II activity compared with all other transfections. We also examined the activity of CaM kinase II without the addition of exogenous CaM (addition of exogenous CaM is recommended, as described in the manufacturer's protocol). There was no significant effect of CaM-peptide under these assay conditions either (Figure 6).

Bottom Line: Importantly, the effect of the CaM-peptide shows selectivity, such that total TG activity is not significantly altered by expression of CaM-peptide nor is the activity of another CaM-dependent enzyme, CaM kinase II.In vitro, recombinant exon 1 of huntingtin with 44 glutamines (htt-exon1-44Q) binds to CaM-agarose; the addition of 10 microM of CaM-peptide significantly decreases the interaction of htt-exon1-44Q and CaM but not the binding between CaM and calcineurin, another CaM-binding protein.These data support the hypothesis that CaM regulates TG-catalyzed modifications of mutant huntingtin and that specific and selective disruption of the CaM-huntingtin interaction is potentially a new target for therapeutic intervention in HD.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Experimental Therapeutics, Loyola University Chicago School of Medicine, Maywood, IL, USA.

ABSTRACT
Huntington's disease (HD) is a neurodegenerative disease caused by mutant huntingtin protein containing an expanded polyglutamine tract, which may cause abnormal protein-protein interactions such as increased association with calmodulin (CaM). We previously demonstrated in HEK293 cells that a peptide containing amino acids 76-121 of CaM (CaM-peptide) interrupted the interaction between CaM and mutant huntingtin, reduced mutant huntingtin-induced cytotoxicity and reduced transglutaminase (TG)-modified mutant huntingtin. We now report that adeno-associated virus (AAV)-mediated expression of CaM-peptide in differentiated neuroblastoma SH-SY5Y cells, stably expressing an N-terminal fragment of huntingtin containing 148 glutamine repeats, significantly decreases the amount of TG-modified huntingtin and attenuates cytotoxicity. Importantly, the effect of the CaM-peptide shows selectivity, such that total TG activity is not significantly altered by expression of CaM-peptide nor is the activity of another CaM-dependent enzyme, CaM kinase II. In vitro, recombinant exon 1 of huntingtin with 44 glutamines (htt-exon1-44Q) binds to CaM-agarose; the addition of 10 microM of CaM-peptide significantly decreases the interaction of htt-exon1-44Q and CaM but not the binding between CaM and calcineurin, another CaM-binding protein. These data support the hypothesis that CaM regulates TG-catalyzed modifications of mutant huntingtin and that specific and selective disruption of the CaM-huntingtin interaction is potentially a new target for therapeutic intervention in HD.

Show MeSH
Related in: MedlinePlus