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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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The level of CaM-bound huntingtin-exon 1 with an expanded polyglutamine repeat (htt-exon1-44Q) in the absence and presence of varying concentrations of CaM-peptide and/or W-5. A. The presence of W-5 did not alter the amount of CaM-bound htt-exon1-44Q. The amount of CaM-bound htt-exon1-44Q was significantly lower when 10 µM of CaM-peptide was present than when W-5 or no CaM peptide was present (control). However, the amount of CaM-bound htt-exon1-44Q was significantly increased when 664 µM W-5 is present along with 10 µM of CaM-peptide. (A) Representative western blot of protein immunoprecipitated using CaM-agarose. Blot was probed for N-terminal mutant huntingtin. B. Quantification of immunoblot depicted graphically. Data shown are the mean ± SEM (n = 3). Two-way ANOVA [Factor: W-5 (F(3,23) = 7.712, P = 0.0021)]; Factor: CaM-peptide (F(1,23) = 157.734, P < 0.0001); W-5 × CaM-peptide (F(3,23) = 1.544, P = 0.2417) and Newman-Keuls comparison. ** indicates P < 0.01 compared with 10 mM (CaM-peptide). * indicates P < 0.05 compared with 10 mM (CaM-peptide). ∧ indicates P < 0.01 compared with 66 mM W-5 + 10 mM (CaM-peptide). ++ indicates P < 0.01 compared with 210 mM + W-5 10 mM (CaM-peptide). + indicates P < 0.05 compared with 210 mM W-5 + 10 mM (CaM-peptide). ## indicates P < 0.01 compared with 664 mM W-5 + 10 mM (CaM-peptide). # indicates P < 0.05 compared with 664 mM W-5 + 10 mM (CaM-peptide). Abbreviations: CaM = calmodulin; htt-exon1-44Q = recombinant exon 1 of huntingtin with 44 glutamines.
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fig08: The level of CaM-bound huntingtin-exon 1 with an expanded polyglutamine repeat (htt-exon1-44Q) in the absence and presence of varying concentrations of CaM-peptide and/or W-5. A. The presence of W-5 did not alter the amount of CaM-bound htt-exon1-44Q. The amount of CaM-bound htt-exon1-44Q was significantly lower when 10 µM of CaM-peptide was present than when W-5 or no CaM peptide was present (control). However, the amount of CaM-bound htt-exon1-44Q was significantly increased when 664 µM W-5 is present along with 10 µM of CaM-peptide. (A) Representative western blot of protein immunoprecipitated using CaM-agarose. Blot was probed for N-terminal mutant huntingtin. B. Quantification of immunoblot depicted graphically. Data shown are the mean ± SEM (n = 3). Two-way ANOVA [Factor: W-5 (F(3,23) = 7.712, P = 0.0021)]; Factor: CaM-peptide (F(1,23) = 157.734, P < 0.0001); W-5 × CaM-peptide (F(3,23) = 1.544, P = 0.2417) and Newman-Keuls comparison. ** indicates P < 0.01 compared with 10 mM (CaM-peptide). * indicates P < 0.05 compared with 10 mM (CaM-peptide). ∧ indicates P < 0.01 compared with 66 mM W-5 + 10 mM (CaM-peptide). ++ indicates P < 0.01 compared with 210 mM + W-5 10 mM (CaM-peptide). + indicates P < 0.05 compared with 210 mM W-5 + 10 mM (CaM-peptide). ## indicates P < 0.01 compared with 664 mM W-5 + 10 mM (CaM-peptide). # indicates P < 0.05 compared with 664 mM W-5 + 10 mM (CaM-peptide). Abbreviations: CaM = calmodulin; htt-exon1-44Q = recombinant exon 1 of huntingtin with 44 glutamines.

Mentions: Thus far, all the experiments performed to examine the effects of CaM-peptide were done in cells where other endogenous proteins could play a role in mediating the action of CaM-peptide. Therefore, an in vitro assay was used to determine if CaM-peptide could directly interfere with the interaction of N-terminal mutant huntingtin and CaM. CaM-agarose was used to immunoprecipitate recombinant purified huntingtin exon 1 with an expanded polyglutamine repeat (htt-exon1-44Q) in the absence and presence of varying concentrations of CaM-peptide. The amount of CaM-bound htt-exon1-44Q was significantly lower when 10 µM of CaM-peptide was present than when CaM-peptide was absent (Figure 7A,B). To determine if CaM-peptide would non-specifically inhibit the interaction of CaM with any CaM-binding protein, we incubated calcineurin with CaM-agarose in the absence and presence of 10 µM of CaM-peptide. The presence of 10 µM of CaM-peptide did not affect the binding of CaM with calcineurin (Figure 7C,D). Next, to investigate the potential site of interaction of CaM-peptide, the CaM antagonist, W-5, was used along with CaM-peptide. W-5 alone did not affect the amount of CaM-bound htt-exon1-44Q, but once again the amount of CaM-bound htt-exon1-44Q was significantly lower when 10 µM of CaM-peptide was present. However, the amount of CaM-bound htt-exon1-44Q was significantly increased when 664 µM W-5 is present along with 10 µM of CaM-peptide (Figure 8).


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

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

The level of CaM-bound huntingtin-exon 1 with an expanded polyglutamine repeat (htt-exon1-44Q) in the absence and presence of varying concentrations of CaM-peptide and/or W-5. A. The presence of W-5 did not alter the amount of CaM-bound htt-exon1-44Q. The amount of CaM-bound htt-exon1-44Q was significantly lower when 10 µM of CaM-peptide was present than when W-5 or no CaM peptide was present (control). However, the amount of CaM-bound htt-exon1-44Q was significantly increased when 664 µM W-5 is present along with 10 µM of CaM-peptide. (A) Representative western blot of protein immunoprecipitated using CaM-agarose. Blot was probed for N-terminal mutant huntingtin. B. Quantification of immunoblot depicted graphically. Data shown are the mean ± SEM (n = 3). Two-way ANOVA [Factor: W-5 (F(3,23) = 7.712, P = 0.0021)]; Factor: CaM-peptide (F(1,23) = 157.734, P < 0.0001); W-5 × CaM-peptide (F(3,23) = 1.544, P = 0.2417) and Newman-Keuls comparison. ** indicates P < 0.01 compared with 10 mM (CaM-peptide). * indicates P < 0.05 compared with 10 mM (CaM-peptide). ∧ indicates P < 0.01 compared with 66 mM W-5 + 10 mM (CaM-peptide). ++ indicates P < 0.01 compared with 210 mM + W-5 10 mM (CaM-peptide). + indicates P < 0.05 compared with 210 mM W-5 + 10 mM (CaM-peptide). ## indicates P < 0.01 compared with 664 mM W-5 + 10 mM (CaM-peptide). # indicates P < 0.05 compared with 664 mM W-5 + 10 mM (CaM-peptide). Abbreviations: CaM = calmodulin; htt-exon1-44Q = recombinant exon 1 of huntingtin with 44 glutamines.
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fig08: The level of CaM-bound huntingtin-exon 1 with an expanded polyglutamine repeat (htt-exon1-44Q) in the absence and presence of varying concentrations of CaM-peptide and/or W-5. A. The presence of W-5 did not alter the amount of CaM-bound htt-exon1-44Q. The amount of CaM-bound htt-exon1-44Q was significantly lower when 10 µM of CaM-peptide was present than when W-5 or no CaM peptide was present (control). However, the amount of CaM-bound htt-exon1-44Q was significantly increased when 664 µM W-5 is present along with 10 µM of CaM-peptide. (A) Representative western blot of protein immunoprecipitated using CaM-agarose. Blot was probed for N-terminal mutant huntingtin. B. Quantification of immunoblot depicted graphically. Data shown are the mean ± SEM (n = 3). Two-way ANOVA [Factor: W-5 (F(3,23) = 7.712, P = 0.0021)]; Factor: CaM-peptide (F(1,23) = 157.734, P < 0.0001); W-5 × CaM-peptide (F(3,23) = 1.544, P = 0.2417) and Newman-Keuls comparison. ** indicates P < 0.01 compared with 10 mM (CaM-peptide). * indicates P < 0.05 compared with 10 mM (CaM-peptide). ∧ indicates P < 0.01 compared with 66 mM W-5 + 10 mM (CaM-peptide). ++ indicates P < 0.01 compared with 210 mM + W-5 10 mM (CaM-peptide). + indicates P < 0.05 compared with 210 mM W-5 + 10 mM (CaM-peptide). ## indicates P < 0.01 compared with 664 mM W-5 + 10 mM (CaM-peptide). # indicates P < 0.05 compared with 664 mM W-5 + 10 mM (CaM-peptide). Abbreviations: CaM = calmodulin; htt-exon1-44Q = recombinant exon 1 of huntingtin with 44 glutamines.
Mentions: Thus far, all the experiments performed to examine the effects of CaM-peptide were done in cells where other endogenous proteins could play a role in mediating the action of CaM-peptide. Therefore, an in vitro assay was used to determine if CaM-peptide could directly interfere with the interaction of N-terminal mutant huntingtin and CaM. CaM-agarose was used to immunoprecipitate recombinant purified huntingtin exon 1 with an expanded polyglutamine repeat (htt-exon1-44Q) in the absence and presence of varying concentrations of CaM-peptide. The amount of CaM-bound htt-exon1-44Q was significantly lower when 10 µM of CaM-peptide was present than when CaM-peptide was absent (Figure 7A,B). To determine if CaM-peptide would non-specifically inhibit the interaction of CaM with any CaM-binding protein, we incubated calcineurin with CaM-agarose in the absence and presence of 10 µM of CaM-peptide. The presence of 10 µM of CaM-peptide did not affect the binding of CaM with calcineurin (Figure 7C,D). Next, to investigate the potential site of interaction of CaM-peptide, the CaM antagonist, W-5, was used along with CaM-peptide. W-5 alone did not affect the amount of CaM-bound htt-exon1-44Q, but once again the amount of CaM-bound htt-exon1-44Q was significantly lower when 10 µM of CaM-peptide was present. However, the amount of CaM-bound htt-exon1-44Q was significantly increased when 664 µM W-5 is present along with 10 µM of CaM-peptide (Figure 8).

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