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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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There were no significant differences in total ex vivo or in situ TG activity in non-htt-SHSY5Y cells or SHSY5Y-htt-N63-148Q cells expressing CaM-peptide, scram-CaM-peptide or GFP. A. There were no significant differences in total ex vivo TG activity in non-htt-SHSY5Y cells expressing either CaM-peptide, scram-CaM-peptide or GFP (control). There was a small but insignificant increase in total ex vivo TG activity in SHSY5Y-htt-N63-148Q cells expressing scram-CaM-peptide or GFP compared with non-htt-SHSY5Y cells and SHSY5Y-htt-N63-148Q cells expressing CaM-peptide. B. Similarly there were no significant differences in total in situ TG activity in non-htt-SHSY5Y cells expressing CaM-peptide, scram-CaM-peptide or GFP. However, total in situ TG activity was significantly less in non-htt-SHSY5Y cells compared with SHSY5Y-N63-htt-148Q cells expressing scram-CaM-peptide or GFP. But, total in situ TG activity in non-htt-SH-SY5Y cells was not significantly different from total TG activity in SH-SY5Y-htt-N63-148Q cells expressing the CaM-peptide. A. Data shown are the mean ± standard error of the mean (SEM; n = 3). Two-way analysis of variance (ANOVA) [Factor: Virus (F(2,12) = 2.613, P = 0.1143)]; Factor: Huntingtin (F(1,12) = 24.16, P < 0.0004); Virus × Huntingtin [F(2,12) = 1.016, P = 0.3911] and Newman–Keuls comparison. B. Data shown are the mean ± SEM (n = 3). Two-way ANOVA [Factor: Virus (F(2,12) = 1.722, p = 0.22)]; Factor: Huntingtin (F(1,12) = 6.139, P < 0.0291); Virus × Huntingtin (F(2,12) = 0.2775, P = 0.7624) and Newman–Keuls comparison. * indicates P < 0.05 compared with SHSY5Y-htt-N63-148Q cells infected with AAV-GFP. # indicates P < 0.05 compared with SHSY5Y-htt-N63-148Q cells infected with AAV-scram-CaM-peptide. Abbreviations: AAV = adeno-associated virus; CaM = calmodulin; GFP = green fluorescent protein; TG = transglutaminase.
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fig05: There were no significant differences in total ex vivo or in situ TG activity in non-htt-SHSY5Y cells or SHSY5Y-htt-N63-148Q cells expressing CaM-peptide, scram-CaM-peptide or GFP. A. There were no significant differences in total ex vivo TG activity in non-htt-SHSY5Y cells expressing either CaM-peptide, scram-CaM-peptide or GFP (control). There was a small but insignificant increase in total ex vivo TG activity in SHSY5Y-htt-N63-148Q cells expressing scram-CaM-peptide or GFP compared with non-htt-SHSY5Y cells and SHSY5Y-htt-N63-148Q cells expressing CaM-peptide. B. Similarly there were no significant differences in total in situ TG activity in non-htt-SHSY5Y cells expressing CaM-peptide, scram-CaM-peptide or GFP. However, total in situ TG activity was significantly less in non-htt-SHSY5Y cells compared with SHSY5Y-N63-htt-148Q cells expressing scram-CaM-peptide or GFP. But, total in situ TG activity in non-htt-SH-SY5Y cells was not significantly different from total TG activity in SH-SY5Y-htt-N63-148Q cells expressing the CaM-peptide. A. Data shown are the mean ± standard error of the mean (SEM; n = 3). Two-way analysis of variance (ANOVA) [Factor: Virus (F(2,12) = 2.613, P = 0.1143)]; Factor: Huntingtin (F(1,12) = 24.16, P < 0.0004); Virus × Huntingtin [F(2,12) = 1.016, P = 0.3911] and Newman–Keuls comparison. B. Data shown are the mean ± SEM (n = 3). Two-way ANOVA [Factor: Virus (F(2,12) = 1.722, p = 0.22)]; Factor: Huntingtin (F(1,12) = 6.139, P < 0.0291); Virus × Huntingtin (F(2,12) = 0.2775, P = 0.7624) and Newman–Keuls comparison. * indicates P < 0.05 compared with SHSY5Y-htt-N63-148Q cells infected with AAV-GFP. # indicates P < 0.05 compared with SHSY5Y-htt-N63-148Q cells infected with AAV-scram-CaM-peptide. Abbreviations: AAV = adeno-associated virus; CaM = calmodulin; GFP = green fluorescent protein; TG = transglutaminase.

Mentions: Next, we examined the effect of expression of the CaM-peptide on total TG activity in differentiated non-htt-SHSY5Y and SHSY5Y-htt-N63-148Q cells. Cells were infected with either AAV-CaM-peptide + GFP, AAV-scram-CaM-peptide + GFP or AAV-GFP (MOI = 50), and harvested forty-eight hours post-infection. TG activity was measured ex vivo based on the incorporation of 5-(biotinamido)pentylamine into the N,N′-dimethylcasein substrate coated onto micro-plates. There were no significant differences in total TG activity between the different infections [AAV-CaM-peptide + GFP, AAV-scram-CaM-peptide + GFP or AAV-GFP (control)] in either non-htt-SHSY5Y cells or SHSY5Y-htt-N63-148Q cells (Figure 5A). There were small but insignificant increases in total TG activity in SHSY5Y-htt-N63-148Q cells expressing scram-CaM-peptide or only GFP, compared with non-htt-SHSY5Y cells expressing either GFP only, scram-CaM-peptide or CaM-peptide (Figure 5A). To determine whether cell lysis had an effect on enzyme activity, we measured total TG activity in situ. Non-htt-SHSY5Y and SHSY5Y-htt-N63-148Q cells were infected with one of the various AAV, and 42 h post-infection, were treated with 5-(biotinamido)pentylamine. Six hours later, cells were harvested and the cell lysates were applied to micro-plates coated with anti-ε-(γ-glutamyl) lysine (81D4) antibody in order to capture proteins that were modified by TG in situ. TG-catalyzed incorporation of 5-(biotinamido)pentylamine into cellular proteins was then determined by incubation with streptavidin-HRP. Similar to the ex vivo assay, there was no significant difference in total TG activity between the various infections [CaM-peptide, scram-CaM-peptide or GFP (control)] in SHSY5Y-htt-N63-148Q cells (Figure 5B). Similarly, there also was no difference in total TG activity in the various infections in non-htt-SHSY5Y cells. However, there was a significant decrease in total TG activity in non-htt-SHSY5Y cells compared with SHSY5Y-htt-N63-148Q cells expressing scram-CaM-peptide or only GFP. Total TG activity in non-htt-SHSY5Y cells was not significantly different from total TG activity in SHSY5Y-htt-N63-148Q cells expressing CaM-peptide (Figure 5B).


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

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

There were no significant differences in total ex vivo or in situ TG activity in non-htt-SHSY5Y cells or SHSY5Y-htt-N63-148Q cells expressing CaM-peptide, scram-CaM-peptide or GFP. A. There were no significant differences in total ex vivo TG activity in non-htt-SHSY5Y cells expressing either CaM-peptide, scram-CaM-peptide or GFP (control). There was a small but insignificant increase in total ex vivo TG activity in SHSY5Y-htt-N63-148Q cells expressing scram-CaM-peptide or GFP compared with non-htt-SHSY5Y cells and SHSY5Y-htt-N63-148Q cells expressing CaM-peptide. B. Similarly there were no significant differences in total in situ TG activity in non-htt-SHSY5Y cells expressing CaM-peptide, scram-CaM-peptide or GFP. However, total in situ TG activity was significantly less in non-htt-SHSY5Y cells compared with SHSY5Y-N63-htt-148Q cells expressing scram-CaM-peptide or GFP. But, total in situ TG activity in non-htt-SH-SY5Y cells was not significantly different from total TG activity in SH-SY5Y-htt-N63-148Q cells expressing the CaM-peptide. A. Data shown are the mean ± standard error of the mean (SEM; n = 3). Two-way analysis of variance (ANOVA) [Factor: Virus (F(2,12) = 2.613, P = 0.1143)]; Factor: Huntingtin (F(1,12) = 24.16, P < 0.0004); Virus × Huntingtin [F(2,12) = 1.016, P = 0.3911] and Newman–Keuls comparison. B. Data shown are the mean ± SEM (n = 3). Two-way ANOVA [Factor: Virus (F(2,12) = 1.722, p = 0.22)]; Factor: Huntingtin (F(1,12) = 6.139, P < 0.0291); Virus × Huntingtin (F(2,12) = 0.2775, P = 0.7624) and Newman–Keuls comparison. * indicates P < 0.05 compared with SHSY5Y-htt-N63-148Q cells infected with AAV-GFP. # indicates P < 0.05 compared with SHSY5Y-htt-N63-148Q cells infected with AAV-scram-CaM-peptide. Abbreviations: AAV = adeno-associated virus; CaM = calmodulin; GFP = green fluorescent protein; TG = transglutaminase.
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Related In: Results  -  Collection

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Show All Figures
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fig05: There were no significant differences in total ex vivo or in situ TG activity in non-htt-SHSY5Y cells or SHSY5Y-htt-N63-148Q cells expressing CaM-peptide, scram-CaM-peptide or GFP. A. There were no significant differences in total ex vivo TG activity in non-htt-SHSY5Y cells expressing either CaM-peptide, scram-CaM-peptide or GFP (control). There was a small but insignificant increase in total ex vivo TG activity in SHSY5Y-htt-N63-148Q cells expressing scram-CaM-peptide or GFP compared with non-htt-SHSY5Y cells and SHSY5Y-htt-N63-148Q cells expressing CaM-peptide. B. Similarly there were no significant differences in total in situ TG activity in non-htt-SHSY5Y cells expressing CaM-peptide, scram-CaM-peptide or GFP. However, total in situ TG activity was significantly less in non-htt-SHSY5Y cells compared with SHSY5Y-N63-htt-148Q cells expressing scram-CaM-peptide or GFP. But, total in situ TG activity in non-htt-SH-SY5Y cells was not significantly different from total TG activity in SH-SY5Y-htt-N63-148Q cells expressing the CaM-peptide. A. Data shown are the mean ± standard error of the mean (SEM; n = 3). Two-way analysis of variance (ANOVA) [Factor: Virus (F(2,12) = 2.613, P = 0.1143)]; Factor: Huntingtin (F(1,12) = 24.16, P < 0.0004); Virus × Huntingtin [F(2,12) = 1.016, P = 0.3911] and Newman–Keuls comparison. B. Data shown are the mean ± SEM (n = 3). Two-way ANOVA [Factor: Virus (F(2,12) = 1.722, p = 0.22)]; Factor: Huntingtin (F(1,12) = 6.139, P < 0.0291); Virus × Huntingtin (F(2,12) = 0.2775, P = 0.7624) and Newman–Keuls comparison. * indicates P < 0.05 compared with SHSY5Y-htt-N63-148Q cells infected with AAV-GFP. # indicates P < 0.05 compared with SHSY5Y-htt-N63-148Q cells infected with AAV-scram-CaM-peptide. Abbreviations: AAV = adeno-associated virus; CaM = calmodulin; GFP = green fluorescent protein; TG = transglutaminase.
Mentions: Next, we examined the effect of expression of the CaM-peptide on total TG activity in differentiated non-htt-SHSY5Y and SHSY5Y-htt-N63-148Q cells. Cells were infected with either AAV-CaM-peptide + GFP, AAV-scram-CaM-peptide + GFP or AAV-GFP (MOI = 50), and harvested forty-eight hours post-infection. TG activity was measured ex vivo based on the incorporation of 5-(biotinamido)pentylamine into the N,N′-dimethylcasein substrate coated onto micro-plates. There were no significant differences in total TG activity between the different infections [AAV-CaM-peptide + GFP, AAV-scram-CaM-peptide + GFP or AAV-GFP (control)] in either non-htt-SHSY5Y cells or SHSY5Y-htt-N63-148Q cells (Figure 5A). There were small but insignificant increases in total TG activity in SHSY5Y-htt-N63-148Q cells expressing scram-CaM-peptide or only GFP, compared with non-htt-SHSY5Y cells expressing either GFP only, scram-CaM-peptide or CaM-peptide (Figure 5A). To determine whether cell lysis had an effect on enzyme activity, we measured total TG activity in situ. Non-htt-SHSY5Y and SHSY5Y-htt-N63-148Q cells were infected with one of the various AAV, and 42 h post-infection, were treated with 5-(biotinamido)pentylamine. Six hours later, cells were harvested and the cell lysates were applied to micro-plates coated with anti-ε-(γ-glutamyl) lysine (81D4) antibody in order to capture proteins that were modified by TG in situ. TG-catalyzed incorporation of 5-(biotinamido)pentylamine into cellular proteins was then determined by incubation with streptavidin-HRP. Similar to the ex vivo assay, there was no significant difference in total TG activity between the various infections [CaM-peptide, scram-CaM-peptide or GFP (control)] in SHSY5Y-htt-N63-148Q cells (Figure 5B). Similarly, there also was no difference in total TG activity in the various infections in non-htt-SHSY5Y cells. However, there was a significant decrease in total TG activity in non-htt-SHSY5Y cells compared with SHSY5Y-htt-N63-148Q cells expressing scram-CaM-peptide or only GFP. Total TG activity in non-htt-SHSY5Y cells was not significantly different from total TG activity in SHSY5Y-htt-N63-148Q cells expressing CaM-peptide (Figure 5B).

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