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Nebulin interactions with actin and tropomyosin are altered by disease-causing mutations.

Marttila M, Hanif M, Lemola E, Nowak KJ, Laitila J, Grönholm M, Wallgren-Pettersson C, Pelin K - Skelet Muscle (2014)

Bottom Line: Both the nebulin super repeats containing the p.Glu2431Lys mutation and nebulin super repeats lacking exon 55 (p.Arg2478_Asp2512del) showed weak affinity for F-actin compared with WT fragments.Of the tropomyosin mutations, only p.Glu41Lys showed weaker affinity for nebulin (super repeat 18).We demonstrate for the first time the existence of direct tropomyosin-nebulin interactions in vitro, and show that nebulin interactions with actin and tropomyosin are altered by disease-causing mutations in nebulin and tropomyosin.

View Article: PubMed Central - HTML - PubMed

Affiliation: The Folkhälsan Institute of Genetics, Biomedicum Helsinki, Helsinki, Finland ; Department of Medical Genetics, Haartman Institute, University of Helsinki, Helsinki, Finland.

ABSTRACT

Background: Nemaline myopathy (NM) is a rare genetic muscle disorder, but one of the most common among the congenital myopathies. NM is caused by mutations in at least nine genes: Nebulin (NEB), α-actin (ACTA1), α-tropomyosin (TPM3), β-tropomyosin (TPM2), troponin T (TNNT1), cofilin-2 (CFL2), Kelch repeat and BTB (POZ) domain-containing 13 (KBTBD13), and Kelch-like family members 40 and 41 (KLHL40 and KLHL41). Nebulin is a giant (600 to 900 kDa) filamentous protein constituting part of the skeletal muscle thin filament. Around 90% of the primary structure of nebulin is composed of approximately 35-residue α-helical domains, which form super repeats that bind actin with high affinity. Each super repeat has been proposed to harbor one tropomyosin-binding site.

Methods: We produced four wild-type (WT) nebulin super repeats (S9, S14, S18, and S22), 283 to 347 amino acids long, and five corresponding repeats with a patient mutation included: three missense mutations (p.Glu2431Lys, p.Ser6366Ile, and p.Thr7382Pro) and two in-frame deletions (p.Arg2478_Asp2512del and p.Val3924_Asn3929del). We performed F-actin and tropomyosin-binding experiments for the nebulin super repeats, using co-sedimentation and GST (glutathione-S-transferase) pull-down assays. We also used the GST pull-down assay to test the affinity of WT nebulin super repeats for WT α- and β-tropomyosin, and for β-tropomyosin with six patient mutations: p.Lys7del, p.Glu41Lys, p.Lys49del, p.Glu117Lys, p.Glu139del and p.Gln147Pro.

Results: WT nebulin was shown to interact with actin and tropomyosin. Both the nebulin super repeats containing the p.Glu2431Lys mutation and nebulin super repeats lacking exon 55 (p.Arg2478_Asp2512del) showed weak affinity for F-actin compared with WT fragments. Super repeats containing the p.Ser6366Ile mutation showed strong affinity for actin. When tested for tropomyosin affinity, super repeats containing the p.Glu2431Lys mutation showed stronger binding than WT proteins to tropomyosin, and the super repeat containing the p.Thr7382Pro mutation showed weaker binding than WT proteins to tropomyosin. Super repeats containing the deletion p.Val3924_Asn3929del showed similar affinity for actin and tropomyosin as that seen with WT super repeats. Of the tropomyosin mutations, only p.Glu41Lys showed weaker affinity for nebulin (super repeat 18).

Conclusions: We demonstrate for the first time the existence of direct tropomyosin-nebulin interactions in vitro, and show that nebulin interactions with actin and tropomyosin are altered by disease-causing mutations in nebulin and tropomyosin.

No MeSH data available.


Related in: MedlinePlus

Nebulin mutations affect binding to tropomyosin. Purified GST-nebulin domains bound to beads were incubated with purified α-tropomyosin and β-tropomyosin, then beads were washed and the bound proteins run in SDS-PAGE gels, and stained with Coomassie Blue. The relative intensity of bound α-tropomyosin and β-tropomyosin was quantified from three independent experiments. The mean value and standard deviations from three experiments are shown in the bar charts to the left, and gel pictures of representative experiments are shown on the right. Nebulin domains containing the p.Thr7382Pro (ex151m) mutation showed significantly weakened affinity for tropomyosin than wild-type (WT) proteins (P = 0.039). P values were calculated using the Kruskal–Wallis test when comparing three groups (S9) and the Mann–Whitney test when comparing two groups (S14, S18, S22). Asterisks indicate significant differences compared with the WT protein.
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Figure 3: Nebulin mutations affect binding to tropomyosin. Purified GST-nebulin domains bound to beads were incubated with purified α-tropomyosin and β-tropomyosin, then beads were washed and the bound proteins run in SDS-PAGE gels, and stained with Coomassie Blue. The relative intensity of bound α-tropomyosin and β-tropomyosin was quantified from three independent experiments. The mean value and standard deviations from three experiments are shown in the bar charts to the left, and gel pictures of representative experiments are shown on the right. Nebulin domains containing the p.Thr7382Pro (ex151m) mutation showed significantly weakened affinity for tropomyosin than wild-type (WT) proteins (P = 0.039). P values were calculated using the Kruskal–Wallis test when comparing three groups (S9) and the Mann–Whitney test when comparing two groups (S14, S18, S22). Asterisks indicate significant differences compared with the WT protein.

Mentions: We also performed tropomyosin-binding experiments for the super repeats using GST pull-down assays (Figure 3). Some of the produced nebulin domains showed degraded fragments, but these were larger in size than tropomyosin (Figure 1B). Super repeat 9, containing the p.Glu2431Lys mutation, showed stronger affinity for tropomyosin but this was not confirmed to be statistically significant. Super repeat 9, containing the in-frame deletion of exon 55 (p.Arg2478_Asp2512del), and super repeat 14, containing the in-frame deletion p.Val3924_Asn3929del, showed slightly, but not statistically significant, stronger affinity for tropomyosin. Super repeat 18, containing the p.Ser6366Ile mutation, showed similar affinity for tropomyosin as WT fragments. The nebulin exon 151, containing super repeat 22 with the missense mutation p.Thr7382Pro, showed significantly weaker affinity for tropomyosin compared with the WT protein fragment (P = 0.039). Tropomyosin affinities for each nebulin super repeat are shown as binding curves (Figure 4).We also tested the affinity of WT nebulin super repeats for WT and six β–tropomyosin mutants (p.Lys7del, p.Glu41Lys, p.Lys49del, p.Glu117Lys, p.Glu139del, and p.Gln147Pro) using GST-pull-down assays. Nebulin super repeat 18 containing the WT exon 122 showed slightly weaker affinity for the β-tropomyosin p.Glu41Lys mutant, but this was not statistically significant using the Kruskal–Wallis test. The other mutant tropomyosins did not show significant differences in binding affinity for WT nebulin compared with WT tropomyosin (Figure 5).


Nebulin interactions with actin and tropomyosin are altered by disease-causing mutations.

Marttila M, Hanif M, Lemola E, Nowak KJ, Laitila J, Grönholm M, Wallgren-Pettersson C, Pelin K - Skelet Muscle (2014)

Nebulin mutations affect binding to tropomyosin. Purified GST-nebulin domains bound to beads were incubated with purified α-tropomyosin and β-tropomyosin, then beads were washed and the bound proteins run in SDS-PAGE gels, and stained with Coomassie Blue. The relative intensity of bound α-tropomyosin and β-tropomyosin was quantified from three independent experiments. The mean value and standard deviations from three experiments are shown in the bar charts to the left, and gel pictures of representative experiments are shown on the right. Nebulin domains containing the p.Thr7382Pro (ex151m) mutation showed significantly weakened affinity for tropomyosin than wild-type (WT) proteins (P = 0.039). P values were calculated using the Kruskal–Wallis test when comparing three groups (S9) and the Mann–Whitney test when comparing two groups (S14, S18, S22). Asterisks indicate significant differences compared with the WT protein.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4126377&req=5

Figure 3: Nebulin mutations affect binding to tropomyosin. Purified GST-nebulin domains bound to beads were incubated with purified α-tropomyosin and β-tropomyosin, then beads were washed and the bound proteins run in SDS-PAGE gels, and stained with Coomassie Blue. The relative intensity of bound α-tropomyosin and β-tropomyosin was quantified from three independent experiments. The mean value and standard deviations from three experiments are shown in the bar charts to the left, and gel pictures of representative experiments are shown on the right. Nebulin domains containing the p.Thr7382Pro (ex151m) mutation showed significantly weakened affinity for tropomyosin than wild-type (WT) proteins (P = 0.039). P values were calculated using the Kruskal–Wallis test when comparing three groups (S9) and the Mann–Whitney test when comparing two groups (S14, S18, S22). Asterisks indicate significant differences compared with the WT protein.
Mentions: We also performed tropomyosin-binding experiments for the super repeats using GST pull-down assays (Figure 3). Some of the produced nebulin domains showed degraded fragments, but these were larger in size than tropomyosin (Figure 1B). Super repeat 9, containing the p.Glu2431Lys mutation, showed stronger affinity for tropomyosin but this was not confirmed to be statistically significant. Super repeat 9, containing the in-frame deletion of exon 55 (p.Arg2478_Asp2512del), and super repeat 14, containing the in-frame deletion p.Val3924_Asn3929del, showed slightly, but not statistically significant, stronger affinity for tropomyosin. Super repeat 18, containing the p.Ser6366Ile mutation, showed similar affinity for tropomyosin as WT fragments. The nebulin exon 151, containing super repeat 22 with the missense mutation p.Thr7382Pro, showed significantly weaker affinity for tropomyosin compared with the WT protein fragment (P = 0.039). Tropomyosin affinities for each nebulin super repeat are shown as binding curves (Figure 4).We also tested the affinity of WT nebulin super repeats for WT and six β–tropomyosin mutants (p.Lys7del, p.Glu41Lys, p.Lys49del, p.Glu117Lys, p.Glu139del, and p.Gln147Pro) using GST-pull-down assays. Nebulin super repeat 18 containing the WT exon 122 showed slightly weaker affinity for the β-tropomyosin p.Glu41Lys mutant, but this was not statistically significant using the Kruskal–Wallis test. The other mutant tropomyosins did not show significant differences in binding affinity for WT nebulin compared with WT tropomyosin (Figure 5).

Bottom Line: Both the nebulin super repeats containing the p.Glu2431Lys mutation and nebulin super repeats lacking exon 55 (p.Arg2478_Asp2512del) showed weak affinity for F-actin compared with WT fragments.Of the tropomyosin mutations, only p.Glu41Lys showed weaker affinity for nebulin (super repeat 18).We demonstrate for the first time the existence of direct tropomyosin-nebulin interactions in vitro, and show that nebulin interactions with actin and tropomyosin are altered by disease-causing mutations in nebulin and tropomyosin.

View Article: PubMed Central - HTML - PubMed

Affiliation: The Folkhälsan Institute of Genetics, Biomedicum Helsinki, Helsinki, Finland ; Department of Medical Genetics, Haartman Institute, University of Helsinki, Helsinki, Finland.

ABSTRACT

Background: Nemaline myopathy (NM) is a rare genetic muscle disorder, but one of the most common among the congenital myopathies. NM is caused by mutations in at least nine genes: Nebulin (NEB), α-actin (ACTA1), α-tropomyosin (TPM3), β-tropomyosin (TPM2), troponin T (TNNT1), cofilin-2 (CFL2), Kelch repeat and BTB (POZ) domain-containing 13 (KBTBD13), and Kelch-like family members 40 and 41 (KLHL40 and KLHL41). Nebulin is a giant (600 to 900 kDa) filamentous protein constituting part of the skeletal muscle thin filament. Around 90% of the primary structure of nebulin is composed of approximately 35-residue α-helical domains, which form super repeats that bind actin with high affinity. Each super repeat has been proposed to harbor one tropomyosin-binding site.

Methods: We produced four wild-type (WT) nebulin super repeats (S9, S14, S18, and S22), 283 to 347 amino acids long, and five corresponding repeats with a patient mutation included: three missense mutations (p.Glu2431Lys, p.Ser6366Ile, and p.Thr7382Pro) and two in-frame deletions (p.Arg2478_Asp2512del and p.Val3924_Asn3929del). We performed F-actin and tropomyosin-binding experiments for the nebulin super repeats, using co-sedimentation and GST (glutathione-S-transferase) pull-down assays. We also used the GST pull-down assay to test the affinity of WT nebulin super repeats for WT α- and β-tropomyosin, and for β-tropomyosin with six patient mutations: p.Lys7del, p.Glu41Lys, p.Lys49del, p.Glu117Lys, p.Glu139del and p.Gln147Pro.

Results: WT nebulin was shown to interact with actin and tropomyosin. Both the nebulin super repeats containing the p.Glu2431Lys mutation and nebulin super repeats lacking exon 55 (p.Arg2478_Asp2512del) showed weak affinity for F-actin compared with WT fragments. Super repeats containing the p.Ser6366Ile mutation showed strong affinity for actin. When tested for tropomyosin affinity, super repeats containing the p.Glu2431Lys mutation showed stronger binding than WT proteins to tropomyosin, and the super repeat containing the p.Thr7382Pro mutation showed weaker binding than WT proteins to tropomyosin. Super repeats containing the deletion p.Val3924_Asn3929del showed similar affinity for actin and tropomyosin as that seen with WT super repeats. Of the tropomyosin mutations, only p.Glu41Lys showed weaker affinity for nebulin (super repeat 18).

Conclusions: We demonstrate for the first time the existence of direct tropomyosin-nebulin interactions in vitro, and show that nebulin interactions with actin and tropomyosin are altered by disease-causing mutations in nebulin and tropomyosin.

No MeSH data available.


Related in: MedlinePlus