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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

Mutations affect the binding of nebulin to F-actin. Nebulin protein domains were incubated with F-actin and centrifuged. Pellet and supernatant fractions were separated, run on SDS-PAGE gels, and stained with Coomassie Blue. The relative abundance of nebulin protein in the pellet 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 mutation p.Ser6366Ile (ex122m) showed significantly strengthened actin affinity (P = 0.048). 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 wild-type (WT) protein.
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Figure 2: Mutations affect the binding of nebulin to F-actin. Nebulin protein domains were incubated with F-actin and centrifuged. Pellet and supernatant fractions were separated, run on SDS-PAGE gels, and stained with Coomassie Blue. The relative abundance of nebulin protein in the pellet 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 mutation p.Ser6366Ile (ex122m) showed significantly strengthened actin affinity (P = 0.048). 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 wild-type (WT) protein.

Mentions: Nebulin super repeat 9, containing the p.Glu2431Lys (exon 54, c.7291G > A) mutation (which is close to a putative tropomyosin-binding site), and nebulin super repeat 9, lacking 35 aa due to deletion of the entire exon 55 (p.Arg2478_Asp2512del, c.7431 + 1916_7536 + 372del), showed weaker affinity for F-actin compared with the WT fragments, but the difference was not confirmed to be statistically significant using the Kruskal–Wallis test (Figure 2). Super repeat 18, containing the p.Ser6366Ile (exon 122, c.19097G > T) mutation at an actin-binding site, showed stronger actin affinity (P = 0.048, Figure 2). Super repeat 14, containing an in-frame deletion of six aa (exon 78, p.Val3924_Asn3929del), and super repeat 22, containing the p.Thr7382Pro (exon 151, c.22144A > C) mutation, showed similar affinity for actin as the WT fragments (Figure 2).


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)

Mutations affect the binding of nebulin to F-actin. Nebulin protein domains were incubated with F-actin and centrifuged. Pellet and supernatant fractions were separated, run on SDS-PAGE gels, and stained with Coomassie Blue. The relative abundance of nebulin protein in the pellet 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 mutation p.Ser6366Ile (ex122m) showed significantly strengthened actin affinity (P = 0.048). 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 wild-type (WT) protein.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Mutations affect the binding of nebulin to F-actin. Nebulin protein domains were incubated with F-actin and centrifuged. Pellet and supernatant fractions were separated, run on SDS-PAGE gels, and stained with Coomassie Blue. The relative abundance of nebulin protein in the pellet 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 mutation p.Ser6366Ile (ex122m) showed significantly strengthened actin affinity (P = 0.048). 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 wild-type (WT) protein.
Mentions: Nebulin super repeat 9, containing the p.Glu2431Lys (exon 54, c.7291G > A) mutation (which is close to a putative tropomyosin-binding site), and nebulin super repeat 9, lacking 35 aa due to deletion of the entire exon 55 (p.Arg2478_Asp2512del, c.7431 + 1916_7536 + 372del), showed weaker affinity for F-actin compared with the WT fragments, but the difference was not confirmed to be statistically significant using the Kruskal–Wallis test (Figure 2). Super repeat 18, containing the p.Ser6366Ile (exon 122, c.19097G > T) mutation at an actin-binding site, showed stronger actin affinity (P = 0.048, Figure 2). Super repeat 14, containing an in-frame deletion of six aa (exon 78, p.Val3924_Asn3929del), and super repeat 22, containing the p.Thr7382Pro (exon 151, c.22144A > C) mutation, showed similar affinity for actin as the WT fragments (Figure 2).

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