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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 and β-tropomyosin mutations studied. (A) The nebulin protein structure and the location of the mutations. The upper part of the figure shows a schematic presentation of the nebulin protein structure and its known protein interaction partners. The lower part of the figure shows a detailed view of the super repeats included in the study (S9, S14, S18, and S22), and the location of the mutations (arrows) and tropomyosin-binding sites (X) in the super repeats. (B) Purified GST (glutathione-S-transferase)-nebulin and tropomyosin. GST-nebulin domains were produced in the Escherichia coli strain BL21 (upper panel), and the α-tropomyosins (Tm3) and β-tropomyosins (Tm2) in insect cells (lower panel). The proteins were purified, run in a SDS-PAGE gel, and stained with Coomassie Blue. The β-tropomyosin mutations p.K49del and p.E139del cause altered protein conformation and thus slower migration in the SDS-PAGE gel [35]. Nomenclature of the mutations in relation to other figures: p.Glu2431Lys = ex54m, p.Arg2478_Asp2512del = ex55del, p.Val3924_Asn3929del = ex78m, p.Ser6366Ile = ex122m, p.Thr7382Pro = ex151m. Abbreviations: Tmod, tropomodulin; M1-M8, M163-M176, M177-M185 simple repeats; S1R1-S1R7, S22R1-S22R7, super repeats of seven simple repeats; Ser, Serine-rich domain, SH3, Src homology domain.
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Figure 1: Nebulin and β-tropomyosin mutations studied. (A) The nebulin protein structure and the location of the mutations. The upper part of the figure shows a schematic presentation of the nebulin protein structure and its known protein interaction partners. The lower part of the figure shows a detailed view of the super repeats included in the study (S9, S14, S18, and S22), and the location of the mutations (arrows) and tropomyosin-binding sites (X) in the super repeats. (B) Purified GST (glutathione-S-transferase)-nebulin and tropomyosin. GST-nebulin domains were produced in the Escherichia coli strain BL21 (upper panel), and the α-tropomyosins (Tm3) and β-tropomyosins (Tm2) in insect cells (lower panel). The proteins were purified, run in a SDS-PAGE gel, and stained with Coomassie Blue. The β-tropomyosin mutations p.K49del and p.E139del cause altered protein conformation and thus slower migration in the SDS-PAGE gel [35]. Nomenclature of the mutations in relation to other figures: p.Glu2431Lys = ex54m, p.Arg2478_Asp2512del = ex55del, p.Val3924_Asn3929del = ex78m, p.Ser6366Ile = ex122m, p.Thr7382Pro = ex151m. Abbreviations: Tmod, tropomodulin; M1-M8, M163-M176, M177-M185 simple repeats; S1R1-S1R7, S22R1-S22R7, super repeats of seven simple repeats; Ser, Serine-rich domain, SH3, Src homology domain.

Mentions: The successful isolation of the nebulin protein for the first time [33], and the generation of knockout mouse models [24,29,34] have helped elucidate the function of this giant molecule. Because of the enormous size of nebulin, functional studies of full-length nebulin are difficult. Hence, we opted for studying protein domains (super repeats) containing mutations known to cause NM (Figure 1). Mutations in all the selected super repeats have been reported to cause NM or distal myopathy (Table 1).


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 and β-tropomyosin mutations studied. (A) The nebulin protein structure and the location of the mutations. The upper part of the figure shows a schematic presentation of the nebulin protein structure and its known protein interaction partners. The lower part of the figure shows a detailed view of the super repeats included in the study (S9, S14, S18, and S22), and the location of the mutations (arrows) and tropomyosin-binding sites (X) in the super repeats. (B) Purified GST (glutathione-S-transferase)-nebulin and tropomyosin. GST-nebulin domains were produced in the Escherichia coli strain BL21 (upper panel), and the α-tropomyosins (Tm3) and β-tropomyosins (Tm2) in insect cells (lower panel). The proteins were purified, run in a SDS-PAGE gel, and stained with Coomassie Blue. The β-tropomyosin mutations p.K49del and p.E139del cause altered protein conformation and thus slower migration in the SDS-PAGE gel [35]. Nomenclature of the mutations in relation to other figures: p.Glu2431Lys = ex54m, p.Arg2478_Asp2512del = ex55del, p.Val3924_Asn3929del = ex78m, p.Ser6366Ile = ex122m, p.Thr7382Pro = ex151m. Abbreviations: Tmod, tropomodulin; M1-M8, M163-M176, M177-M185 simple repeats; S1R1-S1R7, S22R1-S22R7, super repeats of seven simple repeats; Ser, Serine-rich domain, SH3, Src homology domain.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
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Figure 1: Nebulin and β-tropomyosin mutations studied. (A) The nebulin protein structure and the location of the mutations. The upper part of the figure shows a schematic presentation of the nebulin protein structure and its known protein interaction partners. The lower part of the figure shows a detailed view of the super repeats included in the study (S9, S14, S18, and S22), and the location of the mutations (arrows) and tropomyosin-binding sites (X) in the super repeats. (B) Purified GST (glutathione-S-transferase)-nebulin and tropomyosin. GST-nebulin domains were produced in the Escherichia coli strain BL21 (upper panel), and the α-tropomyosins (Tm3) and β-tropomyosins (Tm2) in insect cells (lower panel). The proteins were purified, run in a SDS-PAGE gel, and stained with Coomassie Blue. The β-tropomyosin mutations p.K49del and p.E139del cause altered protein conformation and thus slower migration in the SDS-PAGE gel [35]. Nomenclature of the mutations in relation to other figures: p.Glu2431Lys = ex54m, p.Arg2478_Asp2512del = ex55del, p.Val3924_Asn3929del = ex78m, p.Ser6366Ile = ex122m, p.Thr7382Pro = ex151m. Abbreviations: Tmod, tropomodulin; M1-M8, M163-M176, M177-M185 simple repeats; S1R1-S1R7, S22R1-S22R7, super repeats of seven simple repeats; Ser, Serine-rich domain, SH3, Src homology domain.
Mentions: The successful isolation of the nebulin protein for the first time [33], and the generation of knockout mouse models [24,29,34] have helped elucidate the function of this giant molecule. Because of the enormous size of nebulin, functional studies of full-length nebulin are difficult. Hence, we opted for studying protein domains (super repeats) containing mutations known to cause NM (Figure 1). Mutations in all the selected super repeats have been reported to cause NM or distal myopathy (Table 1).

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