title: Nebulin interactions with actin and tropomyosin are altered by disease-causing mutations.
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.
authors.affiliation: The Folkhälsan Institute of Genetics, Biomedicum Helsinki, Helsinki, Finland.|Department of Medical Genetics, Haartman Institute, University of Helsinki, Helsinki, Finland.
authors.forename: Minttu
authors.initials: M
authors.lastname: Marttila
authors.affiliation: The Folkhälsan Institute of Genetics, Biomedicum Helsinki, Helsinki, Finland.|Department of Medical Genetics, Haartman Institute, University of Helsinki, Helsinki, Finland.
authors.forename: Mubashir
authors.initials: M
authors.lastname: Hanif
authors.affiliation: The Folkhälsan Institute of Genetics, Biomedicum Helsinki, Helsinki, Finland.|Department of Medical Genetics, Haartman Institute, University of Helsinki, Helsinki, Finland.
authors.forename: Elina
authors.initials: E
authors.lastname: Lemola
authors.affiliation: Molecular Neurogenetics Laboratory, Centre for Medical Research, University of Western Australia, Nedlands, Australia.
authors.forename: Kristen J
authors.initials: KJ
authors.lastname: Nowak
authors.affiliation: The Folkhälsan Institute of Genetics, Biomedicum Helsinki, Helsinki, Finland.|Department of Medical Genetics, Haartman Institute, University of Helsinki, Helsinki, Finland.
authors.forename: Jenni
authors.initials: J
authors.lastname: Laitila
authors.affiliation: Department of Biosciences, Division of Biochemistry and Biotechnology, University of Helsinki, Helsinki, Finland.
authors.forename: Mikaela
authors.initials: M
authors.lastname: Grönholm
authors.affiliation: The Folkhälsan Institute of Genetics, Biomedicum Helsinki, Helsinki, Finland.|Department of Medical Genetics, Haartman Institute, University of Helsinki, Helsinki, Finland.
authors.forename: Carina
authors.initials: C
authors.lastname: Wallgren-Pettersson
authors.affiliation: Department of Biosciences, Division of Genetics, University of Helsinki, Helsinki, Finland.
authors.forename: Katarina
authors.initials: K
authors.lastname: Pelin
keywords: Actin; Congenital myopathy; Nebulin; Nemaline (rod) myopathy; Tropomyosin and protein binding
references.citation: Nat Genet. 1995 Jun;10(2):249
references.pmid: 7663526
references.citation: Biochemistry. 2001 Feb 6;40(5):1171-86
references.pmid: 11170442
references.citation: Biochem J. 2012 Feb 15;442(1):231-9
references.pmid: 22084935
references.citation: Curr Biol. 2002 Mar 5;12(5):383-8
references.pmid: 11882289
references.citation: Brain. 2013 Jun;136(Pt 6):1718-31
references.pmid: 23715096
references.citation: J Struct Biol. 2010 May;170(2):334-43
references.pmid: 19944167
references.citation: J Mol Biol. 1995 Apr 28;248(2):308-15
references.pmid: 7739042
references.citation: Am J Hum Genet. 2013 Jul 11;93(1):6-18
references.pmid: 23746549
references.citation: Neuromuscul Disord. 1999 Dec;9(8):564-72
references.pmid: 10619714
references.citation: Eur J Hum Genet. 2004 Sep;12(9):744-51
references.pmid: 15266303
references.citation: Neuromuscul Disord. 2002 Feb;12(2):151-8
references.pmid: 11738357
references.citation: Neuromuscul Disord. 2011 Aug;21(8):556-62
references.pmid: 21724397
references.citation: Biochem Biophys Res Commun. 2002 Aug 16;296(2):300-4
references.pmid: 12163017
references.citation: Neuromuscul Disord. 2002 Oct;12(7-8):680-6
references.pmid: 12207938
references.citation: Nat Genet. 1999 Oct;23(2):208-12
references.pmid: 10508519
references.citation: Am J Hum Genet. 2000 Oct;67(4):814-21
references.pmid: 10952871
references.citation: J Med Genet. 2013 Jun;50(6):383-92
references.pmid: 23572184
references.citation: Am J Hum Genet. 2013 Dec 5;93(6):1108-17
references.pmid: 24268659
references.citation: Hum Mutat. 2014 Jul;35(7):779-90
references.pmid: 24692096
references.citation: J Biomed Biotechnol. 2010;2010:108495
references.pmid: 20467585
references.citation: Brain. 2007 Jun;130(Pt 6):1465-76
references.pmid: 17525139
references.citation: Biophys J. 2009 Mar 4;96(5):1856-65
references.pmid: 19254544
references.citation: J Muscle Res Cell Motil. 2013 Aug;34(3-4):165-9
references.pmid: 23719967
references.citation: J Cell Sci. 2010 Feb 1;123(Pt 3):384-91
references.pmid: 20053633
references.citation: Neurology. 2009 Oct 6;73(14):1159-61
references.pmid: 19805734
references.citation: J Cell Biol. 2006 Jun 19;173(6):905-16
references.pmid: 16769824
references.citation: EMBO J. 1994 Apr 15;13(8):1782-9
references.pmid: 8168478
references.citation: Neuromuscul Disord. 2013 Jan;23(1):56-65
references.pmid: 23010307
references.citation: FASEB J. 2011 Jun;25(6):1903-13
references.pmid: 21350120
references.citation: J Biomed Biotechnol. 2010;2010:968139
references.pmid: 20589077
references.citation: Mol Biol Cell. 2008 May;19(5):1837-47
references.pmid: 18272787
references.citation: Hum Mol Genet. 2009 Jul 1;18(13):2359-69
references.pmid: 19346529
references.citation: Neuromuscul Disord. 2002 Oct;12(7-8):674-9
references.pmid: 12207937
references.citation: Adv Exp Med Biol. 2008;642:28-39
references.pmid: 19181091
references.citation: EMBO J. 2006 Aug 23;25(16):3843-55
references.pmid: 16902413
references.citation: Am J Hum Genet. 2010 Dec 10;87(6):842-7
references.pmid: 21109227
references.citation: J Biol Chem. 2003 Jan 31;278(5):3089-97
references.pmid: 12446728
references.citation: Eur J Paediatr Neurol. 2005;9(1):27-8
references.pmid: 15701564
references.citation: FEBS Lett. 1991 May 6;282(2):313-6
references.pmid: 2037050
references.citation: Am J Hum Genet. 2007 Jan;80(1):162-7
references.pmid: 17160903
references.citation: J Mol Med (Berl). 2008 Nov;86(11):1197-204
references.pmid: 18574571
references.citation: Hum Mutat. 2006 Sep;27(9):946-56
references.pmid: 16917880
references.citation: Proc Natl Acad Sci U S A. 1999 Mar 2;96(5):2305-10
references.pmid: 10051637
references.citation: Skelet Muscle. 2011 Jun 20;1(1):23
references.pmid: 21798101