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Congenital myopathy-related mutations in tropomyosin disrupt regulatory function through altered actin affinity and tropomodulin binding.

ABSTRACT: Tropomyosin (Tpm) binds along actin filaments and regulates myosin binding to control muscle contraction. Tropomodulin binds to the pointed end of a filament and regulates actin dynamics, which maintains the length of a thin filament. To define the structural determinants of these Tpm functions, we examined the effects of two congenital myopathy mutations, A4V and R91C, in the Tpm gene, TPM3, which encodes the Tpm3.12 isoform, specific for slow-twitch muscle fibers. Mutation A4V is located in the tropomodulin-binding, N-terminal region of Tpm3.12. R91C is located in the actin-binding period 3 and directly interacts with actin. The A4V and R91C mutations resulted in a 2.5-fold reduced affinity of Tpm3.12 homodimers for F-actin in the absence and presence of troponin, and a two-fold decrease in actomyosin ATPase activation in the presence of Ca2+ . Actomyosin ATPase inhibition in the absence of Ca2+ was not affected. The Ca2+ sensitivity of ATPase activity was decreased by R91C, but not by A4V. In vitro, R91C altered the ability of tropomodulin 1 (Tmod1) to inhibit actin polymerization at the pointed end of the filaments, which correlated with the reduced affinity of Tpm3.12-R91C for Tmod1. Molecular dynamics simulations of Tpm3.12 in complex with F-actin suggested that both mutations reduce the affinity of Tpm3.12 for F-actin binding by perturbing the van der Waals energy, which may be attributable to two different molecular mechanisms-a reduced flexibility of Tpm3.12-R91C and an increased flexibility of Tpm3.12-A4V.

SUBMITTER: Moraczewska J

PROVIDER: S-EPMC7202179 | biostudies-literature | 2019 May

REPOSITORIES: biostudies-literature

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Congenital myopathy-related mutations in tropomyosin disrupt regulatory function through altered actin affinity and tropomodulin binding.

Moraczewska Joanna J Robaszkiewicz Katarzyna K Śliwinska Małgorzata M Czajkowska Marta M Ly Thu T Kostyukova Alla A Wen Han H Zheng Wenjun W

The FEBS journal 20190305 10

Tropomyosin (Tpm) binds along actin filaments and regulates myosin binding to control muscle contraction. Tropomodulin binds to the pointed end of a filament and regulates actin dynamics, which maintains the length of a thin filament. To define the structural determinants of these Tpm functions, we examined the effects of two congenital myopathy mutations, A4V and R91C, in the Tpm gene, TPM3, which encodes the Tpm3.12 isoform, specific for slow-twitch muscle fibers. Mutation A4V is located in th ...[more]

PMID: 30768849

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Project description:BackgroundNemaline 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.MethodsWe 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.ResultsWT 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).ConclusionsWe 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.

Project description:BackgroundCongenital myopathy (CM) is a group of clinically and genetically heterogeneous muscle disorders, characterized by muscle weakness and hypotonia from birth. Currently, no definite treatment exists for CM. A de novo mutation in Tropomyosin 3-TPM3(E151G) was identified from a boy diagnosed with CM, previously TPM3(E151A) was reported to cause CM. However, the role of TPM3(E151G) in CM is unknown.MethodsHistopathological, swimming behavior, and muscle endurance were monitored in TPM3 wild-type and mutant transgenic fish, modelling CM. Gene expression profiling of muscle of the transgenic fish were studied through RNAseq, and mitochondria respiration was investigated.ResultsWhile TPM3(WT) and TPM3(E151A) fish show normal appearance, amazingly a few TPM3(E151G) fish display either no tail, a crooked body in both F0 and F1 adults. Using histochemical staining for the muscle biopsy, we found TPM3(E151G) displays congenital fiber type disproportion and TPM3(E151A) resembles nemaline myopathy. TPM3(E151G) transgenic fish dramatically swimming slower than those in TPM3(WT) and TPM3(E151A) fish measured by DanioVision and T-maze, and exhibit weaker muscle endurance by swimming tunnel instrument. Interestingly, L-carnitine treatment on TPM3(E151G) transgenic larvae significantly improves the muscle endurance by restoring the basal respiration and ATP levels in mitochondria. With RNAseq transcriptomic analysis of the expression profiling from the muscle specimens, it surprisingly discloses large downregulation of genes involved in pathways of sodium, potassium, and calcium channels, which can be rescued by L-carnitine treatment, fatty acid metabolism was differentially dysregulated in TPM3(E151G) fish and rescued by L-carnitine treatment.ConclusionsThese results demonstrate that TPM3(E151G) and TPM3(E151A) exhibit different pathogenicity, also have distinct gene regulatory profiles but the ion channels were downregulated in both mutants, and provides a potential mechanism of action of TPM3 pathophysiology. Our results shed a new light in the future development of potential treatment for TPM3-related CM.

Project description:Purpose: Congenital myopathies (CM) are a group of clinically and genetically heterogeneous muscle disorders, characterized by muscle weakness and hypotonia from birth. Currently, no definite treatment exists for CM. A novel and de novo mutation in tropomyosin 3 [TPM3(E151G)] was identified from a 7-year-old boy with clinically diagnosed CM. TPM3(E151A) mutation was previously reported as causing CM. In this study, we created transgenic zebrafish to investigate the pathogenicity and find potential treatment. The goals of this study are to describe the RNA contents in muscle specimens derived from TPM3(WT), TPM3(E151A), TPM3(E152G) at embryos and adult stages and to compare the differences TPM3(E151A), TPM3(E151G) mutants and TPM3(WT). The biological function of L-carnitine treatment TPM3(E151G) versus without treatment are also predicted and evaluated. Methods: Muscle mRNA profiles of 6 months adult zebrafish from TPM3(WT), TPM3(E151A) and TPM3(E151G) were extracted and performed deep sequencing, in duplicate, using Illumina Novaseq6000. We also obtained the muscle mRNA profiles of 2 day's zebrafish embryos from TPM3(WT), TPM3(E151A), TPM3(E151G) and TPM3(E151G) treated with L-carnitine. RNA profiles of adult and embryos were generated by deep sequencing, using Illumina NovaSeq 6000. Results: After acquiring of clean reads, we mapped about 40 million sequence reads per sample to the zebrafish genome (GRCz11). Saturation rate analysis was then performed evaluating the gene coverage rate along with read depth increase. The transcripts mapping to difference functional region of the genome was then analyzed. Circos software was used to map the consistency of samples with zebrafish genome on a chromosomal level. The quantification of transcript intensity was analyzed using Fragments Per Kilobase of exon modelper Million mapped fragments (FPKM). Conclusions: Our study represents the first detailed analysis of zebrafish muscle transcriptomes, with biologic replicates, generated by RNA-seq technology. The optimized data analysis workflows reported here should provide a framework for comparative investigations of expression profiles between TPM3(WT), TPM3(E151A) and TPM3(E151G) and L-carnitine treatment. Our results show that NGS offers a comprehensive and more accurate quantitative and qualitative evaluation of mRNA content within a cell or tissue. We conclude that RNA-seq based transcriptome characterization would expedite genetic network analyses and permit the dissection of complex biologic functions.

Dataset Information

Congenital myopathy-related mutations in tropomyosin disrupt regulatory function through altered actin affinity and tropomodulin binding.

Publications

Congenital myopathy-related mutations in tropomyosin disrupt regulatory function through altered actin affinity and tropomodulin binding.

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