Mutations Q93H and E97K in TPM2 Disrupt Ca-Dependent Regulation of Actin Filaments.

Małgorzata Śliwinska,Piotr Wasąg,Katarzyna Robaszkiewicz,Joanna Moraczewska

doi:10.3390/ijms22084036

Małgorzata Śliwinska, Piotr Wasąg + Show 2 more

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https://doi.org/10.3390/ijms22084036

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Abstract

Tropomyosin is a two-chain coiled coil protein, which together with the troponin complex controls interactions of actin with myosin in a Ca2+-dependent manner. In fast skeletal muscle, the contractile actin filaments are regulated by tropomyosin isoforms Tpm1.1 and Tpm2.2, which form homo- and heterodimers. Mutations in the TPM2 gene encoding isoform Tpm2.2 are linked to distal arthrogryposis and congenital myopathy—skeletal muscle diseases characterized by hyper- and hypocontractile phenotypes, respectively. In this work, in vitro functional assays were used to elucidate the molecular mechanisms of mutations Q93H and E97K in TPM2. Both mutations tended to decrease actin affinity of homo-and heterodimers in the absence and presence of troponin and Ca2+, although the effect of Q93H was stronger. Changes in susceptibility of tropomyosin to trypsin digestion suggested that the mutations diversified dynamics of tropomyosin homo- and heterodimers on the filament. The presence of Q93H in homo- and heterodimers strongly decreased activation of the actomyosin ATPase and reduced sensitivity of the thin filament to [Ca2+]. In contrast, the presence of E97K caused hyperactivation of the ATPase and increased sensitivity to [Ca2+]. In conclusion, the hypo- and hypercontractile phenotypes associated with mutations Q93H and E97K in Tpm2.2 are caused by defects in Ca2+-dependent regulation of actin–myosin interactions.

Highlights

Tropomyosin isoforms are proteins associated with most actin filaments present in different compartments of eukaryotic cells
We demonstrated for the first time that the mutation Q93H, linked to congenital myopathies (CM), severely decreased the ability of tropomyosin-troponin to activate interactions between actin and myosin in the presence of Ca2+ and rendered the thin filament less sensitive to activating
To examine the functional effects of the mutations in tropomyosin variants found in muscle cells, we tested three forms of tropomyosin dimers: (1) Tpm2.2 homodimer with substitutions in both chains; (2) Tpm2.2 heterodimer of wild type and mutant Tpm2.2; and (3) Tpm1.1/Tpm2.2 heterodimer formed by folding wild type or mutant Tpm2.2 with wild type Tpm1.1

Summary

Introduction

Tropomyosin isoforms are proteins associated with most actin filaments present in different compartments of eukaryotic cells. They stabilize the filaments while at the same time acting as gatekeepers that control the access of numerous actin-binding proteins to the filament [1,2]. Tropomyosin is an elongated two-chain coiled coil stabilized by hydrophobic interactions between residues located at the interface between two parallel α-helices. Strong interchain contacts are possible due to a pattern of seven residues (marked as abcdefg) that repeat along the sequence. The tropomyosin sequence contains six to seven pseudorepeats between 39 and 41 amino acids long (Figure 1), in which the N-terminal half of the repeat harbors amino acid residues that directly interact with actin. Due to the head-to-tail interactions between adjacent molecules, tropomyosin binds along the actin helix to form uninterrupted cable-like polymers on both sides of the filament [3,4,5,6,7]

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