Joanna Moraczewska

Functional effects of congenital myopathy-related mutations in gamma-tropomyosin gene.

Missense mutations in human TPM3 gene encoding γ-tropomyosin expressed in slow muscle type 1 fibers, were associated with three types of congenital myopathies-nemaline myopathy, cap disease and congenital fiber type disproportion. Functional effects of the following substitutions: Leu100Met, Ala156Thr, Arg168His, Arg168Cys, Arg168Gly, Lys169Glu, and Arg245Gly, were examined in biochemical assays using recombinant tropomyosin mutants and native proteins isolated from skeletal muscle. Most, but not all, mutations decreased the affinity of tropomyosin for actin alone and in complex with troponin (±Ca(2+)). All studied tropomyosin mutants reduced Ca-induced activation but had no effect on the inhibition of actomyosin cross-bridges. Ca(2+)-sensitivity of the actomyosin interactions, as well as cooperativity of myosin-induced activation of the thin filament was affected by individual tropomyosin mutants with various degrees. Decreased motility of the reconstructed thin filaments was a result of combined functional defects caused by myopathy-related tropomyosin mutants. We conclude that muscle weakness and structural abnormalities observed in TPM3-related congenital myopathies result from reduced capability of the thin filament to fully activate actin-myosin cross-bridges.

Effect of actin C-terminal modification on tropomyosin isoforms binding and thin filament regulation

Tropomyosins, a family of actin-binding regulatory proteins, are present in muscle and non-muscle cells. Multiple tropomyosin (TM) isoforms differ in actin affinity and regulatory properties, but little is known about the molecular bases of these differences. The C-terminus of actin stabilizes contacts between actin subunits in the filament and interacts with myosin and regulatory proteins. The goal of this work was to reveal how structural changes in actin and differences between TM isoforms affect binding between these proteins and affect thin filament regulation. Actin proteolytically truncated by three C-terminal amino acids exhibited 1.2–1.5 fold reduced affinity for non-muscle and smooth muscle tropomyosin isoforms. The truncation increased the cooperativity of myosin S1-induced tropomyosin binding for short tropomyosins (TM5a and TM1b9a), but it was neutral for long isoforms (smTM and TM2). Actin modification affected regulation of actomyosin ATPase activity in the presence of all tropomyosins by shifting the filament into a more active state. We conclude that the integrity of the actin C-terminus is important for actin–tropomyosin interactions, however the increased affinity of tropomyosin binding in the S1-induced state of the filament appears not to be involved in the tropomyosin isoform-dependent mechanism of the actomyosin ATPase activation.

Tropomyosin isoforms differentially modulate the regulation of actin filament polymerization and depolymerization by cofilins.

The specific functions of actin filaments located in the contractile and cytoskeletal compartments of muscle cells depend on the stability and dynamic polymerization/depolymerization of filaments. Tropomyosins and cofilins control the length and dynamic rearrangement of the filaments, although the mechanisms regulating actin dynamics are not well understood. In the present study, we used in vitro assays to examine the regulation of two cofilin isoforms, constitutive cofilin‐1 and muscle cofilin‐2, by the muscle homodimer Tpm1.1, muscle heterodimer Tpm1.1/Tpm2.2, and the cytoskeletal Tpm3.1. Depolymerization from the pointed end induced by the muscle‐specific cofilin‐2 was inhibited by all tropomyosins, whereas the muscle isoforms were most effective. By contrast, depolymerization by cofilin‐1 was inhibited by Tpm3.1 and Tpm1.1, but not by Tpm1.1/Tpm2.2. Polymerization of G‐actin was inhibited by cofilin‐2, whereas cofilin‐1 had no effect. All three tropomyosins switched on the inhibiting activity of cofilin‐1; however, Tpm3.1 and Tpm1.1 were much more efficient. Cofilin‐2‐induced inhibition of polymerization was affected neither by Tpm1.1, nor by Tpm3.1, but partly relieved by Tpm1.1/Tpm2.2. Cofilins removed tropomyosin isoforms from the filament with different efficiencies, which correlated with the cooperativities of cofilin binding to the F‐actin/tropomyosin complex. Because neither zero‐length, nor long‐arm cross‐linking between tropomyosin and cofilin isoforms was observed, the effects of tropomyosin isoforms on the activities of cofilins were executed allosterically. The results reveal that isoform‐specific interactions with actin filament permit tropomyosins to discriminate between cofilin isoforms and to differentially regulate their activities.

CacyBP/SIP as a novel modulator of the thin filament.

The CacyBP/SIP protein interacts with several targets, including actin. Since the majority of actin filaments are associated with tropomyosin, in this work we characterized binding of CacyBP/SIP to the actin-tropomyosin complex and examined the effects of CacyBP/SIP on actin filament functions. By using reconstituted filaments composed of actin and AEDANS-labeled tropomyosin, we observed that binding of CacyBP/SIP caused an increase in tropomyosin fluorescence intensity indicating the occurrence of conformational changes within the filament. We also found that CacyBP/SIP bound directly to tropomyosin and that these proteins did not compete with each other for binding to actin. Electron microscopy showed that in the absence of tropomyosin CacyBP/SIP destabilized actin filaments, but tropomyosin reversed this effect. Actin-activated myosin S1 ATPase activity assays, performed using a colorimetric method, indicated that CacyBP/SIP reduced ATPase activity and that the presence of tropomyosin enhanced this inhibitory effect. Thus, our results suggest that CacyBP/SIP, through its interaction with both actin and tropomyosin, regulates the organization and functional properties of the thin filament.

Different positions of tropomyosin isoforms on actin filament are determined by specific sequences of end‐to‐end overlaps

Tropomyosins are dimeric rod‐like proteins which polymerize along actin filaments and regulate interactions with other actin‐binding proteins. Homologous sequences responsible for the binding of tropomyosin to consecutive actin monomers repeat along tropomyosin and are called actin‐binding periods. In this work, the localization of tropomyosin isoforms on actin alone and on actin–myosin complex was evaluated by measuring Förster resonance energy transfer (FRET) distances between a donor (AEDANS) attached to either the N‐terminal actin‐binding period 1 or to the central actin‐binding period 5 and an acceptor (DABMI) bound to actins Cys374. The recombinant α‐tropomyosin isoforms – TM2, TM5a, and TM1b9a, used in this study, had various amino acid sequences of the N‐ and C‐termini forming the end‐to‐end overlap. Although the sequences of actin‐binding period 5 of the three isoforms were identical, the donor–acceptor distances calculated for each isoform varied between 38.6 and 41.5 Å. Differences in FRET distances between the three tropomyosin isoforms labeled in actin‐binding period 1 varied between 34.8 and 40.2 Å. Rigor binding of myosin heads to actin increased all measured distances. The degree and cooperativity of myosin‐induced shift was different for each of the isoforms and actin‐binding periods. The structural differences correlate with cooperative regulation of actin‐activated S1 ATPase by the three tropomyosins. The results indicate that amino acid sequences of the end‐to‐end overlap determine specific orientation of tropomyosin isoform on actin. This can be important for steric and cooperative regulation of the actin filament and determine functional specificity of multiple tropomyosin isoforms present in eucaryotic cells.

Role of Actin C-Terminus in Regulation of Striated Muscle Thin Filament

In striated muscle, regulation of actin-myosin interactions depends on a series of conformational changes within the thin filament that result in a shifting of the tropomyosin-troponin complex between distinct locations on actin. The major factors activating the filament are Ca2+ and strongly bound myosin heads. Many lines of evidence also point to an active role of actin in the regulation. Involvement of the actin C-terminus in binding of tropomyosin-troponin in different activation states and the regulation of actin-myosin interactions were examined using actin modified by proteolytic removal of three C-terminal amino acids. Actin C-terminal modification has no effect on the binding of tropomyosin or tropomyosin-troponin + Ca2+, but it reduces tropomyosin-troponin affinity in the absence of Ca2+. In contrast, myosin S1 induces binding of tropomyosin to truncated actin more readily than to native actin. The rate of actin-activated myosin S1 ATPase activity is reduced by actin truncation both in the absence and presence of tropomyosin. The Ca2+-dependent regulation of the ATPase activity is preserved. Without Ca2+ the ATPase activity is fully inhibited, but in the presence of Ca2+ the activation does not reach the level observed for native actin. The results suggest that through long-range allosteric interactions the actin C-terminus participates in the thin filament regulation.

Impaired tropomyosin-troponin interactions reduce activation of the actin thin filament.

Tropomyosin and troponin are bound to the actin filament to control the contraction of striated muscle in the Ca-dependent manner. The interactions between both regulatory proteins important for the regulation process are not fully understood. To gain more insight into the mechanisms of the thin filament regulation by skeletal α-tropomyosin and troponin, we analyzed effects of seven myopathy-related substitutions: Leu99Met, Ala155Thr, Arg167Gly, Arg167Cys, Arg167His, Lys168Glu, and Arg244Gly. All substitutions reduced Ca-dependent activation of the actomyosin ATPase. The effects of mutations in Arg167 and Lys168 were the most severe. The amino acid substitutions did not significantly affect troponin binding to the whole filament, but reduced 1.2-2.8 fold the affinity of troponin to tropomyosin alone. The excimer fluorescence of N-(1-pyrene)iodoacetamide, a probe attached to the central Cys190, demonstrated that substitutions located near the troponin core domain-binding region strongly affected conformational changes accompanying the tropomyosin-troponin interactions. The thermal stability of all tropomyosin mutants was lower than the stability of the wild type tropomyosin, with TM reduced by 5.3-8.5°C. Together the analyses demonstrated that the myopathy-causing mutations affected tropomyosin structure and led to changes in interactions between tropomyosin and troponin, which impaired the transition of the thin filament from the inactive off to the active on state.

Deviations in conformational rearrangements of thin filaments and myosin caused by the Ala155Thr substitution in hydrophobic core of tropomyosin

Effects of the Ala155Thr substitution in hydrophobic core of tropomyosin Tpm1.1 on conformational rearrangements of the components of the contractile system (Tpm1.1, actin and myosin heads) were studied by polarized fluorimetry technique at different stages of the actomyosin ATPase cycle. The proteins were labelled by fluorescent probes and incorporated into ghost muscle fibres. The substitution violated the blocked and closed states of thin filaments stimulating abnormal displacement of tropomyosin to the inner domains of actin, switching actin on and increasing the relative number of the myosin heads in strong-binding state. Furthermore, the mutant tropomyosin disrupted the major function of troponin to alter the distribution of the different functional states of thin filaments. At low Ca2+ troponin did not effectively switch thin filament off and the myosin head lost the ability to drive the spatial arrangement of the mutant tropomyosin. The information about tropomyosin flexibility obtained from the fluorescent probes at Cys190 indicates that this tropomyosin is generally more rigid, that obviously prevents tropomyosin to bend and adopt the appropriate conformation required for proper regulation.

Differential binding of tropomyosin isoforms to actin modified with m-maleimidobenzoyl-N-hydroxysuccinimide ester and fluorescein-5-isothiocyanate

Differential interactions of tropomyosin (TM) isoforms with actin can be important for determination of the thin filament functions. A mechanism of tropomyosin binding to actin was studied by comparing interactions of five αTM isoforms with actin modified with m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) and with fluorescein-5-isothiocyanate (FITC). MBS attachment sites were revealed with mass spectrometry methods. We found that the predominant actin fraction was cross-linked by MBS within subdomain 3. A smaller fraction of the modified actin was cross-linked within subdomain 2 and between subdomains 2 and 1. Moreover, investigated actins carried single labels in subdomains 1, 2, and 3. Such extensive modification caused a large decrease in actin affinity for skeletal and smooth muscle tropomyosins, nonmuscle TM2, and chimeric TM1b9a. In contrast, binding of nonmuscle isoform TM5a was less affected. Isoform’s affinity for actin modified in subdomain 2 by binding of FITC to Lys61 was intermediate between the affinity for native actin and MBS-modified actin except for TM5a, which bound to FITC–actin with similar affinity as to actin modified with MBS. The analysis of binding curves according to the McGhee–von Hippel model revealed that binding to an isolated site, as well as cooperativity of binding to a contiguous site, was affected by both actin modifications in a TM isoform-specific manner.

Cofilin – a protein controlling dynamics of actin filaments

Cofilins are evolutionary conserved proteins present in all Eukaryotic cells. Their primary function is dynamic reorganization of actin cytoskeleton. Two cofilin isoforms are known: cofilin 1, present in all studied non-muscle cells and in embryonic muscle cells, and cofilin 2, which dominates in mature skeletal and cardiac muscles. Polypeptide chains of both isoforms fold into a structure homological to a conservative ADF (actin depolymerizing factor) domain, which is characteristic of actin depolymerizing factor. In cofilin molecule two actin-binding sites were found. One site binds monomeric and filamentous actin, the second one interacts only with the filament. Binding of cofilin to actin filament causes a change in the orientation of subunits, which results in filament severing. This increases number of ends which can either elongate or shorten the filament, depending on the conditions. Cofilin interactions with monomeric actin decreases availability of polymerization-competent actin subunits. Cofilin activity is controlled by phosphorylation, binding membrane phospholipids, local pH and oxidative stress. Under conditions of oxidative stress oxidation of cysteine residues leads to formation of dimers, which are able to cross-link actin filaments. Stable actin-cofilin rods save cellular ATP, which is not used during active polymerization process. This facilitates faster cell recovery from the stress. The final cellular reaction on the environmental stimuli is a resultant of cofilin activity and activities of other actin-binding proteins, which function either synergistically or antagonistically. Due to the central role in the regulation of actin filaments dynamics, cofilin is involved in development of cancer, neurodegenerative diseases, congenital myopathies and cardiomyopathies.