Joanna Moraczewska

Publications of Joanna Moraczewska

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

  Authors: Małgorzata Sliwińska, Magdalena Zukowska, Danuta Borys, Joanna Moraczewska

  Cytoskeleton (Hoboken, N.J.). 05/2011; 68(5):300-12.

  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 ofTropomyosins 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 actin's Cys374. The recombinant -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.

• [Congenital myopathies - skeletal muscle diseases related to disorder of actin filament structure and functions].

  Authors: Katarzyna Robaszkiewicz, Joanna Moraczewska

  Postȩpy higieny i medycyny doświadczalnej (Online). 01/2011; 65:347-56.

  Congenital myopathies are clinically and genetically heterogeneous disorders characterized by muscle structural abnormalities, muscle weakness and deformities. The clinical spectrum of the diseaseCongenital myopathies are clinically and genetically heterogeneous disorders characterized by muscle structural abnormalities, muscle weakness and deformities. The clinical spectrum of the disease ranges from severe cases with early death to adult-onset cases with slow progression. In the skeletal muscle fibers, the specific structural changes are rod-shaped structures present in the sarcoplasm (nemaline myopathy – NM) or nuclei (intranuclear rod myopathy – IRM), cap-like structures peripherally located within muscle fibers (cap disease – CD), accumulations of actin filaments (actin myopathy – AM), changes in the fiber type proportion and size (congenital fiber type disproportion – CFTD), irregularity of Z-lines and abnormal localization of myofiber nuclei. Mutations in several genes encoding muscle proteins have been linked to congenital myopathy. These genes include a-skeletal actin (ACTA1), tropomyosin (TPM2 and TPM3), troponin (TNNT1) and nebulin (NEB). In vitro and in vivo studies show that mutations identified within these genes have varying impacts on thin filament protein structure, which affect polymerization and stabilization of actin filament, actin cellular localization and regulation of actin-myosin activity. Many lines of evidence suggest that mutated proteins have "toxic" effects. Unfortunately, there is no existing simple correlation between the degree of protein disruption, muscle pathologies and disease severity.

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

  Authors: Radosław Skórzewski, Katarzyna Robaszkiewicz, Justyna Jarzębińska, Piotr Suder, Jerzy Silberring, Joanna Moraczewska

  Analytical biochemistry. 08/2009;

  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 byDifferential 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 alphaTM 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.

• [Role of tropomyosin isoforms in diversification of actin filaments functions]

  Authors: Joanna Moraczewska

  Postepy biochemii. 01/2009; 55(2):201-6.

  Actin interactions with myosin as well as dynamic polymerization and depolymerization of actin filaments underlie various forms of cellular motility. Both mechanisms are under control of regulatoryActin interactions with myosin as well as dynamic polymerization and depolymerization of actin filaments underlie various forms of cellular motility. Both mechanisms are under control of regulatory proteins, among which tropomyosins take a central position. Tropomyosin isoforms can be tissue-specific or constitutive, expressed in different cell types. Tropomyosin expression pattern depends on the cell type and developmental stage. Several tropomyosin isoforms can simultanously co-exist in a single cell, and they are subjected to sorting to different cellular compartments. Due to specific localization, tropomyosins determine interactions of the filaments with actin binding proteins, and ascertain various filaments functions.

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

  Authors: Radosław Skórzewski, Małgorzata Sliwińska, Danuta Borys, Apolinary Sobieszek, Joanna Moraczewska

  Biochimica et biophysica acta. 12/2008;

  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, butTropomyosins, 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.

• Role of actin C-terminus in regulation of striated muscle thin filament.

  Authors: Malgorzata Sliwinska, Radoslaw Skórzewski, Joanna Moraczewska

  Biophysical journal. 03/2008; 94(4):1341-7.

  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 complexIn 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 Ca(2+) 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 + Ca(2+), but it reduces tropomyosin-troponin affinity in the absence of Ca(2+). 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 Ca(2+)-dependent regulation of the ATPase activity is preserved. Without Ca(2+) the ATPase activity is fully inhibited, but in the presence of Ca(2+) 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.

• The DNase-I binding loop of actin may play a role in the regulation of actin-myosin interaction by tropomyosin/troponin.

  Authors: Joanna Moraczewska, Joanna Gruszczynska-Biegala, Maria J Redowicz, Sofia Yu Khaitlina, Hanna Strzelecka-Golaszewska

  The Journal of biological chemistry. 08/2004; 279(30):31197-204.

  Various lines of evidence suggest that communication between tropomyosin and myosin in the regulation of vertebrate-striated muscle contraction involves yet unknown changes in actin conformation.Various lines of evidence suggest that communication between tropomyosin and myosin in the regulation of vertebrate-striated muscle contraction involves yet unknown changes in actin conformation. Possible participation of loop 38-52 in this communication has recently been questioned based on unimpaired Ca(2+) regulation of myosin interaction, in the presence of the tropomyosin-troponin complex, with actin cleaved by subtilisin between Met(47) and Gly(48). We have compared the effects of actin cleavage by subtilisin and by protease ECP32, between Gly(42) and Val(43), on its interaction with myosin S1 in the presence and absence of tropomyosin or tropomyosin-troponin. Both individual modifications reduced activation of S1 ATPase by actin to a similar extent. The effect of ECP cleavage, but not of subtilisin cleavage, was partially reversed by stabilization of interprotomer contacts with phalloidin, indicating different pathways of signal transmission from the N- and C-terminal parts of loop 38-52 to myosin binding sites. ECP cleavage diminished the affinity to tropomyosin and reduced its inhibition of acto-S1 ATPase at low S1 concentrations, but increased the tropomyosin-mediated cooperative enhancement of the ATPase by S1 binding to actin. These effects were reversed by phalloidin. Subtilisin-cleaved actin more closely resembled unmodified actin than the ECP-modified actin. Limited proteolysis of the modified and unmodified F-actins revealed an allosteric effect of ECP cleavage on the conformation of the actin subdomain 4 region that is presumably involved in tropomyosin binding. Our results point to a possible role of the N-terminal part of loop 38-52 of actin in communication between tropomyosin and myosin through changes in actin structure.

• Structural determinants of cooperativity in acto-myosin interactions.

  Authors: Joanna Moraczewska

  Acta biochimica Polonica. 02/2002; 49(4):805-12.

  Regulation of muscle contraction is a very cooperative process. The presence of tropomyosin on the thin filament is both necessary and sufficient for cooperativity to occur. Data recently obtainedRegulation of muscle contraction is a very cooperative process. The presence of tropomyosin on the thin filament is both necessary and sufficient for cooperativity to occur. Data recently obtained with various tropomyosin isoforms and mutants help us to understand better the structural requirements in the thin filament for cooperative protein interactions. Forming an end-to-end overlap between neighboring tropomyosin molecules is not necessary for the cooperativity of the thin filament activation. When direct contacts between tropomyosin molecules are disrupted, the conformational changes in the filament are most probably transmitted cooperatively through actin subunits, although the exact nature of these changes is not known. The function of tropomyosin ends, alternatively expressed in various isoforms, is to confer specific actin affinity. Tropomyosin's affinity or actin is directly related to the size of the apparent cooperative unit defined as the number of actin subunits turned into the active state by binding of one myosin head. Inner sequences of tropomyosin, particularly actin-binding periods 3 to 5, play crucial role in myosin-induced activation of the thin filament. A plausible mechanism of tropomyosin function in this process is that inner tropomyosin regions are either specifically recognized by myosin or they define the right actin conformation required for tropomyosin movement from its blocking position.