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.

Molecular mechanisms of deregulation of the thin filament associated with the R167H and K168E substitutions in tropomyosin Tpm1.1.

Point mutations R167H and K168E in tropomyosin Tpm1.1 (TM) disturb Ca2+-dependent regulation of the actomyosin ATPase. To understand mechanisms of this defect we studied multistep changes in mobility and spatial arrangement of tropomyosin, actin and myosin heads during the ATPase cycle in reconstituted ghost fibres using the polarized fluorescence microscopy. It was found that both mutations disturbed the mode of troponin operation in the fibres. At high Ca2+, troponin increased the fraction of actin monomers that were in the "switched on" state, but both mutant tropomyosins were shifted toward the outer actin domains, which decreased the fraction of strongly bound myosin heads throughout the ATPase cycle. At low Ca2+, the R167H-TM was located close to the outer actin domains, which reduced the number of strongly-bound myosin heads. However, under these conditions troponin increased the number of actin monomers that were switched on. The K168E-TM was displaced far to the outer actin domains and troponin binding decreased the fraction of switched on actin monomers, but the proportion of the strongly bound myosin heads was abnormally high. Thus, the mutations differently disturbed transmission of conformational changes between troponin, tropomyosin and actin, which is essential for the Са2+-dependent regulation of the thin filament.

Abnormal movement of tropomyosin and response of myosin heads and actin during the ATPase cycle caused by the Arg167His, Arg167Gly and Lys168Glu mutations in TPM1 gene.

Amino acid substitutions: Arg167His, Arg167Gly and Lys168Glu, located in a consensus actin-binding site of the striated muscle tropomyosin Tpm1.1 (TM), were used to investigate mechanisms of the thin filament regulation. The azimuthal movement of TM strands on the actin filament and the responses of the myosin heads and actin subunits during the ATPase cycle were studied using fluorescence polarization of muscle fibres. The recombinant wild-type and mutant TMs labelled with 5-IAF, 1,5-IAEDANS-labelled S1and FITC-phalloidin F-actin were incorporated into the ghost muscle fibres to acquire information on the orientation of the probes relative to the fibre axis. The substitutions Arg167Gly and Lys168Glu shifted TM strands into the actin filament centre, whereas Arg167His moved TM towards the periphery of the filament. In the presence of Arg167Gly-TM and Lys168Glu-TM the fraction of actin monomers that were switched on and the number of the myosin heads strongly bound to F-actin were abnormally high even under conditions close to relaxation. In contrast, Arg167His-TM decreased the fraction of switched on actin and reduced the formation of strongly bound myosin heads throughout the ATPase cycle. We concluded that the altered TM-actin contacts destabilized the thin filament and affected the actin-myosin interactions.

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.

[Economic prerequisites of active influenza prevention]. / Ekonomiczne przeslanki stosowania czynnej profilaktyki grypy.

Influenza and other acute infections of the upper respiratory tract are characterized by a high morbidity and mortality. As a result, they entail not only human but also economic consequences. A typical treatment is nonspecific and conservative. It consists of treatment with nonsteroid antiinflammatory drugs, antitussive drugs, hydratation, etc. Influenza is very often associated with complications especially in high risk groups (children, the elderly, chronically-ill people). The cost of treatment increases because of raising costs of pharmacotherapy and the increased absence from work. Vaccination is a safe and morbidity-diminishing method.

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 actin­myosin 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 isoforms­TM2, 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 donor­acceptor 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.