Characterization of two tropomyosin isoforms from the fast skeletal muscle of bluefin tuna Thunnus thynnusorientalis

Fast skeletal muscle tropomyosin (TM) of tunas is composed of nearly equimolar amount of two isoforms designated α-TM and β-TM expediently based on their migration behavior in SDS-PAGE, whereas corresponding TMs from the other fish species are homogenous (α-type). The presence of β-TM is thus specific to tunas so far. The amino acid sequence of β-TM from bluefin tuna Thunnus thynnus orientalis, which has not been revealed to date unlike α-TM, was successfully obtained in this study by cDNA cloning. The coding region of β-TM cDNA comprised of an open reading frame of 855 bp encoding 284 amino acid residues, like most of the TMs. Unexpectedly, the sequence of β-TM showed high similarity to those of other vertebrate α-type TMs including tuna α-TM. Phylogenetic analysis also showed that β-TM has the closest relationship with α-TM of tuna. This fact was quite unlike the relation of mammalian α- and β-TMs. Based on the distribution of amino acid substitutions, it was suggested that tuna TM isoforms are the products of different genes. By thermodynamic analysis of native and reconstituted TMs, it was demonstrated that β-TM is less thermostable than α-TM. Proteolytic digestion also supported the lower stability of the former.     

Effect of polyamines on the structure, thermal stability and 2,2,2-trifluoroethanol-induced aggregation of alpha-chymotrypsin

Naturally occurring polyamines are known to interact with a variety of biomolecules and critically involve in some important physiological processes. They have also been shown to influence protein aggregation in vitro in some cases. The aim of the present study was to investigate how polyamines may influence the structure and thermal stability of alpha-chymotrypsin and modulate alcohol-induced aggregation of this protein. Various techniques, including turbidity measurements, tensiometry, DSC, intrinsic fluorescence and far- and near-UV circular dichroism spectroscopy were used to examine the effect of putrescine and spermidine on alpha-chymotrypsin. While slight changes in the secondary and tertiary structure of the protein was observed, a clear stabilizing effect against its thermal unfolding was achieved. Moreover, the polyamines were found to inhibit TFE-induced aggregation at 32% TFE and promote formation of non-native alpha-helices in the protein structure. Based on the observed increase in surface tension induced by polyamines, it is suggested that their effects on enhancing thermal stability and alcohol-induced alpha-helices formation may be due to their kosmotropic properties.     

Effect of 2‐hydroxypropyl‐β‐cyclodextrin on thermal stability and aggregation of glycogen phosphorylase b from rabbit skeletal muscle

The study of the kinetics of thermal aggregation of glycogen phosphorylase b (Phb) from rabbit skeletal muscles by dynamic light scattering at 48°C showed that 2‐hydroxypropyl‐β‐cyclodextrin (HP‐β‐CD) accelerated the aggregation process and induced the formation of the larger protein aggregates. The reason of the accelerating effect of HP‐β‐CD is destabilization of the protein molecule under action of HP‐β‐CD. This conclusion was supported by the data on differential scanning calorimetry and the kinetic data on thermal inactivation of Phb. It is assumed that destabilization of the Phb molecule is due to preferential binding of HP‐β‐CD to intermediates of protein unfolding in comparison with the original native state. The conclusion regarding the ability of the native Phb for binding of HP‐β‐CD was substantiated by the data on the enzyme inhibition by HP‐β‐CD. © 2010 Wiley Periodicals, Inc. Biopolymers 93: 986–993, 2010.     

mRNA expression of genes involved in fatty acid utilization in skeletal muscle and white adipose tissues of sows during lactation

Rodents are able to lower fatty acid utilization in liver and muscle during lactation in order to spare fatty acids for the production of milk triacylglycerols, an effect which is mediated by a down-regulation of peroxisome proliferator-activated receptor α (PPARα). The present study was performed to investigate whether similar fatty acid sparing effects are developing in lactating sows. We considered PPARα and its target genes involved in fatty acid utilization in biopsy samples from muscle and adipose tissue of lactating compared to non-lactating sows. In muscle, PPARα target genes involved in fatty acid utilization were up-regulated during lactation indicating that the fatty acid utilization in muscle was increased. Activation of PPARα was probably due to increased concentrations of non-esterified fatty acids in plasma observed in the lactating sows. In contrast to muscle, PPARα and its target genes involved in β-oxidation in white adipose tissue were down-regulated in early lactation. Overall, the present study shows that sows, unlike rats, are not able to reduce the fatty acid utilization in muscle in order to spare fatty acids for milk production. However, fatty acid oxidation in adipose tissue is lowered during early lactation, an effect that might be helpful to conserve fatty acids released from adipose tissue for the delivery into other tissues, including mammary gland, via the blood.     

The role of TGF‐β1 during skeletal muscle regeneration

The injury of adult skeletal muscle initiates series of well‐coordinated events that lead to the efficient repair of the damaged tissue. Any disturbances during muscle myolysis or reconstruction may result in the unsuccessful regeneration, characterised by strong inflammatory response and formation of connective tissue, that is, fibrosis. The switch between proper regeneration of skeletal muscle and development of fibrosis is controlled by various factors. Amongst them are those belonging to the transforming growth factor β family. One of the TGF‐β family members is TGF‐β1, a multifunctional cytokine involved in the regulation of muscle repair via satellite cells activation, connective tissue formation, as well as regulation of the immune response intensity. Here, we present the role of TGF‐β1 in myogenic differentiation and muscle repair. The understanding of the mechanisms controlling these processes can contribute to the better understanding of skeletal muscle atrophy and diseases which consequence is fibrosis disrupting muscle function.     

A novel serine protease complexed with α2-macroglobulin from skeletal muscle of lizard fish (Saurida undosquamis)

A novel fish muscle serine protease named muscle soluble serine protease (MSSP) was purified from the soluble fraction of lizard fish (Saurida undosquamis: Synodontidae) muscle by ammonium sulfate fractionation followed by four steps of column chromatographies. In native-PAGE, the purified enzyme appeared as a single band with an estimated mol. mass of approximately 380 kDa by gel filtration. In SDS-PAGE under reducing conditions, the purified enzyme migrated as two protein bands at 110 and 100 kDa, named subunits A and B, respectively. The 20 residues of N-terminal amino acid sequence of subunit B showed 70% of homology to β-chain of carp α₂-macroglobulin-1. Moreover, both subunits A and B showed immunoreactivity with anti carp α₂-macroglobulin antibody. Purified MSSP was inactivated by Pefabloc SC, aprotinin, benzamidine and TLCK, but not by α₁-antitrypsin. After acid treatment (pH 2, 24 h), however, the enzyme activity eluted at 14 kDa from Sephacryl S-200 carried out under acidic conditions was inhibited by α₁-antitrypsin. Lizard fish MSSP most rapidly hydrolyzed Boc-Val-Pro-Arg-MCA and Boc-Gln-Arg-Arg-MCA, but did not hydrolyzed Suc-Leu-Leu-Val-Tyr-MCA and Suc-Ala-Ala-Pro-Phe-MCA, and was not suppressed either by E-64, pepstatin A and ethylenediaminetetraacetic acid (EDTA). These results indicate that the purified MSSP is a serine protease complexed with α₂-macroglobulin, and the entrapped protease was dissociated by the acid treatment. Purified and free MSSPs were most active at pH 10.0 and 9.0, respectively. Purified MSSP degraded myofibrillar proteins and casein but time courses of degradation of these substrates by the enzyme differed.     

Cellular and genetic models of H6PDH and 11β‐HSD1 function in skeletal muscle

Glucocorticoids are important for skeletal muscle energy metabolism, regulating glucose utilization, insulin sensitivity, and muscle mass. Nicotinamide adenine dinucleotide phosphate‐dependent 11β‐hydroxysteroid dehydrogenase type 1 (11β‐HSD1)‐mediated glucocorticoid activation in the sarcoplasmic reticulum (SR) is integral to mediating the detrimental effects of glucocorticoid excess in muscle. 11β‐Hydroxysteroid dehydrogenase type 1 activity requires glucose‐6‐phosphate transporter (G6PT)‐mediated G6P transport into the SR for its metabolism by hexose‐6‐phosphate dehydrogenase (H6PDH) for NADPH generation. Here, we examine the G6PT/H6PDH/11β‐HSD1 triad in differentiating myotubes and explore the consequences of muscle‐specific knockout of 11β‐HSD1 and H6PDH. 11β‐Hydroxysteroid dehydrogenase type 1 expression and activity increase with myotube differentiation and in response to glucocorticoids. Hexose‐6‐phosphate dehydrogenase shows some elevation in expression with differentiation and in response to glucocorticoid, while G6PT appears largely unresponsive to these particular conditions. When examining 11β‐HSD1 muscle‐knockout mice, we were unable to detect significant decrements in activity, despite using a well‐validated muscle‐specific Cre transgene and confirming high‐level recombination of the floxed HSD11B1 allele. We propose that the level of recombination at the HSD11B1 locus may be insufficient to negate basal 11β‐HSD1 activity for a protein with a long half‐life. Hexose‐6‐phosphate dehydrogenase was undetectable in H6PDH muscle‐knockout mice, which display the myopathic phenotype seen in global KO mice, validating the importance of SR NADPH generation. We envisage these data and models finding utility when investigating the muscle‐specific functions of the 11β‐HSD1/G6PT/H6PDH triad.     

Insights into thermal resistance of proteins from the intrinsic stability of their α-helices

To investigate the role of α helices in protein thermostability, we compared energy characteristics of α helices from thermophilic and mesophilic proteins belonging to four protein families of known three-dimensional structure, for at least one member of each family. The changes in intrinsic free energy of α-helix formation were estimated using the statistical mechanical theory for describing helix/coil transitions in peptide helices [Munoz, V., Serrano, L. Nature Struc. Biol. 1:399–409, 1994; Munoz, V., Serrano, L. J. Mol. Biol. 245:275–296, 1995; Munoz, V., Serrano, L. J. Mol. Biol. 245:297–308, 1995]. Based on known sequences of mesophilic and thermophilic RecA proteins we found that (1) a high stability of α helices is necessary but is not a sufficient condition for thermostability of RecA proteins, (2) the total helix stability, rather than that of individual helices, is the factor determining protein thermostability, and (3) two facets of intrahelical interactions, the intrinsic helical propensities of amino acids and the side chain–side chain interactions, are the main contributors to protein thermostability. Similar analysis applied to families of L-lactate dehydrogenases, seryl-tRNA synthetases, and aspartate amino transferases led us to conclude that an enhanced total stability of α helices is a general feature of many thermophilic proteins. The magnitude of the observed decrease in intrinsic free energy on α-helix formation of several thermoresistant proteins was found to be sufficient to explain the experimentally determined increase of their thermostability. Free energies of intrahelical interactions of different RecA proteins calculated at three temperatures that are thought to be close to its normal environmental conditions were found to be approximately equal. This indicates that certain flexibility of RecA protein structure is an essential factor for protein function. All RecA proteins analyzed fell into three temperature-dependent classes of similar α-helix stability (ΔG_int = 45.0 ± 2.0 kcal/mol). These classes were consistent with the natural origin of the proteins. Based on the sequences of protein α helices with optimized arrangement of stabilizing interactions, a natural reserve of RecA protein thermoresistance was estimated to be sufficient for conformational stability of the protein at nearly 200°C. Proteins 29:309–320, 1997. © 1997 Wiley-Liss, Inc.     

Secreted protein acidic and rich in cysteine internalization and its age‐related alterations in skeletal muscle progenitor cells

Aging causes phenotypic changes in skeletal muscle progenitor cells (Skm‐PCs), such as reduced myogenesis and increased adipogenesis due to alterations in their environment or niche. Secreted protein acidic and rich in cysteine (SPARC), which is secreted into the niche of Skm‐PCs, inhibits adipogenesis and promotes myogenesis. We have previously reported that Skm‐PC responsiveness to SPARC declines with age, although the mechanism underlying this decline is unknown. In this study, we found that SPARC is internalized by Skm‐PCs and that this uptake increases with age. Internalization is dependent on integrin‐α5, a cell surface SPARC‐binding molecule, and clathrin‐mediated endocytosis. We also demonstrated that internalized SPARC is transported to Rab7‐positive endosomes. Skm‐PCs from old rats exhibited increased clathrin expression and decreased Rab7 expression exclusively in MyoD‐negative cells. In loss‐of‐function analyses, clathrin knockdown increased the anti‐adipogenic effect of SPARC, whereas Rab7 knockdown reduced it, indicating that alterations in SPARC internalization may mediate the age‐related decline in its anti‐adipogenic effect. These results provide insights into age‐related SPARC resistance in Skm‐PCs, which may lead to sarcopenia.     

NMR studies on thermal stability of α‐helix conformation of melittin in pure ethanol and ethanol–water mixture solvents

Thermal stability of the α‐helix conformation of melittin in pure ethanol and ethanol–water mixture solvents has been investigated by using NMR spectroscopy. With increase in water concentration of the mixture solvents (from 0 wt% to ~71.5 wt%) as well as temperature (from room temperature to 60 °C), the intramolecular hydrogen bonds formed in melittin are destabilized and the α‐helix is partially uncoiled. Further, the hydrogen bonds are found to be more thermally stable in pure ethanol than in pure methanol, suggesting that their stability is enhanced with increase in the size of the alkyl groups of alcohol molecules. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

Peroxisome proliferator‐activated receptor γ coactivator 1α regulates mitochondrial calcium homeostasis,sarcoplasmic reticulum stress,and cell death to mitigate skeletal muscle aging

Age‐related impairment of muscle function severely affects the health of an increasing elderly population. While causality and the underlying mechanisms remain poorly understood, exercise is an efficient intervention to blunt these aging effects. We thus investigated the role of the peroxisome proliferator‐activated receptor γ coactivator 1α (PGC‐1α), a potent regulator of mitochondrial function and exercise adaptation, in skeletal muscle during aging. We demonstrate that PGC‐1α overexpression improves mitochondrial dynamics and calcium buffering in an estrogen‐related receptor α‐dependent manner. Moreover, we show that sarcoplasmic reticulum stress is attenuated by PGC‐1α. As a result, PGC‐1α prevents tubular aggregate formation and cell death pathway activation in old muscle. Similarly, the pro‐apoptotic effects of ceramide and thapsigargin were blunted by PGC‐1α in muscle cells. Accordingly, mice with muscle‐specific gain‐of‐function and loss‐of‐function of PGC‐1α exhibit a delayed and premature aging phenotype, respectively. Together, our data reveal a key protective effect of PGC‐1α on muscle function and overall health span in aging.     

Ethanol-induced conformational transitions in holo-α-lactalbumin: Spectral and calorimetric studies

Conformational transitions of holo-α-lactalbumin in a hydro-ethanolic cosolvent system was studied by spectrofluorescence, CD in near- and far-uv regions, and high-sensitivity differential scanning calorimetry. Experimental results allow us to propose that in isothermal conditions α-lactalbumin undergoes a number of conformational transitions with increasing ethanol concentration: N ⇔ I ⇔ D ⇔ H . The existence of I -state was deduced from spectrofluorometric and near-uv CD data. In this state the aromatic chromophores of the amino acid side chains are more accessible to the solvent displaying higher local mobility. The H -state was detected from far-uv CD spectra as a state corresponding to the content of α-helices higher than originally found in native protein. However, calorimetric measurements provide data revealing only the two-state mechanism of α-lactalbumin unfolding in both water and in aqueous ethanol solutions. This indicates that the energy levels of N - and I -states as well as of D - and H -states are similar. Thermodynamics of the unfolding of α-lactalbumin in hydro-ethanolic solutions was analyzed with the help of the linear model of solvent denaturation. Unfolding increments of enthalpy, entropy, and Gibbs energy of transfer of the protein from a reference aqueous solution to hydro-ethanolic solutions of different concentrations were determined from the calorimetric data. They are linear functions of molar ethanol fraction. The slope of the unfolding increment of Gibbs energy of transfer was calculated from data on transfer of amino acid residues taking into account the average solvent accessibility of amino acid residues in the native structure of small globular proteins, using the additive group contribution method. © 1998 John Wiley & Sons, Inc. Biopoly 46: 253–265, 1998