The interaction of actin monomers with myosin heads and other muscle proteins.

The simplest interacting unit of actomyosin, viz., single myosin heads (subfragment 1) with actin monomers, has been studied at physiological ionic strength, by isolating the actin molecules from each other on a solid support. The interaction is characterized by a binding constant of 10(5) to 10(6) M-1 in the temperature range 4-30degrees C. It is endothermic with a standard enthalpy of 24 +/- 10 kcal mol-1, and a standard entropy of 110 +/- 40 eu. It is thus, like many protein-protein association processes, entropy-driven. Despite the high affinity of the association, which is comparable in its binding constant to that of subfragment 1 with F-actin, there is only very small activation of myosin ATPase. The ionic-strength dependence of the interaction shows unusual features. Binding of the proteins of the relaxing system to the monomeric actin was also examined: troponin binds both in the presence and absence of calcium ions, but neither tropomyosin nor the tropomyosin-troponin complex was found to bind significantly. Monomeric actin has also been examined as a function of ionic strength by spectroscopic methods; it appears that conformational differences between the G and the F state are the consequence of polymerization, and not of the change in ionic strength required to being the conversion about.     

Tetrahymena actin: copolymerization with skeletal muscle actin and interactions with muscle actin-binding proteins

We have previously shown that actin from Tetrahymena pyriformis has a very divergent primary structure (Hirono, M., Endoh, H., Okada, N., Numata, O., & Watanabe, Y. (1987) J. Mol. Biol. 194, 181-192) and that though it shares essential properties with skeletal muscle actin, it does not interact at all with phalloidin or DNase I (Hirono, M., Kumagai, Y., Numata, O., & Watanabe, Y. (1989) Proc. Natl. Acad. Sci. U.S. 86, 75-79). In this study, we investigated the copolymerization of this actin with skeletal muscle actin by direct observation of the heteropolymers formed from the two actins by means of electron microscopy. We also examined the binding of actin-binding proteins from skeletal muscle or smooth muscle to Tetrahymena actin by means of a cosedimentation assay. The results show that (i) Tetrahymena actin copolymerizes with skeletal muscle actin and that (ii) muscle myosin subfragment 1 binds to it in the absence of ATP, like skeletal muscle actin. However, it was also shown that (iii) muscle alpha-actinin hardly binds to Tetrahymena actin and that (iv) muscle tropomyosin does not bind to it at all. The results show that Tetrahymena actin has both properties similar and dissimilar to those of skeletal muscle actin.     

Regulation of alpha-smooth muscle actin and other polypeptides in proliferating and density-arrested vascular smooth muscle cells

We have examined alpha-smooth muscle actin (alpha-SM actin) protein and mRNA levels in proliferating and density-arrested rabbit vascular smooth muscle cells (SMC) and also studied overall polypeptide synthesis in these cells by two-dimensional (2-D) gel electrophoresis. Of the approximately 1,000 cellular polypeptides resolved by 2-D gel analysis, we consistently detected increased expression of 12 polypeptides in growth-arrested SMC. These polypeptides, with apparent molecular weights of 24,000 to 55,000 exhibited relative increases of between fourfold to greater than tenfold. Three of these polypeptides were expressed at undetectable levels in proliferating SMC. We also detected 12 secreted polypeptides that were expressed at higher levels in growth-arrested SMC. More changes were associated with the secreted polypeptides, since they represented approximately 4% of the total resolved secreted polypeptides, while only 1% of the cellular polypeptides were increased in high-density growth-arrested cells. Under these conditions we observed no change in relative alpha-SM actin protein content as determined by 2-D gel analysis and Western blots. This was corroborated by high levels of alpha-SM actin mRNA levels in both proliferating and high-density growth-arrested SMC. These results indicate rabbit vascular SMC maintain a high level of expression of a smooth muscle differentiation marker (alpha-SM actin) in a proliferation- and density-independent manner. We also examined polypeptide synthesis in SMC isolated by enzymatic digestion of the aorta vs. cells isolated by the explant method. We found that although overall protein patterns were remarkably similar, several differences were observed. These differences were not due to increased contamination by fibroblasts, since both enzymatically- and explant-derived SMC contained high levels of alpha-SM actin as determined by immunofluorescence and by Northern analysis.     

Erythrocyte actin and spectrin. Interactions with muscle contractile and regulatory proteins

Actin and spectrin were isolated from washed red blood cell membranes. Spectrin bound and polymerized erythrocyte actin in the absence of potassium. Spectrin coated onto polystyrene latex particles bound 8–9 mol of erythrocyte actin per mol of spectrin when actin was in its depolymerized state. Spectrin enhanced the interaction of erythrocyte actin with muscle myosin as manifested by changes in Mg²⁺-ATPase activity. A similar enhancement also was observed with muscle α-actinin while muscle tropomyosin abolished these effects. The data suggest that spectrin may play the role of polymerizing factor as well as the anchoring site for erythrocyte actin just as α-actinin is the anchoring site for actin filaments in muscle and other non-muscle cells.     

Regulation of actin and other proteins in the differentiation of myeloid leukemic cells.

The regulation of cytoplasmic proteins in mutants of mouse myeloid leukemic cells, differing in their competence to be induced to differentiate by the normal macrophage- and granulocyte-inducing protein (MGI) and the steroid inducer dexamethasone, was analyzed using SDS-polyacrylamide gel electrophoresis of ³⁵S-methionine-labeled proteins. Before induction, no consistent differences in the pattern of cytoplasmic proteins were found between clones with different capabilities to differentiate.Four MGI⁺D⁺ clones, which are induced by MGI for Fc and C3 rosettes, the synthesis and secretion of lysozyme, and the formation of mature macrophages and granulocytes, all showed the same nine prominent changes in cytoplasmic proteins after induction. Five of these changes were either an increase or a decrease in proteins present in uninduced cells; four proteins appeared to be newly synthesized. One of the proteins that increased after induction was identified as actin. The pattern of cytoplasmic proteins from MGI-induced MGI⁺D⁺ clones more closely resembled that of normal peritoneal macrophages and granulocytes than the pattern of the uninduced clones. The relationship of these protein changes to cell differentiation was further substantiated by the finding that MGI⁺D^? cells, which can be induced by MGI for Fc and C3 rosettes and lysozyme, but not for mature cells, showed only four cytoplasmic protein changes which were quantitatively less than those found for MGI⁺D⁺ clones. An MGI^?D^? clone which was not inducible for any differentiation-associated properties by MGI showed no alteration in protein synthesis. Thus in all the clones studied, there was a correlation between the number and extent of protein changes and the degree of MGI-induced differentiation.In MGI⁺D⁺ clones, some of the differentiation-associated properties induced by MGI can be induced by the steroid hormone dexamethasone. Of the nine protein changes induced by MGI, six were also induced by dexamethasone, and no changes were induced by dexamethasone which were not also induced by MGI. These results, which were also shown by two-dimensional polyacrylamide gel electrophoresis, indicate that in cells which can respond to both MGI and dexamethasone, the proteins induced by dexamethasone were a subset of those induced by MGI.     

Proteomic studies of human and other vertebrate muscle proteins

This review summarizes results of some systemic studies of muscle proteins of humans and some other vertebrates. The studies, started after introduction of two-dimensional gel electrophoresis of OFarrell, were significantly extended during development of proteomics, a special branch of functional genomics. Special attention is paid to analysis of characteristic features of strategy for practical realization of the systemic approach during three main stages of these studies: pre-genomic, genomic (with organizational registration of proteomics), and post-genomic characterized by active use of structural genomics data. Proteomic technologies play an important role in detection of changes in isoforms of various muscle proteins (myosins, troponins, etc.). These changes possibly reflecting tissue specificity of gene expression may underline functional state of muscle tissues under normal and pathological conditions, and such proteomic analysis is now used in various fields of medicine.Translated from Biokhimiya, Vol. 69, No. 11, 2004, pp. 1574–1591.Original Russian Text Copyright © 2004 by Shishkin, Kovalyov, Kovalyova     

Thyrotropin modifies the synthesis of actin and other proteins during thyroid cell culture

Primary cultures of dog thyroid cells have been used to study the effects of thyrotropin on the synthesis of proteins. The cells were cultured for 4 days in serum-free and thyrotropin-free conditions. Thyrotropin was then added for varying periods of time (6-96 h). In the absence of thyrotropin, the cells have an elongated flattened aspect. Exposure to thyrotropin for 6-24 h produces retraction and rounding up of cells whereas cells incubated with thyrotropin for longer periods of time have an epithelial cuboidal shape. After varying periods of culture the cells were labelled with [35S]methionine for 6 h and then analyzed by one- and two-dimensional gel electrophoresis, followed by autoradiography. The results were as follows. After exposure to thyrotropin for 32 h and 48 h, the synthesis of about 18 proteins was increased while that of about 14 others was decreased. After 6 h the labelling of three and five of these proteins was already increased or decreased, respectively. Some of the proteins whose synthesis is modified in the presence of thyrotropin were identified. Actin synthesis was markedly decreased with a maximum 24-48 h after the addition of thyrotropin. A modification in the ratio between alpha and beta tubulins was also observed together with very large changes in a group of proteins having both the relative molecular mass (30 000-40 000) and the isoelectric points of tropomyosins. Forskolin and cholera toxin caused the same qualitative and quantitative changes as thyrotropin; this suggests that the regulation by thyrotropin of the synthesis of several thyroid cell proteins is mediated by cAMP. In conclusion, the data obtained in this work might help to explain the molecular mechanisms by which thyrotropin (and cAMP) triggers the changes in cell shape which occur during thyroid cell culture. They also indicate that one of the main effects of thyrotropin takes place at the level of several proteins which belong to the cytoskeleton and which are involved in the definition of the cytostructure of the thyroid cells.     

Simultaneous expression of skeletal muscle and heart actin proteins in various striated muscle tissues and cells. A quantitative determination of the two actin isoforms

A procedure was developed to determine the percentage of skeletal muscle actin and cardiac actin present in different striated muscle tissues. The method was applied to 2 mg of actin mixtures isolated from various origins. All samples show simultaneous expression of both striated muscle isoactins, with the cardiac actin being the major form (congruent to 80%) in 11-day-old chick embryonic leg muscle, decreasing to approximately 50% values in the late fetal stage of chicken, mouse, and in fused mouse muscle cell cultures and becoming the minor species (less than 5%) in adult skeletal muscle tissues. We also find a significant amount (up to 20%) of the skeletal muscle isoform in adult heart (ventricle) of porcine, bovine, and human origin and no differences in muscle actin ratios in human atrium and ventriculum cells. Similarly, no significant variation in the actin ratios was observed between a normal heart and a heart from a patient with hereditary obstructive myopathy. For those cells and tissues where comparison with levels of mRNA was possible we mostly find a good correlation between the relative ratios of expression of cardiac and skeletal actin proteins and mRNAs.     

3-Methylhistidine turnover in the whole body, and the contribution of skeletal muscle and intestine to urinary 3-methylhistidine excretion in the adult rat.

The tissue origin of 3-methylhistidine (N tau-methylhistidine) was investigated in adult female rats. The decay of labelling of urinary 3-methylhistidine was compared with the labelling of protein-bound 3-methylhistidine in skeletal muscle and intestine after the injection of [methyl-14C]methionine. The decay curve for urinary 3-methylhistidine was much steeper than that in muscle or intestine, falling to values lower than those in either tissue after 30 days. The lack of decay of labelling in muscle during the first 30 days is shown to result from the persistence of label in the precursor S-adenosylmethionine. The relative labelling of urinary, skeletal-muscle and intestinal 3-methylhistidine cannot be explained in terms of skeletal muscle accounting for a major proportion of urinary 3-methylhistidine. Measurements were also made of the steady-state synthesis rate of protein-bound 3-methylhistidine in intestinal smooth muscle in vivo in adult female rats. This involved measurement of the overall rate of protein synthesis and measurement of the relative rates of synthesis of 3-methylhistidine and of mixed protein. The synthesis rate of 3-methylhistidine was 29.1%/day, compared with the overall rate of 77.1%/day for mixed, non-mucosal intestinal protein. Measurement of the amount of 3-methylhistidine in skeletal muscle (0.632 +/- 0.024 mumol/g) and in the whole body (0.332 +/- 0.013 mumol/g) indicate that, although the muscle pool is 86% of the total, because of its slow turnover rate of 1.1-1.6%/day, it only accounts for 38-52% of the observed excretion. Measurements of the mass of the intestine (9.95 g/250 g body wt.) and protein-bound 3-methylhistidine content (0.160 mumol/g of tissue) indicate a pool size of 1.59 mumol/250 micrograms rat. Thus 463 nmol of the urinary excretion/day would originate from the intestine, 22% of the total. The tissue source of the remaining urinary excretion is not identified, but other non-muscle sources constituting about 10% of the whole-body pool could account for this with turnover rates of only 6%/day, a much lower value than the turnover rate in the intestine.     

Retention of actin synthesis in liver under conditions that inhibit synthesis of almost all other proteins

As briefly reported [(1986) Fed. Proc. 45, 1771, Abstr. 1690], rats fed a protein-free diet for a few days often show a marked inhibition of protein synthesis in liver cytosol. However the synthesis of a protein of molecular mass approximately 42 kDa is fully retained. We show here on the basis of its molecular mass, number of bands on isoelectric focusing, isoelectric point and immunological reactivity that this protein is actin and also that actin mRNA is not degraded by micrococcal nuclease under conditions which degrade the bulk of other mRNAs.     

TRPC3-interacting triadic proteins in skeletal muscle

The expression of TRPC3 (canonical-type transient receptor potential cation channel type 3) is tightly regulated during skeletal muscle cell differentiation, and a functional interaction between TRPC3 and RyR1 [(ryanodine receptor type 1), an SR (sarcoplasmic reticulum) Ca2+-release channel] regulates the gain of SR Ca2+ release during EC (excitation-contraction) coupling. However, it has not been possible to demonstrate direct protein-protein interactions between TRPC3 and RyR1. To identify possible candidate(s) for a linker protein(s) between TRPC3 and RyR1 in skeletal muscle, in the present study we performed MALDI-TOF (matrix-assisted laser-desorption ionization-time-of-flight) MS analysis of a cross-linked triadic protein complex from rabbit skeletal triad vesicles and co-immunoprecipitation assays using primary mouse skeletal myotubes. From these studies, we found that six triadic proteins, that are known to regulate RyR1 function and/or EC coupling [TRPC1, JP2 (junctophilin 2), homer, mitsugumin 29, calreticulin and calmodulin], interacted directly with TRPC3 in a Ca2+-independent manner. However we again found no direct interaction between TRPC3 and RyR1. TRPC1 was identified as a potential physical link between TRPC3 and RyR1, as it interacted with both TRPC3 and RyR1, and JPs showed subtype-specific interactions with both RyR1 and TRPC3 (JP1-RyR1 and JP2-TRPC3). These results support the hypothesis that TRPC3 and RyR1 are functionally engaged via linker proteins in skeletal muscle.