Biomedical subjects

S B Marston

Publications and source records attributed to S B Marston.

At least 19 recordsLinked to original sources

PubMed2005

Structure, properties, and probable physiological role of small heat shock protein with molecular mass 20 kD (Hsp20, HspB6).

This review is devoted to critical analysis of data concerning the structure and functions of small heat shock proteins with apparent molecular mass 20 kD (Hsp20). We describe the structure of Hsp20, its phosphorylation by different protein kinases, interaction of Hsp20 with other small heat shock proteins, and chaperone activity of Hsp20. The distribution of Hsp20 in different animal tissues and the factors affecting expression of Hsp20 are also described. Data on the possible involvement of Hsp20 in regulation of platelet aggregation and glucose transport are presented and analyzed. Special attention is paid to literature data describing probable regulatory effect of Hsp20 on contraction of smooth muscle. Two hypotheses postulating direct effect of Hsp20 on actomyosin interaction or its effect on cytoskeleton are compared and analyzed. The most recent data on the effect of Hsp20 on apoptosis and contractile activity of cardiomyocytes are also presented.

Animals↗

PubMed2005

Force-velocity relationship of single actin filament interacting with immobilised myosin measured by electromagnetic technique.

The effect of applying an external load to actin filaments moving in the in vitro motility assay is studied. Bead-tailed actin filaments were made by polymerising actin onto 2.8 microm diameter Dynabeads conjugated with gelsolin-G actin. These were introduced into a motility cell coated with 100 microg/ml rabbit fast skeletal myosin in the presence of ATP and 0.5% methylcellulose. The motility cell was inserted between the pole-pieces of an electromagnet and the fluorescent beads and filaments were observed. The force-current relationship of the electromagnet was determined from the velocity of free beads in viscous solution and Stokes' equation. The magnet produced up to 6 pN force on the Dynabeads at 1 A. Many bead-tailed actin filaments stuck to the surface, but the beads that did move moved at the same speed as unloaded f-actin in the same cell. Bead-tailed filaments slowed down under an increasing magnetic load, eventually stalled and then slid backward under increasing load before detaching from the surface. Single-filament force-velocity curves were constructed and a stalling force of about 0.6 pN/mm of actin filament estimated.

Actin Cytoskeleton↗

PubMed2002

Structure and properties of small heat shock proteins (sHsp) and their interaction with cytoskeleton proteins.

The modern classification of small heat shock proteins (sHsp) is presented and peculiarities of their primary structure and the mechanism of formation of oligomeric complexes are described. Data on phosphorylation of sHsp by different protein kinases are presented and the effect of phosphorylation on oligomeric state and chaperone activity of sHsp is discussed. Intracellular location of sHsp under normal and stress conditions is described and it is emphasized that under certain condition sHsp interact with different elements of cytoskeleton. The literature concerning the effect of sHsp on polymerization of actin in vitro is analyzed. An attempt is made to compare effects of sHsp on polymerization of actin in vitro with the results obtained on living cells under normal conditions and after heat shock or hormone action. The literature concerning possible effects of sHsp on cell motility is also analyzed.

Amino Acid Sequence↗

PubMed2001

Actomyosin cross-linking by caldesmon in non-muscle cells.

The role of myosin-binding in cytoskeletal arrangement of non-muscle low molecular weight caldesmon (l-caldesmon) was studied. The N-terminal myosin-binding domain of caldesmon N152 colocalized with myosin in transiently transfected chicken fibroblasts. When added exogenously to the Triton-insoluble cytoskeleton, N152 enhanced l-caldesmon displacement by exogenous C-terminal actin-binding fragment (H1). Thus, a significant fraction of l-caldesmon cross-links actin and myosin. In contrast, in epithelioid HeLa cells most of l-caldesmon was only actin-bound as H1 alone was enough for its displacement. Phosphorylation by mitogen-activated protein kinase reduced the capability of H1 to displace endogenous l-caldesmon, suggesting it may represent a regulatory mechanism for actin-caldesmon interaction in vivo.

Actins↗

PubMed2001

Replacement of Lys-75 of calmodulin affects its interaction with smooth muscle caldesmon.

The interaction of smooth muscle caldesmon with synthetic calmodulin (SynCam) and its five mutants with replacement of Lys-75 was analyzed by means of intrinsic Trp fluorescence, zero-length crosslinking and by caldesmon-induced inhibition of actomyosin ATPase activity. SynCam and its double mutant with replacement K75P and simultaneous insertion of KGK between residues 80 and 81 have a comparably low affinity to caldesmon and the probability of crosslinking of this mutant to caldesmon was the lowest among all mutants analyzed. SynCam and its double mutant (K75P+KGK) induced nearly complete reversion of caldesmon inhibition of actomyosin ATPase activity with half-maximal reversion achieved at about 1 microM. Two mutants, K75A and K75V, with partially stabilized less positive central domain have higher affinity to caldesmon. These mutants induce 80-85% reversion of caldesmon inhibition of actomyosin ATPase and the half-maximal reversion was achieved at about 0.3-0.4 microM. Two last mutants, K75P and K75E, with distorted central domain have high affinity to caldesmon and the probability of crosslinking of K75P to caldesmon was the highest among calmodulin mutants tested. These mutants induced complete reversion of caldesmon inhibition with half-maximal effect observed at 0.3-0.4 microM. We suggest that the length, flexibility and charge of the central domain affect binding of calmodulin mutants and their ability to reverse caldesmon-induced inhibition of actomyosin ATPase activity.

Amino Acid Substitution↗

PubMed2001

C-terminal actin-binding sites of smooth muscle caldesmon switch actin between conformational states.

Caldesmon is a component of the thin filaments of smooth muscles where it is believed to play an essential role in regulating the thin filaments' interaction with myosin and hence contractility. We studied the effects of caldesmon and two recombinant fragments CaDH1 (residues 506-793) and CaDH2 (residues 683-767) on the structure of actin-tropomyosin by making measurements of the fluorescence polarisation of probes specifically attached to actin. CaDH1, like the parent molecule caldesmon, is an inhibitor of actin-tropomyosin interaction with myosin whilst CaDH2 is an activator. The F-actin in permeabilised and myosin free rabbit skeletal muscle 'ghost' fibres was labelled by tetramethyl rhodamine-isothiocyanate (TRITC)-phalloidin or fluorescein-5'-isothiocyanate (FITC) at lysine 61. Fluorescence polarisation measurements were made and the parameters Phi(A), Phi(E), Theta(1/2) and Nu were calculated. Phi(A) and Phi(E) are angles between the fiber axis and the absorption and emission dipoles, respectively; Theta(1/2) is the angle between the F-actin filament axis and the fiber axis; Nu is the relative number of randomly oriented fluorophores. Actin-tropomyosin interaction with myosin subfragment-1 induced changes in the parameters of the polarised fluorescence that are typical of strong binding of myosin to actin and of the 'on' conformational state of actin. Caldesmon and CaDH1 (as well as troponin in the absence of Ca(2+)) diminished the effect of S-1, whereas CaDH2 (as well as troponin in the presence of Ca(2+)) enhanced the effect of S1. Thus the structural evidence correlates with biochemical evidence that C-terminal actin-binding sites of caldesmon can modulate the structural transition of actin monomers between 'off' (caldesmon and CaDH1) and 'on' (S-1 and CaDH2) states in a manner analogous to troponin.

Actins↗

PubMed2001

Cardiac and skeletal myopathies: can genotype explain phenotype?

The inherited muscle diseases, skeletal muscle nemaline myopathy and cardiac muscle hypertrophic myopathy (HCM) have been recognised for decades. Recently it has become apparent that mutations in almost any protein component of the sarcomere could cause myopathy. Thus changes in many sarcomeric protein genes can produce a common phenotype. Several recent publications indicate the opposite property: mutations in one sarcomeric protein can produce different muscle disease phenotypes. The most dramatic example of this property is actin, mutations in which are associated with hypertrophic cardiomyopathy, dilated cardiomyopathy, nemaline myopathy and actin myopathy.

Actin Cytoskeleton↗

PubMed2001

Arthroscopic stabilization of type II SLAP lesions using an absorbable tack.

PURPOSE: To document the outcomes of arthroscopic stabilization of Snyder type II SLAP (superior labrum, anterior and posterior) lesions, using a bioabsorbable tack. TYPE OF STUDY: A case series. METHODS: Twenty-five SLAP lesions were repaired arthroscopically using a bioabsorbable tack. There were 22 recreational, 2 high school, and 1 professional athlete in this group. Shoulder function was surveyed at a mean follow-up of 35 months (range, 24 to 51 months) using the UCLA and ASES shoulder scoring algorithms. RESULTS: Shoulder function improved in 24 of the 25 cases. Follow-up UCLA scores averaged 32 points with 9 patients scoring as excellent, 13 good, 2 fair, and 1 poor, for an overall success rate of 88%. ASES shoulder scores similarly improved from a preoperative average of 45 points to a postoperative average of 92. All but 2 of the athletes had returned to their preinjury level of sports participation. CONCLUSIONS: Detachment of the superior labrum from the glenoid is recognized as a problematic injury in throwing athletes and others who engage in repetitive overhead activities. We conclude from our experience that using an absorbable tack to repair type II SLAP lesions is an effective treatment, even in athletes with high demands and expectations for shoulder function.

Absorbable Implants↗

PubMed2000

A simple method for measuring the relative force exerted by myosin on actin filaments in the in vitro motility assay: evidence that tropomyosin and troponin increase force in single thin filaments.

We have studied the effect of an internal load on the movement of actin filaments over a bed of heavy meromyosin (HMM) in the in vitro motility assay. Immobilized alpha-actinin can bind to actin filaments reversibly and ultimately stop the filaments from moving. Above a critical concentration of alpha-actinin, thin filament velocity rapidly diminished to zero. The fraction of thin motile filaments decreased linearly to zero with increasing alpha-actinin concentration. The concentration of alpha-actinin needed to stop all filaments from moving (0.8 microg/ml with actin) was very consistent both within and between experiments. In the present study we have defined the 'index of retardation' as the concentration of alpha-actinin needed to stop all filament movement, and we propose that this index is a measure of the isometric force exerted by HMM on actin filaments. When we measured the effect of immobilized alpha-actinin on motility in the presence of 10 mM P(i) we found that the index of retardation was 0.62+/-0.07 (n=3) times that in the absence of P(i). This observation is in agreement with the reduction of isometric tension in chemically-skinned muscle due to P(i). In a series of comparative experiments we observed that tropomyosin and troponin increase the index of retardation and that the degree of increase depends upon the tropomyosin isoform studied. The index of retardation of actin is increased 1.8-fold by skeletal-muscle tropomyosin, and 3-fold by both cardiac-muscle and smooth-muscle tropomyosin. In the presence of troponin the index of retardation is 2.9-3.4-fold greater than that of actin with all tropomyosin isoforms.

Actins↗

PubMed2000

A novel Ca2+ binding protein associated with caldesmon in Ca2+-regulated smooth muscle thin filaments: evidence for a structurally altered form of calmodulin.

Smooth muscle thin filaments are made up of actin, tropomyosin, the inhibitory protein caldesmon and a Ca2+-binding protein. Thin filament activation of myosin MgATPase is Ca2+-regulated but thin filaments assembled from smooth muscle actin, tropomyosin and caldesmon plus brain or aorta calmodulin are not Ca2+-regulated at 25 degrees C/50 mM KCl. We isolated the Ca2+-binding protein (CaBP) from smooth muscle thin filaments by DEAE fast-flow chromatography in 6 M urea and phenyl sepharose chromatography using sheep aorta as our starting material. CaBP combines with smooth muscle actin, tropomyosin and caldesmon to reconstitute a normally regulated thin filament at 25 degrees C/50 mM KCl. It reverses caldesmon inhibition at pCa5 under conditions where CaM is largely inactive, it binds to caldesmon when complexed with actin and tropomyosin rather than displacing it and it binds to caldesmon independently of [Ca2+]. Amino acid sequencing, and electrospray mass spectrometry show the CaBP is identical to CaM. Structural probes indicate it is different: calmodulin increases caldesmon tryptophan fluorescence but CaBP does not. The distribution of charged species in electrospray mass spectrometry and nozzle skimmer fragmentation patterns are different indicating a less stable N-terminal lobe for CaBP. Brief heating abolishes these special properties of the CaBP. Mass spectrometry in aqueous buffer showed no evidence for the presence of any covalent or non-covalently bound adduct. The only remaining conclusion is that CaBP is calmodulin locked in a metastable altered state.

Actin Cytoskeleton↗

PubMed2000

[Effect of the C-terminal actin-binding sites of caldesmon on the interaction of actin with myosin].

TRITC-phalloidin or FITC-labeled F-actin of ghost muscle fibers was bound to tropomyosin and C-terminal recombinant fragments of caldesmon CaDH1 (residues 506-793) or CaDH2 (residues 683-767). After that the fibers were decorated with myosin subfragment 1. In the absence of caldesmon fragments, subfragment 1 interaction with F-actin caused changes in parameters of polarized fluorescence, that were typical of "strong" binding of myosin heads to F-actin and of the "switched on" conformational state of actin. CaDH1 inhibited, whereas CaDH2 activated the effect of subfragment 1. It is suggested that C-terminal part of caldesmon may modulate the transition of F-actin subunits from the "switched on" to the "switched off" state.

Actins↗

PubMed1999

The interface between caldesmon domain 4b and subdomain 1 of actin studied by nuclear magnetic resonance spectroscopy.

The ability of caldesmon to inhibit actomyosin ATPase activity involves the interaction of three nonsequential segments of caldesmon domain 4 (amino acids 600-756) with actin. Two of these contacts are located in the C-terminal half of this region of caldesmon which has been designated domain 4b (658-756). To investigate the spatial relationship between the two sites and to determine whether their corresponding contacts on actin are sequentially distinct, we have used NMR spectroscopy to compare the actin binding properties of the minimal inhibitory peptide LW30 comprising residues 693-722 with those of the recombinant domain 4b constructs 658C (658-756) and Cg1 (a mutant of 658C in which the sequence (691)Glu-Trp-Leu-Thr-Lys-Thr(696) is changed to Pro-Gly-His-Tyr-Asn-Asn). Cg1 retains dual-sited actin attachment but displays lowered actin affinity. In the presence of tropomyosin, domain 4b-actin contacts were stronger but not qualitatively different, indicating that tropomyosin affected the conformational equilibrium of caldesmon binding. Simultaneous dual-sited attachment of domain 4b to actin is enabled by the conformational properties of the site-spanning sequence common to 658C, Cg1, and LW30 as reflected in the corresponding NOE and other NMR spectral parameters. A backbone turn region ((713)Gly-Asp-Val-Ser(716)) preceded by an extended segment (Ser(702)-Pro-Ala-Pro-Lys-Pro) acts to constrain the relative disposition of the flanking actin contact sites of domain 4b. In tests with a library of actin peptides, only the C-terminus, 350-375, bound to 658C and LW30. The use of Cu(2+) as a paramagnetic spectral probe bound to the unique His-371 provided evidence of a well-defined geometry for the complex between LW30 and actin residues 350-375 with the N-terminal, site B of domain 4b close to the C-terminal residues of actin. The data are discussed in the context of the potentiation of inhibitory activity by tropomyosin.

Actins↗

PubMed1999

Evidence against the regulation of caldesmon inhibitory activity by p42/p44erk mitogen-activated protein kinase in vitro and demonstration of another caldesmon kinase in intact gizzard smooth muscle.

The effect of direct phosphorylation by recombinant p44erk1 mitogen-activated protein kinase on the inhibitory activity of caldesmon and its C-terminal fragment H1 was studied in vitro. Neither inhibition of actin-tropomyosin activated ATPase of heavy meromyosin by caldesmon or H1, nor inhibition of the actin-tropomyosin motility over heavy meromyosin by H1 was significantly affected by the phosphorylation while only a moderate effect on the actin-activated component of heavy meromyosin ATPase inhibition was observed. Phosphopeptide mapping of caldesmon immunoprecipitated from [32P]PO4-labelled intact gizzard strips revealed that it is predominantly phosphorylated at mitogen-activated protein kinase sites in unstimulated tissue and that it is stimulated for 1 h with phorbol 12,13-dibutyrate. We find that phorbol 12,13-dibutyrate also induces a transitory phosphorylation of caldesmon peaking at 15 min after addition and this phosphorylation is not attributed to mitogen-activated protein kinase, protein kinase C, Ca2+/calmodulin-dependent kinase II or casein kinase II. We suggest that a yet unidentified kinase, rather than mitogen-activated protein kinase, may be involved in regulation of the caldesmon function in vivo.

Animals↗

PubMed1999

Control of shortening speed in single guinea-pig taenia coli smooth muscle cells by Ca2+, phosphorylation and caldesmon.

We studied the effect of caldesmon peptides on the regulation of shortening of single guinea-pig taenia coli cells permeabilised with saponin. When contraction was initiated by Ca2+ and MgATP shortening rate at pCa 4.5 was 0.17+/-0.04 cell lengths s-1 and half-maximal rate was at pCa 5.6. Following thiophosphorylation with 1 mM adenosine 5'-O-(3-thiotriphosphate) (ATP[gamma-S]) at pCa 4.5 for 10 min, on addition of ATP these cells contracted at of 0.25+/-0.04 cell lengths s-1 independently of pCa. If thiophosphorylated cells were preincubated with H1 (domains 3 and 4 of caldesmon), shortening speed was reduced (ID50=2 microM). Shortening speed was also reduced by 658C (domain 4b) at higher concentrations (ID50=400 microM). H13 (domain 4a), which does not block weak binding but inhibits actin-tropomyosin, inhibited cell shortening (ID50=6 microM). H2, which blocks weak binding but does not inhibit actin-tropomyosin, did not inhibit shortening. Western blots of the cells showed that the peptides were tightly bound within the cell but the native caldesmon was not displaced. These results indicate that exogenous caldesmon peptides added to smooth muscle cells may be incorporated into the thin filaments and produce effects on shortening, as expected if it were involved in tropomyosin-dependent inhibition of the actin filament in the cell.

Animals↗

PubMed1999

Functional analysis of human cardiac troponin by the in vitro motility assay: comparison of adult, foetal and failing hearts.

OBJECTIVE: Human cardiac development and heart failure are associated with altered troponin isoform expression and phosphorylation. As the functional effects of these changes in troponin are unknown, we isolated troponin from human foetal, normal adult and failing adult hearts and investigated their regulatory function. METHODS: Human cardiac troponin was assayed for regulatory function by in vitro motility assay and for protein content by SDS PAGE and immunoblotting. RESULTS: Human cardiac troponin regulated movement of actin-tropomyosin filaments over a bed of immobilised heavy meromyosin. At pCa 9, troponin from foetal and adult hearts reduced the fraction of filaments moving from 90% to less than 15% with a modest (25-30%) decrease in velocity. At pCa 5, troponin from normal adult hearts increased filament velocity by up to 47 +/- 3% with no change in the fraction of filaments moving. Foetal troponin increased velocity by only 4 +/- 6% and the effect of troponin from failing hearts was between these values at 31 +/- 5%. Foetal hearts showed different troponin I and T isoform expression compared with adult hearts. No differences in troponin isoform expression were demonstrated between normal and failing adult hearts. CONCLUSIONS: Functioning troponin and tropomyosin may be isolated from human heart and their properties investigated by in vitro motility assay. Both functional and isoform expression differences exist between foetal and adult cardiac troponin. The regulatory function of troponin from adults with end stage heart failure is different from normal adult troponin. These data suggest a role for altered troponin function in human cardiac development and heart failure.

Actins↗

PubMed1998

Characterization of the functional properties of smooth muscle caldesmon domain 4a: evidence for an independent inhibitory actin-tropomyosin binding domain.

Recent analysis has shown the presence of three sequences in the C-terminal 170 amino acids of human caldesmon (domain 4) which are involved in actin binding and tropomyosin-dependent inhibition of actomyosin ATPase. Two are in domain 4b (amino acids 715-793) and one is in domain 4a (amino acids 636-714). In the present work we have compared recombinant peptides containing either domain 4a or domain 4b to address the question as to whether domain 4a alone has any inhibitory activity. We have produced three new recombinant fragments containing domain 4a: H10 [622-708], H12 [506-708] and H13 [622-726] and we have characterized their functional properties. All three fragments bound to actin and tropomyosin. Caldesmon, but not domain 4b, was able to displace the fragments H10, H12 and H13 from actin. Thus the isolated caldesmon domain 4a peptides bind to the same region on actin as in the whole molecule while domains 4a and 4b occupy different sites on the actin molecule. Unlike domain 4b, none of the domain 4a fragments inhibited the actomyosin ATPase in the absence of tropomyosin. However both domain 4a and 4b fragments displayed an inhibitory activity in the presence of tropomyosin. H13 and H12 were more potent inhibitors than H10. Ca2+-calmodulin bound to H13 and reversed the inhibitory activity of this fragment but did not bind to H10 and H12. We conclude that domain 4a can act as an independent inhibitory actin-tropomyosin binding domain, but its properties are very different from the extreme C-terminal domain 4b.

Actins↗

PubMed1998

Structure-activity studies of the regulatory interaction of the 10 kilodalton C-terminal fragment of caldesmon with actin and the effect of mutation of caldesmon residues 691-696.

We have used isotope-edited nuclear magnetic resonance spectroscopy, binding studies, and ATPase activity assays to investigate the interaction with F-actin of the 10 kDa C-terminal 658C fragment of chicken gizzard caldesmon and two site-directed mutants of this fragment. Simultaneous dual-sited contacts with F-actin are observed for the segments of the 658C sequence flanking tryptophan residues 692 and 722. Competition experiments showed that both 658C contacts with actin are displaced by substoichiometric concentrations of the short inhibitory region of troponin-I indicative of different binding sites on actin for these regions of troponin-I and caldesmon. Substitution of caldesmon serine-702 by aspartic acid within the spacer region linking the two actin contacts of 658C led to weaker binding but with retention of equivalent affinity for each interaction site. Differential binding affinity of the two sites was achieved by replacement of the sequence Glu691-Trp-Leu-Thr-Lys-Thr696 by Pro-Gly-His-Tyr-Asn-Asn. Consistent with these data, the concentration of this Cg1 mutant required to achieve 50% inhibition of actin-tropomyosin-activated myosin ATPase was 4-fold greater than found for the 658C fragment. Although calmodulin binding to Cg1 was observed, calmodulin proved ineffective in relieving the inhibition induced by the binding of this mutant to actin. These results are discussed in light of the actin contacts which are involved in the inhibitory activity possessed by different regions of the C-terminus of caldesmon.

Actins↗

PubMed1998

Characterisation of the effects of mutation of the caldesmon sequence 691glu-trp-leu-thr-lys-thr696 to pro-gly-his-tyr-asn-asn on caldesmon-calmodulin interaction.

We have investigated the functional properties of a mutant (Cg1) derived from the C-terminal 99 amino acids of chicken caldesmon, 658-756 (658C) where the sequence 691glu-trp-leu-thr-lys-thr696 is changed to pro-gly-his-tyr-asn-asn. Cg1 bound Ca2+-calmodulin with (1/7)th of the affinity as compared to 658C or whole caldesmon. NMR titrations indicate that the contacts of Ca2+-calmodulin with the Trp-722 region of the peptide are retained but that those at the mutated site are lost. Most importantly Ca2+-calmodulin is not able to reverse the Cg1-induced inhibition. We conclude that the interaction of calmodulin with this caldesmon sequence is crucial for the reversal of caldesmon inhibition of actin-tropomyosin activation of myosin ATPase. The results are interpreted in terms of multisite attachment of actin and Ca2+-calmodulin to overlapping sequences in caldesmon domain 4b.

Animals↗