S. B. Marston

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.

Three-dimensional image reconstruction of reconstituted smooth muscle thin filaments: effects of caldesmon.

Caldesmon inhibits actomyosin ATPase and filament sliding in vitro, and therefore may play a role in modulating smooth and non-muscle motile activities. A bacterially expressed caldesmon fragment, 606C, which consists of the C-terminal 150 amino acids of the intact molecule, possesses the same inhibitory properties as full-length caldesmon and was used in our structural studies to examine caldesmon function. Three-dimensional image reconstruction was carried out from electron micrographs of negatively stained, reconstituted thin filaments consisting of actin and smooth muscle tropomyosin both with and without added 606C. Helically arranged actin monomers and tropomyosin strands were observed in both cases. In the absence of 606C, tropomyosin adopted a position on the inner edge of the outer domain of actin monomers, with an apparent connection to sub-domain 1 of actin. In 606C-containing filaments that inhibited acto-HMM ATPase activity, tropomyosin was found in a different position, in association with the inner domain of actin, away from the majority of strong myosin binding sites. The effect of caldesmon on tropomyosin position therefore differs from that of troponin on skeletal muscle filaments, implying that caldesmon and troponin act by different structural mechanisms.

What is latch? New ideas about tonic contraction in smooth muscle

Smooth muscles have traditionally been classified as phasic or tonic, the tonic muscles being those which maintain a steady tension indefinitely with a low consumption of energy. Until ten years ago it was considered that the differences between smooth muscle types reflected different innervation or excitation-contraction coupling. However, recent work makes it clear that the contractile apparatus itself is adapted in tonic muscles.

Nemaline myopathy with stiffness and hypertonia associated with an ACTA1 mutation

Nemaline myopathy, known to be caused by mutations in 7 genes, including skeletal muscle α-actin ( ACTA1 ),1 is characterized by muscle weakness, hypotonia, and nemaline rods in muscle biopsy. Here we report a patient with nemaline rods but the opposite phenotype of hypercontractility. ### Case report. The John Radcliffe Hospital ethics review board approved the study. The first child of nonconsanguineous European parents presented at 6 weeks of age with an apneic episode thought due to bilateral strangulated inguinal hernias and an umbilical hernia. However, after herniotomy, rigidity and recurrent apneas requiring mechanical ventilation continued. The patient was born by elective Caesarean section at 39 weeks, with no perinatal complications, although the mother, with hindsight, thought there might have been stiffness in the weeks after birth as she had difficulty clothing him. Polyhydramnios had been noted. On examination, he had very stiff and hypertrophic abdominal and proximal limb muscles and elbow and knee contractures. Deep tendon reflexes showed brisk contraction and slow relaxation and percussion myotonia could be elicited. However, EMG in multiple muscles showed no evidence of myotonia. He also had normal nerve conduction studies, MRI, EEG, and cardiac assessment, including echocardiography. Neurometabolic investigations were also normal including serum and CSF lactate, CSF neurotransmitters, urinary catecholamines, carnitine profile, white cell enzymes, and screening for mucopolysaccharidosis. Serum creatine kinase was slightly raised (370 IU, range 24–195). A sleep study showed hypoventilation and carbon dioxide retention. Hyperekplexia, paroxysmal episodic pain disorder, neuromyotonia, and episodic ataxia were considered, though none exactly matched the phenotype because of the underlying stiffness, but relevant testing ( ARX , FRAXA , GLRA1 , KCN1A , POLG , SCN9A , anti GAD, and VGKC antibody studies) was negative. Pharmacologic treatment including phenytoin, carbamazepine, flecainide, baclofen, dantrolene, and acetazolamide was ineffective. He continued having recurrent episodes of stiffness, where he stopped …

K7del is a common TPM2 gene mutation associated with nemaline myopathy and raised myofibre calcium sensitivity

Mutations in the TPM2 gene, which encodes β-tropomyosin, are an established cause of several congenital skeletal myopathies and distal arthrogryposis. We have identified a TPM2 mutation, p.K7del, in five unrelated families with nemaline myopathy and a consistent distinctive clinical phenotype. Patients develop large joint contractures during childhood, followed by slowly progressive skeletal muscle weakness during adulthood. The TPM2 p.K7del mutation results in the loss of a highly conserved lysine residue near the N-terminus of β-tropomyosin, which is predicted to disrupt head-to-tail polymerization of tropomyosin. Recombinant K7del-β-tropomyosin incorporates poorly into sarcomeres in C2C12 myotubes and has a reduced affinity for actin. Two-dimensional gel electrophoresis of patient muscle and primary patient cultured myotubes showed that mutant protein is expressed but incorporates poorly into sarcomeres and likely accumulates in nemaline rods. In vitro studies using recombinant K7del-β-tropomyosin and force measurements from single dissected patient myofibres showed increased myofilament calcium sensitivity. Together these data indicate that p.K7del is a common recurrent TPM2 mutation associated with mild nemaline myopathy. The p.K7del mutation likely disrupts head-to-tail polymerization of tropomyosin, which impairs incorporation into sarcomeres and also affects the equilibrium of the troponin/tropomyosin-dependent calcium switch of muscle. Joint contractures may stem from chronic muscle hypercontraction due to increased myofibrillar calcium sensitivity while declining strength in adulthood likely arises from other mechanisms, such as myofibre decompensation and fatty infiltration. These results suggest that patients may benefit from therapies that reduce skeletal muscle calcium sensitivity, and we highlight late muscle decompensation as an important cause of morbidity.

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.

Phosphorylation of aorta caldesmon by endogeneous proteolytic fragments of protein kinase C

SummaryEndogenous caldesmon kinase activity in sheep aorta smooth muscle was purified and characterized. The enzyme was identified as a proteolytic fragment of protein kinase C by cross-reactivity with anti-protein kinase C antibodies, autophosphorylation, substrate specificity and the primary structure of the sites of phosphorylation on caldesmon. The enzyme phosphorylated aorta caldesmon both in native thin filaments and in the isolated state. Up to 2.9 mols of phosphate per mol of caldesmon were transferred. Prolonged incubation of caldesmon with the kinase resulted in phosphorylation of Ser-127, Ser-587, Ser-600, Ser-657, Ser-686, and Ser-726 (numbering corresponds to chicken gizzard caldesmon sequence). Ser-600 and Ser-587 were the major sites of phosphorylation containing more than 30% of phosphate transferred. Phosphorylation did not significantly affect the interaction of caldesmon with Ca2+-calmodulin. However, phosphorylation of both intact caldesmon and of its C-terminal fragment (658C), containing residues 658–756, significantly decreased their ability to inhibit acto-heavy meromyosin ATPase. This seems to be partially due to a decrease in the binding of caldesmon and 658C to actin-tropomyosin and partly due to an uncoupling of the binding-inhibition relationship.

Structure and properties of avian small heat shock protein with molecular weight 25 kDa

The primary structure of chicken small heat shock protein (sHsp) with apparent molecular weight 25 kDa was refined and it was shown that this protein has conservative primary structure 74RALSRQLSSG(83) at Ser77 and Ser81, which are potential sites of phosphorylation. Recombinant wild-type chicken Hsp25, its three mutants, 1D (S15D), 2D (S77D+S81D) and 3D (S15D+S77D+S81D), as well as delR mutant with the primary structure 74RALS-ELSSG(82) at potential sites of phosphorylation were expressed and purified. It has been shown that the avian tissues contain three forms of Hsp25 having pI values similar to that of the wild-type protein, 1D and 2D mutants that presumably correspond to nonphosphorylated, mono- and di-phosphorylated forms of Hsp25. Recombinant wild-type protein, its 1D mutant and Hsp25, isolated from chicken gizzard, form stable high molecular weight oligomeric complexes. The delR, 2D and 3D mutants tend to dissociate and exist in the form of a mixture of high and low molecular weight oligomers. Point mutations mimicking phoshorylation decrease chaperone activity of Hsp25 measured by reduction of dithiothreitol induced aggregation of alpha-lactalbumin, but increase the chaperone activity of Hsp25 measured by heat induced aggregation of alcohol dehydrogenase. It is concluded that avian Hsp25 has a more stable quaternary structure than its mammalian counterparts and mutations mimicking phosphorylation differently affect chaperone activity of avian Hsp25, depending on the nature of target protein and the way of denaturing.

Role of Tropomyosin in the Regulation of Contraction in Smooth Muscle

Smooth muscle contraction is due to the interaction ofmyosin filaments with thin filaments. Thin filaments are composed of actin, tropomyosin, caldesmon and calmodulin in ratios 14:2:1:1. Tissue specific isoforms of act and beta tropomyosin are expressed in smooth muscle. Compared with skeletal muscle tropomyosin, the cooperative activation of actomyosin is enhanced by smooth muscle tropomyosin: cooperative unit size is 10 and the equilibrium between on and off states is shifted towards the on state. The smooth muscle-specific actin-bindingprotein caldesmon, together with calmodulin regulates the activity of the thin filament in response to Ca2+. Caldesmon and calmodulin control the tropomyosin-mediated transition between on and offactivity states.

Identification of functioning regulatory sites and a new myosin binding site in the C-terminal 288 amino acids of caldesmon expressed from a human clone

SummaryA partial clone of caldesmon, coding for the C-terminal 288 amino acids, was isolated from a human fetal liver cDNA library and sequenced. Expression of the clone in Escherichia coli produced a peptide called H1 (Mr 32 549), which inhibited tropomyosin-enhanced actomyosin Mg2+-ATPase activity by 90% with half maximal inhibition at 0.03–0.04 mol H1 per mol actin. The inhibition could be reversed by Ca2+-calmodulin. H1 bound actin, Ca2+-calmodulin and tropomyosin and smooth muscle myosin with high affinities. This latter finding shows the presence of a second myosin-binding site in caldesmon. This was confirmed in thrombic digests of native sheep aorta and chicken gizzard caldesmon.