Paul C. Leavis

Thin filament proteins and thin filament-linked regulation of vertebrate muscle contraction.

Recent developments in the field of myofibrillar proteins will be reviewed. Consideration will be given to the proteins that participate in the contractile process itself as well as to those involved in Ca-dependent regulation of striated (skeletal and cardiac) and smooth muscle. The relation of protein structure to function will be emphasized and the relation of various physiologically and histochemically defined fiber types to the proteins found in them will be discussed.

[10] Solute quenching of protein fluorescence

Publisher Summary This chapter considers various ways of presenting and interpreting data using model calculations based on the modified Stern–Volmer equation. It provides detail information about the experimental methods for the purpose of obtaining meaningful quenching data. A quencher molecule can deactivate an excited fluorophor at the instant of excitation by forming a nonfluorescent, or “dark,” complex in the ground state, or by being present within a certain “sphere of action” at the time of excitation. Before considering heterogeneous systems, certain effects that can cause deviations from the simple Stem–Volmer law are also described in the chapter. Solute quenching of protein fluorescence is a technique that can yield information regarding the exposure of protein-bound intrinsic or extrinsic fluorophors. It is a dynamic method, because the presence of solute alters the fluorescence lifetime and thereby the steady-state intensity, without altering the electronic energy levels that are involved.

Cytoskeletal remodeling in differentiated vascular smooth muscle is actin isoform dependent and stimulus dependent

Dynamic remodeling of the actin cytoskeleton plays an essential role in the migration and proliferation of vascular smooth muscle cells. It has been suggested that actin remodeling may also play an important functional role in nonmigrating, nonproliferating differentiated vascular smooth muscle (dVSM). In the present study, we show that contractile agonists increase the net polymerization of actin in dVSM, as measured by the differential ultracentrifugation of vascular smooth muscle tissue and the costaining of single freshly dissociated cells with fluorescent probes specific for globular and filamentous actin. Furthermore, induced alterations of the actin polymerization state, as well as actin decoy peptides, inhibit contractility in a stimulus-dependent manner. Latrunculin pretreatment or actin decoy peptides significantly inhibit contractility induced by a phorbol ester or an alpha-agonist, but these procedures have no effect on contractions induced by KCl. Aorta dVSM expresses alpha-smooth muscle actin, beta-actin, nonmuscle gamma-actin, and smooth muscle gamma-actin. The incorporation of isoform-specific cell-permeant synthetic actin decoy peptides, as well as isoform-specific probing of cell fractions and two-dimensional gels, demonstrates that actin remodeling during alpha-agonist contractions involves the remodeling of primarily gamma-actin and, to a lesser extent, beta-actin. Taken together, these results show that net isoform- and agonist-dependent increases in actin polymerization regulate vascular contractility.

A peptide derived from the human leptin molecule is a potent inhibitor of the leptin receptor function in rabbit endometrial cells

In this article we show that rabbit endometrial cells express leptin receptor and that human leptin triggers phosphorylation of signal transducer and activator of transcription 3 and up-regulates the expression of interleukin-1 receptor type I as was previously found in human endometrial cells. Interestingly, leptin also upregulates the secretion of leukemia inhibitory factor and expression of its receptor by rabbit endometrial cells. Analysis of a structural model of the leptin-leptin receptor complex suggested that helices I and III of the human leptin structure were likely sites of interaction with the cytokine binding domain of leptin receptor. Accordingly, we synthesized a peptide (LPA-2) comprising helix III (residues 70–95) and investigated its ability to inhibit leptin receptor function. The effects of LPA-2 were assayed in rabbit endometrial cells, and an antileptin receptor antibody and a scrambled version of LPA-2 were used as positive and negative controls, respectively. LPA-2 binds specifically and with high affinity (Ki ∼ 0.6×10−10M) to leptin receptor and is a potent inhibitor of its functions in rabbit endometrial cells. Because leukemia inhibitory factor and interleukin-1 have been implicated in embryo implantation, our results raise the possibility that the LPA-2-induced inhibition of leptin receptor may be exploited to study the actions of leptin in endometrium and in other tissues under conditions characterized by abnormal leptin production.

Calcium binding to cardiac troponin C

Abstract The binding of Ca2+ to cardiac troponin C was studied by determining changes in the fluorescence and circular dichroism of the protein and by following changes in the free Ca2+ concentration by means of a Ca2+-specific electrode. Cardiac troponin C contains three Ca2+-binding sites which fall into two classes —two sites with a higher affinity and one with a lower affinity. The higher-affinity sites also bind Mg2+ which competes with the Ca2+.

The nature of the trifluoperazine binding sites on calmodulin and troponin-C.

We have employed 1H-nuclear magnetic resonance spectroscopy to study the interaction of the drug trifluoperazine with calmodulin and troponin-C. Distinct trifluoperazine-binding sites exist in the N- and C-terminal halves of both proteins. Each site consists of a group of hydrophobic side-chains brought into proximity by the Ca2+-dependent juxtaposition of two alpha-helical segments of the protein, each, in turn, belonging to a different Ca2+-binding site in the protein half. The trifluoperazine-induced inhibition of the biological activating ability of calmodulin appears to result from conformational restrictions conferred upon the protein by the bound drug.

Leptin upregulates β3-integrin expression and interleukin-1β upregulates leptin and leptin receptor expression in human endometrial epithelial cell cultures

Human endometrium and endometrial epithelial cells (EECs) either cultured alone or cocultured with human embryos express leptin and leptin receptor. This study compares the effect of leptin with that of interleukin-1β (IL-1β) on the expression of β3-EEC integrin, a marker of endometrial receptivity. Both cytokines increased the expression of β3-EEC at concentrations in the range of 0.06–3 nM; however, leptin exhibited a significantly greater effect than IL-1β. We also determined the regulatory effects of IL-1β on leptin secretion and on the expression of leptin and leptin receptor at the protein level in both EEC and endometrial stromal cell (ESC) cultures. In EEC cultures, IL-1β upregulated secretion of leptin and expression of both leptin and leptin receptors. No effect of IL-1β was found in the ESC cultures. However, leptin exhibited marginal upregulation of leptin receptor. The upregulation of β3-integrin and leptin/leptin receptor expression by IL-1β in EEC cultures indicates that both cytokines may be implicated in embryonic-maternal cross-talk during the early phase of human implantation. Our present data also raise the possibility that leptin is an endometrial molecular effector of IL-1β action on β3-integrin upregulation. Thus, a new role for leptin in human reproduction as an autocrine/paracrine regulator of endometrial receptivity is proposed.

Structure of a longitudinal actin dimer assembled by tandem w domains: implications for actin filament nucleation.

Actin filament nucleators initiate polymerization in cells in a regulated manner. A common architecture among these molecules consists of tandem WASP homology 2 domains (W domains) that recruit three to four actin subunits to form a polymerization nucleus. We describe a low-resolution crystal structure of an actin dimer assembled by tandem W domains, where the first W domain is cross-linked to Cys374 of the actin subunit bound to it, whereas the last W domain is followed by the C-terminal pointed end-capping helix of thymosin β4. While the arrangement of actin subunits in the dimer resembles that of a long-pitch helix of the actin filament, important differences are observed. These differences result from steric hindrance of the W domain with intersubunit contacts in the actin filament. We also determined the structure of the first W domain of Vibrio parahaemolyticus VopL cross-linked to actin Cys374 and show it to be nearly identical with non-cross-linked W-Actin structures. This result validates the use of cross-linking as a tool for the study of actin nucleation complexes, whose natural tendency to polymerize interferes with most structural methods. Combined with a biochemical analysis of nucleation, the structures may explain why nucleators based on tandem W domains with short inter-W linkers have relatively weak activity, cannot stay bound to filaments after nucleation, and are unlikely to influence filament elongation. The findings may also explain why nucleation-promoting factors of the Arp2/3 complex, which are related to tandem-W-domain nucleators, are ejected from branch junctions after nucleation. We finally show that the simple addition of the C-terminal pointed end-capping helix of thymosin β4 to tandem W domains can change their activity from actin filament nucleation to monomer sequestration.

Proton magnetic resonance studies on proteolytic fragments of troponin-C. Structural homology with the native molecule

Comparison of proton magnetic resonance spectra of a tryptic and a thrombin fragment of troponin-C with that of the native protein has identified the domain of the molecule influenced by Ca2+ binding to the lower affinity regions I and II of troponin-C. The binding of Ca2+ to these sites results in a subtle alteration of the tertiary fold of the N-terminal half of troponin-C involving weakened contacts between several hydrophobic groups. The role and kinetics of the movements within the troponin-C molecule associated with binding at the regulatory sites are discussed.

Terbium binding to troponin C: binding stoichiometry and structural changes induced in the protein.

Abstract Terbium (Tb3+) binding to skeletal muscle troponin C was studied by fluorescence spectroscopy and circular dichroism. Titrations indicate that Tb3+, like Ca2+, preferentially binds to the two high affinity Ca2+-Mg2+ sites (III and IV) inducing structural changes similar to those induced by Ca2+. Tb3+ readily displaces Ca2+ from these sites suggesting a K(Tb3+) ≥ 109 M−1 In 6 M urea, both Ca2+ and Tb3+ bind preferentially to a single site on troponin C. The spectral changes suggest this to be site III.