Philip Graceffa

The role of free radicals in asbestos-induced diseases

Asbestos exposure causes pulmonary fibrosis and malignant neoplasms by mechanisms that remain uncertain. In this review, we explore the evidence supporting the hypothesis that free radicals and other reactive oxygen species (ROS) are an important mechanism by which asbestos mediates tissue damage. There appears to be at least two principal mechanisms by which asbestos can induce ROS production; one operates in cell-free systems and the other involves mediation by phagocytic cells. Asbestos and other synthetic mineral fibers can generate free radicals in cell-free systems containing atmospheric oxygen. In particular, the hydroxyl radical often appears to be involved, and the iron content of the fibers has an important role in the generation of this reactive radical. However, asbestos also appears to catalyze electron transfer reactions that do not require iron. Iron chelators either inhibit or augment asbestos-catalyzed generation of the hydroxyl radical and/or pathological changes, depending on the chelator and the nature of the asbestos sample used. The second principal mechanism for asbestos-induced ROS generation involves the activation of phagocytic cells. A variety of mineral fibers have been shown to augment the release of reactive oxygen intermediates from phagocytic cells such as neutrophils and alveolar macrophages. The molecular mechanisms involved are unclear but may involve incomplete phagocytosis with subsequent oxidant release, stimulation of the phospholipase C pathway, and/or IgG-fragment receptor activation. Reactive oxygen species are important mediators of asbestos-induced toxicity to a number of pulmonary cells including alveolar macrophages, epithelial cells, mesothelial cells, and endothelial cells. Reactive oxygen species may contribute to the well-known synergistic effects of asbestos and cigarette smoke on the lung, and the reasons for this synergy are discussed. We conclude that there is strong evidence supporting the premise that reactive oxygen species and/or free radicals contribute to asbestos-induced and cigarette smoke/asbestos-induced lung injury and that strategies aimed at reducing the oxidant stress on pulmonary cells may attenuate the deleterious effects of asbestos.

Crystal Structure of Monomeric Actin in the ATP State STRUCTURAL BASIS OF NUCLEOTIDE-DEPENDENT ACTIN DYNAMICS

A nucleotide-dependent conformational change regulates actin filament dynamics. Yet, the structural basis of this mechanism remains controversial. The x-ray crystal structure of tetramethylrhodamine-5-maleimide-actin with bound AMPPNP, a non-hydrolyzable ATP analog, was determined to 1.85-Å resolution. A comparison of this structure to that of tetramethylrhodamine-5-maleimide-actin with bound ADP, determined previously under similar conditions, reveals how the release of the nucleotide γ-phosphate sets in motion a sequence of events leading to a conformational change in subdomain 2. The side chain of Ser-14 in the catalytic site rotates upon Pi release, triggering the rearrangement of the loop containing the methylated His-73, referred to as the sensor loop. This in turn causes a transition in the DNase I-binding loop in subdomain 2 from a disordered loop in ATP-actin to an ordered α-helix in ADP-actin. Despite this conformational change, the nucleotide cleft remains closed in ADP-actin, similar to ATP-actin. An analysis of the existing structures of members of the actin superfamily suggests that the cleft is open in the nucleotide-free state.

Crystal structures of the vitamin D-binding protein and its complex with actin: Structural basis of the actin-scavenger system

Actin is the most abundant protein in eukaryotic cells, but its release from cells into blood vessels can be lethal, being associated with clinical situations including hepatic necrosis and septic shock. A homeostatic mechanism, termed the actin-scavenger system, is responsible for the depolymerization and removal of actin from the circulation. During the first phase of this mechanism, gelsolin severs the actin filaments. In the second phase, the vitamin D-binding protein (DBP) traps the actin monomers, which accelerates their clearance. We have determined the crystal structures of DBP by itself and complexed with actin to 2.1 Å resolution. Similar to its homologue serum albumin, DBP consists of three related domains. Yet, in DBP a strikingly different organization of the domains gives rise to a large actin-binding cavity. After complex formation the three domains of DBP move slightly to “clamp” onto actin subdomain 3 and to a lesser extent subdomain 1. Contacts between actin and DBP throughout their extensive 3,454-Å2 intermolecular interface involve a mixture of hydrophobic, electrostatic, and solvent-mediated interactions. The area of actin covered by DBP within the complex approximately equals the sum of those covered by gelsolin and profilin. Moreover, certain interactions of DBP with actin mirror those observed in the actin-gelsolin complex, which may explain how DBP can compete effectively with gelsolin for actin binding. Formation of the strong actin–DBP complex proceeds with limited conformational changes to both proteins, demonstrating how DBP has evolved to become an effective actin-scavenger protein.

Phosphorylation Dependence of Hsp27 Multimeric Size and Molecular Chaperone Function

The molecular chaperone Hsp27 exists as a distribution of large oligomers that are disassembled by phosphorylation at Ser-15, -78, and -82. It is controversial whether the unphosphorylated Hsp27 or the widely used triple Ser-to-Asp phospho-mimic mutant is the more active molecular chaperone in vitro. This question was investigated here by correlating chaperone activity, as measured by the aggregation of reduced insulin or α-lactalbumin, with Hsp27 self-association as monitored by analytical ultracentrifugation. Furthermore, because the phospho-mimic is generally assumed to reproduce the phosphorylated molecule, the size and chaperone activity of phosphorylated Hsp27 were compared with that of the phospho-mimic. Hsp27 was triply phosphorylated by MAPKAP-2 kinase, and phosphorylation was tracked by urea-PAGE. An increasing degree of suppression of insulin or α-lactalbumin aggregation correlated with a decreasing Hsp27 self-association, which was the least for phosphorylated Hsp27 followed by the mimic followed by the unphosphorylated protein. It was also found that Hsp27 added to pre-aggregated insulin did not reverse aggregation but did inhibit these aggregates from assembling into even larger aggregates. This chaperone activity appears to be independent of Hsp27 phosphorylation. In conclusion, the most active chaperone of insulin and α-lactalbumin was the Hsp27 (elongated) dimer, the smallest Hsp27 subunit observed under physiological conditions. Next, the Hsp27 phospho-mimic is only a partial mimic of phosphorylated Hsp27, both in self-association and in chaperone function. Finally, the efficient inhibition of insulin aggregation by Hsp27 dimer led to the proposal of two models for this chaperone activity.

Mammal-specific, ERK-dependent, Caldesmon Phosphorylation in Smooth Muscle QUANTITATION USING NOVEL ANTI-PHOSPHOPEPTIDE ANTIBODIES

Extracellular signal-regulated kinases (ERKs) phosphorylate the high molecular mass isoform of the actin-binding protein caldesmon (h-CaD) at two sites (Ser759 and Ser789) during smooth muscle stimulation. To investigate the role of phosphorylation at these sites, antibodies were generated against phosphopeptides analogous to the sequences around Ser759 and Ser789. Affinity-purified antibodies were phosho- and sequence-specific. The major site of phosphorylation in h-CaD in porcine carotid arterial muscle strips was at Ser789; however, the amount of phosphate did not vary appreciably with either KCl or phorbol ester stimulation. Phosphorylation at Ser759 of h-CaD was almost undetectable (<0.005 mol of phosphate/mol of protein). Moreover, phosphorylation of the low molecular mass isoform of the protein (l-CaD) at the site analogous to Ser789 was greater in serum-stimulated cultured smooth muscle cells than in serum-starved cells. Serum-stimulated l-CaD phosphorylation was attenuated by the protein kinase inhibitor PD98059. These data 1) identify Ser789 of h-CaD as the major site of ERK-dependent phosphorylation in carotid arteries; 2) show that the level of phosphorylation at Ser789 is relatively constant following carotid arterial muscle stimulation, despite an increase in total protein phosphate content; and 3) suggest a functional role for ERK-dependent l-CaD phosphorylation in cell division.

Phytic Acid, an Iron Chelator, Attenuates Pulmonary Inflammation and Fibrosis in Rats after Intratracheal Instillation of Asbestos

Reactive oxygen species, especially iron-catalyzed hydroxyl radicals (·OH), are implicated in the pathogenesis of asbestos-induced pulmonary toxicity. We previously demonstrated that phytic acid, an iron chelator, reduces amosite asbestos-induced OH generation, DNA strand break formation, and injury to cultured pulmonary epithelial cells (268 [1995, Am. J. Physiol. (Lung Cell. Mol. Physiol.) 12: L471-480]). To determine whether phytic acid diminishes pulmonary inflammation and fibrosis in rats after a single intratracheal (it) instillation of amosite asbestos, Sprague-Dawley rats were given either saline (1 ml), amosite asbestos (5 mg; 1 ml saline), or amosite treated with phytic acid (500 μM) for 24 hr and then instilled. At various times after asbestos exposure, the rats were euthanized and the lungs were lavaged and examined histologically. A fibrosis score was determined from trichrome-stained specimens. As compared to controls, asbestos elicited a significant pulmonary inflammatory response, as evidenced by an increase (∼ 2-fold) in bronchoalveolar lavage (BAL) cell counts at 1 wk and the percentage of BAL neutrophils (PMNs) and giant cells at 2 wk (0.1 vs 6.5% and 1.3 vs 6.1%, respectively; p < 0.05). Asbestos significantly increased the fibrosis score at 2 wk (0 ± 0 vs 5 ± 1; p < 0.05). The inflammatory and fibrotic changes were, as expected, observed in the respiratory bronchioles and terminal alveolar duct bifurcations. The increased percentage of BAL PMNs and giant cells persisted at 4 wk, as did the fibrotic changes. Compared to asbestos alone, phytic acid-treated asbestos elicited significantly less BAL PMNs (6.5 vs 1.0%; p < 0.05) and giant cells (6.1 vs 0.2%; p < 0.05) and caused significantly less fibrosis (5 vs 0.8; p < 0.05) 2 wk after exposure. We conclude that asbestos causes pulmonary inflammation and fibrosis in rats after it instillation and that phytic acid reduces these effects. These data support the role of iron-catalyzed free radicals in causing pulmonary toxicity from asbestos in vivo.

Evidence for interaction between smooth muscle tropomyosin and caldesmon

The viscosity of chicken gizzard smooth muscle tropomyosin is enhanced 4.7‐fold in the absence of salt and 1.43‐fold in 0.1 M salt by the presence of stoichiometric amounts of gizzard caldesmon, indicating that the two proteins interact under these conditions. Since the thin filament regulation of smooth muscle contraction by caldesmon requires the presence of tropomyosin, these results suggest that the direct interaction between tropomyosin and caldesmon on the thin filament plays a role in this regulation.

Modes of Caldesmon Binding to Actin SITES OF CALDESMON CONTACT AND MODULATION OF INTERACTIONS BY PHOSPHORYLATION

Smooth muscle caldesmon binds actin and inhibits actomyosin ATPase activity. Phosphorylation of caldesmon by extracellular signal-regulated kinase (ERK) reverses this inhibitory effect and weakens actin binding. To better understand this function, we have examined the phosphorylation-dependent contact sites of caldesmon on actin by low dose electron microscopy and three-dimensional reconstruction of actin filaments decorated with a C-terminal fragment, hH32K, of human caldesmon containing the principal actin-binding domains. Helical reconstruction of negatively stained filaments demonstrated that hH32K is located on the inner portion of actin subdomain 1, traversing its upper surface toward the C-terminal segment of actin, and forms a bridge to the neighboring actin monomer of the adjacent long pitch helical strand by connecting to its subdomain 3. Such lateral binding was supported by cross-linking experiments using a mutant isoform, which was capable of cross-linking actin subunits. Upon ERK phosphorylation, however, the mutant no longer cross-linked actin to polymers. Three-dimensional reconstruction of ERK-phosphorylated hH32K indeed indicated loss of the interstrand connectivity. These results, together with fluorescence quenching data, are consistent with a phosphorylation-dependent conformational change that moves the C-terminal end segment of caldesmon near the phosphorylation site but not the upstream region around Cys595, away from F-actin, thus neutralizing its inhibitory effect on actomyosin interactions. The binding pattern of hH32K suggests a mechanism by which unphosphorylated, but not ERK-phosphorylated, caldesmon could stabilize actin filaments and resist F-actin severing or depolymerization in both smooth muscle and nonmuscle cells.

Spin labeling of protein sulfhydryl groups by spin trapping a sulfur radical: Application to bovine serum albumin and myosin☆

Reaction of sulfhydryl-containing compounds, RSH, with Ce4+ in the presence of the spin trap phenyl-N-t-butylnitrone results in the appearance of a nitroxide ESR spectrum, which is greatly diminished if the sulfhydryl group is blocked prior to reaction. The spectra have short lifetimes which can be increased two- to fivefold to half-lives of 5-60 min by prior flushing of the solutions with nitrogen. For small molecules, such as cysteine, N-acetylcysteine, glutathione, and 2-mercaptoethanol, the spectrum is that of a freely rotating nitroxide while for the proteins, bovine serum albumin and myosin, the spectrum is characteristic of a strongly immobilized nitroxide spin label rigidly attached to the protein. Since Ce4+ is reported to oxidize the sulfhydryl group via the thiyl radical, RS, the following reactions are proposed to account for the formation of the nitroxide: (formula; see text) These reactions permit the spin labeling of sulfhydryl proteins such that the nitroxide is much closer to the point of attachment than when using conventional spin-labeling methods.

Actin polymerization in differentiated vascular smooth muscle cells requires vasodilator-stimulated phosphoprotein

Our group has previously shown that vasoconstrictors increase net actin polymerization in differentiated vascular smooth muscle cells (dVSMC) and that increased actin polymerization is linked to contractility of vascular tissue (Kim et al., Am J Physiol Cell Physiol 295: C768-778, 2008). However, the underlying mechanisms are largely unknown. Here, we evaluated the possible functions of the Ena/vasodilator-stimulated phosphoprotein (VASP) family of actin filament elongation factors in dVSMC. Inhibition of actin filament elongation by cytochalasin D decreases contractility without changing myosin light-chain phosphorylation levels, suggesting that actin filament elongation is necessary for dVSM contraction. VASP is the only Ena/VASP protein highly expressed in aorta tissues, and VASP knockdown decreased smooth muscle contractility. VASP partially colocalizes with alpha-actinin and vinculin in dVSMC. Profilin, known to associate with G actin and VASP, also colocalizes with alpha-actinin and vinculin, potentially identifying the dense bodies and the adhesion plaques as hot spots of actin polymerization. The EVH1 domain of Ena/VASP is known to target these proteins to their sites of action. Introduction of an expressed EVH1 domain as a dominant negative inhibits stimulus-induced increases in actin polymerization. VASP phosphorylation, known to inhibit actin polymerization, is decreased during phenylephrine stimulation in dVSMC. We also directly visualized, for the first time, rhodamine-labeled actin incorporation in dVSMC and identified hot spots of actin polymerization in the cell cortex that colocalize with VASP. These results indicate a role for VASP in actin filament assembly, specifically at the cell cortex, that modulates contractility in dVSMC.