Introduction MicroRNAs are small noncoding RNA molecules that negatively regulate gene expression via degradation or translational repression of their targeted mRNAs. It is known that aberrant microRNA expression can play important roles in cancer, but the role of microRNAs in autoimmune diseases is only beginning to emerge. In this study, the expression of selected microRNAs is examined in rheumatoid arthritis.

Jasplakinolide paradoxically stabilizes actin filaments in vitro, but in vivo it can disrupt actin filaments and induce polymerization of monomeric actin into amorphous masses. A detailed analysis of the effects of jasplakinolide on the kinetics of actin polymerization suggests a resolution to this paradox. Jasplakinolide markedly enhances the rate of actin filament nucleation. This increase corresponds to a change in the size of actin oligomer capable of nucleating filament growth from four to approximately three subunits, which is mechanistically consistent with the localization of the jasplakinolide-binding site at an interface of three actin subunits. Because jasplakinolide both decreases the amount of sequestered actin (by lowering the critical concentration of actin) and augments nucleation, the enhancement of polymerization by jasplakinolide is amplified in the presence of actin-monomer sequestering proteins such as thymosin ␤ 4 . Overall, the kinetic parameters in vitro define the mechanism by which jasplakinolide induces polymerization of monomeric actin in vivo. Expected consequences of jasplakinolide function are consistent with the experimental observations and include de novo nucleation resulting in disordered polymeric actin and in insufficient monomeric actin to allow for remodeling of stress fibers.Jasplakinolide is a cyclic peptide isolated from the marine sponge, Jaspis johnstoni, that we have previously shown to bind to and stabilize filamentous actin in vitro (1). In vivo data suggests that jasplakinolide-treated prostate cancer cells have both decreased labeling of F-actin and decreased amounts of rhodamine-phalloidin bound to cell extracts (2), results that could be explained by the observation that jasplakinolide and phalloidin bind competitively to actin (1). In addition, however, in vivo data also convincingly show that jasplakinolide disrupts actin filaments with alterations in cellular architecture (2, 3), an effect that cannot be explained simply by competitive binding. We now present kinetic data characterizing the steady state and time-dependent in vitro interactions between jasplakinolide and actin that provide a plausible explanation for the effects of jasplakinolide on actin distribution in cultured cells. EXPERIMENTAL PROCEDURESMaterials-Rabbit skeletal muscle actin was prepared from frozen muscle (Pel-Freez, Rogers, AR) in buffer G (5.0 mM Tris, 0.2 mM ATP, 0.2 mM dithiothreitol, 0.1 mM CaCl 2 , and 0.01% sodium azide, pH 7.8) (4). Non-muscle actin from bovine brain was prepared by the method of Ruscha and Himes (5). Muscle and non-muscle pyrenyl-labeled actins 1 were prepared with 0.67-0.95 mol of label/mol of protein using the method of Kouyama and Mihashi (6). Labeled and unlabeled actins were further purified by gel filtration on Superose 12 (Amersham Pharmacia Biotech). Thymosin ␤ 4 cDNA was a gift from Dr. Vivian Nachmias and was inserted in a pET-11a vector, expressed in BL21(DE3) Escherichia coli, and purified as described previously (7). Jasplakinolide was a gift from ...

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2 SUMMARYLatrunculin A is used extensively as an agent to sequester monomeric actin in living cells. We hypothesize that additional activities of latrunculin A may be important for its biological activity. Our data are consistent with the formation of a one to one stoichiometric complex with equilibrium dissociation constant of 0.2 to 0.4 µM, and provide no evidence that the actin-latrunculin A complex participates in the elongation of actin filaments. Profilin and latrunculin A bind independently to actin, whereas binding of thymosin β 4 to actin is inhibited by latrunculin A.Potential implications of this differential effect on actin-binding proteins are discussed. From a structural perspective, if latrunculin A binds to actin at a site that sterically influences binding by thymosin β 4 , then the observation that latrunculin A inhibits nucleotide exchange on actin implies an allosteric effect on the nucleotide binding cleft. Alternatively, if as previously postulated, latrunculin A binds in the nucleotide cleft of actin, then its ability to inhibit binding by thymosin β 4 is a surprising result that suggests that significant allosteric changes affect the thymosin β 4 binding site. We show that latrunculin A and actin form a crystalline structure with orthorhombic space group P2 1 2 1 2 1 and diffraction to 3.10 Å. A highresolution structure with optimized crystallization conditions should provide insight regarding these remarkable allosteric properties.

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Swinholide A, isolated from the marien sponge Theonella swinhoei, is a 44-carbon ring dimeric dilactone macrolide with a 2-fold axis of symmetry. Recent studies have elucidated its unusual structure and shown that it has potent cytotoxic activity. We now report that swinholide A disrupts the actin cytoskeleton of cells grown in culture, sequesters actin dimers in vitro in both polymerizing and non-polymerizing buffers with a binding stoichiometry of one swinholide A molecule per actin dimer, and rapidly severs F-actin in vitro with high cooperativity. These unique properties are sufficient to explain the cytotoxicity of swinholide A. They also suggest that swinholide A might be a model for studies of the mechanism of action of F-actin severing proteins and be therapeutically useful in conditions where filamentous actin contributes to pathologically high viscosities.

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An antiparallel actin dimer has been proposed to be an intermediate species during actin filament nucleation. We now show that latrunculin A, a marine natural product that inhibits actin polymerization, arrests polylysine-induced nucleation at the level of an antiparallel dimer, resulting in its accumulation. These dimers, when composed of pyrene-labeled actin subunits, give rise to a fluorescent excimer, permitting detection during polymerization in vitro. We report the crystallographic structure of the polylysine-actin-latrunculin A complex at 3.5-Å resolution. The non-crystallographic contact is consistent with a dimeric structure and confirms the antiparallel orientation of its subunits. The crystallographic contacts reveal that the mobile DNase I binding loop of one subunit of a symmetry-related antiparallel actin dimer is partially stabilized in the interface between the two subunits of a second antiparallel dimer. These results provide a potential explanation for the paradoxical nucleation of actin filaments that have exclusively parallel subunits by a dimer containing antiparallel subunits.Actin filament nucleation occurs very slowly de novo, but it occurs rapidly as a necessary step in actin-based motility (1). The formation of a dimer from monomeric subunits is the most thermodynamically unfavorable nucleation step with an estimated equilibrium dissociation constant of 4.6 M (in contrast to 0.6 mM for conversion of dimer to trimer) in a recent molecular dynamic simulation of nucleation (2). The formation of an effective nucleus may be accelerated in vivo by an actin-binding protein such as gelsolin, which can stabilize dimeric actin, or by a protein complex such as Arp2/3 that is thought to contain two actin-like molecules constrained in an orientation that promotes nucleation (3, 4). Antiparallel actin dimers have been identified as a precursor to actin filament polymerization by covalent cross-linking during polymerization induced with divalent cations (5). A gelsolin-actin complex capable of nucleating filament growth at the slow growing, pointed end of filaments has also been shown by covalent cross-linking to contain two actin subunits in the antiparallel configuration (6). The assumption of an antiparallel configuration of subunits is based on evidence that Cys-374 in the C terminus of actin is the only residue involved in the cross-linking reaction. In contrast, when polymerization is complete, intrafilament cross-linking yields a parallel dimer. More recently, electron microscopy has revealed that newly formed actin filaments show evidence of incorporation of antiparallel dimers. This incorporation results in a branched filament network, implying that the dimers have nucleating activity (7). Interestingly, analysis of a Listeria model of cell motility using high-resolution laser tracking provides evidence that filaments elongate in 5.4 nm steps, consistent with in vivo incorporation of dimeric actin (8).In the current work, we provide evidence that polylysine nucleates actin polymerization by e...