Toxin Induced Conformational and Dinamical Changes in Actin Filaments

Phalloidin, a cyclic peptide fromAmanita phalloides can tightly bind to actin filaments and stabilizes their structure (Faulstich et al., 1977; Miyamoto et al., 1986). Phalloidin stabilized actin filaments were extensively used in in vitro studies. Fluorescent derivatives of phalloidin (e.g.: rhodamine-phalloidin, fluorescein-phalloidin, eosin-phalloidin) were also applied to visualize the architecture of the actin cytoskeleton by fluorescence microscopic methods in intracellular studies. The effect of another cyclic peptide, jasplakinolide from a marine sponge (Jaspis johnstoni) on actin filaments is understood to a much lesser extent than that of phalloidin. It is known that jasplakinolide binds to F-actin, and stabilize it’s structure in vitro (Bubb et al., 1994). Jasplakinolide accelerates actin polymerization (Bubb et al., 1994), promotes actin polymerization under nonpolymerizing conditions. Although phalloidin can stabilize actin oligomers, similar effect from jasplakinolide was not observed (Spector et al., 1999). Jasplakinolide and phalloidin competitively bind to actin filament, which suggest that the binding sites of the two drugs are identical or similar in high extent (Senderowicz et al., 1995). Another important difference between the two drugs is that in contrast to phalloidin, jasplakinolide readily enters cells. It appears that the effect of jasplakinolide is similar to that of phalloidin in some aspects, while other effects are different for the two toxins. In this study we compared the effect of phalloidin and jasplakinolide on the dynamic properties and thermal stability of actin filaments.6

A simple model for the cooperative stabilisation of actin filaments by phalloidin and jasplakinolide

AbstractThe stabilisation of magnesium actin filaments by phalloidin and jasplakinolide was studied using the method of differential scanning calorimetry. The results showed that actin could adapt three conformations in the presence of drugs. One conformation was adapted in direct interaction with the drug, while another conformation was identical to that observed in the absence of drugs. A third conformation was induced through allosteric inter-protomer interactions. The effect of both drugs propagated cooperatively along the actin filaments. The number of the cooperative units determined by using a quantitative model was larger for jasplakinolide (15 actin protomers) than for phalloidin (7 protomers)

Az aktin citoszkeleton szabályozásának molekuláris aspektusai = Molecular Aspects of the Actin Cytoskeleton

Munkánk során számos - az aktin és az aktin citoszkeleton működésében fontos szerepet játszó - fehérje molekuláris tulajdonságait és kölcsönhatásait leírtuk. Folytattuk az aktin és miozin fehérjék kölcsönhatásának feltérképezését. Nemzetközi kooperációk keretei között gyors kinetikai módszereket alkalmazva jellemeztünk rovar (Drosophila) izomból származó miozint. Fluoreszcencia spektroszkópiai és kalorimetriai módszerekkel tanulmányoztuk az aktin kölcsönhatását toxikus peptidekkel (falloidin és jasplakinolid). A miozinok működésében meghatározó szerepet játszó nukleotidok, azokon belül is az ADP szerepét írtuk le részletesen egy összefoglaló cikkben. Vizsgáltuk a nem izom eredetű miozinok közül a patkányból származó miozin IX kinetikai tulajdonságait. Egy másik vizsgálatsorozatban a különböző emlős állatokból származó lassú és gyors miozin izoformák tulajdonságait írtuk le kinetikai módszerek segítségével. Ugyancsak nemzetközi együttműködés keretein belül tanulmányoztuk az aktinnak a forminokkal, a forminokon belül is az ún. Diaphanous Related forminokkal való kölcsönhatását. Megállapítottuk azt is, hogy az emlős (egér) sejtekből származó formin fragmentumok megváltoztatják az aktin filamentumok dinamikai tulajdonságait, konformációját. | We characterised a number of actin-binding proteins during the project. We described the dynamic aspects of the interaction between actin and myosin from rabbit skeletal muscle by using spectroscopic methods. Within the framework of international collaboration the properties of Drosophila flight muscle myosin was also investigated by rapid kinetic methods. We described the effect of toxic peptides (phalloidin and jasplakinolide) on the actin filaments by spectroscopic and calorimetric methods. By using rapid kinetic methods we studied the behaviour of a non-muscle myosin, rat myosin IX. In a separate study we described the differences between mysoin isoforms from various mammalian species. In another international collaboration we studied the interaction between actin and mammalian formins. We also showed by using specrtroscopic methods that these formin fragments change the dynamic and conformational properties of actin filaments

Az aktin kölcsönhatása aktin-kötő fehérjékkel és peptidekkel: fluoreszcencia spektroszkópiai vizsgálatok = The Interaction of Actin with Actin-Binding Proteins and Peptides as Revealed by Spectroscopic Methods

Az OTKA K60968 pályázat keretei között az eredeti terveinknek megfelelően tanulmányoztuk az aktin monomereknek és filamentumoknak más fehérjékkel és peptidekkel való kölcsönhatásait. A kutatások során elsősorban fluoreszcencia spektroszkópiai módszereket alkalmaztunk, de az adott kérdéskörtől függően ezen módszerek eredményeit kiegészítettük elektron paramágneses rezonancia spektroszkópiai és kalorimetriai vizsgálatokkal is. Részletes vizsgálatokban jellemeztük az aktin filamentumoknak a forminokkal való kölcsönhatását, és megállapítottuk, hogy a forminok kötődésével a filamentumok szerkezete lazábbá válik. Azt is megfigyeltük, hogy a forminok által kiváltott konformációs módosulásokat a tropomiozin vagy a miozin kötődése megszünteti. Tanulmányoztunk és leírtunk továbbá egy eddig nem jellemzett formin családot, a DAAM forminokat. Ezen vizsgálataink mellett jellemeztük és értelmeztük az aktinnak a kölcsönhatását egyes mérgező toxinokkal, valamint új megfigyeléseket tettünk az aktin-miozin kölcsönhatás szerkezeti és kinetikai sajátságait illetően is. | According to the project plans we have studied the interactions between actin monomers / filaments and actin binding proteins and peptides. In these investigations we applied fluorescence spectroscopic methods, in conjunction with electron paramagnetic resonance and calorimetric assays. We described in details the interaction between actin filaments and formin, and found that the binding of formins made the actin filaments ore flexible. We also observed that the formin induced conformational changes were reversed by the binding of tropomyosin or myosin. We studied and described a novel formin family, the DAAM formins. Furthermore, we described the interactions between actin and toxic actin-binding peptides, and also characterised the interactions of myosin with actin in terms of the conformational and kinetic properties

The Role of the I-BAR Domain in the Filopodia Formation

No abstract available

Neuronal Dopamine D3 Receptors: Translational Implications for Preclinical Research and CNS Disorders

Dopamine (DA), as one of the major neurotransmitters in the central nervous system (CNS) and periphery, exerts its actions through five types of receptors which belong to two major subfamilies such as D1-like (i.e., D1 and D5 receptors) and D2-like (i.e., D2, D3 and D4) receptors. Dopamine D3 receptor (D3R) was cloned 30 years ago, and its distribution in the CNS and in the periphery, molecular structure, cellular signaling mechanisms have been largely explored. Involvement of D3Rs has been recognized in several CNS functions such as movement control, cognition, learning, reward, emotional regulation and social behavior. D3Rs have become a promising target of drug research and great efforts have been made to obtain high affinity ligands (selective agonists, partial agonists and antagonists) in order to elucidate D3R functions. There has been a strong drive behind the efforts to find drug-like compounds with high affinity and selectivity and various functionality for D3Rs in the hope that they would have potential treatment options in CNS diseases such as schizophrenia, drug abuse, Parkinson’s disease, depression, and restless leg syndrome. In this review, we provide an overview and update of the major aspects of research related to D3Rs: distribution in the CNS and periphery, signaling and molecular properties, the status of ligands available for D3R research (agonists, antagonists and partial agonists), behavioral functions of D3Rs, the role in neural networks, and we provide a summary on how the D3R-related drug research has been translated to human therapy

Myopathy-causing actin mutations promote defects in serum-response factor signalling

Mutations in the gene encoding skeletal muscle alpha-actin (ACTA1) account for approx. 20% of patients with the muscular disorder nemaline myopathy. Nemaline myopathy is a muscular wasting disease similar to muscular dystrophy, but distinguished by deposits of actin and actin-associated proteins near the z-line of the sarcomere. Approx. one-third of the over 140 myopathy actin mutations have been characterized either biochemically or in cultured cells to determine their effects on the actin cytoskeleton. However, the actin defects causing myopathy are likely to be heterogeneous, with only a few common trends observed among the actin mutants, such as reduced polymerization capacity or an inability to fold properly. Notably, the transcriptional programme regulated by serum-response factor, which is instrumental in muscle development and maintenance, is directly controlled by the balance of actin assembly and disassembly in cells. In the present study, we explored the impact of myopathy mutations in actin on the control of the transcriptional response by serum-response factor and found that the majority of mutants examined have altered serum-response factor signalling. We propose that altered serum-response factor signalling could be a major factor in actin-based nemaline myopathy, and that this area could be exploited to develop therapies for sufferers

N-WASP coordinates the delivery and F-actin-mediated capture of MT1-MMP at invasive pseudopods

Metastasizing tumor cells use matrix metalloproteases, such as the transmembrane collagenase MT1-MMP, together with actin-based protrusions, to break through extracellular matrix barriers and migrate in dense matrix. Here we show that the actin nucleation–promoting protein N-WASP (Neural Wiskott-Aldrich syndrome protein) is up-regulated in breast cancer, and has a pivotal role in mediating the assembly of elongated pseudopodia that are instrumental in matrix degradation. Although a role for N-WASP in invadopodia was known, we now show how N-WASP regulates invasive protrusion in 3D matrices. In actively invading cells, N-WASP promoted trafficking of MT1-MMP into invasive pseudopodia, primarily from late endosomes, from which it was delivered to the plasma membrane. Upon MT1-MMP’s arrival at the plasma membrane in pseudopodia, N-WASP stabilized MT1-MMP via direct tethering of its cytoplasmic tail to F-actin. Thus, N-WASP is crucial for extension of invasive pseudopods into which MT1-MMP traffics and for providing the correct cytoskeletal framework to couple matrix remodeling with protrusive invasion

Bacterial and host determinants of MAL activation upon EPEC infection: the roles of Tir, ABRA, and FLRT3

Infection of host cells by pathogenic microbes triggers signal transduction pathways leading to a multitude of host cell responses including actin cytoskeletal re-arrangements and transcriptional programs. The diarrheagenic pathogens Enteropathogenic E. coli (EPEC) and the related Enterohemorrhagic E. coli (EHEC) subvert the host-cell actin cytoskeleton to form attaching and effacing lesions on the surface of intestinal epithelial cells by injecting effector proteins via a type III secretion system. Here we use a MAL translocation assay to establish the effect of bacterial pathogens on host cell signaling to transcription factor activation. MAL is a cofactor of Serum response factor (SRF), a transcription factor with important roles in the regulation of the actin cytoskeleton. We show that EPEC induces nuclear accumulation of MAL-GFP. The translocated intimin receptor is essential for this process and phosphorylation of Tyrosine residues 454 and 474 is important. Using an expression screen we identify FLRT3, C22orf28 and TESK1 as novel activators of SRF. Importantly we demonstrate that ABRA (actin-binding Rho-activating protein, also known as STARS) is necessary for EPEC-induced nuclear accumulation of MAL and the novel SRF activator FLRT3, is a component of this pathway. We further demonstrate that ABRA is important for structural maintenance of EPEC pedestals. Our results uncover novel components in pathogen-activated cytoskeleton signalling to MAL activation