The Effect of Formins on the Polymerisation and Dynamic Properties of Actin Filaments

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Forminok hatása az aktin filamentum konformációjára = The effect of formin fragments on the conformation of actin filaments

A pályázat keretei között végzett vizsgálatainkban fluoreszcencia spektroszkópiai, gyors-kinetika és EPR módszereket alkalmaztunk motor fehérjék, és az aktin citoszkeleton felépítésében meghatározó szerepet betöltő aktin nukleációs faktorok tanulmányozására. Kinetikai módszerekkel jellemeztük a speciális és eddig ritkán vizsgált XIV-es miozint, valamint részletesen leírtuk és összevetettük különböző fajokból származó emlős izom miozin izoformák tulajdonságait. Megmutattuk, hogy az aktin nukleációs faktorok közé tartozó forminok kötődése fellazítja az aktin filamentumok szerkezetét. EPR vizsgálataink arra is rávilágítottak, hogy ezek a konformációs módosulások heterogén és összetett módon valósulnak meg. Azt is megmutattuk, hogy forminok által kiváltott konformációs változások visszafordíthatóak tropomiozin kötődésével. | The investigations involved in the project were based on the applications of fluorescence spectroscopic, rapid kinetic and EPR methods. We have studied the kinetic and conformational properties of motor proteins and actin nucleation factors. We described the kinetic behaviour of a special class of myosins, the myosin XIV, and also many isoforms isolated from mammalian muscles of various species. We sowed that the binding of formins - a family of actin nucleation factors - to the actin filaments made the structure of the filaments looser. The EPR data showed that these conformational modifications are complex and heterogeneous. Further studies provided evidence, that the forming-induced structural changes could be reversed by the binding of tropomyosin

Celiac disease-specific prolamin peptide content of wheat relatives and wild species determined by ELISA assays and bioinformatics analyses

Enzyme-linked immunosorbent assays (ELISAs) are widely used to determine gluten contamination in gluten-free and low gluten food samples. ELISA assays developed using monoclonal antibodies against known toxic peptides have an advantage in the identification of toxic prolamin content in protein extracts of different food samples, as well as raw materials. R5 and G12 monoclonal antibodies specific for two known toxic peptides used in commercially available gluten ELISA assays were applied to test toxic peptide contents in wheat relatives and wild wheat species with different genome composition and complexity. Although the R5 peptide content showed some correlation with ploidy levels in Triticum species, there was a high variance among Aegilops species. Some of the analysed diploid Aegilops species showed extremely high R5 peptide contents. Based on the bioinformatics analyses, the R5 peptide was present in most of the sulphur rich prolamins in all the analysed species, whereas the G12 epitope was exclusively present in alpha gliadins. High variation was detected in the position and frequency of epitopes in sequences originating from the same species, thus highlighting the importance of genotypic variation within species. Identification of new prolamin alleles of wheat relatives and wild wheat species is of great importance in order to find germplasm for special end-use quality purposes as well as development of food with reduced toxicity

Identification of key effects causing weak performance of allergen analysis in processed food matrices

The weaker performance of generally used analytical methods for allergen analysis in processed foods can be connected to protein denaturation. To understand the nature of protein denaturation processes, experimental but realistic model matrices (corn starch based mixture, hydrated dough, and heat treated cookies) were developed that contain a defined amount of milk, egg, soy, and wheat proteins individually or in combination. The protein subunit composition was investigated in every processing phase, i.e. after mixing, dough formation, and baking. SDS-PAGE measurements were carried out to monitor the protein distribution of sample food matrices in non-reducing and reducing gels. The results clearly show that the highly decreased protein solubility is caused by denaturation, aggregation, or complex formation, which are the most significant factors in poorer analytical performances. Solubility can only partly be improved with the application of reducing agents or surfactants, and the rate of improvement is depending on the proteins and the matrices

Tropomyosins Regulate the Severing Activity of Gelsolin in Isoform-Dependent and Independent Manners

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Celiac disease specific prolamin peptide content of wheat relatives and wild species determined by ELISA assays and bioinformatics analyses.

Enzyme-linked immunosorbent assays (ELISAs) are widely used to determine gluten contamination in gluten-free and low gluten food samples. ELISA assays developed using monoclonal antibodies against known toxic peptides have an advantage in the identification of toxic prolamin content in protein extracts of different food samples, as well as raw materials. R5 and G12 monoclonal antibodies specific for two known toxic peptides used in commercially available gluten ELISA assays were applied to test toxic peptide contents in wheat relatives and wild wheat species with different genome composition and complexity. Although the R5 peptide content showed some correlation with ploidy levels in Triticum species, there was a high variance among Aegilops species. Some of the analysed diploid Aegilops species showed extremely high R5 peptide contents. Based on the bioinformatics analyses, the R5 peptide was present in most of the sulphur rich prolamins in all the analysed species, whereas the G12 epitope was exclusively present in alpha gliadins. High variation was detected in the position and frequency of epitopes in sequences originating from the same species, thus highlighting the importance of genotypic variation within species. Identification of new prolamin alleles of wheat relatives and wild wheat species is of great importance in order to find germplasm for special end-use quality purposes as well as development of food with reduced toxicity

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 formin DAAM is required for coordination of the actin and microtubule cytoskeleton in axonal growth cones

Directed axonal growth depends on correct coordination of the actin and microtubule cytoskeleton in the growth cone. However, despite the relatively large number of proteins implicated in actin−microtubule crosstalk, the mechanisms whereby actin polymerization is coupled to microtubule stabilization and advancement in the peripheral growth cone remained largely unclear. Here, we identified the formin Dishevelled-associated activator of morphogenesis (DAAM) as a novel factor playing a role in concerted regulation of actin and microtubule remodeling in Drosophila melanogaster primary neurons. In vitro, DAAM binds to F-actin as well as to microtubules and has the ability to crosslink the two filament systems. Accordingly, DAAM associates with the neuronal cytoskeleton, and a significant fraction of DAAM accumulates at places where the actin filaments overlap with that of microtubules. Loss of DAAM affects growth cone and microtubule morphology, and several aspects of microtubule dynamics; and biochemical and cellular assays revealed a microtubule stabilization activity and binding to the microtubule tip protein EB1. Together, these data suggest that, besides operating as an actin assembly factor, DAAM is involved in linking actin remodeling in filopodia to microtubule stabilization during axonal growth