Metastabil állapotok feltérképezése a fehérjék nyomás-hőmérséklet fázisdiagramján = Exploring metastable states on the pressure-temperature phase diagram of proteins

Munkánk fő célkitűzése a fehérjék nyomás-hőmérséklet fázisdiagramjának vizsgálata, a fázisdiagram metastabil régióinak felderítése, valamint a metastabil tartományok kinetikai jellemzése volt. Az első évben meghatároztuk a tojásfehérje-lizozim, a mioglobin, apomioglobin, tormaperoxidáz és az alfa krisztallin denaturációs nyomás- és hőmérséklet-adatait. A részletes mérésekre a lizozimot szemeltük ki. A második évben megmértük a lizozim nyomásdenaturációs pontjait különböző hőmérsékleteken. Kiszámoltuk a fázisátalakulás termodinamikai paramétereit. Összehasonlításként tanulmányoztuk a tetramer hemoglobin denaturációját ill. disszociációját, valamint a két doménből álló foszfoglicerát kináz ill. a három doménes humán szérumalbumin fehérje széttekeredését is, amelynél újdonságnak számít, hogy a félretekeredett forma még a natívnál is stabilabbnak tűnik. A harmadik évben a lizozim nyomásdenaturációja utáni visszatekeredés során mértük a másodlagos szerkezet újraalakulásának és a közben formálódó aggregátumok megjelenésének kinetikáját. Megállapítottuk, hogy ezek a folyamatok nem írhatók le egyszerű kétállapotú rendszert feltételezve. Egy több kompartmentes modellt dolgoztunk ki, amelyik leírja az aggregáció többlépcsős jellegét. A kinetikai állandók nyomás- és hőmérsékletfüggéséből megállapítottuk az aktivációs térfogatot és az aktivációs energiát. Eredményeinket eddig 6 cikkben publikáltuk Ezeken kívül még két cikk van előkészületben a felsorolt kinetikai eredményekből. | The main aim of the work was the study of the pressure-temperature phase diagram of the proteins, in order to explore metastable regions on the diagram, and to characterize the kinetics of the structural changes in these metastable regions. In the first year the denaturation temperatures and pressures were detected for hen egg white lysozyme, myoglobin, apomyoglobin, horseradish peroxidase and alpha crystallin. Lysozyme was selected for further detailed study. In the second year the pressure denaturation of lysozyme was measured at different temperatures, in order to construct the phase diagram. The thermodynamic parameters of the phase transitions were determined. For comparison the pressure denaturation of the tetrameric hemoglobin, the two-domain phosphoglycerate-kinase and the three-domain human serum albumin were also measured. In the latter case an interesting feature has been found, namely that the misfolded form was more stable than the native one. In the third year we measured the kinetics of the refolding and aggregation after a pressure unfolding. These processes could not be described by a simple two-state model. A multi-compartment model was developed to describe the successive steps of the process. We calculated the activation volume and activation energy, using the pressure and temperature dependence of the kinetic constants. The results have been published in 6 articles in peer-reviewed journals. Two other papers are in preparation

Uniformly curated signaling pathways reveal tissue-specific cross-talks and support drug target discovery

Motivation: Signaling pathways control a large variety of cellular processes. However, currently, even within the same database signaling pathways are often curated at different levels of detail. This makes comparative and cross-talk analyses difficult. Results: We present SignaLink, a database containing 8 major signaling pathways from Caenorhabditis elegans, Drosophila melanogaster, and humans. Based on 170 review and approx. 800 research articles, we have compiled pathways with semi-automatic searches and uniform, well-documented curation rules. We found that in humans any two of the 8 pathways can cross-talk. We quantified the possible tissue- and cancer-specific activity of cross-talks and found pathway-specific expression profiles. In addition, we identified 327 proteins relevant for drug target discovery. Conclusions: We provide a novel resource for comparative and cross-talk analyses of signaling pathways. The identified multi-pathway and tissue-specific cross-talks contribute to the understanding of the signaling complexity in health and disease and underscore its importance in network-based drug target selection. Availability: http://SignaLink.orgComment: 9 pages, 4 figures, 2 tables and a supplementary info with 5 Figures and 13 Table

Stress-induced rearrangements of cellular networks: consequences for protection and drug design

The complexity of the cells can be described and understood by a number of networks such as protein-protein interaction, cytoskeletal, organelle, signalling, gene transcription and metabolic networks. All these networks are highly dynamic producing continuous rearrangements in their links, hubs, network-skeleton and modules. Here we describe the adaptation of cellular networks after various forms of stress causing perturbations, congestions and network damage. Chronic stress decreases link-density, decouples or even quarantines modules, and induces an increased competition between network hubs and bridges. Extremely long or strong stress may induce a topological phase transition in the respective cellular networks, which switches the cell to a completely different mode of cellular function. We summarize our initial knowledge on network restoration after stress including the role of molecular chaperones in this process. Finally, we discuss the implications of stress-induced network rearrangements in diseases and ageing, and propose therapeutic approaches both to increase the robustness and help the repair of cellular networks.Comment: 9 pages, 1 table, 2 figures, invited paper of FEBS Letters Cellular Stress special issu

Chaperone-like activity of alpha-crystallin is enhanced by high-pressure treatment.

alpha-Crystallin, an oligomeric protein in vertebrate eye lens, is a member of the small heat-shock protein family. Several papers pointed out that its chaperone-like activity could be enhanced by increasing the temperature. We demonstrate in the present study that structural perturbations by high hydrostatic pressures up to 300 MPa also enhance this activity. In contrast with temperature-induced changes, the pressure-induced enhancement is reversible. After pressure release, the extra activity is lost with a relaxation time of 2.0+/-0.5 h. Structural alterations contributing to the higher activity were studied with IR and fluorescence spectroscopy, and light-scattering measurements. The results suggest that while the secondary structure barely changes under pressure, the interactions between the subunits weaken, the oligomers dissociate, the area of accessible hydrophobic surfaces significantly increases and the environment of tryptophan residues becomes slightly more polar. It seems that structural flexibility and the total surface area of the oligomers are the key factors in the chaperone capacity, and that the increase in the chaperone activity does not require the increase in the oligomer size as was assumed previously [Burgio, Kim, Dow and Koretz (2000) Biochem. Biophys. Res. Commun. 268, 426-432]. After pressure release, the structure of subunits are reorganized relatively quickly, whereas the oligomer size reaches its original value slowly with a relaxation time of 33+/-4 h. In our interpretation, both the fast and slow structural rearrangements have an impact on the functional relaxation

The small heat-shock proteins HSPB2 and HSPB3 form well-defined heterooligomers in a unique 3 to 1 subunit ratio

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