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Budapest, Hungary

Fuxreiter M.,Weizmann Institute of Science | Simon I.,Institute of Enzymology | Bondos S.,Texas A&M University
Trends in Biochemical Sciences | Year: 2011

Traditionally, specific DNA recognition is thought to rely on static contacts with the bases or phosphates. Recent results, however, indicate that residues far outside the binding context can crucially influence selectivity or binding affinity via transient, dynamic interactions with the DNA binding interface. These regions usually do not adopt a well-defined structure, even when bound to DNA, and thus form a fuzzy complex. Here, we propose the existence of a dynamic DNA readout mechanism, wherein distant segments modulate conformational preferences, flexibility or spacing of the DNA binding motifs or serve as competitive partners. Despite their low sequence similarity, these intrinsically disordered regions are often conserved at the structural level, and exploited for regulation of the transcription machinery via protein-protein interactions, post-translational modifications or alternative splicing. © 2011 Elsevier Ltd. Source

Pietrosemoli N.,CSIC - National Center for Biotechnology | Pietrosemoli N.,Rice University | Pancsa R.,Vrije Universiteit Brussel | Tompa P.,Vrije Universiteit Brussel | Tompa P.,Institute of Enzymology
PLoS Computational Biology | Year: 2013

Vesicle trafficking systems play essential roles in the communication between the organelles of eukaryotic cells and also between cells and their environment. Endocytosis and the late secretory route are mediated by clathrin-coated vesicles, while the COat Protein I and II (COPI and COPII) routes stand for the bidirectional traffic between the ER and the Golgi apparatus. Despite similar fundamental organizations, the molecular machinery, functions, and evolutionary characteristics of the three systems are very different. In this work, we compiled the basic functional protein groups of the three main routes for human and yeast and analyzed them from the structural disorder perspective. We found similar overall disorder content in yeast and human proteins, confirming the well-conserved nature of these systems. Most functional groups contain highly disordered proteins, supporting the general importance of structural disorder in these routes, although some of them seem to heavily rely on disorder, while others do not. Interestingly, the clathrin system is significantly more disordered (∼23%) than the other two, COPI (∼9%) and COPII (∼8%). We show that this structural phenomenon enhances the inherent plasticity and increased evolutionary adaptability of the clathrin system, which distinguishes it from the other two routes. Since multi-functionality (moonlighting) is indicative of both plasticity and adaptability, we studied its prevalence in vesicle trafficking proteins and correlated it with structural disorder. Clathrin adaptors have the highest capability for moonlighting while also comprising the most highly disordered members. The ability to acquire tissue specific functions was also used to approach adaptability: clathrin route genes have the most tissue specific exons encoding for protein segments enriched in structural disorder and interaction sites. Overall, our results confirm the general importance of structural disorder in vesicle trafficking and suggest major roles for this structural property in shaping the differences of evolutionary adaptability in the three routes. © 2013 Pietrosemoli et al. Source

Agocs G.,Semmelweis University | Solymosi K.,Eotvos Lorand University | Varga A.,Institute of Enzymology | Modos K.,Semmelweis University | And 4 more authors.
FEBS Letters | Year: 2010

Amyloid deposits, which accumulate in numerous diseases, are the final stage of multi-step protein conformational-conversion and oligomerization processes. The underlying molecular mechanisms are not fully understood, and particularly little is known about the reverse reaction. Here we show that phosphoglycerate kinase amyloid fibrils can be converted back into native protein. We achieved recovery with 60% efficiency, which is comparable to the success rate of the unfolding-refolding studies, and the recovered enzyme was folded, stable and fully active. The key intermediate stages in the recovery process are fibril disassembly and unfolding followed by spontaneous protein folding. © 2010 Federation of European Biochemical Societies. Source

Bisio A.,University of Trento | Zamborszky J.,University of Trento | Zamborszky J.,Institute of Enzymology | Zaccara S.,University of Trento | And 8 more authors.
Oncotarget | Year: 2014

The p53 and NFκB sequence-specific transcription factors play crucial roles in cell proliferation and survival with critical, even if typically opposite, effects on cancer progression. To investigate a possible crosstalk between p53 and NFκB driven by chemotherapy-induced responses in the context of an inflammatory microenvironment, we performed a proof of concept study using MCF7 cells. Transcriptome analyses upon single or combined treatments with doxorubicin (Doxo, 1.5μM) and the NFκB inducer TNF-alpha (TNFα, 5ng/ml) revealed 432 upregulated (log2 FC > 2), and 390 repressed genes (log2 FC < -2) for the Doxo+TNFα treatment. 239 up-regulated and 161 repressed genes were synergistically regulated by the double treatment. Annotation and pathway analyses of Doxo+TNFα selectively up-regulated genes indicated strong enrichment for cell migration terms. A panel of genes was examined by qPCR coupled to p53 activation by Doxo, 5-Fluoruracil and Nutlin-3α, or to p53 or NFκB inhibition. Transcriptome data were confirmed for 12 of 15 selected genes and seven (PLK3, LAMP3, ETV7, UNC5B, NTN1, DUSP5, SNAI1) were synergistically up-regulated after Doxo+TNFα and dependent both on p53 and NFκB. Migration assays consistently showed an increase in motility for MCF7 cells upon Doxo+TNFα. A signature of 29 Doxo+TNFα highly synergistic genes exhibited prognostic value for luminal breast cancer patients, with adverse outcome correlating with higher relative expression. We propose that the crosstalk between p53 and NFκB can lead to the activation of specific gene expression programs that may impact on cancer phenotypes and potentially modify the efficacy of cancer therapy. Source

Barabas O.,Institute of Enzymology | Barabas O.,U.S. National Institutes of Health | Barabas O.,Structural and Computational Biology Unit | Nemeth V.,Institute of Enzymology | And 10 more authors.
Nucleic Acids Research | Year: 2013

Enzymatic synthesis and hydrolysis of nucleoside phosphate compounds play a key role in various biological pathways, like signal transduction, DNA synthesis and metabolism. Although these processes have been studied extensively, numerous key issues regarding the chemical pathway and atomic movements remain open for many enzymatic reactions. Here, using the Mason-Pfizer monkey retrovirus dUTPase, we study the dUTPase-catalyzed hydrolysis of dUTP, an incorrect DNA building block, to elaborate the mechanistic details at high resolution. Combining mass spectrometry analysis of the dUTPase-catalyzed reaction carried out in and quantum mechanics/molecular mechanics (QM/MM) simulation, we show that the nucleophilic attack occurs at the α-phosphate site. Phosphorus-31 NMR spectroscopy (31P-NMR) analysis confirms the site of attack and shows the capability of dUTPase to cleave the dUTP analogue α,β-imido-dUTP, containing the imido linkage usually regarded to be non-hydrolyzable. We present numerous X-ray crystal structures of distinct dUTPase and nucleoside phosphate complexes, which report on the progress of the chemical reaction along the reaction coordinate. The presently used combination of diverse structural methods reveals details of the nucleophilic attack and identifies a novel enzyme-product complex structure. © 2013 The Author(s). Source

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