Binolfi A.,CONICET |
Fernandez C.O.,CONICET |
Fernandez C.O.,Max Planck Institute for Biophysical Chemistry |
Fernandez C.O.,Research Center for the Molecular Physiology of the Brain |
And 4 more authors.
Proteins: Structure, Function and Bioinformatics | Year: 2012
Thioredoxins (TRXs) constitute attractive α/β scaffolds for investigating molecular recognition. The interaction between the recombinant fragment spanning the sequence 1-93 of full-length TRX (TRX1-93) and the synthetic peptide comprising residues 94-108 (TRX94-108), plus a C-terminal tyrosine tag (the numbering scheme used in entry pdb 2TRX is used throughout the article, two complementary moieties of E. coli TRX, brings about the consolidation of a native-like complex. Despite its reduced thermodynamic stability, this complex is able to acquire fine structural features remarkably similar to those characteristic of full-length TRX, namely, hydrodynamic behavior, assessed by diffusion-ordered spectroscopy (DOSY)-NMR; the pattern of secondary structure, as revealed by three-bond HNHα coupling constants and secondary shifts for Hα/CO/Cα/Cβ; native-like tertiary structural signatures revealed by near-UV circular dichroism (CD) spectroscopy. The complex exhibits a relaxation behavior compatible with that expected for a native-like structure. However, heteronuclear nuclear Overhauser effect (NOE)s reveal an enhanced dynamics for the complex by comparison with full-length TRX. Furthermore, higher R2 values for residues 43-50 and 74-89 would likely result from an exchange process modulated by the peptide at the interface region. The slow kinetics of the consolidation reaction was followed by CD and real-time NMR. Equilibrium titration experiments by NMR yield a KD value of 1.4 ± 1.0 μM and a second low-affinity (>150 μM) binding event in the vicinity of the active site. Molecular dynamics simulations of both the isolated fragment TRX1-93 and the complex suggest the destabilization of α2 and α3 helical elements and the persistence of β-structure in the absence of TRX94-108. Altogether, structural and dynamic evidence presented herein points to the key role played by the C-terminal helix in establishing the overall fold. This critical switch module endows reduced TRX with the ability to act as a cooperative folding unit. © 2012 Wiley Periodicals, Inc.
Doeppner T.R.,University of Duisburg - Essen |
Doeppner T.R.,University of Gottingen |
Doehring M.,University of Gottingen |
Bretschneider E.,University of Gottingen |
And 8 more authors.
Acta Neuropathologica | Year: 2013
MicroRNAs (miRNAs) are highly conserved non-coding RNAs modulating gene expression via mRNA binding. Recent work suggests an involvement of miRNAs in cardiovascular diseases including stroke. As such, the brain-abundant miR-124 and its transcriptional repressor RE1-silencing transcription factor (REST) do not only have elementary roles in the developing and the adult brain, but also alter expression upon cerebral ischemia. However, the therapeutic potential of miR-124 against stroke and the mechanisms involved remain elusive. Here, we analyzed the therapeutic potential of ectopic miR-124 against stroke and its underlying mechanisms with regard to the interaction between miR-124 and REST. Our results show that viral vector-mediated miR-124 delivery increased the resistance of cultured oxygen-glucose-deprived cortical neurons in vitro and reduced brain injury as well as functional impairment in mice submitted to middle cerebral artery occlusion. Likewise, miR-124 induced enhanced neurovascular remodeling leading to increased angioneurogenesis 8 weeks post-stroke. While REST abundance increased upon stroke, the increase was prevented by miR-124 despite a so far unknown negative feedback loop between miR-124 and REST. Rather, miR-124 decreased the expression of the deubiquitinating enzyme Usp14, which has two conserved miR-124-binding sites in the 3′UTR of its mRNA, and thereby mediated reduced REST levels. The down-regulation of REST by miR-124 was also mimicked by the Usp14 inhibitor IU-1, suggesting that miR-124 promotes neuronal survival under ischemic conditions via Usp14-dependent REST degradation. Ectopic miR-124 expression, therefore, appears as an attractive and novel tool in stroke treatment, mediating neuroprotection via a hitherto unknown mechanism that involves Usp14-dependent REST degradation. © 2013 Springer-Verlag Berlin Heidelberg.
Rezaei-Ghaleh N.,Max Planck Institute for Biophysical Chemistry |
Andreetto E.,TU Munich |
Yan L.-M.,TU Munich |
Kapurniotu A.,TU Munich |
And 2 more authors.
PLoS ONE | Year: 2011
Assembly of amyloid-beta peptide (Aβ) into cytotoxic oligomeric and fibrillar aggregates is believed to be a major pathologic event in Alzheimer's disease (AD) and interfering with Aβ aggregation is an important strategy in the development of novel therapeutic approaches. Prior studies have shown that the double N-methylated analogue of islet amyloid polypeptide (IAPP) IAPP-GI, which is a conformationally constrained IAPP analogue mimicking a non-amyloidogenic IAPP conformation, is capable of blocking cytotoxic self-assembly of Aβ. Here we investigate the interaction of IAPP-GI with Aβ40 and Aβ42 using NMR spectroscopy. The most pronounced NMR chemical shift changes were observed for residues 13-20, while residues 7-9, 15-16 as well as the C-terminal half of Aβ - that is both regions of the Aβ sequence that are converted into β-strands in amyloid fibrils - were less accessible to solvent in the presence of IAPP-GI. At the same time, interaction of IAPP-GI with Aβ resulted in a concentration-dependent co-aggregation of Aβ and IAPP-GI that was enhanced for the more aggregation prone Aβ42 peptide. On the basis of the reduced toxicity of the Aβ peptide in the presence of IAPP-GI, our data are consistent with the suggestion that IAPP-GI redirects Aβ into nontoxic "off-pathway" aggregates. © 2011 Rezaei-Ghaleh et al.
Woehler A.,Research Center for the Molecular Physiology of the Brain |
Woehler A.,Max Planck Institute for Biophysical Chemistry |
Woehler A.,University of Gottingen |
Wlodarczyk J.,Nencki Institute of Experimental Biology |
And 2 more authors.
Biophysical Journal | Year: 2010
Molecular sensors based on intramolecular Förster resonance energy transfer (FRET) have become versatile tools to monitor regulatory molecules in living tissue. However, their use is often compromised by low signal strength and excessive noise. We analyzed signal/noise (SNR) aspects of spectral FRET analysis methods, with the following conclusions: The most commonly used method (measurement of the emission ratio after a single short wavelength excitation) is optimal in terms of signal/noise, if only relative changes of this uncalibrated ratio are of interest. In the case that quantitative data on FRET efficiencies are required, these can be calculated from the emission ratio and some calibration parameters, but at reduced SNR. Lux-FRET, a recently described method for spectral analysis of FRET data, allows one to do so in three different ways, each based on a ratio of two out of three measured fluorescence signals (the donor and acceptor signal during a short-wavelength excitation and the acceptor signal during long wavelength excitation). Lux-FRET also allows for calculation of the total abundance of donor and acceptor fluorophores. The SNR for all these quantities is lower than that of the plain emission ratio due to unfavorable error propagation. However, if ligand concentration is calculated either from lux-FRET values or else, after its calibration, from the emission ratio, SNR for both analysis modes is very similar. Likewise, SNR values are similar, if the noise of these quantities is related to the expected dynamic range. We demonstrate these relationships based on data from an Epac-based cAMP sensor and discuss how the SNR changes with the FRET efficiency and the number of photons collected. © 2010 by the Biophysical Society.
Cho M.-K.,Max Planck Institute for Biophysical Chemistry |
Kim H.-Y.,Max Planck Institute for Biophysical Chemistry |
Fernandez C.O.,National University of Rosario |
Becker S.,Max Planck Institute for Biophysical Chemistry |
And 2 more authors.
Protein Science | Year: 2011
The major component of neural inclusions that are the pathological hallmark of Parkinson's disease are amyloid fibrils of the protein α-synuclein (aS). Here we investigated if the disease-related mutation A30P not only modulates the kinetics of aS aggregation, but also alters the structure of amyloid fibrils. To this end we optimized the method of quenched hydrogen/deuterium exchange coupled to NMR spectroscopy and performed two-dimensional proton-detected highresolution magic angle spinning experiments. The combined data indicate that the A30P mutation does not cause changes in the number, location and overall arrangement of β-strands in amyloid fibrils of aS. At the same time, several residues within the fibrillar core retain nano-second dynamics. We conclude that the increased pathogenicity related to the familial A30P mutation is unlikely to be caused by a mutation-induced change in the conformation of aS aggregates. Published by Wiley-Blackwell. © 2010 The Protein Society.
Tonges L.,University of Gottingen |
Ostendorf T.,University of Gottingen |
Lamballe F.,French National Center for Scientific Research |
Genestine M.,French National Center for Scientific Research |
And 7 more authors.
Journal of Neurochemistry | Year: 2011
Hepatocyte growth factor (HGF) is known to promote the survival and foster neuritic outgrowth of different subpopulations of CNS neurons during development. Together with its corresponding receptor c-mesenchymal-epithelial transition factor (Met), it is expressed in the developing and the adult murine, rat and human CNS. We have studied the role of HGF in paradigms of retinal ganglion cell (RGC) regeneration and cell death in vitro and in vivo. After application of recombinant HGF in vitro, survival of serum-deprived RGC-5 cells and of growth factor-deprived primary RGC was significantly increased. This was shown to be correlated to the phosphorylation of c-Met and subsequent activation of serine/threonine protein kinase Akt and MAPK downstream signalling pathways involved in neuronal survival. Furthermore, neurite outgrowth of primary RGC was stimulated by HGF. In vivo, c-Met expression in RGC was up-regulated after optic nerve axotomy lesion. Here, treatment with HGF significantly improved survival of axotomized RGC and enhanced axonal regeneration after optic nerve crush. Our data demonstrates that exogenously applied HGF has a neuroprotective and regeneration-promoting function for lesioned CNS neurons. We provide strong evidence that HGF may represent a trophic factor for adult CNS neurons, which may play a role as therapeutic target in the treatment of neurotraumatic and neurodegenerative CNS disorders. © 2011 International Society for Neurochemistry.
Gorinski N.,Hannover Medical School |
Kowalsman N.,Hebrew University of Jerusalem |
Renner U.,Research Center for the Molecular Physiology of the Brain |
Wirth A.,Hannover Medical School |
And 6 more authors.
Molecular Pharmacology | Year: 2012
Experimental evidence suggests that most members of class A G-protein coupled receptors (GPCRs) can form homomers and heteromers in addition to functioning as single monomers. In particular, serotonin (5-HT) receptors were shown to homodimerize and heterodimerize with other GPCRs, although the details and the physiological role of the oligomerization has not yet been fully elucidated. Here we used computational modeling of the 5-HT1A receptor monomer and dimer to predict residues important for dimerization. Based on these results, we carried out rationally designed site-directed mutagenesis. The ability of the mutants to dimerize was evaluated using different FRET-based approaches. The reduced levels of acceptor photobleaching-Fö rster resonance energy transfer (FRET) and the lower number of monomers participating in oligomers, as assessed by lux-FRET, confirmed the decreased ability of the mutants to dimerize and the involvement of the predicted contacts (Trp1754.64, Tyr1985.41, Arg1514.40, and Arg1524.41) at the interface. This information was reintroduced as constraints for computational protein-protein docking to obtain a high-quality dimer model. Analysis of the refined model as well as molecular dynamics simulations of wild-type (WT) and mutant dimers revealed compensating interactions in dimers composed of WT and W175A mutant. This provides an explanation for the requirement of mutations of Trp1754.64 in both homomers for disrupting dimerization. Our iterative computational-experimental study demonstrates that transmembrane domains TM4/TM5 can form an interaction interface in 5-HT1A receptor dimers and indicates that specific amino acid interactions maintain this interface. The mutants and the optimized model of the dimer structure may be used in functional studies of serotonin dimers. Copyright © 2012 The American Society for Pharmacology and Experimental Therapeutics.
Doeppner T.R.,University of Duisburg - Essen |
Kaltwasser B.,University of Duisburg - Essen |
Elali A.,University of Duisburg - Essen |
Zechariah A.,University of Duisburg - Essen |
And 3 more authors.
Journal of Cerebral Blood Flow and Metabolism | Year: 2011
Hepatocyte growth factor (HGF) is an interesting candidate for acute stroke treatment as shown by continuous infusion or gene delivery protocols. However, little is known about HGF-mediated long-term effects. The present study therefore analyzed long-term effects of an acute intrastriatal HGF treatment (5 g) after a 45-minute stroke, with regard to brain injury and neurologic recovery. Hepatocyte growth factor induced long-term neuroprotection as assessed by infarct volume and neuronal cell death analysis for as long as 4 weeks after stroke, which was associated with sustained neurologic recovery as evidenced by corner-turn and tight-rope tests. Analyzing underlying mechanisms of HGF-induced sustained neuroprotection, enhanced cell proliferation followed by increased neuronal differentiation of neural precursor cells (NPCs) was observed in the ischemic striatum of HGF-treated mice, which persisted for up to 4 weeks. In line with this, HGF promoted neurosphere formation as well as proliferation of NPC and decreased caspase-3-dependent hypoxic injury in vitro. Preservation of blood-brain barrier integrity 24 hours after stroke was furthermore noticed in animals receiving HGF, which was associated with the inhibition of matrix metalloproteases (MMP)-2 and MMP-9 at 4 and 24 hours, respectively. We suggest that sustained recruitment of proliferating cells together with improved neurovascular remodeling provides an explanation for HGF-induced long-term neuroprotection. © 2011 ISCBFM All rights reserved.
Kumar S.,University of Bonn |
Rezaei-Ghaleh N.,Max Planck Institute for Biophysical Chemistry |
Terwel D.,University of Bonn |
Thal D.R.,University of Ulm |
And 10 more authors.
EMBO Journal | Year: 2011
Alzheimer's disease (AD) is the most common form of dementia and associated with progressive deposition of amyloid β 2-peptides (Aβ 2) in the brain. Aβ 2 derives by sequential proteolytic processing of the amyloid precursor protein by β 2-and β 3-secretases. Rare mutations that lead to amino-acid substitutions within or close to the Aβ 2 domain promote the formation of neurotoxic Aβ 2 assemblies and can cause early-onset AD. However, mechanisms that increase the aggregation of wild-type Aβ 2 and cause the much more common sporadic forms of AD are largely unknown. Here, we show that extracellular Aβ 2 undergoes phosphorylation by protein kinases at the cell surface and in cerebrospinal fluid of the human brain. Phosphorylation of serine residue 8 promotes formation of oligomeric Aβ 2 assemblies that represent nuclei for fibrillization. Phosphorylated Aβ 2 was detected in the brains of transgenic mice and human AD brains and showed increased toxicity in Drosophila models as compared with non-phosphorylated Aβ 2. Phosphorylation of Aβ 2 could represent an important molecular mechanism in the pathogenesis of the most common sporadic form of AD. © 2011 European Molecular Biology Organization.
Binolfi A.,National University of Rosario |
Valiente-Gabioud A.A.,National University of Rosario |
Duran R.,Institute Pasteur Of Montevideo Ipm |
Duran R.,Institute Investigaciones Biologicas Clemente Estable IIBCE |
And 5 more authors.
Journal of the American Chemical Society | Year: 2011
The aggregation of α-synuclein (AS) is selectively enhanced by copper in vitro, and the interaction is proposed to play a potential role in vivo. In this work, we report the structural, residue-specific characterization of Cu(I) binding to AS and demonstrate that the protein is able to bind Cu(I) with relatively high affinity in a coordination environment that involves the participation of Met1 and Met5 residues. This knowledge is a key to understanding the structural-aggregation basis of the copper-catalyzed oxidation of AS. © 2010 American Chemical Society.