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Dive into the research topics where Ilka Wittig is active.

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Featured researches published by Ilka Wittig.


Nature Protocols | 2006

Blue native PAGE

Ilka Wittig; Hans-Peter Braun; Hermann Schägger

Blue native PAGE (BN-PAGE) can be used for one-step isolation of protein complexes from biological membranes and total cell and tissue homogenates. It can also be used to determine native protein masses and oligomeric states and to identify physiological protein–protein interactions. Native complexes are recovered from gels by electroelution or diffusion and are used for 2D crystallization and electron microscopy or analyzed by in-gel activity assays or by native electroblotting and immunodetection. In this protocol, we describe methodology to perform BN-PAGE followed by (i) native extraction or native electroblotting of separated proteins, or (ii) a second dimension of tricine-SDS-PAGE or modified BN-PAGE, or (iii) a second dimension of isoelectric focusing (IEF) followed by a third dimension of tricine-SDS-PAGE for the separation of subunits of complexes. These protocols for 2D and 3D PAGE can be completed in 2 and 3 days.


Molecular & Cellular Proteomics | 2007

High Resolution Clear Native Electrophoresis for In-gel Functional Assays and Fluorescence Studies of Membrane Protein Complexes

Ilka Wittig; Michael Karas; Hermann Schägger

Clear native electrophoresis and blue native electrophoresis are microscale techniques for the isolation of membrane protein complexes. The Coomassie Blue G-250 dye, used in blue native electrophoresis, interferes with in-gel fluorescence detection and in-gel catalytic activity assays. This problem can be overcome by omitting the dye in clear native electrophoresis. However, clear native electrophoresis suffers from enhanced protein aggregation and broadening of protein bands during electrophoresis and therefore has been used rarely. To preserve the advantages of both electrophoresis techniques we substituted Coomassie dye in the cathode buffer of blue native electrophoresis by non-colored mixtures of anionic and neutral detergents. Like Coomassie dye, these mixed micelles imposed a charge shift on the membrane proteins to enhance their anodic migration and improved membrane protein solubility during electrophoresis. This improved clear native electrophoresis offers a high resolution of membrane protein complexes comparable to that of blue native electrophoresis. We demonstrate the superiority of high resolution clear native electrophoresis for in-gel catalytic activity assays of mitochondrial complexes I–V. We present the first in-gel histochemical staining protocol for respiratory complex III. Moreover we demonstrate the special advantages of high resolution clear native electrophoresis for in-gel detection of fluorescent labeled proteins labeled by reactive fluorescent dyes and tagged by fluorescent proteins. The advantages of high resolution clear native electrophoresis make this technique superior for functional proteomics analyses.


Proceedings of the National Academy of Sciences of the United States of America | 2012

Structure of the yeast F1Fo-ATP synthase dimer and its role in shaping the mitochondrial cristae

Karen M. Davies; Claudio Anselmi; Ilka Wittig; José D. Faraldo-Gómez; Werner Kühlbrandt

We used electron cryotomography of mitochondrial membranes from wild-type and mutant Saccharomyces cerevisiae to investigate the structure and organization of ATP synthase dimers in situ. Subtomogram averaging of the dimers to 3.7 nm resolution revealed a V-shaped structure of twofold symmetry, with an angle of 86° between monomers. The central and peripheral stalks are well resolved. The monomers interact within the membrane at the base of the peripheral stalks. In wild-type mitochondria ATP synthase dimers are found in rows along the highly curved cristae ridges, and appear to be crucial for membrane morphology. Strains deficient in the dimer-specific subunits e and g or the first transmembrane helix of subunit 4 lack both dimers and lamellar cristae. Instead, cristae are either absent or balloon-shaped, with ATP synthase monomers distributed randomly in the membrane. Computer simulations indicate that isolated dimers induce a plastic deformation in the lipid bilayer, which is partially relieved by their side-by-side association. We propose that the assembly of ATP synthase dimer rows is driven by the reduction in the membrane elastic energy, rather than by direct protein contacts, and that the dimer rows enable the formation of highly curved ridges in mitochondrial cristae.


Nature Genetics | 2010

Exome sequencing identifies ACAD9 mutations as a cause of complex I deficiency

Tobias B. Haack; Katharina Danhauser; Birgit Haberberger; Jonathan Hoser; Valentina Strecker; Detlef Boehm; Graziella Uziel; Eleonora Lamantea; Federica Invernizzi; Joanna Poulton; Boris Rolinski; Arcangela Iuso; Saskia Biskup; Thorsten Schmidt; Hans W. Mewes; Ilka Wittig; Thomas Meitinger; Massimo Zeviani; Holger Prokisch

An isolated defect of respiratory chain complex I activity is a frequent biochemical abnormality in mitochondrial disorders. Despite intensive investigation in recent years, in most instances, the molecular basis underpinning complex I defects remains unknown. We report whole-exome sequencing of a single individual with severe, isolated complex I deficiency. This analysis, followed by filtering with a prioritization of mitochondrial proteins, led us to identify compound heterozygous mutations in ACAD9, which encodes a poorly understood member of the mitochondrial acyl-CoA dehydrogenase protein family. We demonstrated the pathogenic role of the ACAD9 variants by the correction of the complex I defect on expression of the wildtype ACAD9 protein in fibroblasts derived from affected individuals. ACAD9 screening of 120 additional complex I–defective index cases led us to identify two additional unrelated cases and a total of five pathogenic ACAD9 alleles.


Antioxidants & Redox Signaling | 2012

Mitochondrion-Derived Reactive Oxygen Species Lead to Enhanced Amyloid Beta Formation

Kristina Leuner; Tanja Schütt; Christopher Kurz; Schamim H. Eckert; Carola Schiller; Angelo Occhipinti; Sören Mai; Marina Jendrach; Gunter P. Eckert; Shane E. Kruse; Richard D. Palmiter; Ulrich Brandt; Stephan Dröse; Ilka Wittig; Michael Willem; Christian Haass; Andreas S. Reichert; Walter E. Müller

AIMS Intracellular amyloid beta (Aβ) oligomers and extracellular Aβ plaques are key players in the progression of sporadic Alzheimers disease (AD). Still, the molecular signals triggering Aβ production are largely unclear. We asked whether mitochondrion-derived reactive oxygen species (ROS) are sufficient to increase Aβ generation and thereby initiate a vicious cycle further impairing mitochondrial function. RESULTS Complex I and III dysfunction was induced in a cell model using the respiratory inhibitors rotenone and antimycin, resulting in mitochondrial dysfunction and enhanced ROS levels. Both treatments lead to elevated levels of Aβ. Presence of an antioxidant rescued mitochondrial function and reduced formation of Aβ, demonstrating that the observed effects depended on ROS. Conversely, cells overproducing Aβ showed impairment of mitochondrial function such as comprised mitochondrial respiration, strongly altered morphology, and reduced intracellular mobility of mitochondria. Again, the capability of these cells to generate Aβ was partly reduced by an antioxidant, indicating that Aβ formation was also ROS dependent. Moreover, mice with a genetic defect in complex I, or AD mice treated with a complex I inhibitor, showed enhanced Aβ levels in vivo. INNOVATION We show for the first time that mitochondrion-derived ROS are sufficient to trigger Aβ production in vitro and in vivo. CONCLUSION Several lines of evidence show that mitochondrion-derived ROS result in enhanced amyloidogenic amyloid precursor protein processing, and that Aβ itself leads to mitochondrial dysfunction and increased ROS levels. We propose that starting from mitochondrial dysfunction a vicious cycle is triggered that contributes to the pathogenesis of sporadic AD.


Proteomics | 2008

Features and applications of blue‐native and clear‐native electrophoresis

Ilka Wittig; Hermann Schägger

1‐D native electrophoresis is used for the separation of individual proteins, protein complexes, and supercomplexes. Stable and labile protein–protein interactions can be identified depending on detergent and buffer conditions. 1‐D native gels are immediately applicable for in‐gel detection of fluorescent‐labeled proteins and for in‐gel catalytic activity assays. 1‐D native gels and blots are used to determine native mass and oligomeric state of membrane proteins. Protein extracts from 1‐D native gels are used for generation of antibodies, for proteomic work, and for advanced structural investigations. 2‐D separation of subunits of protein complexes by SDS‐PAGE is mostly used for immunological and proteomic studies. Following the discussion of these general features, specific applications of native electrophoresis techniques in various research fields are highlighted: immunological and receptor studies, biogenesis and assembly of membrane protein complexes, protein import into organelles, dynamics of proteasomes, proteome and subproteome investigations, the identification and quantification of mitochondrial alterations in apoptosis, carcinogenesis, and neurodegenerative disorders like Parkinsons disease, Alzheimers disease, and the vast variety of mitochondrial encephalomyopathies.


Molecular and Cellular Biology | 2009

Human Ind1, an Iron-Sulfur Cluster Assembly Factor for Respiratory Complex I

Alex D. Sheftel; Oliver Stehling; Antonio J. Pierik; Daili J. A. Netz; Stefan Kerscher; Hans-Peter Elsässer; Ilka Wittig; Janneke Balk; Ulrich Brandt; Roland Lill

ABSTRACT Respiratory complex I (NADH:ubiquinone oxidoreductase) is a large mitochondrial inner membrane enzyme consisting of 45 subunits and 8 iron-sulfur (Fe/S) clusters. While complex I dysfunction is the most common reason for mitochondrial diseases, the assembly of complex I and its Fe/S cofactors remains elusive. Here, we identify the human mitochondrial P-loop NTPase, designated huInd1, that is critically required for the assembly of complex I. huInd1 can bind an Fe/S cluster via a conserved CXXC motif in a labile fashion. Knockdown of huInd1 in HeLa cells by RNA interference technology led to strong decreases in complex I protein and activity levels, remodeling of respiratory supercomplexes, and alteration of mitochondrial morphology. In addition, huInd1 depletion resulted in massive decreases in several subunits (NDUFS1, NDUFV1, NDUFS3, and NDUFA13) of the peripheral arm of complex I, with the concomitant appearance of a 450-kDa subcomplex representing part of the membrane arm. By a novel radiolabeling technique, the amount of iron associated with complex I was also shown to reflect the dependence of this enzyme on huInd1 for assembly. Together, these data identify huInd1 as a new assembly factor for human respiratory complex I with a possible role in the delivery of one or more Fe/S clusters to complex I subunits.


Biochimica et Biophysica Acta | 2009

Supramolecular organization of ATP synthase and respiratory chain in mitochondrial membranes.

Ilka Wittig; Hermann Schägger

Mitochondrial ATP synthase is mostly isolated in monomeric form, but in the inner mitochondrial membrane it seems to dimerize and to form higher oligomeric structures from dimeric building blocks. Following a period of electron microscopic single particle analyses that revealed an angular orientation of the membrane parts of monomeric ATP synthases in the dimeric structures, and after extensive studies of the monomer-monomer interface, the focus now shifts to the potentially dynamic state of the oligomeric structures, their potential involvement in metabolic regulation of mitochondria and cells, and to newly identified interactions like physical associations of complexes IV and V. Similarly, larger structures like respiratory strings that have been postulated to form from individual respiratory complexes and their supercomplexes, the respirasomes, come into the focus. Progress by structural investigations is paralleled by insights into the functional roles of respirasomes including substrate channelling and stabilization of individual complexes. Cardiolipin was found to be important for the structural stability of respirasomes which in turn is required to maintain cells and tissues in a healthy state. Defects in cardiolipin remodeling cause devastating diseases like Barth syndrome. Novel species-specific roles of respirasomes for the stability of respiratory complexes have been identified, and potential additional roles may be deduced from newly observed interactions of respirasomes with components of the protein import machinery and with the ADP/ATP translocator.


Journal of Medical Genetics | 2012

Molecular diagnosis in mitochondrial complex I deficiency using exome sequencing

Tobias B. Haack; Birgit Haberberger; Eva-Maria Frisch; Thomas Wieland; Arcangela Iuso; Matteo Gorza; Valentina Strecker; Elisabeth Graf; Johannes A. Mayr; U. Herberg; Julia B. Hennermann; Thomas Klopstock; Klaus A. Kuhn; Uwe Ahting; Wolfgang Sperl; Ekkehard Wilichowski; Georg F. Hoffmann; Marketa Tesarova; Hana Hansikova; Jiri Zeman; Barbara Plecko; Massimo Zeviani; Ilka Wittig; Tim M. Strom; Markus Schuelke; Peter Freisinger; Thomas Meitinger; Holger Prokisch

Background Next generation sequencing has become the core technology for gene discovery in rare inherited disorders. However, the interpretation of the numerous sequence variants identified remains challenging. We assessed the application of exome sequencing for diagnostics in complex I deficiency, a disease with vast genetic heterogeneity. Methods Ten unrelated individuals with complex I deficiency were selected for exome sequencing and sequential bioinformatic filtering. Cellular rescue experiments were performed to verify pathogenicity of novel disease alleles. Results The first filter criterion was ‘Presence of known pathogenic complex I deficiency variants’. This revealed homozygous mutations in NDUFS3 and ACAD9 in two individuals. A second criterion was ‘Presence of two novel potentially pathogenic variants in a structural gene of complex I’, which discovered rare variants in NDUFS8 in two unrelated individuals and in NDUFB3 in a third. Expression of wild-type cDNA in mutant cell lines rescued complex I activity and assembly, thus providing a functional validation of their pathogenicity. Using the third criterion ‘Presence of two potentially pathogenic variants in a gene encoding a mitochondrial protein’, loss-of-function mutations in MTFMT were discovered in two patients. In three patients the molecular genetic correlate remained unclear and follow-up analysis is ongoing. Conclusion Appropriate in silico filtering of exome sequencing data, coupled with functional validation of new disease alleles, is effective in rapidly identifying disease-causative variants in known and new complex I associated disease genes.


Journal of Biological Chemistry | 2003

Sequence-specific Peptide Aptamers, Interacting with the Intracellular Domain of the Epidermal Growth Factor Receptor, Interfere with Stat3 Activation and Inhibit the Growth of Tumor Cells

Claudia Buerger; Kerstin Nagel-Wolfrum; Christian Kunz; Ilka Wittig; Karin Butz; Felix Hoppe-Seyler; Bernd Groner

Receptor tyrosine kinases of the epidermal growth factor (EGF) receptor family regulate essential cellular functions such as proliferation, survival, migration, and differentiation but also play central roles in the etiology and progression of tumors. We have identified short peptide sequences from a random peptide library integrated into the thioredoxin scaffold protein, which specifically bind to the intracellular domain of the EGF receptor (EGFR). These molecules have the potential to selectively inhibit specific aspects of EGF receptor signaling and might become valuable as anticancer agents. Intracellular expression of the aptamer encoding gene construct KDI1 or introduction of bacterially expressed KDI1 via a protein transduction domain into EGFR-expressing cells results in KDI1·EGF receptor complex formation, a slower proliferation, and reduced soft agar colony formation. Aptamer KDI1 did not summarily block the EGF receptor tyrosine kinase activity but selectively interfered with the EGF-induced phosphorylation of the tyrosine residues 845, 1068, and 1148 as well as the phosphorylation of tyrosine 317 of p46 Shc. EGF-induced phosphorylation of Stat3 at tyrosine 705 and Stat3-dependent transactivation were also impaired. Transduction of a short synthetic peptide aptamer sequence not embedded into the scaffold protein resulted in the same impairment of EGF-induced Stat3 activation.

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Juliana Heidler

Goethe University Frankfurt

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Hermann Schägger

Goethe University Frankfurt

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Ulrich Brandt

Radboud University Nijmegen

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Heinrich Heide

Goethe University Frankfurt

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Valentina Strecker

Goethe University Frankfurt

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Stefan Dröse

Goethe University Frankfurt

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Mirco Steger

Goethe University Frankfurt

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Bernhard Brüne

Goethe University Frankfurt

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Irmgard Tegeder

Goethe University Frankfurt

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