Publications by authors named "Ulrich Brandt"

119 Publications

CEDAR, an online resource for the reporting and exploration of complexome profiling data.

Biochim Biophys Acta Bioenerg 2021 Mar 17;1862(7):148411. Epub 2021 Mar 17.

Center for Molecular and Biomolecular Informatics, Radboud Institute for Molecular Life Sciences, Radboud University Medical Center, Nijmegen, the Netherlands. Electronic address:

Complexome profiling is an emerging 'omics' approach that systematically interrogates the composition of protein complexes (the complexome) of a sample, by combining biochemical separation of native protein complexes with mass-spectrometry based quantitation proteomics. The resulting fractionation profiles hold comprehensive information on the abundance and composition of the complexome, and have a high potential for reuse by experimental and computational researchers. However, the lack of a central resource that provides access to these data, reported with adequate descriptions and an analysis tool, has limited their reuse. Therefore, we established the ComplexomE profiling DAta Resource (CEDAR, www3.cmbi.umcn.nl/cedar/), an openly accessible database for depositing and exploring mass spectrometry data from complexome profiling studies. Compatibility and reusability of the data is ensured by a standardized data and reporting format containing the "minimum information required for a complexome profiling experiment" (MIACE). The data can be accessed through a user-friendly web interface, as well as programmatically using the REST API portal. Additionally, all complexome profiles available on CEDAR can be inspected directly on the website with the profile viewer tool that allows the detection of correlated profiles and inference of potential complexes. In conclusion, CEDAR is a unique, growing and invaluable resource for the study of protein complex composition and dynamics across biological systems.
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http://dx.doi.org/10.1016/j.bbabio.2021.148411DOI Listing
March 2021

Investigation of central energy metabolism-related protein complexes of ANME-2d methanotrophic archaea by complexome profiling.

Biochim Biophys Acta Bioenerg 2021 01 28;1862(1):148308. Epub 2020 Sep 28.

Institute for Wetland and Water Research, Radboud University, Heyendaalseweg 135, 6525 AJ Nijmegen, the Netherlands. Electronic address:

The anaerobic oxidation of methane is important for mitigating emissions of this potent greenhouse gas to the atmosphere and is mediated by anaerobic methanotrophic archaea. In a 'Candidatus Methanoperedens BLZ2' enrichment culture used in this study, methane is oxidized to CO with nitrate being the terminal electron acceptor of an anaerobic respiratory chain. Energy conservation mechanisms of anaerobic methanotrophs have mostly been studied at metagenomic level and hardly any protein data is available at this point. To close this gap, we used complexome profiling to investigate the presence and subunit composition of protein complexes involved in energy conservation processes. All enzyme complexes and their subunit composition involved in reverse methanogenesis were identified. The membrane-bound enzymes of the respiratory chain, such as FH:quinone oxidoreductase, membrane-bound heterodisulfide reductase, nitrate reductases and Rieske cytochrome bc complex were all detected. Additional or putative subunits such as an octaheme subunit as part of the Rieske cytochrome bc complex were discovered that will be interesting targets for future studies. Furthermore, several soluble proteins were identified, which are potentially involved in oxidation of reduced ferredoxin produced during reverse methanogenesis leading to formation of small organic molecules. Taken together these findings provide an updated, refined picture of the energy metabolism of the environmentally important group of anaerobic methanotrophic archaea.
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http://dx.doi.org/10.1016/j.bbabio.2020.148308DOI Listing
January 2021

Effect of vitamin D supplementation on N-glycan branching and cellular immunophenotypes in MS.

Ann Clin Transl Neurol 2020 09 23;7(9):1628-1641. Epub 2020 Aug 23.

Charité - Universitätsmedizin Berlin, Corporate member of Freie Universität Berlin, Humboldt - Universität zu Berlin, and Berlin Institute of Health, NeuroCure Cluster of Excellence, Berlin, Germany.

Objective: To investigate the effect of cholecalciferol (vitamin D3) supplementation on peripheral immune cell frequency and N-glycan branching in patients with relapsing-remitting multiple sclerosis (RRMS).

Methods: Exploratory analysis of high-dose (20 400 IU) and low-dose (400 IU) vitamin D3 supplementation taken every other day of an 18-month randomized controlled clinical trial including 38 RRMS patients on stable immunomodulatory therapy (NCT01440062). We investigated cholecalciferol treatment effects on N-glycan branching using L-PHA stain (phaseolus vulgaris leukoagglutinin) at 6 months and frequencies of T-, B-, and NK-cell subpopulations at 12 months with flow cytometry.

Results: High-dose supplementation did not change CD3+ T cell subsets, CD19+ B cells subsets, and NK cells frequencies, except for CD8+ T regulatory cells, which were reduced in the low-dose arm compared to the high-dose arm at 12 months. High-dose supplementation decreased N-glycan branching on T and NK cells, measured as L-PHA mean fluorescence intensity (MFI). A reduction of N-glycan branching in B cells was not significant. In contrast, low-dose supplementation did not affect N-glycan branching. Changes in N-glycan branching did not correlate with cell frequencies.

Interpretation: Immunomodulatory effect of vitamin D may involve regulation of N-glycan branching in vivo. Vitamin D3 supplementation did at large not affect the frequencies of peripheral immune cells.
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http://dx.doi.org/10.1002/acn3.51148DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7480923PMC
September 2020

Novel defect in phosphatidylinositol 4-kinase type 2-alpha (PI4K2A) at the membrane-enzyme interface is associated with metabolic cutis laxa.

J Inherit Metab Dis 2020 11 26;43(6):1382-1391. Epub 2020 Jun 26.

Translational Metabolic Laboratory, Department of Laboratory Medicine, Radboud University Medical Center, Nijmegen, The Netherlands.

Inherited cutis laxa, or inelastic, sagging skin is a genetic condition of premature and generalised connective tissue ageing, affecting various elastic components of the extracellular matrix. Several cutis laxa syndromes are inborn errors of metabolism and lead to severe neurological symptoms. In a patient with cutis laxa, a choreoathetoid movement disorder, dysmorphic features and intellectual disability we performed exome sequencing to elucidate the underlying genetic defect. We identified the amino acid substitution R275W in phosphatidylinositol 4-kinase type IIα, caused by a homozygous missense mutation in the PI4K2A gene. We used lipidomics, complexome profiling and functional studies to measure phosphatidylinositol 4-phosphate synthesis in the patient and evaluated PI4K2A deficient mice to define a novel metabolic disorder. The R275W residue, located on the surface of the protein, is involved in forming electrostatic interactions with the membrane. The catalytic activity of PI4K2A in patient fibroblasts was severely reduced and lipid mass spectrometry showed that particular acyl-chain pools of PI4P and PI(4,5)P were decreased. Phosphoinositide lipids play a major role in intracellular signalling and trafficking and regulate the balance between proliferation and apoptosis. Phosphatidylinositol 4-kinases such as PI4K2A mediate the first step in the main metabolic pathway that generates PI4P, PI(4,5)P and PI(3,4,5)P . Although neurologic involvement is common, cutis laxa has not been reported previously in metabolic defects affecting signalling. Here we describe a patient with a complex neurological phenotype, premature ageing and a mutation in PI4K2A, illustrating the importance of this enzyme in the generation of inositol lipids with particular acylation characteristics.
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http://dx.doi.org/10.1002/jimd.12255DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7687218PMC
November 2020

TMEM70 functions in the assembly of complexes I and V.

Biochim Biophys Acta Bioenerg 2020 08 7;1861(8):148202. Epub 2020 Apr 7.

Department of Paediatrics, Radboud Centre for Mitochondrial Medicine, Radboud University Medical Centre, Nijmegen, the Netherlands.

Protein complexes from the oxidative phosphorylation (OXPHOS) system are assembled with the help of proteins called assembly factors. We here delineate the function of the inner mitochondrial membrane protein TMEM70, in which mutations have been linked to OXPHOS deficiencies, using a combination of BioID, complexome profiling and coevolution analyses. TMEM70 interacts with complex I and V and for both complexes the loss of TMEM70 results in the accumulation of an assembly intermediate followed by a reduction of the next assembly intermediate in the pathway. This indicates that TMEM70 has a role in the stability of membrane-bound subassemblies or in the membrane recruitment of subunits into the forming complex. Independent evidence for a role of TMEM70 in OXPHOS assembly comes from evolutionary analyses. The TMEM70/TMEM186/TMEM223 protein family, of which we show that TMEM186 and TMEM223 are mitochondrial in human as well, only occurs in species with OXPHOS complexes. Our results validate the use of combining complexome profiling with BioID and evolutionary analyses in elucidating congenital defects in protein complex assembly.
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http://dx.doi.org/10.1016/j.bbabio.2020.148202DOI Listing
August 2020

A salvage pathway maintains highly functional respiratory complex I.

Nat Commun 2020 04 2;11(1):1643. Epub 2020 Apr 2.

Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD) and Center for Molecular Medicine (CMMC), University of Cologne, 50931, Cologne, Germany.

Regulation of the turnover of complex I (CI), the largest mitochondrial respiratory chain complex, remains enigmatic despite huge advancement in understanding its structure and the assembly. Here, we report that the NADH-oxidizing N-module of CI is turned over at a higher rate and largely independently of the rest of the complex by mitochondrial matrix protease ClpXP, which selectively removes and degrades damaged subunits. The observed mechanism seems to be a safeguard against the accumulation of dysfunctional CI arising from the inactivation of the N-module subunits due to attrition caused by its constant activity under physiological conditions. This CI salvage pathway maintains highly functional CI through a favorable mechanism that demands much lower energetic cost than de novo synthesis and reassembly of the entire CI. Our results also identify ClpXP activity as an unforeseen target for therapeutic interventions in the large group of mitochondrial diseases characterized by the CI instability.
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http://dx.doi.org/10.1038/s41467-020-15467-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7118099PMC
April 2020

Complexome analysis of the nitrite-dependent methanotroph Methylomirabilis lanthanidiphila.

Biochim Biophys Acta Bioenerg 2019 09 31;1860(9):734-744. Epub 2019 Jul 31.

Microbial Physiology Group, Max Planck Institute for Marine Microbiology, Bremen, Germany. Electronic address:

The atmospheric concentration of the potent greenhouse gases methane and nitrous oxide (NO) has increased drastically during the last century. Methylomirabilis bacteria can play an important role in controlling the emission of these two gases from natural ecosystems, by oxidizing methane to CO and reducing nitrite to N without producing NO. These bacteria have an anaerobic metabolism, but are proposed to possess an oxygen-dependent pathway for methane activation. Methylomirabilis bacteria reduce nitrite to NO, and are proposed to dismutate NO into O and N by a putative NO dismutase (NO-D). The O produced in the cell can then be used to activate methane by a particulate methane monooxygenase. So far, the metabolic model of Methylomirabilis bacteria was based mainly on (meta)genomics and physiological experiments. Here we applied a complexome profiling approach to determine which of the proposed enzymes are actually expressed in Methylomirabilis lanthanidiphila. To validate the proposed metabolic model, we focused on enzymes involved in respiration, as well as nitrogen and carbon transformation. All complexes suggested to be involved in nitrite-dependent methane oxidation, were identified in M. lanthanidiphila, including the putative NO-D. Furthermore, several complexes involved in nitrate reduction/nitrite oxidation and NO reduction were detected, which likely play a role in detoxification and redox homeostasis. In conclusion, complexome profiling validated the expression and composition of enzymes hypothesized to be involved in the energy, methane and nitrogen metabolism of M. lanthanidiphila, thereby further corroborating their unique metabolism involved in the environmentally relevant process of nitrite-dependent methane oxidation.
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http://dx.doi.org/10.1016/j.bbabio.2019.07.011DOI Listing
September 2019

Adaptations of an ancient modular machine.

Authors:
Ulrich Brandt

Science 2019 01;363(6424):230-231

Radboud Institute for Molecular Life Sciences, Department of Pediatrics, Radboud University Medical Center, Geert Grooteplein-Zuid 10, Route 772, 6525 GA Nijmegen, Netherlands.

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http://dx.doi.org/10.1126/science.aaw0493DOI Listing
January 2019

COmplexome Profiling ALignment (COPAL) reveals remodeling of mitochondrial protein complexes in Barth syndrome.

Bioinformatics 2019 09;35(17):3083-3091

CMBI, Radboud Centre for Mitochondrial Medicine, Radboud Institute for Molecular Life Sciences, Radboud University Medical Center, Nijmegen, The Netherlands.

Motivation: Complexome profiling combines native gel electrophoresis with mass spectrometry to obtain the inventory, composition and abundance of multiprotein assemblies in an organelle. Applying complexome profiling to determine the effect of a mutation on protein complexes requires separating technical and biological variations from the variations caused by that mutation.

Results: We have developed the COmplexome Profiling ALignment (COPAL) tool that aligns multiple complexome profiles with each other. It includes the abundance profiles of all proteins on two gels, using a multi-dimensional implementation of the dynamic time warping algorithm to align the gels. Subsequent progressive alignment allows us to align multiple profiles with each other. We tested COPAL on complexome profiles from control mitochondria and from Barth syndrome (BTHS) mitochondria, which have a mutation in tafazzin gene that is involved in remodeling the inner mitochondrial membrane phospholipid cardiolipin. By comparing the variation between BTHS mitochondria and controls with the variation among either, we assessed the effects of BTHS on the abundance profiles of individual proteins. Combining those profiles with gene set enrichment analysis allows detecting significantly affected protein complexes. Most of the significantly affected protein complexes are located in the inner mitochondrial membrane (mitochondrial contact site and cristae organizing system, prohibitins), or are attached to it (the large ribosomal subunit).

Availability And Implementation: COPAL is written in python and is available from http://github.com/cmbi/copal.

Supplementary Information: Supplementary data are available at Bioinformatics online.
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http://dx.doi.org/10.1093/bioinformatics/btz025DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6735710PMC
September 2019

Locking loop movement in the ubiquinone pocket of complex I disengages the proton pumps.

Nat Commun 2018 10 29;9(1):4500. Epub 2018 Oct 29.

Radboud Institute for Molecular Life Sciences, Department of Pediatrics, Radboud University Medical Center, Geert Grooteplein-Zuid 10, 6525, GA, Nijmegen, The Netherlands.

Complex I (proton-pumping NADH:ubiquinone oxidoreductase) is the largest enzyme of the mitochondrial respiratory chain and a significant source of reactive oxygen species (ROS). We hypothesized that during energy conversion by complex I, electron transfer onto ubiquinone triggers the concerted rearrangement of three protein loops of subunits ND1, ND3, and 49-kDa thereby generating the power-stoke driving proton pumping. Here we show that fixing loop TMH1-2 to the nearby subunit PSST via a disulfide bridge introduced by site-directed mutagenesis reversibly disengages proton pumping without impairing ubiquinone reduction, inhibitor binding or the Active/Deactive transition. The X-ray structure of mutant complex I indicates that the disulfide bridge immobilizes but does not displace the tip of loop TMH1-2. We conclude that movement of loop TMH1-2 located at the ubiquinone-binding pocket is required to drive proton pumping corroborating one of the central predictions of our model for the mechanism of energy conversion by complex I proposed earlier.
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http://dx.doi.org/10.1038/s41467-018-06955-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6206036PMC
October 2018

Cryo-EM structure of respiratory complex I at work.

Elife 2018 10 2;7. Epub 2018 Oct 2.

Cluster of Excellence Macromolecular Complexes, Goethe University Frankfurt, Frankfurt, Germany.

Mitochondrial complex I has a key role in cellular energy metabolism, generating a major portion of the proton motive force that drives aerobic ATP synthesis. The hydrophilic arm of the L-shaped ~1 MDa membrane protein complex transfers electrons from NADH to ubiquinone, providing the energy to drive proton pumping at distant sites in the membrane arm. The critical steps of energy conversion are associated with the redox chemistry of ubiquinone. We report the cryo-EM structure of complete mitochondrial complex I from the aerobic yeast both in the deactive form and after capturing the enzyme during steady-state activity. The site of ubiquinone binding observed during turnover supports a two-state stabilization change mechanism for complex I.
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http://dx.doi.org/10.7554/eLife.39213DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6168287PMC
October 2018

Barth syndrome cells display widespread remodeling of mitochondrial complexes without affecting metabolic flux distribution.

Biochim Biophys Acta Mol Basis Dis 2018 11 1;1864(11):3650-3658. Epub 2018 Sep 1.

Laboratory Genetic Metabolic Diseases, Amsterdam UMC, University of Amsterdam, Amsterdam Gastroenterology & Metabolism, Amsterdam Cardiovascular Sciences, Meibergdreef 9, 1105 AZ Amsterdam, the Netherlands. Electronic address:

Barth syndrome (BTHS) is a rare X-linked disorder that is characterized by cardiac and skeletal myopathy, neutropenia and growth abnormalities. The disease is caused by mutations in the tafazzin (TAZ) gene encoding an enzyme involved in the acyl chain remodeling of the mitochondrial phospholipid cardiolipin (CL). Biochemically, this leads to decreased levels of mature CL and accumulation of the intermediate monolysocardiolipin (MLCL). At a cellular level, this causes mitochondrial fragmentation and reduced stability of the respiratory chain supercomplexes. However, the exact mechanism through which tafazzin deficiency leads to disease development remains unclear. We therefore aimed to elucidate the pathways affected in BTHS cells by employing proteomic and metabolic profiling assays. Complexome profiling of patient skin fibroblasts revealed significant effects for about 200 different mitochondrial proteins. Prominently, we found a specific destabilization of higher order oxidative phosphorylation (OXPHOS) supercomplexes, as well as changes in complexes involved in cristae organization and CL trafficking. Moreover, the key metabolic complexes 2-oxoglutarate dehydrogenase (OGDH) and branched-chain ketoacid dehydrogenase (BCKD) were profoundly destabilized in BTHS patient samples. Surprisingly, metabolic flux distribution assays using stable isotope tracer-based metabolomics did not show reduced flux through the TCA cycle. Overall, insights from analyzing the impact of TAZ mutations on the mitochondrial complexome provided a better understanding of the resulting functional and structural consequences and thus the pathological mechanisms leading to Barth syndrome.
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http://dx.doi.org/10.1016/j.bbadis.2018.08.041DOI Listing
November 2018

Bi-allelic Mutations in the Mitochondrial Ribosomal Protein MRPS2 Cause Sensorineural Hearing Loss, Hypoglycemia, and Multiple OXPHOS Complex Deficiencies.

Am J Hum Genet 2018 04 22;102(4):685-695. Epub 2018 Mar 22.

Department of Clinical Genomics, Mayo Clinic, Rochester, MN 55905, USA. Electronic address:

Biogenesis of the mitochondrial oxidative phosphorylation system, which produces the bulk of ATP for almost all eukaryotic cells, depends on the translation of 13 mtDNA-encoded polypeptides by mitochondria-specific ribosomes in the mitochondrial matrix. These mitoribosomes are dual-origin ribonucleoprotein complexes, which contain mtDNA-encoded rRNAs and tRNAs and ∼80 nucleus-encoded proteins. An increasing number of gene mutations that impair mitoribosomal function and result in multiple OXPHOS deficiencies are being linked to human mitochondrial diseases. Using exome sequencing in two unrelated subjects presenting with sensorineural hearing impairment, mild developmental delay, hypoglycemia, and a combined OXPHOS deficiency, we identified mutations in the gene encoding the mitochondrial ribosomal protein S2, which has not previously been implicated in disease. Characterization of subjects' fibroblasts revealed a decrease in the steady-state amounts of mutant MRPS2, and this decrease was shown by complexome profiling to prevent the assembly of the small mitoribosomal subunit. In turn, mitochondrial translation was inhibited, resulting in a combined OXPHOS deficiency detectable in subjects' muscle and liver biopsies as well as in cultured skin fibroblasts. Reintroduction of wild-type MRPS2 restored mitochondrial translation and OXPHOS assembly. The combination of lactic acidemia, hypoglycemia, and sensorineural hearing loss, especially in the presence of a combined OXPHOS deficiency, should raise suspicion for a ribosomal-subunit-related mitochondrial defect, and clinical recognition could allow for a targeted diagnostic approach. The identification of MRPS2 as an additional gene related to mitochondrial disease further expands the genetic and phenotypic spectra of OXPHOS deficiencies caused by impaired mitochondrial translation.
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http://dx.doi.org/10.1016/j.ajhg.2018.02.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5985281PMC
April 2018

Biallelic variants in WARS2 encoding mitochondrial tryptophanyl-tRNA synthase in six individuals with mitochondrial encephalopathy.

Hum Mutat 2017 12 6;38(12):1786-1795. Epub 2017 Oct 6.

Department of Pediatrics, Radboud Center for Mitochondrial Medicine, Radboud University Medical Center, Nijmegen, The Netherlands.

Mitochondrial protein synthesis involves an intricate interplay between mitochondrial DNA encoded RNAs and nuclear DNA encoded proteins, such as ribosomal proteins and aminoacyl-tRNA synthases. Eukaryotic cells contain 17 mitochondria-specific aminoacyl-tRNA synthases. WARS2 encodes mitochondrial tryptophanyl-tRNA synthase (mtTrpRS), a homodimeric class Ic enzyme (mitochondrial tryptophan-tRNA ligase; EC 6.1.1.2). Here, we report six individuals from five families presenting with either severe neonatal onset lactic acidosis, encephalomyopathy and early death or a later onset, more attenuated course of disease with predominating intellectual disability. Respiratory chain enzymes were usually normal in muscle and fibroblasts, while a severe combined respiratory chain deficiency was found in the liver of a severely affected individual. Exome sequencing revealed rare biallelic variants in WARS2 in all affected individuals. An increase of uncharged mitochondrial tRNA and a decrease of mtTrpRS protein content were found in fibroblasts of affected individuals. We hereby define the clinical, neuroradiological, and metabolic phenotype of WARS2 defects. This confidently implicates that mutations in WARS2 cause mitochondrial disease with a broad spectrum of clinical presentation.
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http://dx.doi.org/10.1002/humu.23340DOI Listing
December 2017

Mutations in ATP6V1E1 or ATP6V1A Cause Autosomal-Recessive Cutis Laxa.

Am J Hum Genet 2017 02 5;100(2):216-227. Epub 2017 Jan 5.

Translational Metabolic Laboratory, Department of Laboratory Medicine, Radboud University Medical Center, Nijmegen 6500 HB, the Netherlands. Electronic address:

Defects of the V-type proton (H) ATPase (V-ATPase) impair acidification and intracellular trafficking of membrane-enclosed compartments, including secretory granules, endosomes, and lysosomes. Whole-exome sequencing in five families affected by mild to severe cutis laxa, dysmorphic facial features, and cardiopulmonary involvement identified biallelic missense mutations in ATP6V1E1 and ATP6V1A, which encode the E1 and A subunits, respectively, of the V domain of the heteromultimeric V-ATPase complex. Structural modeling indicated that all substitutions affect critical residues and inter- or intrasubunit interactions. Furthermore, complexome profiling, a method combining blue-native gel electrophoresis and liquid chromatography tandem mass spectrometry, showed that they disturb either the assembly or the stability of the V-ATPase complex. Protein glycosylation was variably affected. Abnormal vesicular trafficking was evidenced by delayed retrograde transport after brefeldin A treatment and abnormal swelling and fragmentation of the Golgi apparatus. In addition to showing reduced and fragmented elastic fibers, the histopathological hallmark of cutis laxa, transmission electron microscopy of the dermis also showed pronounced changes in the structure and organization of the collagen fibers. Our findings expand the clinical and molecular spectrum of metabolic cutis laxa syndromes and further link defective extracellular matrix assembly to faulty protein processing and cellular trafficking caused by genetic defects in the V-ATPase complex.
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http://dx.doi.org/10.1016/j.ajhg.2016.12.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5294668PMC
February 2017

Compound heterozygosity for severe and hypomorphic mutations cause non-syndromic LHON-like optic neuropathy.

J Med Genet 2017 05 28;54(5):346-356. Epub 2016 Dec 28.

Laboratory of Genetics in Ophthalmology (LGO), INSERM UMR1163, Institute of Genetic Diseases, Imagine, Paris Descartes University, Paris, France.

Background: Non-syndromic hereditary optic neuropathy (HON) has been ascribed to mutations in mitochondrial fusion/fission dynamics genes, nuclear and mitochondrial DNA-encoded respiratory enzyme genes or nuclear genes of poorly known mitochondrial function. However, the disease causing gene remains unknown in many families. The objective of the present study was to identify the molecular cause of non-syndromic LHON-like disease in siblings born to non-consanguineous parents of French origin.

Methods: We used a combination of genetic analysis (gene mapping and whole-exome sequencing) in a multiplex family of non-syndromic HON and of functional analyses in patient-derived cultured skin fibroblasts and the yeast .

Results: We identified compound heterozygote disease-causing mutations (p.Tyr53Cys; p.Tyr308Cys). Studies using patient-derived cultured skin fibroblasts revealed mildly decreased NDUFS2 and complex I abundance but apparently normal respiratory chain activity. In the yeast ortholog , the mutations resulted in absence of complex I and moderate reduction in nicotinamide adenine dinucleotide-ubiquinone oxidoreductase activity, respectively.

Conclusions: Biallelism for mutations causing severe complex I deficiency has been previously reported to cause Leigh syndrome with optic neuropathy. Our results are consistent with the view that compound heterozygosity for severe and hypomorphic mutations can cause non-syndromic HON. This observation suggests a direct correlation between the severity of mutations and that of the disease and further support that there exist a genetic overlap between non-syndromic and syndromic HON due to defective mitochondrial function.
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http://dx.doi.org/10.1136/jmedgenet-2016-104212DOI Listing
May 2017

Cryo-EM structure of respiratory complex I reveals a link to mitochondrial sulfur metabolism.

Biochim Biophys Acta 2016 12 30;1857(12):1935-1942. Epub 2016 Sep 30.

Max Planck Institute of Biophysics, Department of Structural Biology, Max-von-Laue-Str. 3, 60438 Frankfurt am Main, Germany. Electronic address:

Mitochondrial complex I is a 1MDa membrane protein complex with a central role in aerobic energy metabolism. The bioenergetic core functions are executed by 14 central subunits that are conserved from bacteria to man. Despite recent progress in structure determination, our understanding of the function of the ~30 accessory subunits associated with the mitochondrial complex is still limited. We have investigated the structure of complex I from the aerobic yeast Yarrowia lipolytica by cryo-electron microscopy. Our density map at 7.9Å resolution closely matches the 3.6-3.9Å X-ray structure of the Yarrowia lipolytica complex. However, the cryo-EM map indicated an additional subunit on the side of the matrix arm above the membrane surface, pointing away from the membrane arm. The density, which is not present in any previously described complex I structure and occurs in about 20 % of the particles, was identified as the accessory sulfur transferase subunit ST1. The Yarrowia lipolytica complex I preparation is active in generating HS from the cysteine derivative 3-mercaptopyruvate, catalyzed by ST1. We thus provide evidence for a link between respiratory complex I and mitochondrial sulfur metabolism.
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http://dx.doi.org/10.1016/j.bbabio.2016.09.014DOI Listing
December 2016

The membrane scaffold SLP2 anchors a proteolytic hub in mitochondria containing PARL and the i-AAA protease YME1L.

EMBO Rep 2016 12 13;17(12):1844-1856. Epub 2016 Oct 13.

Institute for Genetics Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), and University of Cologne, Cologne, Germany

The SPFH (stomatin, prohibitin, flotillin, HflC/K) superfamily is composed of scaffold proteins that form ring-like structures and locally specify the protein-lipid composition in a variety of cellular membranes. Stomatin-like protein 2 (SLP2) is a member of this superfamily that localizes to the mitochondrial inner membrane (IM) where it acts as a membrane organizer. Here, we report that SLP2 anchors a large protease complex composed of the rhomboid protease PARL and the i-AAA protease YME1L, which we term the SPY complex (for SLP2-PARL-YME1L). Association with SLP2 in the SPY complex regulates PARL-mediated processing of PTEN-induced kinase PINK1 and the phosphatase PGAM5 in mitochondria. Moreover, SLP2 inhibits the stress-activated peptidase OMA1, which can bind to SLP2 and cleaves PGAM5 in depolarized mitochondria. SLP2 restricts OMA1-mediated processing of the dynamin-like GTPase OPA1 allowing stress-induced mitochondrial hyperfusion under starvation conditions. Together, our results reveal an important role of SLP2 membrane scaffolds for the spatial organization of IM proteases regulating mitochondrial dynamics, quality control, and cell survival.
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http://dx.doi.org/10.15252/embr.201642698DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5283581PMC
December 2016

The Assembly Pathway of Mitochondrial Respiratory Chain Complex I.

Cell Metab 2017 01 6;25(1):128-139. Epub 2016 Oct 6.

OXPHOS Biogenesis Group, Radboud Center for Mitochondrial Medicine, Department of Pediatrics, Radboud University Medical Center, Geert-Grooteplein Zuid 10, 6525 GA Nijmegen, the Netherlands. Electronic address:

Mitochondrial complex I is the largest integral membrane enzyme of the respiratory chain and consists of 44 different subunits encoded in the mitochondrial and nuclear genome. Its biosynthesis is a highly complicated and multifaceted process involving at least 14 additional assembly factors. How these subunits assemble into a functional complex I and where the assembly factors come into play is largely unknown. Here, we applied a dynamic complexome profiling approach to elucidate the assembly of human mitochondrial complex I and its further incorporation into respiratory chain supercomplexes. We delineate the stepwise incorporation of all but one subunit into a series of distinct assembly intermediates and their association with known and putative assembly factors, which had not been implicated in this process before. The resulting detailed and comprehensive model of complex I assembly is fully consistent with recent structural data and the remarkable modular architecture of this multiprotein complex.
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http://dx.doi.org/10.1016/j.cmet.2016.09.002DOI Listing
January 2017

The m-AAA Protease Associated with Neurodegeneration Limits MCU Activity in Mitochondria.

Mol Cell 2016 10 15;64(1):148-162. Epub 2016 Sep 15.

Institute for Genetics, Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), Center for Molecular Medicine (CMMC), University of Cologne, 50931 Cologne, Germany; Max Planck Institute for Biology of Aging, 50931 Cologne, Germany. Electronic address:

Mutations in subunits of mitochondrial m-AAA proteases in the inner membrane cause neurodegeneration in spinocerebellar ataxia (SCA28) and hereditary spastic paraplegia (HSP7). m-AAA proteases preserve mitochondrial proteostasis, mitochondrial morphology, and efficient OXPHOS activity, but the cause for neuronal loss in disease is unknown. We have determined the neuronal interactome of m-AAA proteases in mice and identified a complex with C2ORF47 (termed MAIP1), which counteracts cell death by regulating the assembly of the mitochondrial Ca uniporter MCU. While MAIP1 assists biogenesis of the MCU subunit EMRE, the m-AAA protease degrades non-assembled EMRE and ensures efficient assembly of gatekeeper subunits with MCU. Loss of the m-AAA protease results in accumulation of constitutively active MCU-EMRE channels lacking gatekeeper subunits in neuronal mitochondria and facilitates mitochondrial Ca overload, mitochondrial permeability transition pore opening, and neuronal death. Together, our results explain neuronal loss in m-AAA protease deficiency by deregulated mitochondrial Ca homeostasis.
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http://dx.doi.org/10.1016/j.molcel.2016.08.020DOI Listing
October 2016

Mutations in Complex I Assembly Factor TMEM126B Result in Muscle Weakness and Isolated Complex I Deficiency.

Am J Hum Genet 2016 Jul 30;99(1):208-16. Epub 2016 Jun 30.

INSERM U1163, Université Paris Descartes - Sorbonne Paris Cité, Institut Imagine, 75015 Paris, France. Electronic address:

Mitochondrial complex I deficiency results in a plethora of often severe clinical phenotypes manifesting in early childhood. Here, we report on three complex-I-deficient adult subjects with relatively mild clinical symptoms, including isolated, progressive exercise-induced myalgia and exercise intolerance but with normal later development. Exome sequencing and targeted exome sequencing revealed compound-heterozygous mutations in TMEM126B, encoding a complex I assembly factor. Further biochemical analysis of subject fibroblasts revealed a severe complex I deficiency caused by defective assembly. Lentiviral complementation with the wild-type cDNA restored the complex I deficiency, demonstrating the pathogenic nature of these mutations. Further complexome analysis of one subject indicated that the complex I assembly defect occurred during assembly of its membrane module. Our results show that TMEM126B defects can lead to complex I deficiencies and, interestingly, that symptoms can occur only after exercise.
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http://dx.doi.org/10.1016/j.ajhg.2016.05.022DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5005453PMC
July 2016

Preface to complex I special issue.

Biochim Biophys Acta 2016 Jul 21;1857(7):861-2. Epub 2016 Apr 21.

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http://dx.doi.org/10.1016/j.bbabio.2016.04.006DOI Listing
July 2016

Structure and function of mitochondrial complex I.

Biochim Biophys Acta 2016 Jul 24;1857(7):902-14. Epub 2016 Feb 24.

Structural Bioenergetics Group, Institute of Biochemistry II, Medical School, Goethe-University, Frankfurt am Main, Germany; Cluster of Excellence Frankfurt "Macromolecular Complexes, Goethe-University, Germany. Electronic address:

Proton-pumping NADH:ubiquinone oxidoreductase (complex I) is the largest and most complicated enzyme of the respiratory chain. Fourteen central subunits represent the minimal form of complex I and can be assigned to functional modules for NADH oxidation, ubiquinone reduction, and proton pumping. In addition, the mitochondrial enzyme comprises some 30 accessory subunits surrounding the central subunits that are not directly associated with energy conservation. Complex I is known to release deleterious oxygen radicals (ROS) and its dysfunction has been linked to a number of hereditary and degenerative diseases. We here review recent progress in structure determination, and in understanding the role of accessory subunits and functional analysis of mitochondrial complex I. For the central subunits, structures provide insight into the arrangement of functional modules including the substrate binding sites, redox-centers and putative proton channels and pump sites. Only for two of the accessory subunits, detailed structures are available. Nevertheless, many of them could be localized in the overall structure of complex I, but most of these assignments have to be considered tentative. Strikingly, redox reactions and proton pumping machinery are spatially completely separated and the site of reduction for the hydrophobic substrate ubiquinone is found deeply buried in the hydrophilic domain of the complex. The X-ray structure of complex I from Yarrowia lipolytica provides clues supporting the previously proposed two-state stabilization change mechanism, in which ubiquinone redox chemistry induces conformational states and thereby drives proton pumping. The same structural rearrangements may explain the active/deactive transition of complex I implying an integrated mechanistic model for energy conversion and regulation. This article is part of a Special Issue entitled Respiratory complex I, edited by Volker Zickermann and Ulrich Brandt.
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http://dx.doi.org/10.1016/j.bbabio.2016.02.013DOI Listing
July 2016

Hodgkin and Reed-Sternberg cells of classical Hodgkin lymphoma are highly dependent on oxidative phosphorylation.

Int J Cancer 2016 May 18;138(9):2231-46. Epub 2016 Jan 18.

Dr. Senckenberg Institute of Pathology, Goethe-University Hospital, Theodor-Stern-Kai 7, Frankfurt Am Main, 60596, Germany.

The metabolic properties of lymphomas derived from germinal center (GC) B cells have important implications for therapeutic strategies. In this study, we have compared metabolic features of Hodgkin-Reed-Sternberg (HRS) cells, the tumor cells of classical Hodgkin's lymphoma (cHL), one of the most frequent (post-)GC-derived B-cell lymphomas, with their normal GC B cell counterparts. We found that the ratio of oxidative to nonoxidative energy conversion was clearly shifted toward oxidative phosphorylation (OXPHOS)-linked ATP synthesis in HRS cells as compared to GC B cells. Mitochondrial mass, the expression of numerous key proteins of oxidative metabolism and markers of mitochondrial biogenesis were markedly upregulated in cHL cell lines and in primary cHL cases. NFkappaB promoted this shift to OXPHOS. Functional analysis indicated that both cell growth and viability of HRS cells depended on OXPHOS. The high rates of OXPHOS correlated with an almost complete lack of lactate production in HRS cells not observed in other GC B-cell lymphoma cell lines. Overall, we conclude that OXPHOS dominates energy conversion in HRS cells, while nonoxidative ATP production plays a subordinate role. Our results suggest that OXPHOS could be a new therapeutic target and may provide an avenue toward new treatment strategies in cHL.
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http://dx.doi.org/10.1002/ijc.29934DOI Listing
May 2016

Evolution and structural organization of the mitochondrial contact site (MICOS) complex and the mitochondrial intermembrane space bridging (MIB) complex.

Biochim Biophys Acta 2016 Jan 23;1863(1):91-101. Epub 2015 Oct 23.

Nijmegen Centre for Mitochondrial Disorders, Radboud University Medical Center, Nijmegen, The Netherlands; Cluster of Excellence Frankfurt "Macromolecular Complexes", Goethe-University, Germany.

We have analyzed the distribution of mitochondrial contact site and cristae organizing system (MICOS) complex proteins and mitochondrial intermembrane space bridging complex (MIB) proteins over (sub)complexes and over species. The MICOS proteins are associated with the formation and maintenance of mitochondrial cristae. Indeed, the presence of MICOS genes in genomes correlates well with the presence of cristae: all cristae containing species have at least one MICOS gene and cristae-less species have none. Mic10 is the most widespread MICOS gene, while Mic60 appears be the oldest one, as it originates in the ancestors of mitochondria, the proteobacteria. In proteobacteria the gene occurs in clusters with genes involved in heme synthesis while the protein has been observed in intracellular membranes of the alphaproteobacterium Rhodobacter sphaeroides. In contrast, Mic23 and Mic27 appear to be the youngest MICOS proteins, as they only occur in opisthokonts. The remaining MICOS proteins, Mic10, Mic19, Mic25 and Mic12, the latter we show to be orthologous to human C19orf70/QIL1, trace back to the root of the eukaryotes. Of the remaining MIB proteins, also DNAJC11 shows a high correlation with the presence of cristae. In mitochondrial protein complexome profiles, the MIB complex occurs as a defined complex and as separate subcomplexes, potentially reflecting various assembly stages. We find three main forms of the complex: A) The MICOS complex, containing all the MICOS proteins, B) a membrane bridging subcomplex, containing in addition SAMM50, MTX2 and the previously uncharacterized MTX3, and C) the complete MIB complex containing in addition DNAJC11 and MTX1.
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http://dx.doi.org/10.1016/j.bbamcr.2015.10.009DOI Listing
January 2016

Statin-Induced Myopathy Is Associated with Mitochondrial Complex III Inhibition.

Cell Metab 2015 Sep;22(3):399-407

Department of Pharmacology and Toxicology, Radboud University Medical Center, Nijmegen 6500HB, the Netherlands; Center for Systems Biology and Bioenergetics, Nijmegen Center for Mitochondrial Disorders, Radboud University Medical Center, Nijmegen 6500HB, the Netherlands. Electronic address:

Cholesterol-lowering statins effectively reduce the risk of major cardiovascular events. Myopathy is the most important adverse effect, but its underlying mechanism remains enigmatic. In C2C12 myoblasts, several statin lactones reduced respiratory capacity and appeared to be strong inhibitors of mitochondrial complex III (CIII) activity, up to 84% inhibition. The lactones were in general three times more potent inducers of cytotoxicity than their corresponding acid forms. The Qo binding site of CIII was identified as off-target of the statin lactones. These findings could be confirmed in muscle tissue of patients suffering from statin-induced myopathies, in which CIII enzyme activity was reduced by 18%. Respiratory inhibition in C2C12 myoblasts could be attenuated by convergent electron flow into CIII, restoring respiration up to 89% of control. In conclusion, CIII inhibition was identified as a potential off-target mechanism associated with statin-induced myopathies.
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http://dx.doi.org/10.1016/j.cmet.2015.08.002DOI Listing
September 2015

Accessory NUMM (NDUFS6) subunit harbors a Zn-binding site and is essential for biogenesis of mitochondrial complex I.

Proc Natl Acad Sci U S A 2015 May 20;112(18):5685-90. Epub 2015 Apr 20.

Structural Bioenergetics Group, Institute of Biochemistry II, Medical School, Goethe University, 60438 Frankfurt am Main, Germany; Cluster of Excellence Frankfurt "Macromolecular Complexes", Goethe University, 60438 Frankfurt am Main, Germany;

Mitochondrial proton-pumping NADH:ubiquinone oxidoreductase (respiratory complex I) comprises more than 40 polypeptides and contains eight canonical FeS clusters. The integration of subunits and insertion of cofactors into the nascent complex is a complicated multistep process that is aided by assembly factors. We show that the accessory NUMM subunit of complex I (human NDUFS6) harbors a Zn-binding site and resolve its position by X-ray crystallography. Chromosomal deletion of the NUMM gene or mutation of Zn-binding residues blocked a late step of complex I assembly. An accumulating assembly intermediate lacked accessory subunit N7BM (NDUFA12), whereas a paralog of this subunit, the assembly factor N7BML (NDUFAF2), was found firmly bound instead. EPR spectroscopic analysis and metal content determination after chromatographic purification of the assembly intermediate showed that NUMM is required for insertion or stabilization of FeS cluster N4.
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http://dx.doi.org/10.1073/pnas.1424353112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4426432PMC
May 2015

Structural biology. Mechanistic insight from the crystal structure of mitochondrial complex I.

Science 2015 Jan;347(6217):44-9

Cluster of Excellence Frankfurt "Macromolecular Complexes," Goethe-University, 60438 Frankfurt am Main, Germany. Nijmegen Center for Mitochondrial Disorders, Radboud University Medical Center, 6525 GA Nijmegen, Netherlands.

Proton-pumping complex I of the mitochondrial respiratory chain is among the largest and most complicated membrane protein complexes. The enzyme contributes substantially to oxidative energy conversion in eukaryotic cells. Its malfunctions are implicated in many hereditary and degenerative disorders. We report the x-ray structure of mitochondrial complex I at a resolution of 3.6 to 3.9 angstroms, describing in detail the central subunits that execute the bioenergetic function. A continuous axis of basic and acidic residues running centrally through the membrane arm connects the ubiquinone reduction site in the hydrophilic arm to four putative proton-pumping units. The binding position for a substrate analogous inhibitor and blockage of the predicted ubiquinone binding site provide a model for the "deactive" form of the enzyme. The proposed transition into the active form is based on a concerted structural rearrangement at the ubiquinone reduction site, providing support for a two-state stabilization-change mechanism of proton pumping.
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http://dx.doi.org/10.1126/science.1259859DOI Listing
January 2015

Generator-specific targets of mitochondrial reactive oxygen species.

Free Radic Biol Med 2015 Jan 29;78:1-10. Epub 2014 Oct 29.

Molecular Bioenergetics Group, Goethe-University, D-60590 Frankfurt am Main, Germany; Clinic of Anaesthesiology, Intensive Care Medicine and Pain Therapy, Goethe-University Hospital, Frankfurt am Main, Germany. Electronic address:

To understand the role of reactive oxygen species (ROS) in oxidative stress and redox signaling it is necessary to link their site of generation to the oxidative modification of specific targets. Here we have studied the selective modification of protein thiols by mitochondrial ROS that have been implicated as deleterious agents in a number of degenerative diseases and in the process of biological aging, but also as important players in cellular signal transduction. We hypothesized that this bipartite role might be based on different generator sites for "signaling" and "damaging" ROS and a directed release into different mitochondrial compartments. Because two main mitochondrial ROS generators, complex I (NADH:ubiquinone oxidoreductase) and complex III (ubiquinol:cytochrome c oxidoreductase; cytochrome bc1 complex), are known to predominantly release superoxide and the derived hydrogen peroxide (H2O2) into the mitochondrial matrix and the intermembrane space, respectively, we investigated whether these ROS generators selectively oxidize specific protein thiols. We used redox fluorescence difference gel electrophoresis analysis to identify redox-sensitive targets in the mitochondrial proteome of intact rat heart mitochondria. We observed that the modified target proteins were distinctly different when complex I or complex III was employed as the source of ROS. These proteins are potential targets involved in mitochondrial redox signaling and may serve as biomarkers to study the generator-dependent dual role of mitochondrial ROS in redox signaling and oxidative stress.
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http://dx.doi.org/10.1016/j.freeradbiomed.2014.10.511DOI Listing
January 2015