Ulrich Brandt

Statin-Induced Myopathy Is Associated with Mitochondrial Complex III Inhibition

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.

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

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.

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

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 thexa0cause 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 Ca2+ 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 Ca2+ overload, mitochondrial permeability transition pore opening, and neuronal death. Together, our results explain neuronal loss in m-AAA protease deficiency by deregulated mitochondrial Ca2+ homeostasis.

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

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.

NOVA: a software to analyze complexome profiling data

SUMMARYnWe introduce nova, a software for the analysis of complexome profiling data. nova supports the investigation of the composition of complexes, cluster analysis of the experimental data, visual inspection and comparison of experiments and many other features.nnnAVAILABILITY AND IMPLEMENTATIONnnova is licensed under the Artistic License 2.0. It is freely available at http://www.bioinformatik.uni-frankfurt.de. nova requires at least Java 7 and runs under Linux, Microsoft Windows and Mac OS.nnnCONTACTnina.koch@bioinformatik.uni-frankfurt.de.

Mutations in ATP6V1E1 or ATP6V1A Cause Autosomal-Recessive Cutis Laxa

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 V1 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.

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

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.

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

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.

Cluster analysis and visualization techniques for large datasets in complexome profiling

Dysfunctional protein complexes are often associated with diseases. To develop effective treatments it is essential to understand the composition, formation and functionality of protein complexes. Novel techniques like complexome profiling give an overview of possible protein-protein interactions in an entire sample. In this approach intact protein complexes are separated using blue-native electrophoresis. The migration patterns of thousands of proteins are then uncovered using quantitative mass spectrometry and compared to find co-migrating proteins. Here, we present the concepts of our visualization approach for large complexome profiling datasets using our software NOVA. In agreement with recent literature we show that the protein NDUFA4, a previously known subunit of complex I of the mitochondrial respiratory chain, is instead a subunit of complex IV.

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

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.