bims-cytox1 2020-03-15 papers

Issue of 2020–03–15
four papers selected by
Gavin McStay, Staffordshire University

Front Genet. 2020 ;11 24

Novel MT-ND Gene Variants Causing Adult-Onset Mitochondrial Disease and Isolated Complex I Deficiency.

Yi Shiau Ng, Kyle Thompson, Daniela Loher, Sila Hopton, Gavin Falkous, Steven A Hardy, Andrew M Schaefer, Sandip Shaunak, Mark E Roberts, James B Lilleker, Robert W Taylor.

Mitochondrial complex I deficiency is associated with a diverse range of clinical phenotypes and can arise due to either mitochondrial DNA (mtDNA) or nuclear gene defects. We investigated two adult patients who exhibited non-syndromic neurological features and evidence of isolated mitochondrial complex I deficiency in skeletal muscle biopsies. The first presented with indolent myopathy, progressive since age 17, while the second developed deafness around age 20 and other relapsing-remitting neurological symptoms since. A novel, likely de novo, frameshift variant in MT-ND6 (m.14512_14513del) and a novel maternally-inherited transversion mutation in MT-ND1 were identified, respectively. Skewed tissue segregation of mutant heteroplasmy level was observed; the mutant heteroplasmy levels of both variants were greater than 70% in muscle homogenate, however, in blood the MT-ND6 variant was undetectable while the mutant heteroplasmy level of the MT-ND1 variant was low (12%). Assessment of complex I assembly by Blue-Native PAGE demonstrated a decrease in fully assembled complex I in the muscle of both cases. SDS-PAGE and immunoblotting showed decreased levels of mtDNA-encoded ND1 and several nuclear encoded complex I subunits in both cases, consistent with functional pathogenic consequences of the identified variants. Pathogenicity of the m.14512_14513del was further corroborated by single-fiber segregation studies.

Keywords: deafness; mitochondrial DNA; muscle biopsy; myopathy; tissue segregation

DOI: https://doi.org/10.3389/fgene.2020.00024

Nat Commun. 2020 Mar 11. 11(1): 1312

Mitochondrial peptide BRAWNIN is essential for vertebrate respiratory complex III assembly.

The emergence of small open reading frame (sORF)-encoded peptides (SEPs) is rapidly expanding the known proteome at the lower end of the size distribution. Here, we show that the mitochondrial proteome, particularly the respiratory chain, is enriched for small proteins. Using a prediction and validation pipeline for SEPs, we report the discovery of 16 endogenous nuclear encoded, mitochondrial-localized SEPs (mito-SEPs). Through functional prediction, proteomics, metabolomics and metabolic flux modeling, we demonstrate that BRAWNIN, a 71 a.a. peptide encoded by C12orf73, is essential for respiratory chain complex III (CIII) assembly. In human cells, BRAWNIN is induced by the energy-sensing AMPK pathway, and its depletion impairs mitochondrial ATP production. In zebrafish, Brawnin deletion causes complete CIII loss, resulting in severe growth retardation, lactic acidosis and early death. Our findings demonstrate that BRAWNIN is essential for vertebrate oxidative phosphorylation. We propose that mito-SEPs are an untapped resource for essential regulators of oxidative metabolism.

DOI: https://doi.org/10.1038/s41467-020-14999-2

Biol Methods Protoc. 2018 ;3(1): bpy008

Utilization of dried and long-term stored polyacrylamide gels for the advanced proteomic profiling of mitochondrial contact sites from rat liver.

Sandra Murphy, Michael Henry, Paula Meleady, Kay Ohlendieck.

Following subcellular fractionation, the complexity of proteins derived from a particular cellular compartment is often evaluated by gel electrophoretic analysis. For the proteomic cataloguing of these distinct protein populations and their biochemical characterization, gel electrophoretic protein separation can be conveniently combined with liquid chromatography mass spectrometry. Here we describe a gel-enhanced liquid chromatography mass spectrometry (GeLC-MS)/MS approach with a new bioanalytical focus on the proteomic profiling of mitochondrial contact sites from rat liver using the highly sensitive Orbitrap Fusion Tribrid mass spectrometer for optimum protein identification following extraction from dried and long-term stored gels. Mass spectrometric analysis identified 964 protein species in the mitochondrial contact site fraction, whereby 459 proteins were identified by ≥3 unique peptides. This included mitochondrial components of the supramolecular complexes that form the ATP synthase, the respiratory chain, ribosomal subunits and the cytochrome P450 system, as well as crucial components of the translocase complexes translocase of the inner membrane (TIM) and translocase of the outer membrane (TOM) of the two mitochondrial membranes. Proteomics also identified contact site markers, such as glutathione transferase, monoamine oxidase and the pore protein voltage dependent anion channel (VDAC)-1. Hence, this report demonstrates that the GeLC-MS/MS method can be used to study complex mixtures of proteins that have been embedded and stored in dried polyacrylamide gels for a long period of time. Careful re-swelling and standard in-gel digestion is suitable to produce peptide profiles from old gels that can be used to extract sophisticated proteomic maps and enable the subsequent bioinformatics analysis of the distribution of protein function and the determination of potential protein clustering within the contact site system.

Keywords: MICOS; TIM; TOM; VDAC; contact sites; mitochondria

DOI: https://doi.org/10.1093/biomethods/bpy008

Mol Genet Genomic Med. 2020 Mar 12. e1199

Systematic analysis of a mitochondrial disease-causing ND6 mutation in mitochondrial deficiency.

Deyu Chen, Qiongya Zhao, Jingting Xiong, Xiaoting Lou, Qinxia Han, Xiujuan Wei, Jie Xie, Xueyun Li, Huaibin Zhou, Lijun Shen, Yanling Yang, Hezhi Fang, Jianxin Lyu.

BACKGROUND: The m.14487T>C mutation is recognized as a diagnostic mutation of mitochondrial disease during the past 16 years, emerging evidence suggests that mutant loads of m.14487T>C and disease phenotype are not closely correlated.
METHODS: Immortalized lymphocytes were generated by coculturing the Epstein-Barr virus and lymphocytes from m.14487T>C carrier Chinese patient with Leigh syndrome. Fifteen cytoplasmic hybrid (cybrid) cell lines were generated by fusing mtDNA lacking 143B cells with platelets donated by patients. Mitochondrial function was systematically analyzed at transcriptomic, metabolomic, and biochemical levels.
RESULTS: Unlike previous reports, we found that the assembly of mitochondrial respiratory chain complexes, mitochondrial respiration, and mitochondrial OXPHOS function was barely affected in cybrid cells carrying homoplastic m.14487T>C mutation. Mitochondrial dysfunction associated transcriptomic and metabolomic reprogramming were not detected in cybrid carrying homoplastic m.14487T>C. However, we found that mitochondrial function was impaired in patient-derived immortalized lymphocytes.
CONCLUSION: Our data revealed that m.14487T>C mutation is insufficient to cause mitochondrial deficiency; additional modifier genes may be involved in m.14487T>C-associated mitochondrial disease. Our results further demonstrated that a caution should be taken by solely use of m.14487T>C mutation for molecular diagnosis of mitochondrial disease.

Keywords: cybrids; mitochondrial disease; mtDNA mutation; transcriptome and metabolic analyses

DOI: https://doi.org/10.1002/mgg3.1199