bims-cytox1 2021-02-14 papers

Issue of 2021–02–14
five papers selected by
Gavin McStay, Staffordshire University

Cells. 2021 Feb 10. pii: 369. [Epub ahead of print]10(2):

Loss of COX4I1 Leads to Combined Respiratory Chain Deficiency and Impaired Mitochondrial Protein Synthesis.

Kristýna Čunátová, David Pajuelo Reguera, Marek Vrbacký, Erika Fernández-Vizarra, Shujing Ding, Ian M Fearnley, Massimo Zeviani, Josef Houštěk, Tomáš Mráček, Petr Pecina.

The oxidative phosphorylation (OXPHOS) system localized in the inner mitochondrial membrane secures production of the majority of ATP in mammalian organisms. Individual OXPHOS complexes form supramolecular assemblies termed supercomplexes. The complexes are linked not only by their function but also by interdependency of individual complex biogenesis or maintenance. For instance, cytochrome c oxidase (cIV) or cytochrome bc1 complex (cIII) deficiencies affect the level of fully assembled NADH dehydrogenase (cI) in monomeric as well as supercomplex forms. It was hypothesized that cI is affected at the level of enzyme assembly as well as at the level of cI stability and maintenance. However, the true nature of interdependency between cI and cIV is not fully understood yet. We used a HEK293 cellular model where the COX4 subunit was completely knocked out, serving as an ideal system to study interdependency of cI and cIV, as early phases of cIV assembly process were disrupted. Total absence of cIV was accompanied by profound deficiency of cI, documented by decrease in the levels of cI subunits and significantly reduced amount of assembled cI. Supercomplexes assembled from cI, cIII, and cIV were missing in COX4I1 knock-out (KO) due to loss of cIV and decrease in cI amount. Pulse-chase metabolic labeling of mitochondrial DNA (mtDNA)-encoded proteins uncovered a decrease in the translation of cIV and cI subunits. Moreover, partial impairment of mitochondrial protein synthesis correlated with decreased content of mitochondrial ribosomal proteins. In addition, complexome profiling revealed accumulation of cI assembly intermediates, indicating that cI biogenesis, rather than stability, was affected. We propose that attenuation of mitochondrial protein synthesis caused by cIV deficiency represents one of the mechanisms, which may impair biogenesis of cI.

Keywords: COX; COX4; OXPHOS; biogenesis interdependency; cI; cIV; cIV assembly; complex I; complexome profiling; knock-out; mitochondria; mitochondrial protein synthesis

DOI: https://doi.org/10.3390/cells10020369

NPJ Genom Med. 2020 Mar 02. 5(1): 7

The special considerations of gene therapy for mitochondrial diseases.

Jesse Slone, Taosheng Huang.

The recent success of gene therapy across multiple clinical trials has inspired a great deal of hope regarding the treatment of previously intractable genetic diseases. This optimism has been extended to the prospect of gene therapy for mitochondrial disorders, which are not only particularly severe but also difficult to treat. However, this hope must be tempered by the reality of the mitochondrial organelle, which possesses specific biological properties that complicate genetic manipulation. In this perspective, we will discuss some of these complicating factors, including the unique pathways used to express and import mitochondrial proteins. We will also present some ways in which these challenges can be overcome by genetic manipulation strategies tailored specifically for mitochondrial diseases.

DOI: https://doi.org/10.1038/s41525-020-0116-5

Neurochem Res. 2021 Feb 12.

Hypermethylation of Mitochondrial Cytochrome b and Cytochrome c Oxidase II Genes with Decreased Mitochondrial DNA Copy Numbers in the APP/PS1 Transgenic Mouse Model of Alzheimer's Disease.

Yingying Xu, Ling Cheng, Jing Sun, Fan Li, Xiangtian Liu, Yan Wei, Min Han, Zhengyu Zhu, Jianzhong Bi, Chao Lai, Yun Wang.

Alzheimer's disease (AD) is the most common cause of dementia. Increasing evidence shows that mitochondrial DNA (mtDNA) methylation plays an essential role in many diseases related to mitochondrial dysfunction. Since mitochondrial impairment is a key feature of AD, mtDNA methylation may also contribute to AD, but few studies have addressed this issue. Methylation changes of the mitochondrial cytochrome b (CYTB) and cytochrome c oxidase II (COX II) genes in AD have not been reported. We analyzed mtDNA methylation changes of the CYTB and COX II genes in an APP/PS1 transgenic mouse model of AD using pyrosequencing. We examined mtDNA copy numbers and the levels of expression by quantitative real-time PCR. Average methylation levels of different CpG sites were ≤ 4.0%. Methylated mtDNA accounted for only a small part of the total mtDNA. We also observed hypermethylation of mitochondrial CYTB and COX II genes with decreased mtDNA copy numbers and expression in the hippocampi of APP/PS1 transgenic mice. mtDNA methylation may play an important role in AD pathology, which may open a new window for AD therapy.

Keywords: Alzheimer’s disease; Electron transport chain; Mitochondrial DNA methylation; Mitoepigenetics; Pyrosequencing

DOI: https://doi.org/10.1007/s11064-020-03192-y

Biosci Biotechnol Biochem. 2020 Dec 28. pii: zbaa119. [Epub ahead of print]

Development of a mito-CRISPR system for generating mitochondrial DNA-deleted strain in Saccharomyces cerevisiae.

Takamitsu Amai, Tomoka Tsuji, Mitsuyoshi Ueda, Kouichi Kuroda.

Mitochondrial dysfunction can occur in a variety of ways, most often due to the deletion or mutation of mitochondrial DNA (mtDNA). The easy generation of yeasts with mtDNA deletion is attractive for analyzing the functions of the mtDNA gene. Treatment of yeasts with ethidium bromide is a well-known method for generating ρ° cells with complete deletion of mtDNA from Saccharomyces cerevisiae. However, the mutagenic effects of ethidium bromide on the nuclear genome cannot be excluded. In this study, we developed a "mito-CRISPR system" that specifically generates ρ° cells of yeasts. This system enabled the specific cleavage of mtDNA by introducing Cas9 fused with the mitochondrial target sequence at the N-terminus and guide RNA into mitochondria, resulting in the specific generation of ρ° cells in yeasts. The mito-CRISPR system provides a concise technology for deleting mtDNA in yeasts.

Keywords: CRISPR-Cas9 system; Saccharomyces cerevisiae; mitochondrial DNA; mitochondrial target sequence; ρ° cells

DOI: https://doi.org/10.1093/bbb/zbaa119

EMBO J. 2021 Feb 12. e106292

Interconnected assembly factors regulate the biogenesis of mitoribosomal large subunit.

Victor Tobiasson, Ondřej Gahura, Shintaro Aibara, Rozbeh Baradaran, Alena Zíková, Alexey Amunts.

Mitoribosomes consist of ribosomal RNA and protein components, coordinated assembly of which is critical for function. We used mitoribosomes from Trypanosoma brucei with reduced RNA and increased protein mass to provide insights into the biogenesis of the mitoribosomal large subunit. Structural characterization of a stable assembly intermediate revealed 22 assembly factors, some of which have orthologues/counterparts/homologues in mammalian genomes. These assembly factors form a protein network that spans a distance of 180 Å, shielding the ribosomal RNA surface. The central protuberance and L7/L12 stalk are not assembled entirely and require removal of assembly factors and remodeling of the mitoribosomal proteins to become functional. The conserved proteins GTPBP7 and mt-EngA are bound together at the subunit interface in proximity to the peptidyl transferase center. A mitochondrial acyl-carrier protein plays a role in docking the L1 stalk, which needs to be repositioned during maturation. Additional enzymatically deactivated factors scaffold the assembly while the exit tunnel is blocked. Together, this extensive network of accessory factors stabilizes the immature sites and connects the functionally important regions of the mitoribosomal large subunit.

Keywords: assembly; mitochondria; mitoribosome; translation; trypanosoma

DOI: https://doi.org/10.15252/embj.2020106292