bims-cytox1 2023-02-26 papers

bims-cytox1

Biomed News

on Cytochrome oxidase subunit 1

Issue of 2023–02–26
fiveteen papers selected by
Gavin McStay, Liverpool John Moores University

The Mba1 homologue of Trypanosoma brucei is involved in the biogenesis of oxidative phosphorylation complexes.
Investigation of oxidative phosphorylation activity and complex composition in mitochondrial disease.
Regulatory principles of human mitochondrial gene expression revealed by kinetic analysis of the RNA life cycle.
High-throughput Assessment of Mitochondrial Protein Synthesis in Mammalian Cells Using Mito-FUNCAT FACS.
Leigh syndrome.
Genetics of mitochondrial diseases: Current approaches for the molecular diagnosis.
Studying Mitochondrial Nucleic Acid Synthesis Utilizing Intact Isolated Mitochondria.
HIG1 domain family member 1A is a crucial regulator of disorders associated with hypoxia.
Isolation and Quality Control of Yeast Mitochondria.
Neuroimaging in mitochondrial disease.
Creation of Mitochondrial Disease Models Using Mitochondrial DNA Editing.
Manipulation of Murine Mitochondrial DNA Heteroplasmy with mtZFNs.
Peripheral neuropathy in mitochondrial disease.
Experimental therapy for mitochondrial diseases.
Many OX PHOS and replication factor mRNAs target mitochondria through specific binding to the organelle surface, independent of cotranslational protein import.

Mol Microbiol. 2023 Feb 24.

The Mba1 homologue of Trypanosoma brucei is involved in the biogenesis of oxidative phosphorylation complexes.

Christoph Wenger, Anke Harsman, Moritz Niemann, Silke Oeljeklaus, Corinne von Känel, Salvatore Calderaro, Bettina Warscheid, André Schneider.

Consistent with other eukaryotes, the Trypanosoma brucei mitochondrial genome encodes mainly hydrophobic core subunits of the oxidative phosphorylation system. These proteins must be co-translationally inserted into the inner mitochondrial membrane and are synthesized by the highly unique trypanosomal mitoribosomes, which have a much higher protein to RNA ratio than any other ribosome. Here, we show that the trypanosomal ortholog of the mitoribosome receptor Mba1 (TbMba1) is essential for normal growth of procyclic trypanosomes but redundant in the bloodstream form, which lacks an oxidative phosphorylation system. Proteomic analyses of TbMba1-depleted mitochondria from procyclic cells revealed reduced levels of many components of the oxidative phosphorylation system, most of which belong to the cytochrome c oxidase (Cox) complex, three subunits of which are mitochondrially encoded. However, the integrity of the mitoribosome and its interaction with the inner membrane were not affected. Pulldown experiments showed that TbMba1 forms a dynamic interaction network that includes the trypanosomal Mdm38/Letm1 ortholog and a trypanosome-specific factor that stabilizes the CoxI and CoxII mRNAs. In summary, our study suggests that the function of Mba1 in the biogenesis of membrane subunits of OXPHOS complexes is conserved among yeast, mammals, and trypanosomes, which belong to two eukaryotic supergroups.

DOI: https://doi.org/10.1111/mmi.15048
Handb Clin Neurol. 2023 ;pii: B978-0-12-821751-1.00008-7. [Epub ahead of print]194 127-139

Investigation of oxidative phosphorylation activity and complex composition in mitochondrial disease.

Kyle Thompson, David A Stroud, David R Thorburn, Robert W Taylor.

  A multidisciplinary approach to the laboratory diagnosis of mitochondrial disease has long been applied, with crucial information provided by deep clinical phenotyping, blood investigations, and biomarker screening as well as histopathological and biochemical testing of biopsy material to support molecular genetic screening. In an era of second and third generation sequencing technologies, traditional diagnostic algorithms for mitochondrial disease have been replaced by gene agnostic, genomic strategies including whole-exome sequencing (WES) and whole-genome sequencing (WGS), increasingly supported by other 'omics technologies (Alston et al., 2021). Whether a primary testing strategy, or one used to validate and interpret candidate genetic variants, the availability of a range of tests aimed at determining mitochondrial function (i.e., the assessment of individual respiratory chain enzyme activities in a tissue biopsy or cellular respiration in a patient cell line) remains an important part of the diagnostic armory. In this chapter, we summarize several disciplines used in the laboratory investigation of suspected mitochondrial disease, including the histopathological and biochemical assessment of mitochondrial function, as well as protein-based techniques to assess the steady-state levels of oxidative phosphorylation (OXPHOS) subunits and assembly of OXPHOS complexes via traditional (immunoblotting) and cutting-edge (quantitative proteomic) approaches.

Keywords:  Mitochondrial disease; Muscle pathology; Next-generation sequencing; Oxidative phosphorylation; Proteomics

DOI:  https://doi.org/10.1016/B978-0-12-821751-1.00008-7
bioRxiv. 2023 Feb 15. pii: 2023.02.09.527880. [Epub ahead of print]

Regulatory principles of human mitochondrial gene expression revealed by kinetic analysis of the RNA life cycle.

Erik McShane, Mary Couvillion, Robert Ietswaart, Gyan Prakash, Brendan M Smalec, Iliana Soto, Autum R Baxter-Koenigs, Karine Choquet, L Stirling Churchman.

Mitochondria play critical roles in cellular metabolism, primarily by serving as the site of assembly and function of the oxidative phosphorylation (OXPHOS) machinery. The OXPHOS proteins are encoded by mitochondrial DNA (mtDNA) and nuclear DNA, which reside and are regulated within separate compartments. To unravel how the two gene expression systems collaborate to produce the OXPHOS complexes, the regulatory principles controlling the production of mtDNA-encoded proteins need to be elucidated. In this study, we performed a quantitative analysis of the mitochondrial messenger RNA (mt-mRNA) life cycle to determine which steps of gene expression experience strong regulatory control. Our analysis revealed that the high accumulation of mt-mRNA despite their rapid turnover was made possible by a 700-fold higher transcriptional output than nuclear-encoded OXPHOS genes. In addition, we observed that mt-mRNA processing and its association with the mitochondrial ribosome occur rapidly and that these processes are linked mechanistically. Based on these data, we developed a model of mtDNA expression that is predictive across human cell lines, revealing that differences in turnover and translation efficiency are the major contributors to mitochondrial-encoded protein synthesis. Applying this framework to a disease model of Leigh syndrome, French-Canadian type, we found that the disease-associated nuclear-encoded gene, LRPPRC , acts predominantly by stabilizing mt-mRNA. Our findings provide a comprehensive view of the intricate regulatory mechanisms governing mtDNA-encoded protein synthesis, highlighting the importance of quantitatively analyzing the mitochondrial RNA life cycle in order to decode the regulatory principles of mtDNA expression.

DOI: https://doi.org/10.1101/2023.02.09.527880
Bio Protoc. 2023 Feb 05. pii: e4602. [Epub ahead of print]13(3):

High-throughput Assessment of Mitochondrial Protein Synthesis in Mammalian Cells Using Mito-FUNCAT FACS.

Hironori Saito, Tatsuya Osaki, Yoshiho Ikeuchi, Shintaro Iwasaki.

  In addition to cytosolic protein synthesis, mitochondria also utilize another translation system that is tailored for mRNAs encoded in the mitochondrial genome. The importance of mitochondrial protein synthesis has been exemplified by the diverse diseases associated with in organello translation deficiencies. Various methods have been developed to monitor mitochondrial translation, such as the classic method of labeling newly synthesized proteins with radioisotopes and the more recent ribosome profiling. However, since these methods always assess the average cell population, measuring the mitochondrial translation capacity in individual cells has been challenging. To overcome this issue, we recently developed mito-fluorescent noncanonical amino acid tagging (FUNCAT) fluorescence-activated cell sorting (FACS), which labels nascent peptides generated by mitochondrial ribosomes with a methionine analog, L-homopropargylglycine (HPG), conjugates the peptides with fluorophores by an in situ click reaction, and detects the signal in individual cells by FACS equipment. With this methodology, the hidden heterogeneity of mitochondrial translation in cell populations can be addressed.

Keywords:   FACS ; FUNCAT ; Mitochondria ; Mitoribosome ; Translation

DOI:  https://doi.org/10.21769/BioProtoc.4602
Handb Clin Neurol. 2023 ;pii: B978-0-12-821751-1.00015-4. [Epub ahead of print]194 43-63

Leigh syndrome.

Shamima Rahman.

  Leigh syndrome, or subacute necrotizing encephalomyelopathy, was initially recognized as a neuropathological entity in 1951. Bilateral symmetrical lesions, typically extending from the basal ganglia and thalamus through brainstem structures to the posterior columns of the spinal cord, are characterized microscopically by capillary proliferation, gliosis, severe neuronal loss, and relative preservation of astrocytes. Leigh syndrome is a pan-ethnic disorder usually with onset in infancy or early childhood, but late-onset forms occur, including in adult life. Over the last six decades it has emerged that this complex neurodegenerative disorder encompasses more than 100 separate monogenic disorders associated with enormous clinical and biochemical heterogeneity. This chapter discusses clinical, biochemical and neuropathological aspects of the disorder, and postulated pathomechanisms. Known genetic causes, including defects of 16 mitochondrial DNA (mtDNA) genes and approaching 100 nuclear genes, are categorized into disorders of subunits and assembly factors of the five oxidative phosphorylation enzymes, disorders of pyruvate metabolism and vitamin and cofactor transport and metabolism, disorders of mtDNA maintenance, and defects of mitochondrial gene expression, protein quality control, lipid remodeling, dynamics, and toxicity. An approach to diagnosis is presented, together with known treatable causes and an overview of current supportive management options and emerging therapies on the horizon.

Keywords:  Diagnosis; Genetics; History; Leigh syndrome; Neuropathology; Pathomechanisms; Subacute necrotizing encephalomyelopathy; Treatment

DOI:  https://doi.org/10.1016/B978-0-12-821751-1.00015-4
Handb Clin Neurol. 2023 ;pii: B978-0-12-821751-1.00011-7. [Epub ahead of print]194 141-165

Genetics of mitochondrial diseases: Current approaches for the molecular diagnosis.

Lea D Schlieben, Holger Prokisch.

  Mitochondrial diseases are a genetically and phenotypically variable set of monogenic disorders. The main characteristic of mitochondrial diseases is a defective oxidative phosphorylation. Both nuclear and mitochondrial DNA encode the approximately 1500 mitochondrial proteins. Since identification of the first mitochondrial disease gene in 1988 a total of 425 genes have been associated with mitochondrial diseases. Mitochondrial dysfunctions can be caused both by pathogenic variants in the mitochondrial DNA or the nuclear DNA. Hence, besides maternal inheritance, mitochondrial diseases can follow all modes of Mendelian inheritance. The maternal inheritance and tissue specificity distinguish molecular diagnostics of mitochondrial disorders from other rare disorders. With the advances made in the next-generation sequencing technology, whole exome sequencing and even whole-genome sequencing are now the established methods of choice for molecular diagnostics of mitochondrial diseases. They reach a diagnostic rate of more than 50% in clinically suspected mitochondrial disease patients. Moreover, next-generation sequencing is delivering a constantly growing number of novel mitochondrial disease genes. This chapter reviews mitochondrial and nuclear causes of mitochondrial diseases, molecular diagnostic methodologies, and their current challenges and perspectives.

Keywords:  Diagnostic; Genetic; Heteroplasmy; Mitochondrial DNA; Mitochondrial disease; Multi-omic; Mutation

DOI:  https://doi.org/10.1016/B978-0-12-821751-1.00011-7
Methods Mol Biol. 2023 ;2615 219-228

Studying Mitochondrial Nucleic Acid Synthesis Utilizing Intact Isolated Mitochondria.

Jelena Misic, Dusanka Milenkovic.

  Mitochondria are eukaryotic organelles of endosymbiotic origin that contain their own genetic material, mitochondrial DNA (mtDNA), and dedicated systems for mtDNA maintenance and expression. MtDNA molecules encode a limited number of proteins that are nevertheless all essential subunits of the mitochondrial oxidative phosphorylation system. Here, we describe protocols to monitor DNA and RNA synthesis in intact, isolated mitochondria. These in organello synthesis protocols are valuable techniques for studying the mechanisms and regulation of mtDNA maintenance and expression.

Keywords:  Mitochondria; Radioactive labeling of nucleic acids; in organello replication and transcription; mtDNA; mtDNA maintenance and expression

DOI:  https://doi.org/10.1007/978-1-0716-2922-2_16
Mitochondrion. 2023 Feb 16. pii: S1567-7249(23)00018-1. [Epub ahead of print]

HIG1 domain family member 1A is a crucial regulator of disorders associated with hypoxia.

Yin-Zhao Jia, Jing Liu, Geng-Qiao Wang, Hao Pan, Tie-Zeng Huang, Ran Liu, Yong Zhang.

  Mitochondria play a central role in cellular energy conversion, metabolism, and cell proliferation. The regulation of mitochondrial function by HIGD1A, which is located on the inner membrane of the mitochondria, is essential to maintain cell survival under hypoxic conditions. In recent years, there have been shown other cellular pathways and mechanisms involving HIGD1A diametrically or through its interaction. As a novel regulator, HIGD1A maintains mitochondrial integrity and enhances cell viability under hypoxic conditions, increasing cell resistance to hypoxia. HIGD1A mainly targets cytochrome c oxidase by regulating downstream signaling pathways, which affects the ATP generation system and subsequently alters mitochondrial respiratory function. In addition, HIGD1A plays a dual role in cell survival in distinct degree hypoxia regions of the tumor. Under mild and moderate anoxic areas, HIGD1A acts as a positive regulator to promote cell growth. However, HIGD1A plays a role in inhibiting cell growth but retaining cellular activity under severe anoxic areas. We speculate that HIGD1A engages in tumor recurrence and drug resistance mechanisms. This review will focus on data concerning how HIGD1A regulates cell viability under hypoxic conditions. Therefore, HIGD1A could be a potential therapeutic target for hypoxia-related diseases.

Keywords:  HIGD1A; apoptosis; cytochrome c oxidase; hypoxia; mitochondria; tumor

DOI:  https://doi.org/10.1016/j.mito.2023.02.009
Methods Mol Biol. 2023 ;2615 41-55

Isolation and Quality Control of Yeast Mitochondria.

Asli Aras Taskin, Daiana Nerina Moretti, F Nora Vögtle, Chris Meisinger.

  The isolation of organelles devoid of other cellular compartments is crucial for studying organellar proteomes and the localization of newly identified proteins, as well as for assessing specific organellar functions. Here, we describe a protocol for the isolation of crude and highly pure mitochondria from Saccharomyces cerevisiae and provide methods for testing the functional integrity of the isolated organelles.

Keywords:  Differential centrifugation; Membrane potential; Mitochondria; Osmotic swelling; Protein import; Saccharomyces cerevisiae; Sucrose-gradient purification

DOI:  https://doi.org/10.1007/978-1-0716-2922-2_4
Handb Clin Neurol. 2023 ;pii: B978-0-12-821751-1.00016-6. [Epub ahead of print]194 173-185

Neuroimaging in mitochondrial disease.

Felix Distelmaier, Thomas Klopstock.

  The anatomic complexity of the brain in combination with its high energy demands makes this organ specifically vulnerable to defects of mitochondrial oxidative phosphorylation. Therefore, neurodegeneration is a hallmark of mitochondrial diseases. The nervous system of affected individuals typically shows selective regional vulnerability leading to distinct patterns of tissue damage. A classic example is Leigh syndrome, which causes symmetric alterations of basal ganglia and brain stem. Leigh syndrome can be caused by different genetic defects (>75 known disease genes) with variable disease onset ranging from infancy to adulthood. Other mitochondrial diseases are characterized by focal brain lesions, which is a core feature of MELAS syndrome (mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes). Apart from gray matter, also white matter can be affected by mitochondrial dysfunction. White matter lesions vary depending on the underlying genetic defect and may progress into cystic cavities. In view of the recognizable patterns of brain damage in mitochondrial diseases, neuroimaging techniques play a key role in diagnostic work-up. In the clinical setting, magnetic resonance imaging (MRI) and MR spectroscopy (MRS) are the mainstay of diagnostic work-up. Apart from visualization of brain anatomy, MRS allows the detection of metabolites such as lactate, which is of specific interest in the context of mitochondrial dysfunction. However, it is important to note that findings like symmetric basal ganglia lesions on MRI or a lactate peak on MRS are not specific, and that there is a broad range of disorders that can mimic mitochondrial diseases on neuroimaging. In this chapter, we will review the spectrum of neuroimaging findings in mitochondrial diseases and discuss important differential diagnoses. Moreover, we will give an outlook on novel biomedical imaging tools that may provide interesting insights into mitochondrial disease pathophysiology.

Keywords:  Brain; Central nervous system; Leigh disease; Magnetic resonance imaging; Neurodegeneration; OXPHOS

DOI:  https://doi.org/10.1016/B978-0-12-821751-1.00016-6
Biomedicines. 2023 Feb 12. pii: 532. [Epub ahead of print]11(2):

Creation of Mitochondrial Disease Models Using Mitochondrial DNA Editing.

Victoria A Khotina, Andrey Y Vinokurov, Mariam Bagheri Ekta, Vasily N Sukhorukov, Alexander N Orekhov.

  Mitochondrial diseases are a large class of human hereditary diseases, accompanied by the dysfunction of mitochondria and the disruption of cellular energy synthesis, that affect various tissues and organ systems. Mitochondrial DNA mutation-caused disorders are difficult to study because of the insufficient number of clinical cases and the challenges of creating appropriate models. There are many cellular models of mitochondrial diseases, but their application has a number of limitations. The most proper and promising models of mitochondrial diseases are animal models, which, unfortunately, are quite rare and more difficult to develop. The challenges mainly arise from the structural features of mitochondria, which complicate the genetic editing of mitochondrial DNA. This review is devoted to discussing animal models of human mitochondrial diseases and recently developed approaches used to create them. Furthermore, this review discusses mitochondrial diseases and studies of metabolic disorders caused by the mitochondrial DNA mutations underlying these diseases.

Keywords:  animal model; cellular model; gene editing; mitochondrial diseases; mitochondrial mutations

DOI:  https://doi.org/10.3390/biomedicines11020532
Methods Mol Biol. 2023 ;2615 329-344

Manipulation of Murine Mitochondrial DNA Heteroplasmy with mtZFNs.

Pavel A Nash, Michal Minczuk.

  Mouse models of mitochondrial DNA mutations hold promise in the development and optimization of mitochondrial gene therapy technology and for gathering pre-clinical data prior to human trials. Their suitability for this purpose stems from the high similarity of human and murine mitochondrial genomes and the increasing availability of rationally designed AAV vectors capable of selectively transducing murine tissues. Our laboratory routinely optimizes mitochondrially targeted zinc finger nucleases (mtZFNs), the compactness of which makes them highly suitable for downstream AAV-based in vivo mitochondrial gene therapy. This chapter discusses the necessary precautions for the robust and precise genotyping of the murine mitochondrial genome as well as the optimization of mtZFNs intended for subsequent use in vivo.

Keywords:  Gene therapy; Heteroplasmy; MEF; Mitochondria; Mouse; Zinc Finger nuclease; mtDNA; mtZFN

DOI:  https://doi.org/10.1007/978-1-0716-2922-2_23
Handb Clin Neurol. 2023 ;pii: B978-0-12-821751-1.00014-2. [Epub ahead of print]194 99-116

Peripheral neuropathy in mitochondrial disease.

Rita Horvath, Jessica Medina, Mary M Reilly, Michael E Shy, Stephan Zuchner.

  Mitochondria are essential for the health and viability of both motor and sensory neurons and their axons. Processes that disrupt their normal distribution and transport along axons will likely cause peripheral neuropathies. Similarly, mutations in mtDNA or nuclear encoded genes result in neuropathies that either stand alone or are part of multisystem disorders. This chapter focuses on the more common genetic forms and characteristic clinical phenotypes of "mitochondrial" peripheral neuropathies. We also explain how these various mitochondrial abnormalities cause peripheral neuropathy. In a patient with a neuropathy either due to a mutation in a nuclear or an mtDNA gene, clinical investigations aim to characterize the neuropathy and make an accurate diagnosis. In some patients, this may be relatively straightforward, where a clinical assessment and nerve conduction studies followed by genetic testing is all that is needed. In others, multiple investigations including a muscle biopsy, CNS imaging, CSF analysis, and a wide range of metabolic and genetic tests in blood and muscle may be needed to establish diagnosis.

Keywords:  Axonal transport; Charcot–Marie–Tooth disease; Mitochondrial DNA; Mitochondrial fusion/fission; Nuclear mitochondrial genes; Respiratory chain enzymes

DOI:  https://doi.org/10.1016/B978-0-12-821751-1.00014-2
Handb Clin Neurol. 2023 ;pii: B978-0-12-821751-1.00013-0. [Epub ahead of print]194 259-277

Experimental therapy for mitochondrial diseases.

Carlo Viscomi, Massimo Zeviani.

  Mitochondrial diseases are extremely heterogeneous genetic disorders due to faulty oxidative phosphorylation (OxPhos). No cure is currently available for these conditions, beside supportive interventions aimed at relieving complications. Mitochondria are under a double genetic control carried out by the mitochondrial DNA (mtDNA) and by nuclear DNA. Thus, not surprisingly, mutations in either genome can cause mitochondrial disease. Although mitochondria are usually associated with respiration and ATP synthesis, they play fundamental roles in a large number of other biochemical, signaling, and execution pathways, each being a potential target for therapeutic interventions. These can be classified as general therapies, i.e., potentially applicable to a number of different mitochondrial conditions, or therapies tailored to a single disease, i.e., personalized approaches, such as gene therapy, cell therapy, and organ replacement. Mitochondrial medicine is a particularly lively research field, and the last few years witnessed a steady increase in the number of clinical applications. This chapter will present the most recent therapeutic attempts emerged from preclinical work and an update of the currently ongoing clinical applications. We think that we are starting a new era in which the etiologic treatment of these conditions is becoming a realistic option.

Keywords:  AAV; Gene therapy; Mitochondrial biogenesis; Mitochondrial disease; Mitophagy; OxPhos; Rapamycin

DOI:  https://doi.org/10.1016/B978-0-12-821751-1.00013-0
J Genet. 2023 ;pii: 20. [Epub ahead of print]102

Many OX PHOS and replication factor mRNAs target mitochondria through specific binding to the organelle surface, independent of cotranslational protein import.

Samit Adhya, Milon Banik.

A large number of nucleus-encoded messenger RNAs (mRNAs) encoding proteins involved in oxidative phosphorylation have been found to be associated with mitochondria in vivo, indicating organelle-specific mRNA targeting. However, the identification of mitochondrion-bound mRNA (Mtb-RNA) has traditionally relied on cumbersome isolations of polysomes from a large number of input cells and is therefore biased in favour of mRNAs associated through nascent targeting peptides emerging from the polysome during cotranslational import of their encoded proteins, and tends to ignore sequence-directed mRNA targeting. We have, therefore, sought to identify and quantify Mtb-RNAs rapidly in small numbers of cells, independently of their polysomal status. We isolated Mtb-RNAs from tissue-cultured cells under different conditions and assayed them by endpoint or real-time polymerase chain reaction (RT-PCR). We observed that (i) different Mtb-RNAs are differentially affected by cycloheximide-induced polysome arrest, indicating possible artifacts of the use of this translation elongation inhibitor; (ii) several Mtb-RNAs have direct affinity for the mitochondrial surface in vitro, indicating the possibility of targeting through mRNA recognition by surface-bound RNA-binding proteins (RBP); and (iii) mRNA-mitochondrion interactions are stabilized by formaldehyde crosslinking. Our results reveal the importance of sequence-directed targeting of mRNAs to mitochondria.