Identification and functional analysis of mitochondrial complex I assembly factor homologues in C. elegans

The biogenesis of mitochondrial NADH:ubiquinone oxidoreductase (complex I) requires several assembly chaperones. These so-called complex I assembly factors have emerged as a new class of human disease genes. Here, we identified putative assembly factor homologues in Caenorhabditis elegans. We demonstrate that two candidates (C50B8.3/NUAF-1, homologue of NDUFAF1 and R07H5.3/NUAF-3, homologue of NDUFAF3) clearly affect complex I function. Assembly factor deficient worms were shorter, showed a diminished brood size and displayed reduced fat content. Our results suggest that mitochondrial complex I biogenesis is evolutionarily conserved. Moreover, Caenorhabditis elegans appears to be a promising model organism to study assembly factor related human diseases.     

Understanding mitochondrial complex I assembly in health and disease

Complex I (NADH:ubiquinone oxidoreductase) is the largest multimeric enzyme complex of the mitochondrial respiratory chain, which is responsible for electron transport and the generation of a proton gradient across the mitochondrial inner membrane to drive ATP production. Eukaryotic complex I consists of 14 conserved subunits, which are homologous to the bacterial subunits, and more than 26 accessory subunits. In mammals, complex I consists of 45 subunits, which must be assembled correctly to form the properly functioning mature complex. Complex I dysfunction is the most common oxidative phosphorylation (OXPHOS) disorder in humans and defects in the complex I assembly process are often observed. This assembly process has been difficult to characterize because of its large size, the lack of a high resolution structure for complex I, and its dual control by nuclear and mitochondrial DNA. However, in recent years, some of the atomic structure of the complex has been resolved and new insights into complex I assembly have been generated. Furthermore, a number of proteins have been identified as assembly factors for complex I biogenesis and many patients carrying mutations in genes associated with complex I deficiency and mitochondrial diseases have been discovered. Here, we review the current knowledge of the eukaryotic complex I assembly process and new insights from the identification of novel assembly factors. This article is part of a Special Issue entitled: Biogenesis/Assembly of Respiratory Enzyme Complexes.     

Assembly of mitochondrial complex I and defects in disease

Isolated complex I deficiency is the most common cause of respiratory chain dysfunction. Defects in human complex I result in energy generation disorders and they are also implicated in neurodegenerative disease and altered apoptotic signaling. Complex I dysfunction often occurs as a result of its impaired assembly. The assembly process of complex I is poorly understood, complicated by the fact that in mammals, it is composed of 45 different subunits and is regulated by both nuclear and mitochondrial genomes. However, in recent years we have gained new insights into complex I biogenesis and a number of assembly factors involved in this process have also been identified. In most cases, these factors have been discovered through their gene mutations that lead to specific complex I defects and result in mitochondrial disease. Here we review how complex I is assembled and the factors required to mediate this process.     

Biogenesis of iron-sulfur clusters in mammalian cells: new insights and relevance to human disease

Iron-sulfur (Fe-S) clusters are ubiquitous cofactors composed of iron and inorganic sulfur. They are required for the function of proteins involved in a wide range of activities, including electron transport in respiratory chain complexes, regulatory sensing, photosynthesis and DNA repair. The proteins involved in the biogenesis of Fe-S clusters are evolutionarily conserved from bacteria to humans, and many insights into the process of Fe-S cluster biogenesis have come from studies of model organisms, including bacteria, fungi and plants. It is now clear that several rare and seemingly dissimilar human diseases are attributable to defects in the basic process of Fe-S cluster biogenesis. Although these diseases –which include Friedreich’s ataxia (FRDA), ISCU myopathy, a rare form of sideroblastic anemia, an encephalomyopathy caused by dysfunction of respiratory chain complex I and multiple mitochondrial dysfunctions syndrome – affect different tissues, a feature common to many of them is that mitochondrial iron overload develops as a secondary consequence of a defect in Fe-S cluster biogenesis. This Commentary outlines the basic steps of Fe-S cluster biogenesis as they have been defined in model organisms. In addition, it draws attention to refinements of the process that might be specific to the subcellular compartmentalization of Fe-S cluster biogenesis proteins in some eukaryotes, including mammals. Finally, it outlines several important unresolved questions in the field that, once addressed, should offer important clues into how mitochondrial iron homeostasis is regulated, and how dysfunction in Fe-S cluster biogenesis can contribute to disease.     

Osteoblastic differentiation improved by bezafibrate-induced mitochondrial biogenesis in deciduous tooth-derived pulp stem cells from a child with Leigh syndrome

Leigh syndrome is a highly heterogeneous condition caused by pathological mutations in either nuclear or mitochondrial DNA regions encoding molecules involved in mitochondrial oxidative phosphorylation, in which many organs including the brain can be affected. Among these organs, a high incidence of poor bone health has been recognized in primary mitochondrial diseases including Leigh syndrome. However, the direct association between mitochondrial dysfunction and poor bone health has not been fully elucidated. Mitochondrial biosynthesis is a potential therapeutic target for this syndrome, as it can ameliorate the impairment of oxidative phosphorylation without altering these gene mutations. A recent study has shown the impaired osteogenesis in the dental pulp stem cells derived from the deciduous teeth of a child with Leigh syndrome, harboring the heteroplasmic mutation G13513A in the mitochondrial DNA region encoding the ND5 subunit of the respiratory chain complex I. The present study aimed to investigate whether mitochondrial biogenesis could be a therapeutic target for improving osteogenesis, using the same stem cells in a patient-specific cellular model. For this purpose, bezafibrate was used because it has been reported to induce mitochondrial biogenesis as well as to improve bone metabolism and osteoporosis. Bezafibrate clearly improved the differentiation of patient-derived stem cells into osteoblasts and the mineralization of differentiated osteoblasts. The mRNA expression of peroxisome proliferator-activated receptor-gamma coactivator-1α, ATP production, and mitochondrial Ca²⁺ levels were all significantly increased by bezafibrate in the patient-derived cells. In addition, the increased amount and morphological shift from the fragmentary to network shape associated with DRP1 downregulation were also observed in the bezafibrate-treated patient-derived cells. These results suggest that mitochondrial biogenesis may be a potential therapeutic target for improving osteogenesis in patients with Leigh syndrome, and bezafibrate may be one of the candidate treatment agents.     

酵母线粒体ATP合酶生物合成及组装机制研究进展

线粒体ATP合酶是线粒体氧化磷酸化的关键酶,其功能缺陷会导致能量代谢障碍相关的线粒体疾病。线粒体ATP合酶是由多个亚基组成的蛋白复合物,其生物合成和组装是个复杂的生物过程。酵母是研究线粒体ATP合酶结构、生物合成和组装机制的模式实验材料之一,且相关研究取得了很多进展。本文概述了国内外用酿酒酵母研究线粒体ATP合酶的结构、调控线粒体ATP合酶亚基生物合成和组装的辅助蛋白及合酶的模块化组装过程的研究进展,以期为线粒体ATP合酶的工作机制及相关线粒体疾病的研究提供理论借鉴和参考依据。     

Brain mitochondrial dysfunction and oxidative damage in Parkinson’s disease

Complex factors contribute to the appearance of Parkinson’s disease (PD), but with a constant mitochondrial involvement. There are two interdependent conditions in PD: brain mitochondrial dysfunction and brain mitochondrial oxidative damage. Mitochondrial dysfunction and reduced complex I activity are recognized in substantia nigra and in frontal cortex in PD patients. The molecular mechanism involved in the inactivation of complex I is likely accounted by the sum of ONOO⁻ mediated reactions, reactions with free radical intermediates of the lipid peroxidation process and amine-aldehyde adduction reactions. The inhibitory effects on complex I lead synergistically to denaturation of the protein structure and to further increases of O₂⁻ and ONOO⁻ production at the vicinity of complex I. An adaptive response in PD patients has been described with increases in mtNOS activity, mitochondrial mass and mitochondrial biogenesis. Mitochondrial dysfunction in the human frontal cortex is to be considered a factor contributing to impaired cognition in PD.     

From NADH to ubiquinone in Neurospora mitochondria

The respiratory chain of the mitochondrial inner membrane includes a proton-pumping enzyme, complex I, which catalyses electron transfer from NADH to ubiquinone. This electron pathway occurs through a series of protein-bound prosthetic groups, FMN and around eight iron-sulfur clusters. The high number of polypeptide subunits of mitochondrial complex I, around 40, have a dual genetic origin. Neurospora crassa has been a useful genetic model to characterise complex I. The characterisation of mutants in specific proteins helped to understand the elaborate processes of the biogenesis, structure and function of the oligomeric enzyme. In the fungus, complex I seems to be dispensable for vegetative growth but required for sexual development. N. crassa mitochondria also contain three to four nonproton-pumping alternative NAD(P)H dehydrogenases. One of them is located in the outer face of the inner mitochondrial membrane, working as a calcium-dependent oxidase of cytosolic NADPH.     

The role of PGC-1 coactivators in aging skeletal muscle and heart

Aging is the progressive decline in cellular, tissue, and organ function. This complex process often manifests as loss of muscular strength, cardiovascular function, and cognitive ability. Mitochondrial dysfunction and decreased mitochondrial biogenesis are believed to participate in metabolic abnormalities and loss of organ function, which will eventually contribute to aging and decreased lifespan. In this review, we discuss what is currently known about mitochondrial dysfunction in the aging skeletal muscle and heart. We focused our discussion on the role of PGC-1 coactivators in the regulation of mitochondrial biogenesis and function and possible therapeutic benefits of increased mitochondrial biogenesis in compensating for mitochondrial dysfunction and circumventing aging and aging-related diseases.     

Molecular base of biochemical complex I deficiency

The oxidative phosphorylation (OXPHOS) system, consisting of five enzyme complexes (I-V) together with 2 electron carriers, has an important role in the energy metabolism of the cell. With 45 subunits, complex I is the first and largest complex of the respiratory chain. It is under bigenomic control and a proper interaction between the mitochondrial and the nuclear genome is important for a good biogenesis and functioning of the complex. Isolated complex I deficiency is the most frequently diagnosed form of mitochondrial disorders caused by the disturbance of the OXPHOS system. It has a wide clinical variety and, at present, in many patients the underlying genetic cause of the complex I deficiency is still not known. In this review, the role of complex I in the oxidative phosphorylation and the localization and function of the different complex I subunits will be described. Furthermore, a brief overview of the assembly process and biochemical studies, performed when a patient is suspected of a mitochondrial disorder is given. Finally, the present knowledge for molecular base of complex I deficiency is described and the findings in a research cohort of patients with complex I deficiency are reported. Identifying new genes encoding proteins involved in complex I biogenesis is challenging and in the near future new powerful techniques will make high throughput screening possible. Progress in elucidating the genetic defect causing complex I deficiencies is important for a better genetic counseling, prenatal diagnostic possibilities and further development of new treatment strategies to cure the complex I deficiencies in the future.     

Dual function of GTPBP6 in biogenesis and recycling of human mitochondrial ribosomes

Translation and ribosome biogenesis in mitochondria require auxiliary factors that ensure rapid and accurate synthesis of mitochondrial proteins. Defects in translation are associated with oxidative phosphorylation deficiency and cause severe human diseases, but the exact roles of mitochondrial translation-associated factors are not known. Here we identify the functions of GTPBP6, a homolog of the bacterial ribosome-recycling factor HflX, in human mitochondria. Similarly to HflX, GTPBP6 facilitates the dissociation of ribosomes in vitro and in vivo. In contrast to HflX, GTPBP6 is also required for the assembly of mitochondrial ribosomes. GTPBP6 ablation leads to accumulation of late assembly intermediate(s) of the large ribosomal subunit containing ribosome biogenesis factors MTERF4, NSUN4, MALSU1 and the GTPases GTPBP5, GTPBP7 and GTPBP10. Our data show that GTPBP6 has a dual function acting in ribosome recycling and biogenesis. These findings contribute to our understanding of large ribosomal subunit assembly as well as ribosome recycling pathway in mitochondria.