No full-text available
To read the full-text of this research,
you can request a copy directly from the authors.
Severe Infantile Encephalomyopathy Caused by a Mutation in COX6B1, a Nucleus-Encoded Subunit of Cytochrome C Oxidase
Abstract
Cytochrome c oxidase (COX) deficiency, one of the most common respiratory-chain defects in humans, has been associated with mutations in either mitochondrial DNA genes or nucleus-encoded proteins that are not part in but promote the biogenesis of COX. Mutations of nucleus-encoded structural subunits were sought for but never found in COX-defective patients, leading to the conjecture that they may be incompatible with extra-uterine survival. We report a disease-associated mutation in one such subunit, COX6B1. Nuclear-encoded COX genes should be reconsidered and included in the diagnostic mutational screening of human disorders related to COX deficiency.
... The first documented case of pathology coupled with nuclear-encoded COX subunit mutation was homozygous Arg20His substitution in COX6B1, associated with infantile encephalomyopathy (Massa et al. 2008). Later, another COX6B1 substitution mutation in the same site (Arg20Cys) was identified in patient with encephalomyopathy, hydrocephalus and hypertrophic cardiomyopathy (Abdulhag et al. 2015). ...
... Described mutations lead to substitution in evolutionary conserved region that is, according to the protein modeling, crucial for COX6B1 protein conformation and its predicted contact with COX2 catalytic core subunit. Biochemical analysis of patient fibroblasts and muscle tissue harbouring Arg20His mutations revealed reduced COX6B1 protein steady-state level and decreased incorporation of mutated COX6B1 subunit into COX enzyme during assembly, recorded by presence of assembly intermediate S3 possibly preceding addition of COX6B1 subunit (Massa et al. 2008). Correspondingly, COX activity was decreased in patient cells while other OXPHOS complexes were not affected. ...
Cytochrome c oxidase (COX), the terminal enzyme of mitochondrial electron transport chain, couples electron transport to oxygen with generation of proton gradient indispensable for the production of vast majority of ATP molecules in mammalian cells. The review summarizes current knowledge of COX structure and function of nuclear-encoded COX subunits, which may modulate enzyme activity according to various conditions. Moreover, some nuclear-encoded subunits posess tissue-specific and development-specific isoforms, possibly enabling fine-tuning of COX function in individual tissues. The importance of nuclear-encoded subunits is emphasized by recently discovered pathogenic mutations in patients with severe mitopathies. In addition, proteins substoichiometrically associated with COX were found to contribute to COX activity regulation and stabilization of the respiratory supercomplexes. Based on the summarized data, a model of three levels of quaternary COX structure is postulated. Individual structural levels correspond to subunits of the i) catalytic center, ii) nuclear-encoded stoichiometric subunits and iii) associated proteins, which may constitute several forms of COX with varying composition and differentially regulated function.
... These Cys in the twin structural domains of COX6B1 (Table 1) are found completely oxidized in human mitochondria [64]. Apart from a stabilization and/or assembly role [35,73], a function in Cu delivery was also proposed for COX6B1 [74]. ...
In eukaryotic cells, mitochondria perform cellular respiration through a series of redox reactions ultimately reducing molecular oxygen to water. The system responsible for this process is the respiratory chain or electron transport system (ETS) composed of complexes I–IV. Due to its function, the ETS is the main source of reactive oxygen species (ROS), generating them on both sides of the mitochondrial inner membrane, i.e. the intermembrane space (IMS) and the matrix. A correct balance between ROS generation and scavenging is important for keeping the cellular redox homeostasis and other important aspects of cellular physiology. However, ROS generated in the mitochondria are important signaling molecules regulating mitochondrial biogenesis and function. The IMS contains a large number of redox sensing proteins, containing specific Cys-rich domains, that are involved in ETS complex biogenesis. The large majority of these proteins function as cytochrome c oxidase (COX) assembly factors, mainly for the handling of copper ions necessary for the formation of the redox reactive catalytic centers. A particular case of ROS-regulated COX assembly factor is COA8, whose intramitochondrial levels are increased by oxidative stress, promoting COX assembly and/or protecting the enzyme from oxidative damage. In this review, we will discuss the current knowledge concerning the role played by ROS in regulating mitochondrial activity and biogenesis, focusing on the COX enzyme and with a special emphasis on the functional role exerted by the redox sensitive Cys residues contained in the COX assembly factors.
... COX6B1 is a subunit of COX complex, and is present in various types of cells, such as HeLa cells and yeast (64). In addition, recent research has reported that COX6B1 dysregulation could have significant effect on the COX functions, possibly resulting into the development of hydrocephalus, cerebromyopathy and other disorders (65,66). The ATP5O expression has been reported to play critical roles in diagnosing and prognosing gastric cancer, and the analysis based on NextBio database reveals a downregulation of ATP5O expression in ccRCC (67,68). ...
Background
The extensive spread of coronavirus disease 2019 (COVID-19) has led to a rapid increase in global mortality. Preeclampsia is a commonly observed pregnancy ailment characterized by high maternal morbidity and mortality rates, in addition to the restriction of fetal growth within the uterine environment. Pregnant individuals afflicted with vascular disorders, including preeclampsia, exhibit an increased susceptibility to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection via mechanisms that have not been fully delineated. Additionally, the intricate molecular mechanisms underlying preeclampsia and COVID-19 have not been fully elucidated. This study aimed to discern commonalities in gene expression, regulators, and pathways shared between COVID-19 and preeclampsia. The objective was to uncover potential insights that could contribute to novel treatment strategies for both COVID-19 and preeclampsia.
Method
Transcriptomic datasets for COVID-19 peripheral blood (GSE152418) and preeclampsia blood (GSE48424) were initially sourced from the Gene Expression Omnibus (GEO) database. Subsequent to that, we conducted a subanalysis by selecting females from the GSE152418 dataset and employed the “Deseq2” package to identify genes that exhibited differential expression. Simultaneously, the “limma” package was applied to identify differentially expressed genes (DEGs) in the preeclampsia dataset (GSE48424). Following that, an intersection analysis was conducted to identify the common DEGs obtained from both the COVID-19 and preeclampsia datasets. The identified shared DEGs were subsequently utilized for functional enrichment analysis, transcription factor (TF) and microRNAs (miRNA) prediction, pathway analysis, and identification of potential candidate drugs. Finally, to validate the bioinformatics findings, we collected peripheral blood mononuclear cell (PBMC) samples from healthy individuals, COVID-19 patients, and Preeclampsia patients. The abundance of the top 10 Hub genes in both diseases was assessed using real-time quantitative polymerase chain reaction (RT-qPCR).
Result
A total of 355 overlapping DEGs were identified in both preeclampsia and COVID-19 datasets. Subsequent ontological analysis, encompassing Gene Ontology (GO) functional assessment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, revealed a significant association between the two conditions. Protein-protein interactions (PPIs) were constructed using the STRING database. Additionally, the top 10 hub genes (MRPL11, MRPS12, UQCRH, ATP5I, UQCRQ, ATP5D, COX6B1, ATP5O, ATP5H, NDUFA6) were selected based on their ranking scores using the degree algorithm, which considered the shared DEGs. Moreover, transcription factor-gene interactions, protein-drug interactions, co-regulatory networks of DEGs and miRNAs, and protein-drug interactions involving the shared DEGs were also identified in the datasets. Finally, RT-PCR results confirmed that 10 hub genes do exhibit distinct expression profiles in the two diseases.
Conclusion
This study successfully identified overlapping DEGs, functional pathways, and regulatory elements between COVID-19 and preeclampsia. The findings provide valuable insights into the shared molecular mechanisms and potential therapeutic targets for both diseases. The validation through RT-qPCR further supports the distinct expression profiles of the identified hub genes in COVID-19 and preeclampsia, emphasizing their potential roles as biomarkers or therapeutic targets in these conditions.
... Those fibroblasts were derived from a patient with a mutation in a nucleus-encoded mitochondrial respiratory chain complex IV subunit COX6B1. This mutation manifests itself with an early-onset encephalopathy with leukodystrophy, myopathy and growth retardation associated with cytochrome c oxidase (COX) deficiency 43 . COX6B1-mutant fibroblasts had lower protein expression of UQCRC1, a subunit of mitochondrial complex III, and COX5B, a subunit of mitochondrial complex IV, indicating mitochondrial defects. ...
Perturbed cellular protein homeostasis (proteostasis) and mitochondrial dysfunction play an important role in neurodegenerative diseases, however, the interplay between these two phenomena remains unclear. Mitochondrial dysfunction leads to a delay in mitochondrial protein import, causing accumulation of non-imported mitochondrial proteins in the cytosol and challenging proteostasis. Cells respond by increasing proteasome activity and molecular chaperones in yeast and C. elegans. Here, we demonstrate that in human cells mitochondrial dysfunction leads to the upregulation of a chaperone HSPB1 and, interestingly, an immunoproteasome-specific subunit PSMB9. Moreover, PSMB9 expression is dependent on the translation elongation factor EEF1A2. These mechanisms constitute a defense response to preserve cellular proteostasis under mitochondrial stress. Our findings define a mode of proteasomal activation through the change in proteasome composition driven by EEF1A2 and its spatial regulation, and are useful to formulate therapies to prevent neurodegenerative diseases.
... COX deficiency is also a feature in patients with mutations in genes encoding mitochondrial gene expression factors, such as LRPPRC, a mitochondrial RNA stabilizing factor; TACO1, a specific translational activator of MT-CO1; or even mitochondrial tRNAs and aminoacyl-tRNA-synthetases [59]. Mutations in nucleusencoded structural subunits were hypothesized to be embryonic-lethal for a long time because none were found until 2008, when mutations in COX6B1 were identified [60]. After that, several other mutations in other nDNA-encoded genes encoding different complex IV structural subunits were described, but the quantity of disease-related genes encoding COX assembly factors outnumbers the former by far. ...
The fruit fly—i.e., Drosophila melanogaster—has proven to be a very useful model for the understanding of basic physiological processes, such as development or ageing. The availability of straightforward genetic tools that can be used to produce engineered individuals makes this model extremely interesting for the understanding of the mechanisms underlying genetic diseases in physiological models. Mitochondrial diseases are a group of yet-incurable genetic disorders characterized by the malfunction of the oxidative phosphorylation system (OXPHOS), which is the highly conserved energy transformation system present in mitochondria. The generation of D. melanogaster models of mitochondrial disease started relatively recently but has already provided relevant information about the molecular mechanisms and pathological consequences of mitochondrial dysfunction. Here, we provide an overview of such models and highlight the relevance of D. melanogaster as a model to study mitochondrial disorders.
... Additional, around 30 nDNA-encoded ancillary factors, including copper chaperones, also called COX-assembly proteins, are involved in the biogenesis and assembly of COX holoenzyme. To date, mutations in the subunits encoded by both genomes have been associated with disorder (MT-CO1 [52], MT-CO2 [53], MT-CO3 [54], COX4I1 [55], COX4I2 [56], COX5A [57], COX6A1 [56,58], COX6A2 [56,59], COX6B1 [60], COX7A1 [56], COX7A2 [56], COX7B [61], COX8A [62], and NDUFA4 or COXFA4 [63]), while the majority of isolated COX deficiencies are caused by mutations in COX assembly factors. Specifically, disorder-causing mutations were found in genes such as SURF1 (the first identified nuclear gene encoding a factor involved in the biogenesis of COX and being mutated in the neurodegenerative Leigh's syndrome with COX deficiency) [64,65]; SCO2/SCO1 (involved in mitochondrial copper pathway) [66,67]; COX10 and COX15 (involved in heme A biosynthesis) [68][69][70]; COX14 (or C12ORF62; involved in COX assembly) [71]; COX20 (or FAM36A; involved in MT-CO2 stabilization) [72]; COA3 (CCDC56 or MITRAC12; involved in MT-CO1 maturation) [73]; PET100 (involved in COX biogenesis) [74]; PET117 (involved in the assembly of MT-CO2) [75]; COA5 (C2ORF64) [76]; COA6 [77]; COA7 [78]; COA8 (previously known as APOPT1) [79]; FASTKD2 [80]; LRPPRC; and TACO1 (essential for COX expression) [81,82]. ...
Mitochondrial disorders represent a heterogeneous group of genetic disorders with variations in severity and clinical outcomes, mostly characterized by respiratory chain dysfunction and abnormal mitochondrial function. More specifically, mutations in the human SCO2 gene, encoding the mitochondrial inner membrane Sco2 cytochrome c oxidase (COX) assembly protein, have been implicated in the mitochondrial disorder fatal infantile cardioencephalomyopathy with COX deficiency. Since an effective treatment is still missing, a protein replacement therapy (PRT) was explored using protein transduction domain (PTD) technology. Therefore, the human recombinant full-length mitochondrial protein Sco2, fused to TAT peptide (a common PTD), was produced (fusion Sco2 protein) and successfully transduced into fibroblasts derived from a SCO2/COX-deficient patient. This PRT contributed to effective COX assembly and partial recovery of COX activity. In mice, radiolabeled fusion Sco2 protein was biodistributed in the peripheral tissues of mice and successfully delivered into their mitochondria. Complementary to that, an mRNA-based therapeutic approach has been more recently considered as an innovative treatment option. In particular, a patented, novel PTD-mediated IVT-mRNA delivery platform was developed and applied in recent research efforts. PTD-IVT-mRNA of full-length SCO2 was successfully transduced into the fibroblasts derived from a SCO2/COX-deficient patient, translated in host ribosomes into a nascent chain of human Sco2, imported into mitochondria, and processed to the mature protein. Consequently, the recovery of reduced COX activity was achieved, thus suggesting the potential of this mRNA-based technology for clinical translation as a PRT for metabolic/genetic disorders. In this review, such research efforts will be comprehensibly presented and discussed to elaborate their potential in clinical application and therapeutic usefulness.
... Mutations in CX6B1 R20, which disrupt a salt bridge with D17 (ref. 25 ), are hypothesized to cause instability of CIV, leading to encephalomyopathy and hypertrophic cardiomyopathy in patients 26 . R20 is likely stabilized by a salt bridge with nearby NDUA4 D60, although this sidechain is only partially resolved in the structure (Extended Data Fig. 2a; PDB: 5Z62). ...
Advancements in cross-linking mass spectrometry bridge the gap between purified systems and native tissue environments, allowing the detection of protein structural interactions in their native state. In this study, we used isobaric quantitative protein interaction reporter (iqPIR) technology to compare the mitochondrial protein interactomes in healthy and failing murine hearts 4 weeks after transverse aortic constriction. The failing heart interactome includes 588 statistically significant cross-linked peptide pairs altered in the disease condition. We observed an increase in the assembly of ketone oxidation oligomers corresponding to an increase in ketone metabolic utilization; remodeling of NDUA4 interaction in Complex IV, likely contributing to impaired mitochondrial respiration; and conformational enrichment of the ADP/ATP carrier ADT1, which is non-functional for ADP/ATP translocation but likely possesses non-selective conductivity. Our application of quantitative cross-linking technology in cardiac tissue provides molecular-level insights into the complex mitochondrial remodeling in heart failure while bringing forth new hypotheses for pathological mechanisms. Caudal, Tang, et al. use isobaric quantitative protein interaction reporter (iqPIR) technology to compare the mitochondrial protein interactome in healthy and failing murine hearts, providing molecular-level insights into complex mitochondrial remodeling in heart failure.
... COX6A mutations at various loci have been associated with various forms of neuropathy. For example, mutations in COX6B1 (p.R20H/C) and a 5 bp deletion (c.247-10_247-6delCACTC, aberrant splicing) in a splicing element of COX6A1 cause severe infantile encephalomyopathy and an axonal form of Charcot-Marie Tooth (CMT) syndrome, respectively (Massa et al., 2008;Tamiya et al., 2014;Abdulhag et al., 2015). ...
Mitochondria are essential organelles for neuronal function and cell survival. Besides the well-known bioenergetics, additional mitochondrial roles in calcium signaling, lipid biogenesis, regulation of reactive oxygen species, and apoptosis are pivotal in diverse cellular processes. The mitochondrial proteome encompasses about 1,500 proteins encoded by both the nuclear DNA and the maternally inherited mitochondrial DNA. Mutations in the nuclear or mitochondrial genome, or combinations of both, can result in mitochondrial protein deficiencies and mitochondrial malfunction. Therefore, mitochondrial quality control by proteins involved in various surveillance mechanisms is critical for neuronal integrity and viability. Abnormal proteins involved in mitochondrial bioenergetics, dynamics, mitophagy, import machinery, ion channels, and mitochondrial DNA maintenance have been linked to the pathogenesis of a number of neurological diseases. The goal of this review is to give an overview of these pathways and to summarize the interconnections between mitochondrial protein dysfunction and neurological diseases.
... A loss-of-function mutation in SURF1, a gene usually associated with LD (see above), has been reported in isolated leukodystrophy, including degeneration of the corticospinal tracts [145]. Moreover, the only case so far reported to be associated with a mutation in the nuclear-encoded COX6B1 subunit [146] showed a combination of early-onset leukodystrophic encephalopathy, myopathy, and growth retardation with COX deficiency. However, a COX-related leukoencephalopathy was found in mutations of APOPT1, now known as COA8 [147]. ...
Mitochondria are cytoplasmic organelles, which generate energy as heat and ATP, the universal energy currency of the cell. This process is carried out by coupling electron stripping through oxidation of nutrient substrates with the formation of a proton-based electrochemical gradient across the inner mitochondrial membrane. Controlled dissipation of the gradient can lead to production of heat as well as ATP, via ADP phosphorylation. This process is known as oxidative phosphorylation, and is carried out by four multiheteromeric complexes (from I to IV) of the mitochondrial respiratory chain, carrying out the electron flow whose energy is stored as a proton-based electrochemical gradient. This gradient sustains a second reaction, operated by the mitochondrial ATP synthase, or complex V, which condensates ADP and Pi into ATP. Four complexes (CI, CIII, CIV, and CV) are composed of proteins encoded by genes present in two separate compartments: the nuclear genome and a small circular DNA found in mitochondria themselves, and are termed mitochondrial DNA (mtDNA). Mutations striking either genome can lead to mitochondrial impairment, determining infantile, childhood or adult neurodegeneration. Mitochondrial disorders are complex neurological syndromes, and are often part of a multisystem disorder. In this paper, we divide the diseases into those caused by mtDNA defects and those that are due to mutations involving nuclear genes; from a clinical point of view, we discuss pediatric disorders in comparison to juvenile or adult-onset conditions. The complementary genetic contributions controlling organellar function and the complexity of the biochemical pathways present in the mitochondria justify the extreme genetic and phenotypic heterogeneity of this new area of inborn errors of metabolism known as ‘mitochondrial medicine’.
... Apart from a stabilization role, a function in Cu delivery to the active center of Cox2/MT-CO2 was also proposed for Cox12/COX6B1 (Ghosh et al., 2016). In any case, this structural subunit is clearly important for completion of the assembly process and for the activity of the enzyme, as mutations destabilizing COX6B1 or Cox12 result in assembly defects and COX deficiency in human and yeast (Massa et al., 2008;Abdulhag et al., 2015). ...
Mitochondria are double-membrane organelles that contain their own genome, the mitochondrial DNA (mtDNA), and reminiscent of its endosymbiotic origin. Mitochondria are responsible for cellular respiration via the function of the electron oxidative phosphorylation system (OXPHOS), located in the mitochondrial inner membrane and composed of the four electron transport chain (ETC) enzymes (complexes I-IV), and the ATP synthase (complex V). Even though the mtDNA encodes essential OXPHOS components, the large majority of the structural subunits and additional biogenetical factors (more than seventy proteins) are encoded in the nucleus and translated in the cytoplasm. To incorporate these proteins and the rest of the mitochondrial proteome, mitochondria have evolved varied, and sophisticated import machineries that specifically target proteins to the different compartments defined by the two membranes. The intermembrane space (IMS) contains a high number of cysteine-rich proteins, which are mostly imported via the MIA40 oxidative folding system, dependent on the reduction, and oxidation of key Cys residues. Several of these proteins are structural components or assembly factors necessary for the correct maturation and function of the ETC complexes. Interestingly, many of these proteins are involved in the metalation of the active redox centers of complex IV, the terminal oxidase of the mitochondrial ETC. Due to their function in oxygen reduction, mitochondria are the main generators of reactive oxygen species (ROS), on both sides of the inner membrane, i.e., in the matrix and the IMS. ROS generation is important due to their role as signaling molecules, but an excessive production is detrimental due to unwanted oxidation reactions that impact on the function of different types of biomolecules contained in mitochondria. Therefore, the maintenance of the redox balance in the IMS is essential for mitochondrial function. In this review, we will discuss the role that redox regulation plays in the maintenance of IMS homeostasis as well as how mitochondrial ROS generation may be a key regulatory factor for ETC biogenesis, especially for complex IV.
... CIV assembly grows with a modular process through the incorporation of modules formed by different subunits and defined by each of the mtDNA-encoded core subunits [130,338,339]. Any subunit of complex IV could carry mutations and rise a mitochondriopathy [337,[340][341][342]. Mutations in the MT-CO1, MT-CO2, and MT-CO3 are causative of COX deficiency and mitochondrial disease with an extreme clinical heterogeneity (Table 5). ...
Oxidative phosphorylation (OxPhos) is the basic function of mitochondria, although the landscape of mitochondrial functions is continuously growing to include more aspects of cellular homeostasis. Thanks to the application of -omics technologies to the study of the OxPhos system, novel features emerge from the cataloging of novel proteins as mitochondrial thus adding details to the mitochondrial proteome and defining novel metabolic cellular interrelations, especially in the human brain. We focussed on the diversity of bioenergetics demand and different aspects of mitochondrial structure, functions, and dysfunction in the brain. Definition such as ‘mitoexome’, ‘mitoproteome’ and ‘mitointeractome’ have entered the field of ‘mitochondrial medicine’. In this context, we reviewed several genetic defects that hamper the last step of aerobic metabolism, mostly involving the nervous tissue as one of the most prominent energy-dependent tissues and, as consequence, as a primary target of mitochondrial dysfunction. The dual genetic origin of the OxPhos complexes is one of the reasons for the complexity of the genotype-phenotype correlation when facing human diseases associated with mitochondrial defects. Such complexity clinically manifests with extremely heterogeneous symptoms, ranging from organ-specific to multisystemic dysfunction with different clinical courses. Finally, we briefly discuss the future directions of the multi-omics study of human brain disorders.
... Some mutations in COX6B1 have been associated with COX deficiency disease. Two individuals with early-onset leukodystrophic encephalopathy, myopathy, and growth retardation carried a homozygous mutation in COX6B1 resulting in an amino acid residue replacement of arginine to histidine at the N-terminus of the protein [95]. Another missense mutation, resulting in an arginine to cysteine change at the same position, was mapped in an individual with encephalomyopathy, hydrocephaly, and cardiomyopathy [96]. ...
Oxidative phosphorylation is a tightly regulated process in mammals that takes place in and across the inner mitochondrial membrane and consists of the electron transport chain and ATP synthase. Complex IV, or cytochrome c oxidase (COX), is the terminal enzyme of the electron transport chain, responsible for accepting electrons from cytochrome c, pumping protons to contribute to the gradient utilized by ATP synthase to produce ATP, and reducing oxygen to water. As such, COX is tightly regulated through numerous mechanisms including protein–protein interactions. The twin CX9C family of proteins has recently been shown to be involved in COX regulation by assisting with complex assembly, biogenesis, and activity. The twin CX9C motif allows for the import of these proteins into the intermembrane space of the mitochondria using the redox import machinery of Mia40/CHCHD4. Studies have shown that knockdown of the proteins discussed in this review results in decreased or completely deficient aerobic respiration in experimental models ranging from yeast to human cells, as the proteins are conserved across species. This article highlights and discusses the importance of COX regulation by twin CX9C proteins in the mitochondria via COX assembly and control of its activity through protein–protein interactions, which is further modulated by cell signaling pathways. Interestingly, select members of the CX9C protein family, including MNRR1 and CHCHD10, show a novel feature in that they not only localize to the mitochondria but also to the nucleus, where they mediate oxygen- and stress-induced transcriptional regulation, opening a new view of mitochondrial-nuclear crosstalk and its involvement in human disease.
The moderate restriction of dietary energy intake (dietary restriction: DR) extends the lifespan and health span of various laboratory animals, suggesting that it delays the aging process inherent in many animal species. Attenuated growth hormone and insulin-like growth factor-1 (IGF-1) signaling caused by mutations also increases the lifespan of mice, even those allowed to feed freely. In nematodes, the Daf16, mammalian Forkhead box O (FoxO) transcription factor, was shown to be required for lifespan extension in response to reduced IGF-1 signaling. Because DR also decreases the plasma concentration of IGF-1 in mammals, the IGF-1–FoxO axis may play a central role in the lifespan extension effect of DR and, thus, retardation of aging. Studies using knockout mice under DR conditions revealed the importance of FoxO1 and nuclear factor erythroid-derived 2-like 2 (Nrf2) in tumor suppression, and FoxO3 in lifespan extension. Human genomic studies also identified a strong association between a FOXO3 single nucleotide polymorphism and longevity. The aging mechanism is the most important risk factor for disease and frailty in aging humans. Therefore, further research on the application of DR to humans, the development of compounds and drugs that mimic the effects of DR, and mechanisms underlying FOXO3 polymorphisms for longevity is highly relevant to extending the human health span.
Increasing evidence has demonstrated that cancer cell metabolism is a critical factor in tumor development and progression; however, its role in glioblastoma (GBM) remains limited. In this study, we classified GBM into three metabolism subtypes (MC1, MC2, and MC3) through cluster analysis of 153 GBM samples from the RNA-sequencing data of The Cancer Genome Atlas (TCGA) based on 2752 metabolism-related genes (MRGs). We further explored the prognostic value, metabolic signatures, immune infiltration, and immunotherapy sensitivity of the three metabolism subtypes. Moreover, the metabolism scoring model was established to quantify the different metabolic characteristics of the patients. Results showed that MC3, which is associated with a favorable survival outcome, had higher proportions of isocitrate dehydrogenase (IDH) mutations and lower tumor purity and proliferation. The MC1 subtype, which is associated with the worst prognosis, shows a higher number of segments and homologous recombination defects and significantly lower mRNA expression-based stemness index (mRNAsi) and epigenetic-regulation-based mRNAsi. The MC2 subtype has the highest T-cell exclusion score, indicating a high likelihood of immune escape. The results were validated using an independent dataset. Five MRGs (ACSL1, NDUFA2, CYP1B1, SLC11A1, and COX6B1) correlated with survival outcomes were identified based on metabolism-related co-expression module analysis. Laboratory-based validation tests further showed the expression of these MRGs in GBM tissues and how their expression influences cell function. The results provide a reference for developing clinical management approaches and treatments for GBM.
Moderate restriction of dietary energy intake, referred to here as dietary restriction (DR), delays aging and extends lifespan in experimental animals compared with a diet of ad libitum feeding (AL) control animals. Basic knowledge of the mechanisms underlying the effects of DR could be applicable to extending the healthspan in humans. This review highlights the importance of forkhead box O (FoxO) transcription factors downstream of the growth hormone‐insulin‐like growth factor 1 signaling in the effects of DR. Our lifespan studies in mice with heterozygous Foxo1 or Foxo3 gene knockout indicated differential roles of FoxO1 and FoxO3 in the tumor‐inhibiting and life‐extending effects of DR. Subsequent studies suggested a critical role of FoxO3 in metabolic and mitochondrial bioenergetic adaptation to DR. Our studies also verified hypothalamic neuropeptide Y (Npy) as a vital neuropeptide showing pleiotropic and sexually dimorphic effects for extending the healthspan in the context of nutritional availability. Npy was necessary for DR to exert its effects in male and female mice; meanwhile, under AL conditions, the loss of Npy prevented obesity and insulin resistance only in female mice. Overnutrition disrupts FoxO‐ and Npy‐associated metabolic and mitochondrial bioenergetic adaptive processes, causing the acceleration of aging and related diseases.
Reactive oxygen species (ROS) can modulate protein function through cysteine oxidation. Identifying protein targets of ROS can provide insight into uncharacterized ROS-regulated pathways. Several redox-proteomic workflows, such as oxidative isotope-coded affinity tags (OxICAT), exist to identify sites of cysteine oxidation. However, determining ROS targets localized within subcellular compartments and ROS hotspots remains challenging with existing workflows. Here, we present a chemoproteomic platform, PL-OxICAT, which combines proximity labeling (PL) with OxICAT to monitor localized cysteine oxidation events. We show that TurboID-based PL-OxICAT can monitor cysteine oxidation events within subcellular compartments such as the mitochondrial matrix and intermembrane space. Furthermore, we use ascorbate peroxidase (APEX)-based PL-OxICAT to monitor oxidation events within ROS hotspots by using endogenous ROS as the source of peroxide for APEX activation. Together, these platforms further hone our ability to monitor cysteine oxidation events within specific subcellular locations and ROS hotspots and provide a deeper understanding of the protein targets of endogenous and exogenous ROS.
This major new edition fulfils the need for a single-volume, up-to-date information resource on the etiology, pathogenesis, diagnosis and treatment of diseases of skeletal muscles, including the muscular dystrophies, mitochondrial myopathies, metabolic myopathies, ion channel disorders, and dysimmune myopathies. As background to the clinical coverage, relevant information on advances in molecular and developmental biology, immunopathology, mitochondrial biology, ion-channel dynamics, cell membrane and signal transduction science, and imaging technology is summarized. Combining essential new knowledge with the fundamentals of history-taking and clinical examination, this extensively illustrated book will continue to be the mainstay for practising physicians and biomedical scientists concerned with muscle disease. Regular updates on the clinical and basic science aspects of muscle disease - written mainly by rising stars of myology - will be published on an accompanying website.
This major new edition fulfils the need for a single-volume, up-to-date information resource on the etiology, pathogenesis, diagnosis and treatment of diseases of skeletal muscles, including the muscular dystrophies, mitochondrial myopathies, metabolic myopathies, ion channel disorders, and dysimmune myopathies. As background to the clinical coverage, relevant information on advances in molecular and developmental biology, immunopathology, mitochondrial biology, ion-channel dynamics, cell membrane and signal transduction science, and imaging technology is summarized. Combining essential new knowledge with the fundamentals of history-taking and clinical examination, this extensively illustrated book will continue to be the mainstay for practising physicians and biomedical scientists concerned with muscle disease. Regular updates on the clinical and basic science aspects of muscle disease - written mainly by rising stars of myology - will be published on an accompanying website.
This major new edition fulfils the need for a single-volume, up-to-date information resource on the etiology, pathogenesis, diagnosis and treatment of diseases of skeletal muscles, including the muscular dystrophies, mitochondrial myopathies, metabolic myopathies, ion channel disorders, and dysimmune myopathies. As background to the clinical coverage, relevant information on advances in molecular and developmental biology, immunopathology, mitochondrial biology, ion-channel dynamics, cell membrane and signal transduction science, and imaging technology is summarized. Combining essential new knowledge with the fundamentals of history-taking and clinical examination, this extensively illustrated book will continue to be the mainstay for practising physicians and biomedical scientists concerned with muscle disease. Regular updates on the clinical and basic science aspects of muscle disease - written mainly by rising stars of myology - will be published on an accompanying website.
This major new edition fulfils the need for a single-volume, up-to-date information resource on the etiology, pathogenesis, diagnosis and treatment of diseases of skeletal muscles, including the muscular dystrophies, mitochondrial myopathies, metabolic myopathies, ion channel disorders, and dysimmune myopathies. As background to the clinical coverage, relevant information on advances in molecular and developmental biology, immunopathology, mitochondrial biology, ion-channel dynamics, cell membrane and signal transduction science, and imaging technology is summarized. Combining essential new knowledge with the fundamentals of history-taking and clinical examination, this extensively illustrated book will continue to be the mainstay for practising physicians and biomedical scientists concerned with muscle disease. Regular updates on the clinical and basic science aspects of muscle disease - written mainly by rising stars of myology - will be published on an accompanying website.
This major new edition fulfils the need for a single-volume, up-to-date information resource on the etiology, pathogenesis, diagnosis and treatment of diseases of skeletal muscles, including the muscular dystrophies, mitochondrial myopathies, metabolic myopathies, ion channel disorders, and dysimmune myopathies. As background to the clinical coverage, relevant information on advances in molecular and developmental biology, immunopathology, mitochondrial biology, ion-channel dynamics, cell membrane and signal transduction science, and imaging technology is summarized. Combining essential new knowledge with the fundamentals of history-taking and clinical examination, this extensively illustrated book will continue to be the mainstay for practising physicians and biomedical scientists concerned with muscle disease. Regular updates on the clinical and basic science aspects of muscle disease - written mainly by rising stars of myology - will be published on an accompanying website.
This major new edition fulfils the need for a single-volume, up-to-date information resource on the etiology, pathogenesis, diagnosis and treatment of diseases of skeletal muscles, including the muscular dystrophies, mitochondrial myopathies, metabolic myopathies, ion channel disorders, and dysimmune myopathies. As background to the clinical coverage, relevant information on advances in molecular and developmental biology, immunopathology, mitochondrial biology, ion-channel dynamics, cell membrane and signal transduction science, and imaging technology is summarized. Combining essential new knowledge with the fundamentals of history-taking and clinical examination, this extensively illustrated book will continue to be the mainstay for practising physicians and biomedical scientists concerned with muscle disease. Regular updates on the clinical and basic science aspects of muscle disease - written mainly by rising stars of myology - will be published on an accompanying website.
This major new edition fulfils the need for a single-volume, up-to-date information resource on the etiology, pathogenesis, diagnosis and treatment of diseases of skeletal muscles, including the muscular dystrophies, mitochondrial myopathies, metabolic myopathies, ion channel disorders, and dysimmune myopathies. As background to the clinical coverage, relevant information on advances in molecular and developmental biology, immunopathology, mitochondrial biology, ion-channel dynamics, cell membrane and signal transduction science, and imaging technology is summarized. Combining essential new knowledge with the fundamentals of history-taking and clinical examination, this extensively illustrated book will continue to be the mainstay for practising physicians and biomedical scientists concerned with muscle disease. Regular updates on the clinical and basic science aspects of muscle disease - written mainly by rising stars of myology - will be published on an accompanying website.
This major new edition fulfils the need for a single-volume, up-to-date information resource on the etiology, pathogenesis, diagnosis and treatment of diseases of skeletal muscles, including the muscular dystrophies, mitochondrial myopathies, metabolic myopathies, ion channel disorders, and dysimmune myopathies. As background to the clinical coverage, relevant information on advances in molecular and developmental biology, immunopathology, mitochondrial biology, ion-channel dynamics, cell membrane and signal transduction science, and imaging technology is summarized. Combining essential new knowledge with the fundamentals of history-taking and clinical examination, this extensively illustrated book will continue to be the mainstay for practising physicians and biomedical scientists concerned with muscle disease. Regular updates on the clinical and basic science aspects of muscle disease - written mainly by rising stars of myology - will be published on an accompanying website.
This major new edition fulfils the need for a single-volume, up-to-date information resource on the etiology, pathogenesis, diagnosis and treatment of diseases of skeletal muscles, including the muscular dystrophies, mitochondrial myopathies, metabolic myopathies, ion channel disorders, and dysimmune myopathies. As background to the clinical coverage, relevant information on advances in molecular and developmental biology, immunopathology, mitochondrial biology, ion-channel dynamics, cell membrane and signal transduction science, and imaging technology is summarized. Combining essential new knowledge with the fundamentals of history-taking and clinical examination, this extensively illustrated book will continue to be the mainstay for practising physicians and biomedical scientists concerned with muscle disease. Regular updates on the clinical and basic science aspects of muscle disease - written mainly by rising stars of myology - will be published on an accompanying website.
This major new edition fulfils the need for a single-volume, up-to-date information resource on the etiology, pathogenesis, diagnosis and treatment of diseases of skeletal muscles, including the muscular dystrophies, mitochondrial myopathies, metabolic myopathies, ion channel disorders, and dysimmune myopathies. As background to the clinical coverage, relevant information on advances in molecular and developmental biology, immunopathology, mitochondrial biology, ion-channel dynamics, cell membrane and signal transduction science, and imaging technology is summarized. Combining essential new knowledge with the fundamentals of history-taking and clinical examination, this extensively illustrated book will continue to be the mainstay for practising physicians and biomedical scientists concerned with muscle disease. Regular updates on the clinical and basic science aspects of muscle disease - written mainly by rising stars of myology - will be published on an accompanying website.
This major new edition fulfils the need for a single-volume, up-to-date information resource on the etiology, pathogenesis, diagnosis and treatment of diseases of skeletal muscles, including the muscular dystrophies, mitochondrial myopathies, metabolic myopathies, ion channel disorders, and dysimmune myopathies. As background to the clinical coverage, relevant information on advances in molecular and developmental biology, immunopathology, mitochondrial biology, ion-channel dynamics, cell membrane and signal transduction science, and imaging technology is summarized. Combining essential new knowledge with the fundamentals of history-taking and clinical examination, this extensively illustrated book will continue to be the mainstay for practising physicians and biomedical scientists concerned with muscle disease. Regular updates on the clinical and basic science aspects of muscle disease - written mainly by rising stars of myology - will be published on an accompanying website.
This major new edition fulfils the need for a single-volume, up-to-date information resource on the etiology, pathogenesis, diagnosis and treatment of diseases of skeletal muscles, including the muscular dystrophies, mitochondrial myopathies, metabolic myopathies, ion channel disorders, and dysimmune myopathies. As background to the clinical coverage, relevant information on advances in molecular and developmental biology, immunopathology, mitochondrial biology, ion-channel dynamics, cell membrane and signal transduction science, and imaging technology is summarized. Combining essential new knowledge with the fundamentals of history-taking and clinical examination, this extensively illustrated book will continue to be the mainstay for practising physicians and biomedical scientists concerned with muscle disease. Regular updates on the clinical and basic science aspects of muscle disease - written mainly by rising stars of myology - will be published on an accompanying website.
This major new edition fulfils the need for a single-volume, up-to-date information resource on the etiology, pathogenesis, diagnosis and treatment of diseases of skeletal muscles, including the muscular dystrophies, mitochondrial myopathies, metabolic myopathies, ion channel disorders, and dysimmune myopathies. As background to the clinical coverage, relevant information on advances in molecular and developmental biology, immunopathology, mitochondrial biology, ion-channel dynamics, cell membrane and signal transduction science, and imaging technology is summarized. Combining essential new knowledge with the fundamentals of history-taking and clinical examination, this extensively illustrated book will continue to be the mainstay for practising physicians and biomedical scientists concerned with muscle disease. Regular updates on the clinical and basic science aspects of muscle disease - written mainly by rising stars of myology - will be published on an accompanying website.
This major new edition fulfils the need for a single-volume, up-to-date information resource on the etiology, pathogenesis, diagnosis and treatment of diseases of skeletal muscles, including the muscular dystrophies, mitochondrial myopathies, metabolic myopathies, ion channel disorders, and dysimmune myopathies. As background to the clinical coverage, relevant information on advances in molecular and developmental biology, immunopathology, mitochondrial biology, ion-channel dynamics, cell membrane and signal transduction science, and imaging technology is summarized. Combining essential new knowledge with the fundamentals of history-taking and clinical examination, this extensively illustrated book will continue to be the mainstay for practising physicians and biomedical scientists concerned with muscle disease. Regular updates on the clinical and basic science aspects of muscle disease - written mainly by rising stars of myology - will be published on an accompanying website.
This major new edition fulfils the need for a single-volume, up-to-date information resource on the etiology, pathogenesis, diagnosis and treatment of diseases of skeletal muscles, including the muscular dystrophies, mitochondrial myopathies, metabolic myopathies, ion channel disorders, and dysimmune myopathies. As background to the clinical coverage, relevant information on advances in molecular and developmental biology, immunopathology, mitochondrial biology, ion-channel dynamics, cell membrane and signal transduction science, and imaging technology is summarized. Combining essential new knowledge with the fundamentals of history-taking and clinical examination, this extensively illustrated book will continue to be the mainstay for practising physicians and biomedical scientists concerned with muscle disease. Regular updates on the clinical and basic science aspects of muscle disease - written mainly by rising stars of myology - will be published on an accompanying website.
This major new edition fulfils the need for a single-volume, up-to-date information resource on the etiology, pathogenesis, diagnosis and treatment of diseases of skeletal muscles, including the muscular dystrophies, mitochondrial myopathies, metabolic myopathies, ion channel disorders, and dysimmune myopathies. As background to the clinical coverage, relevant information on advances in molecular and developmental biology, immunopathology, mitochondrial biology, ion-channel dynamics, cell membrane and signal transduction science, and imaging technology is summarized. Combining essential new knowledge with the fundamentals of history-taking and clinical examination, this extensively illustrated book will continue to be the mainstay for practising physicians and biomedical scientists concerned with muscle disease. Regular updates on the clinical and basic science aspects of muscle disease - written mainly by rising stars of myology - will be published on an accompanying website.
This major new edition fulfils the need for a single-volume, up-to-date information resource on the etiology, pathogenesis, diagnosis and treatment of diseases of skeletal muscles, including the muscular dystrophies, mitochondrial myopathies, metabolic myopathies, ion channel disorders, and dysimmune myopathies. As background to the clinical coverage, relevant information on advances in molecular and developmental biology, immunopathology, mitochondrial biology, ion-channel dynamics, cell membrane and signal transduction science, and imaging technology is summarized. Combining essential new knowledge with the fundamentals of history-taking and clinical examination, this extensively illustrated book will continue to be the mainstay for practising physicians and biomedical scientists concerned with muscle disease. Regular updates on the clinical and basic science aspects of muscle disease - written mainly by rising stars of myology - will be published on an accompanying website.
This major new edition fulfils the need for a single-volume, up-to-date information resource on the etiology, pathogenesis, diagnosis and treatment of diseases of skeletal muscles, including the muscular dystrophies, mitochondrial myopathies, metabolic myopathies, ion channel disorders, and dysimmune myopathies. As background to the clinical coverage, relevant information on advances in molecular and developmental biology, immunopathology, mitochondrial biology, ion-channel dynamics, cell membrane and signal transduction science, and imaging technology is summarized. Combining essential new knowledge with the fundamentals of history-taking and clinical examination, this extensively illustrated book will continue to be the mainstay for practising physicians and biomedical scientists concerned with muscle disease. Regular updates on the clinical and basic science aspects of muscle disease - written mainly by rising stars of myology - will be published on an accompanying website.
This major new edition fulfils the need for a single-volume, up-to-date information resource on the etiology, pathogenesis, diagnosis and treatment of diseases of skeletal muscles, including the muscular dystrophies, mitochondrial myopathies, metabolic myopathies, ion channel disorders, and dysimmune myopathies. As background to the clinical coverage, relevant information on advances in molecular and developmental biology, immunopathology, mitochondrial biology, ion-channel dynamics, cell membrane and signal transduction science, and imaging technology is summarized. Combining essential new knowledge with the fundamentals of history-taking and clinical examination, this extensively illustrated book will continue to be the mainstay for practising physicians and biomedical scientists concerned with muscle disease. Regular updates on the clinical and basic science aspects of muscle disease - written mainly by rising stars of myology - will be published on an accompanying website.
This major new edition fulfils the need for a single-volume, up-to-date information resource on the etiology, pathogenesis, diagnosis and treatment of diseases of skeletal muscles, including the muscular dystrophies, mitochondrial myopathies, metabolic myopathies, ion channel disorders, and dysimmune myopathies. As background to the clinical coverage, relevant information on advances in molecular and developmental biology, immunopathology, mitochondrial biology, ion-channel dynamics, cell membrane and signal transduction science, and imaging technology is summarized. Combining essential new knowledge with the fundamentals of history-taking and clinical examination, this extensively illustrated book will continue to be the mainstay for practising physicians and biomedical scientists concerned with muscle disease. Regular updates on the clinical and basic science aspects of muscle disease - written mainly by rising stars of myology - will be published on an accompanying website.
This major new edition fulfils the need for a single-volume, up-to-date information resource on the etiology, pathogenesis, diagnosis and treatment of diseases of skeletal muscles, including the muscular dystrophies, mitochondrial myopathies, metabolic myopathies, ion channel disorders, and dysimmune myopathies. As background to the clinical coverage, relevant information on advances in molecular and developmental biology, immunopathology, mitochondrial biology, ion-channel dynamics, cell membrane and signal transduction science, and imaging technology is summarized. Combining essential new knowledge with the fundamentals of history-taking and clinical examination, this extensively illustrated book will continue to be the mainstay for practising physicians and biomedical scientists concerned with muscle disease. Regular updates on the clinical and basic science aspects of muscle disease - written mainly by rising stars of myology - will be published on an accompanying website.
This major new edition fulfils the need for a single-volume, up-to-date information resource on the etiology, pathogenesis, diagnosis and treatment of diseases of skeletal muscles, including the muscular dystrophies, mitochondrial myopathies, metabolic myopathies, ion channel disorders, and dysimmune myopathies. As background to the clinical coverage, relevant information on advances in molecular and developmental biology, immunopathology, mitochondrial biology, ion-channel dynamics, cell membrane and signal transduction science, and imaging technology is summarized. Combining essential new knowledge with the fundamentals of history-taking and clinical examination, this extensively illustrated book will continue to be the mainstay for practising physicians and biomedical scientists concerned with muscle disease. Regular updates on the clinical and basic science aspects of muscle disease - written mainly by rising stars of myology - will be published on an accompanying website.
Pathogenic variants of zinc finger C4H2-type containing (ZC4H2) on the X chromosome cause a group of genetic diseases termed ZC4H2-associated rare disorders (ZARD), including Wieacker-Wolff Syndrome (WRWF) and Female-restricted Wieacker-Wolff Syndrome (WRWFFR). In the current study, a de novo c.352C>T (p.Gln118*) mutation in ZC4H2 (NM_018684.4) was identified in a female neonate born with severe arthrogryposis multiplex congenita (AMC) and Pierre-Robin sequence (cleft palate and micrognathia). Plasmids containing the wild-type (WT), mutant-type (MT) ZC4H2, or GFP report gene (N) were transfected in 293T cell lines, respectively. RT-qPCR and western blot analysis showed that ZC4H2 protein could not be detected in the 293T cells transfected with MT ZC4H2. The RNA seq results revealed that the expression profile of the MT group was similar to that of the N group but differed significantly from the WT group, indicating that the c.352C>T mutation resulted in the loss of function of ZC4H2. Differentially expressed genes (DEGs) enrichment analysis showed that c.352C>T mutation inhibited the expression levels of a series of genes involved in the oxidative phosphorylation pathway. Subsequently, expression levels of ZC4H2 were knocked down in neural stem cells (NSCs) derived from induced pluripotent stem cells (iPSCs) by lentiviral-expressed small hairpin RNAs (shRNAs) against ZC4H2. The results also demonstrated that decreasing the expression of ZC4H2 significantly reduced the growth of NSCs by affecting the expression of genes related to the oxidative phosphorylation signaling pathway. Taken together, our results strongly suggest that ZC4H2 c.352C>T (p.Gln118*) mutation resulted in the loss of protein function and caused WRWFFR.
Cytochrome c oxidase 6B1 (COX6B1) is one of the less characterized subunits of the mitochondrial electron transport chain complex IV (CIV). Here, we studied the pathobiochemical and respiratory functions of Cox12 (yeast ortholog of COX6B1) using Saccharomyces cerevisiae BY4741 (cox12Δ) cells deficient by the Cox12 protein. The cells exhibited severe growth deficiency in the respiratory glycerol-ethanol medium, which could be reverted by complementation with the yeast COX12 or human COX6B1 genes. Cox12 with arginine 17 residue substituted by histidine (R17H) or cysteine (R17C) (mutations analogous to those observed in human patients) failed to complement the loss of Cox12 function. When cox12Δ cells were grown in rich respiratory/fermentative galactose medium, no changes in the expression of individual respiratory chain subunits were observed. Blue native PAGE/Western blotting analysis using antibodies against Rip1 and Cox1, which are specific components of complexes III (CIII) and IV (CIV), respectively, revealed no noticeable decrease in the native CIII2CIV2 and CIII2CIV1 supercomplexes (SCs). However, the association of the respiratory SC factor 2 (Rcf2) and Cox2 subunit within the SCs of cox12Δ cells was reduced, while the specific activity of CIV was downregulated by 90%. Both basal respiration and succinate-ADP stimulated state 3 respiration, as well as the mitochondrial membrane potential, were decreased in cox12Δ cells. Furthermore, cox12Δ cells and cells synthesizing Cox12 mutants R17H and R17C showed higher sensitivity to the H2O2-induced oxidative stress compared to the wild-type (WT) cells. In silico structural modeling of the WT yeast SCs revealed that Cox12 forms a network of interactions with Rcf2 and Cox2. Together, our results establish that Cox12 is essential for the CIV activity.
The cytochrome c-oxidase (COX) enzyme, also known as mitochondrial complex IV (MT-C4D), is a transmembrane protein complex found in mitochondria. COX deficiency is one of the most frequent causes of electron transport chain defects in humans. Therefore, high energy demand organs and tissues are affected in patients with mutations in the COX15 gene, with variable phenotypic expressiveness.
We describe the case of a male newborn with hypertrophic cardiomyopathy and serum and cerebrospinal fluid hyperlacticaemia, whose exome sequencing revealed two variants in a compound heterozygous state: c.232G>A;p.(Gly78Arg), classified as likely pathogenic, and c.452C>G;p.(Ser151Ter), as pathogenic; the former never previously described in the literature.
The mitochondrial intermembrane space (IMS) is the most constricted sub-mitochondrial compartment, housing only about 5% of the mitochondrial proteome, and yet is endowed with the largest variability of protein import mechanisms. In this review, we summarize our current knowledge of the major IMS import pathway based on the oxidative protein folding pathway and discuss the stunning variability of other IMS protein import pathways. As IMS-localized proteins only have to cross the outer mitochondrial membrane, they do not require energy sources like ATP hydrolysis in the mitochondrial matrix or the inner membrane electrochemical potential which are critical for import into the matrix or insertion into the inner membrane. We also explore several atypical IMS import pathways that are still not very well understood and are guided by poorly defined or completely unknown targeting peptides. Importantly, many of the IMS proteins are linked to several human diseases, and it is therefore crucial to understand how they reach their normal site of function in the IMS. In the final part of this review, we discuss current understanding of how such IMS protein underpin a large spectrum of human disorders.
The increasing application of next generation sequencing approaches to the analysis of human exome and whole genome data has enabled the identification of novel variants and new genes involved in mitochondrial diseases. The ability of surviving in the absence of oxidative phosphorylation (OXPHOS) and mitochondrial genome makes the yeast Saccharomyces cerevisiae an excellent model system for investigating the role of these new variants in mitochondrial-related conditions and dissecting the molecular mechanisms associated with these diseases. The aim of this review was to highlight the main advantages offered by this model for the study of mitochondrial diseases, from the validation and characterisation of novel mutations to the dissection of the role played by genes in mitochondrial functionality and the discovery of potential therapeutic molecules. The review also provides a summary of the main contributions to the understanding of mitochondrial diseases emerged from the study of this simple eukaryotic organism.
Mitochondria are ubiquitous intracellular organelles found in almost all eukaryotes and involved in various aspects of cellular life, with a primary role in energy production. The interest in this organelle has grown stronger with the discovery of their link to various pathologies, including cancer, aging and neurodegenerative diseases. Indeed, dysfunctional mitochondria cannot provide the required energy to tissues with a high-energy demand, such as heart, brain and muscles, leading to a large spectrum of clinical phenotypes. Mitochondrial defects are at the origin of a group of clinically heterogeneous pathologies, called mitochondrial diseases, with an incidence of 1 in 5000 live births. Primary mitochondrial diseases are associated with genetic mutations both in nuclear and mitochondrial DNA (mtDNA), affecting genes involved in every aspect of the organelle function. As a consequence, it is difficult to find a common cause for mitochondrial diseases and, subsequently, to offer a precise clinical definition of the pathology. Moreover, the complexity of this condition makes it challenging to identify possible therapies or drug targets.
Mitochondrial (mt) dysfunction is linked to rare diseases (RDs) such as respiratory chain complex (RCC) deficiency, MELAS and ARSACS. Yet, how altered mt protein networks contribute to these ailments remains understudied. In this perspective article, we identified 21 mt proteins from public repositories that associate with RCC deficiency, MELAS or ARSACS, engaging in a relatively small number of protein-protein interactions (PPIs), underscoring the need for advanced proteomic and interactomic platforms to uncover the complete scope of mt connectivity to RDs. Accordingly, we discuss innovative untargeted label-free proteomics in identifying RD-specific mt or other macromolecular assemblies, and mapping of protein networks in complex tissue, organoid and stem cell-differentiated neurons. Further, tag-and label-based proteomics, genealogical proteomics, and combinatorial affinity purification-mass spectrometry, along with advancements in detecting and integrating transient PPIs with single-cell proteomics and transcriptomics, collectively offer seminal follow-ups to enrich for RD-relevant networks, with implications in RD precision medicine.
Cytochrome c oxidase (COX) deficiency is characterized by a high degree of genetic and phenotypic heterogeneity, partly reflecting the extreme structural complexity, multiple post-translational modification, variable, tissue-specific composition, and the high number of and intricate connections among the assembly factors of this enzyme. In fact, decreased COX specific activity can manifest with different degrees of severity, affect the whole organism or specific tissues, and develop a wide spectrum of disease natural history, including disease onsets ranging from birth to late adulthood. More than 30 genes have been linked to COX deficiency, but the list is still incomplete and in fact constantly updated. We here discuss the current knowledge about COX in health and disease, focusing on genetic aetiology and link to clinical manifestations. In addition, information concerning either fundamental biological features of the enzymes or biochemical signatures of its defects have been provided by experimental in vivo models, including yeast, fly, mouse and fish, which expanded our knowledge on the functional features and the phenotypical consequences of different forms of COX deficiency.
Mitochondrial disorders are amongst the most frequent inborn errors of metabolism, their primary cause being the dysfunction of the oxidative phosphorylation system (OXPHOS). OXPHOS is composed of the electron transport chain (ETC), formed by four multimeric enzymes and two mobile electron carriers, plus an ATP synthase (also called complex V). The ETC performs the redox reactions involved in cellular respiration while generating the proton motive force used by complex V to synthesize ATP. OXPHOS biogenesis involves multiple steps, starting from the expression of genes encoded in physically separated genomes, namely the mitochondrial and nuclear DNA, to the coordinated assembly of components and cofactors building each individual complex and eventually the supercomplexes. The genetic cause underlying around half of the diagnosed mitochondrial disease cases is currently known. Many of these cases result from pathogenic variants in genes encoding structural subunits or additional factors directly involved in the assembly of the ETC complexes. Here we review the historical and most recent findings concerning the clinical phenotypes and the molecular pathological mechanisms underlying this particular group of disorders.
This chapter discusses the cytochrome oxidase from beef heart mitochondria. Cytochrome c oxidase is mostly assayed by the spectrophotometric method. The rate of oxidation of ferrocytochrome c is measured by following the decrease in the absorbency of its α-band at 550 mμ. The activity of cytochrome c oxidase may be defined in terms of the first-order velocity constant. The oxidation-reduction components of isolated cytochrome c oxidase are cytochrome a, cytochrome a3, and copper. Cytochrome c oxidase can also be assayed by measuring oxygen uptake either manometrically or polarographically. Two procedures for purifying cytochrome c oxidase from beef heart mitochondria are described. The enzyme obtained by procedure I is less pure based on its activity and spectrum. Preparations of cytochrome oxidase are stored best in 0.25M sucrose (pH 7.0-7.5) at –15° at a protein concentration of 1 mg/ml. The composition of cytochrome oxidase prepared by procedure II is tabulated. Ferrocytochrome c donates electrons directly to cytochrome oxidase. The reaction as measured spectrophotometrically obeys first-order reaction kinetics. Cytochrome oxidase is inhibited by cyanide, azide, hydroxylamine, and sodium sulfide.
We have cloned and sequenced COX12, the nuclear gene for subunit VIb of Saccharomyces cerevisiae cytochrome c oxidase. This subunit, which was previously not found in cytochrome c oxidase purified from S. cerevisiae, has a deduced amino acid sequence which is 41% identical to the sequences of subunits VIb of bovine and human cytochrome c oxidases. The chromosomal copy of COX12 was replaced with a plasmid-derived copy of COX12, in which the coding region for the suspected cytochrome oxidase subunit was replaced with the yeast URA3 gene. The resulting Ura+ deletion strain grew poorly at room temperature and was unable to grow at 37 degrees C on ethanol/glycerol medium, whereas growth was normal at both temperatures on dextrose. This temperature-dependent, petite phenotype of the deletion strain was complemented to wild-type growth with a single copy plasmid carrying COX12. Cytochrome c oxidase activity in mitochondrial membranes from the cox12 deletion strain is decreased to 5-15% of that in membranes from the wild-type parent, and this activity is restored to normal when the cox12 deletion strain is complemented by the plasmid-borne COX12. Optical spectra of mitochondrial membranes from the cox12 deletion strain revealed that optically detectable cytochrome c oxidase is assembled at room temperature and at 37 degrees C, although the heme a + a3 absorption is diminished approximately 50%. The N-terminal amino acid sequence of the protein encoded by COX12 is identical to the N-terminal sequence of a subunit found in yeast cytochrome c oxidase purified by a new procedure (Taanman, J.-W., and Capaldi, R. A. (1992) J. Biol. Chem. 267, 22481-22485). We conclude that COX12 encodes a subunit of yeast cytochrome c oxidase which is essential during assembly for full cytochrome c oxidase activity but apparently can be removed after the oxidase is assembled, with retention of oxidase activity. This is the first instance in which deletion of a subunit of cytochrome c oxidase results in assembly of optically detectable cytochrome c oxidase but having markedly diminished activity.
COX deficiency is believed to be the most common defect in neonates and infants with mitochondrial diseases. To explore the causes of this group of disorders, we examined 25 mitochondrial genes (three COX subunit genes and 22 tRNA genes) and 10 nuclear COX subunit genes for disease associated mutations using PCR-SSCP and direct sequencing of polymorphic SSCP fragments. DNA from one patient with severe COX deficiency and with consanguineous parents was entirely sequenced. The patient population consisted of 21 unrelated index patients with mitochondrial disorders and predominant (n=7) or isolated (n=14) COX deficiency. We detected two distinct tRNA(Ser)(UCN) mutations, which have been recently described in single kindreds, in a subgroup of four patients with COX deficiency, deafness, myoclonic epilepsy, ataxia, and mental retardation. Besides a number of nucleotide variants, a single novel missense mutation, which may contribute to the disease phenotype, was found in the mitochondrial encoded COX 1 gene (G6480A). Mutations in nuclear encoded COX subunit genes were not detected in this study.
Yeast and bovine cytochrome c oxidases (COX) are composed of 12 and 13 different polypeptides, respectively. In both cases, the three subunits constituting the catalytic core are encoded by mitochondrial DNA. The other subunits are all products of nuclear genes that are translated on cytoplasmic ribosomes and imported through different transport routes into mitochondria. Biogenesis of the functional complex depends on the expression of all the structural and more than two dozen COX-specific genes. The latter impinge on all aspects of the biogenesis process. Here we review the current state of information about the functions of the COX-specific gene products and of their relationship to human COX deficiencies.
The biogenesis and maintenance of mitochondria relies on a sizable number of proteins. Many of these proteins are organized into complexes, which are located in the mitochondrial inner membrane. Blue Native polyacrylamide gel electrophoresis (BN-PAGE) is a method for the isolation of intact protein complexes. Although it was initially used to study mitochondrial respiratory chain enzymes, it can also be applied to other protein complexes. The use of BN-PAGE has increased exponentially over the past few years and new applications have been developed. Here we review how to set up the basic system and outline modifications that can be applied to address specific research questions. Increasing the upper mass limit of complexes that can be separated by BN-PAGE can be achieved by using agarose instead of acrylamide. BN-PAGE can also be used to study assembly of mitochondrial protein complexes. Other applications include in-gel measurements of enzyme activity by histochemical staining and preparative native electrophoresis to isolate a protein complex. Finally, new ways of identifying protein spots in Blue Native gels using mass spectrometry are briefly discussed.
In this chapter we have provided instructions for transforming yeast by a number of variations of the LiAc/SS-DNA/PEG method for a number of different applications. The rapid transformation protocol is used when small numbers of transformants are required. The high efficiency transformation protocol is used to generate large numbers of transformants or to deliver DNA constructs or oligonucleotides into the yeast cell. The large-scale transformation protocol is primarily applicable to the analysis of complex plasmid DNA libraries, such as those required for the yeast two-hybrid system. The microtiter plate versions of the rapid and high efficiency transformation protocols can be applied to high-throughput screening technologies.
Cytochrome c oxidase contains two redox-active copper centers (Cu(A) and Cu(B)) and two redox-active heme A moieties. Assembly of the enzyme relies on several assembly factors in addition to the constituent subunits and prosthetic groups. We studied fibroblast cultures from patients carrying mutations in the assembly factors COX10, SCO1, or SURF1. COX10 is involved in heme A biosynthesis. SCO1 is required for formation of the Cu(A) center. The function of SURF1 is unknown. Immunoblot analysis of native gels demonstrated severely decreased levels of holoenzyme in the patient cultures compared with controls. In addition, the blots revealed the presence of five subassemblies: three subassemblies involving the core subunit MTCO1 but apparently no other subunits; a subassembly containing subunits MTCO1, COX4, and COX5A; and a subassembly containing at least subunits MTCO1, MTCO2, MTCO3, COX4, and COX5A. As some of the subassemblies correspond to known assembly intermediates of human cytochrome c oxidase, we think that these subassemblies are probably assembly intermediates that accumulate in patient cells. The MTCO1.COX4.COX5A subassembly was not detected in COX10-deficient cells, which suggests that heme A incorporation into MTCO1 occurs prior to association of MTCO1 with COX4 and COX5A. SCO1-deficient cells contained accumulated levels of the MTCO1.COX4.COX5A subassembly, suggesting that MTCO2 associates with the MTCO1.COX4.COX5A subassembly after the Cu(A) center of MTCO2 is formed. Assembly in SURF1-deficient cells appears to stall at the same stage as in SCO1-deficient cells, pointing to a role for SURF1 in promoting the association of MTCO2 with the MTCO1.COX4.COX5A subassembly.
Cytochrome c oxidase (COX), the terminal enzyme of the mitochondrial respiratory chain, is one of the key functional and regulatory sites of the mammalian energy metabolism. Owing to the importance of the enzyme, pathogenetic mutations affecting COX frequently result in severe, often fatal metabolic disorders. No satisfactory therapy is currently available so that the treatment remains largely symptomatic and does not improve the course of the disease. While only few genetic defects of COX are caused by mutations in mitochondrial genome, during the last five years a large number of pathogenetic mutations in nuclear genes have been discovered. All these mutations are located in genes encoding COX-specific assembly proteins including SURF1, SCO1, SCO2, COX10, and COX15. Despite the identification of increasing number of mutations, their precise etiopathogenetic mechanisms, which are necessary for the development of future therapeutic protocols, still remain to be elucidated. This review summarizes recent developments, including our efforts in elucidation of the molecular basis of human mitochondrial diseases due to specific defects of COX with special focus on SURF1 assembly protein.
Mega2, the manipulation environment for genetic analysis, transparently allows users to process genetic data for family-based
or case/control studies accurately and efficiently. In addition to data validation checks, Mega2 provides analysis setup capabilities
for a broad choice of commonly used genetic analysis programs, including SimWalk2, ASPEX, GeneHunter, SLINK, SIMULATE, S.A.G.E.,
SOLAR, Vitesse, Allegro, PREST, PAP, Loki, Merlin and MENDEL.
Availability: http://watson.hgen.pitt.edu/register/
Contact: dweeks{at}watson.hgen.pitt.edu
Supplementary information: http://watson.hgen.pitt.edu/docs/mega2_html/mega2.html
The biogenesis of eukaryotic COX (cytochrome c oxidase) requires several accessory proteins in addition to structural subunits and prosthetic groups. We have analysed the assembly state of COX and SCO2 protein levels in various tissues of six patients with mutations in SCO2 and SURF1. SCO2 is a copper-binding protein presumably involved in formation of the Cu(A) centre of the COX2 subunit. The function of SURF1 is unknown. Immunoblot analysis of native gels demonstrated that COX holoenzyme is reduced to 10-20% in skeletal muscle and brain of SCO2 and SURF1 patients and to 10-30% in heart of SCO2 patients, whereas liver of SCO2 patients' contained normal holoenzyme levels. The steady-state levels of mutant SCO2 protein ranged from 0 to 20% in different SCO2 patient tissues. In addition, eight distinct COX subcomplexes and unassembled subunits were found, some of them identical with known assembly intermediates of the human enzyme. Heart, brain and skeletal muscle of SCO2 patients contained accumulated levels of the COX1.COX4.COX5A subcomplex, three COX1-containing subcomplexes, a COX4.COX5A subcomplex and two subcomplexes composed of only COX4 or COX5A. The accumulation of COX1.COX4.COX5A subcomplex, along with the virtual absence of free COX2, suggests that the lack of the Cu(A) centre may result in decreased stability of COX2. The appearance of COX4.COX5A subcomplex indicates that association of these nucleus-encoded subunits probably precedes their addition to COX1 during the assembly process. Finally, the consequences of SCO2 and SURF1 mutations suggest the existence of tissue-specific functional differences of these proteins that may serve different tissue-specific requirements for the regulation of COX biogenesis.
Mitochondrial dysfunction is involved in many neurodegenerative disorders in humans. Here we report mutations in a gene (designated levy) that codes for subunit VIa of cytochrome c oxidase (COX). The mutations were identified by the phenotype of temperature-induced paralysis and showed the additional phenotypes of decreased COX activity, age-dependent bang-induced paralysis, progressive neurodegeneration, and reduced life span. Germ-line transformation using the levy(+) gene rescued the mutant flies from all phenotypes including neurodegeneration. The data from levy mutants reveal a COX-mediated pathway in Drosophila, disruption of which leads to mitochondrial encephalomyopathic effects including neurodegeneration, motor dysfunction, and premature death. The data present the first case of a mutation in a nuclear-encoded structural subunit of COX that causes mitochondrial encephalomyopathy rather than lethality, whereas several previous attempts to identify such mutations have not been successful. The levy mutants provide a genetic model to understand the mechanisms underlying COX-mediated mitochondrial encephalomyopathies and to explore possible therapeutic interventions.
Deficiency of cytochrome c oxidase (COX) is associated with significant pathology in humans. However, the consequences for organogenesis and early development
are not well understood. We have investigated these issues using a zebrafish model. COX deficiency was induced using morpholinos
to reduce expression of CoxVa, a structural subunit, and Surf1, an assembly factor, both of which impaired COX assembly. Reduction
of COX activity to 50% resulted in developmental defects in endodermal tissue, cardiac function, and swimming behavior. Cellular
investigations revealed different underlying mechanisms. Apoptosis was dramatically increased in the hindbrain and neural
tube, and secondary motor neurons were absent or abnormal, explaining the motility defect. In contrast, the heart lacked apoptotic
cells but showed increasingly poor performance over time, consistent with energy deficiency. The zebrafish model has revealed
tissue-specific responses to COX deficiency and holds promise for discovery of new therapies to treat mitochondrial diseases
in humans.
We investigated the interaction between cytochrome c oxidase and its substrate cytochrome c by catalyzing the covalent linkage of the two proteins to yield 1 : 1 covalent enzyme–substrate complexes under conditions of low ionic strength. In addition to the ‘traditional’ oxidized complex formed between oxidized cytochrome c and the oxidized enzyme we prepared complexes under steady‐state reducing conditions. Whereas for the ‘oxidized’ complex cytochrome c became bound exclusively to subunit II of the enzyme, for the ‘steady‐state’ complex cytochrome c became bound to subunit II and two low molecular mass subunits, most likely VIb and IV.
For both complexes we investigated: (a) the ability of the covalently bound cytochrome c to relay electrons into the enzyme, and (b) the ability of the covalently bound enzyme to catalyze the oxidation of unbound (exogenous) ferrocytochrome c . Steady‐state spectral analysis (400–630 nm) combined with stopped‐flow studies, confirmed that the bound cytochrome c mediated the efficient transfer of electrons from the reducing agent ascorbate to the enzyme. In the case of the latter, the half life for the ascorbate reduction of the bound cytochrome c and that for the subsequent transfer of electrons to haem a were both < 5 ms. In contrast the covalent complexes, when reduced, were found to be totally unreactive towards oxidized cytochrome c oxidase confirming that the previously observed reduction of haem a within the complexes occurred via intramolecular rather than intermolecular electron transfer.
Additionally, stopped‐flow analysis at 550 nm showed that haem a within both covalent complexes catalyzed the oxidation of exogenous ferrocytochrome c : The second order rate constant for the traditional complex was 0.55×10 ⁶ m ⁻¹ ·s ⁻¹ while that for the steady‐state was 0.27×10 ⁶ m ⁻¹ ·s ⁻¹ . These values were approximately 25–50% of those observed for 1 : 1 electrostatic complexes of similar concentrations. These results combined with those of the ascorbate and the electrophoresis studies suggest that electrons are able to enter cytochrome c oxidase via two independent pathways. We propose that during enzyme turnover the enzyme cycles between two conformers, one with a substrate binding site at subunit II and the other along the interface of subunits II, IV and VIb. Structural analysis suggests that Glu112, Glu113, Glu114 and Asp125 of subunit IV and Glu40, Glu54, Glu78, Asp35, Asp49, Asp73 and Asp74 of subunit VIb are residues that might possibly be involved.
Thirteen different polypeptide subunits, each in one copy, five phosphatidyl ethanolamines and three phosphatidyl glycerols, two hemes A, three Cu ions, one Mg ion, and one Zn ion are detectable in the crystal structure of bovine heart cytochrome c oxidase in the fully oxidized form at 2.8 A resolution. A propionate of hems a, a peptide unit (-CO-NH-), and an imidazole bound to CuA are hydrogen-bonded sequentially, giving a facile electron transfer path from CUA to heme a. The O2 binding and reduction site, heme a3, is 4.7 A apart from CuB. Two possible proton transfer paths from the matrix side to the cytosolic side are located in subunit I, including hydrogen bonds and internal cavities likely to contain randomly oriented water molecules. Neither path includes the O2 reduction site. The O2 reduction site has a proton transfer path from the matrix side possibly for protons for producing water. The coordination geometry of CuB and the location of Tyr244 in subunit I at the end of the scalar proton path suggests a hydroperoxo species as the two electron reduced intermediate in the O2 reduction process.
The assembly of cytochrome-c oxidase was studied in human cells cultured in the presence of inhibitors of mitochondrial or cytosolic protein synthesis. Mitochondrial fractions were resolved using two-dimensional PAGE (blue native PAGE and tricine/SDS/PAGE) and subsequent western blots were developed with monoclonal antibodies against specific subunits of cytochrome-c oxidase. Proteins were also visualized using metabolic labeling followed by two-dimensional electrophoresis and fluorography. These techniques allowed identification of two assembly intermediates of cytochrome-c oxidase. Assembly of the 13 subunits of cytochrome-c oxidase starts with the association of subunit I with subunit IV. Then a larger subcomplex is formed, lacking only subunits VIa and either VIIa or VIIb.
A 2-month-old boy had progressive generalized weakness, hypotonia, and respiratory insufficiency requiring assisted ventilation. At age 3 1/2 months, he started having seizures and recurrent pulmonary infections; he died at age 7 months. Serum lactate was chronically elevated, but there was no aminoaciduria. Histochemical and ultrastructural studies of muscle biopsies at ages 2 and 3 months showed excessive mitochondria, lipid, and glycogen; a third biopsy at 6 months showed marked increase in perimysial fibrous and fat tissue. Cytochrome c oxidase activity was 7% of normal in the first biopsy and undetectable in the others. Cytochrome spectra of mitochondria isolated from postmortem muscle showed complete lack of cytochrome aa3. Antibodies were obtained against cytochrome c oxidase purified from normal human heart. Immunotitration and enzyme-linked immunosorbent assay (ELISA) showed decreased immunologically reactive enzyme protein in the patient's muscle, but SDS-PAGE electrophoresis of immunoprecipitates of muscle mitochondrial extracts showed the presence of all cytochrome c oxidase subunits. These data suggest that decreased synthesis of one or more subunits may result in markedly decreased concentration of electrophoretically normal complex IV in skeletal muscle.
Increasing the glucose concentration from 0.1 to 10% in exponentially growing cultures of Kluyveromyces lactis CBS 2359 does not repress the antimycin-sensitive respiration (QO2 of 80 μl O2·h-1·mg-1 dry weight) but raises the antimycin-insensitive respiration from 3 to 12 μl O2·h-1·mg-1 dry weight. Antimycin A inhibits the growth of K. lactis on a variety of substrates with the exception of glucose at concentrations equal to or higher than 1% where substantial antimycin-insensitive respiratory rates are induced. It can be concluded that a minimal antimycin-insensitive QO2 is necessary for cellular growth when the normal respiratory pathway is not functional.
The antimycin-insensitive respiration elicited by growth in high glucose concentrations is poorly inhibited by hydroxamate and is inhibited by 50% by 90 μm azide or 1mm cyanide. These concentrations are much higher than those necessary to inhibit cytochrome c oxidase which is not involved in the antimycin-insensitive respiration as was demonstrated by spectral measurements. A pigment absorbing at 555 nm is specifically reduced after addition of glucose to antimycin-inhibited cells. The same pigment is reoxidized by further addition of high concentrations of sodium azide indicating its participation in the antimycin-insensitive, azide-sensitive respiration.
The crystal structure of bovine heart cytochrome c oxidase at 2.8 A resolution with an R value of 19.9 percent reveals 13 subunits, each different from the other, five phosphatidyl ethanolamines, three phosphatidyl glycerols and two cholates, two hemes A, and three copper, one magnesium, and one zinc. Of 3606 amino acid residues in the dimer, 3560 have been converged to a reasonable structure by refinement. A hydrogen-bonded system, including a propionate of a heme A (heme a), part of peptide backbone, and an imidazole ligand of CuA, could provide an electron transfer pathway between CuA and heme a. Two possible proton pathways for pumping, each spanning from the matrix to the cytosolic surfaces, were identified, including hydrogen bonds, internal cavities likely to contain water molecules, and structures that could form hydrogen bonds with small possible conformational change of amino acid side chains. Possible channels for chemical protons to produce H2O, for removing the produced water, and for O2, respectively, were identified.
The introduction of stochastic methods in pedigree analysis has enabled geneticists to tackle computations intractable by standard deterministic methods. Until now these stochastic techniques have worked by running a Markov chain on the set of genetic descent states of a pedigree. Each descent state specifies the paths of gene flow in the pedigree and the founder alleles dropped down each path. The current paper follows up on a suggestion by Elizabeth Thompson that genetic descent graphs offer a more appropriate space for executing a Markov chain. A descent graph specifies the paths of gene flow but not the particular founder alleles traveling down the paths. This paper explores algorithms for implementing Thompson's suggestion for codominant markers in the context of automatic haplotyping, estimating location scores, and computing gene-clustering statistics for robust linkage analysis. Realistic numerical examples demonstrate the feasibility of the algorithms.
This chapter describes the use of blue native electrophoresis-polyacrylamide gel electrophoresis (BN-PAGE) and second-dimension Tricine-sodium dodecyl sulfate (SDS)-PAGE techniques for the microscale isolation and quantification of the protein subunits of oxidative phosphorylation (OX-PHOS) complexes from human tissues, for the determination of OX-PHOS defects in mitochondrial encephalomyopathies, and for the analysis of possible OX-PHOS defects in Alzheimer's and Parkinson's diseases. In contrast to other methods used for studies of human OX-PHOS enzymes, the electrophoretic techniques provide information about the quantity of correctly assembled multiprotein complexes. The preparation of mitochondria, especially from liver and brain, is problematic. To avoid varying protein losses with different samples, a method is developed for direct use of homogenized tissue. The experiments testing reproducibility and near-quantitative recovery of proteins after two-dimensional separation as a prerequisite for the quantification of OX-PHOS complexes, and the characteristics and limitations of the electrophoretic techniques, are described in the chapter. The psrotocols for the analysis of platelets and cells are also addressed in the chapter.
Structural and functional studies on cytochrome c oxidase before the crystal structures appeared, are summarized to show the importance of X-ray crystal structure at the atomic resolution for understanding mechanism of the enzyme reaction, O2 reduction coupled with proton pumping. Crystal structure of bovine heart enzyme at the fully oxidized state at 2.8 A resolution are reviewed to evaluate of the contribution for understanding the enzyme reaction mechanism. Effects of detergent structure on the crystallization conditions of the bovine heart cytochrome c oxidase, which was critical for obtaining the X-ray crystal structure, are presented to propose a possible mechanism of crystallization of multicomponent membrane proteins.
Cytochrome c oxidase deficiency is the most common biochemical defect associated with Leigh's syndrome. The genetic defect responsible for this deficiency has not been identified in any patient with Leigh's syndrome. Given that this disorder appears to be inherited as an autosomal recessive trait, this would suggest prima facie that one of the nuclear DNA-encoded cytochrome c oxidase subunits is affected. We report the first detailed sequence analysis of all 10 cytochrome c oxidase nuclear complementary DNAs and the cytochrome c oxidase mitochondrial genes in a Leigh's syndrome patient with cytochrome c oxidase deficiency. No pathological mutations were identified in any of the cytochrome c oxidase structural genes.
Thirteen different polypeptide subunits, each in one copy, five phosphatidyl ethanolamines and three phosphatidyl glycerols, two hemes A, three Cu ions, one Mg ion, and one Zn ion are detectable in the crystal structure of bovine heart cytochrome c oxidase in the fully oxidized form at 2.8 Å resolution. A propionate of hems a, a peptide unit (–CO–NH–), and an imidazole bound to CuA are hydrogen-bonded sequentially, giving a facile electron transfer path from CuA to heme a. The O2 binding and reduction site, heme a
3, is 4.7 Å apart from CuB. Two possible proton transfer paths from the matrix side to the cytosolic side are located in subunit I, including hydrogen bonds and internal cavities likely to contain randomly oriented water molecules. Neither path includes the O2 reduction site. The O2 reduction site has a proton transfer path from the matrix side possibly for protons for producing water. The coordination geometry of CuB and the location of Tyr244 in subunit I at the end of the scalar proton path suggests a hydroperoxo species as the two electron reduced intermediate in the O2 reduction process.
Leigh disease associated with cytochrome c oxidase deficiency (LD[COX-]) is one of the most common disorders of the mitochondrial respiratory chain, in infancy and childhood. No mutations in any of the genes encoding the COX-protein subunits have been identified in LD(COX-) patients. Using complementation assays based on the fusion of LD(COX-) cell lines with several rodent/human rho0 hybrids, we demonstrated that the COX phenotype was rescued by the presence of a normal human chromosome 9. Linkage analysis restricted the disease locus to the subtelomeric region of chromosome 9q, within the 7-cM interval between markers D9S1847 and D9S1826. Candidate genes within this region include SURF-1, the yeast homologue (SHY-1) of which encodes a mitochondrial protein necessary for the maintenance of COX activity and respiration. Sequence analysis of SURF-1 revealed mutations in numerous DNA samples from LD(COX-) patients, indicating that this gene is responsible for the major complementation group in this important mitochondrial disorder.
Phospholipase A(2) from Crotalus atrox hydrolyzes all of the phospholipids that are associated with purified, detergent-solubilized cytochrome c oxidase; less than 0.05 mol cardiolipin (CL)(1) remains bound per mol enzyme. Coincident with the hydrolysis of cardiolipin is a reversible decrease of 45-50% in the electron transport activity of the dodecylmaltoside-solubilized enzyme. Full activity is recoverable (90-98%) by addition of exogenous cardiolipin, but not by either phosphatidylcholine or phosphatidylethanolamine. Unexpectedly, cleavage of cardiolipin causes the dissociation of both subunits VIa and VIb from the enzyme. These are the two subunits that form the major protein-protein contacts between the two monomeric units within the dimeric complex. Although hydrolysis of CL by phospholipase A(2) and loss of these subunits is linked, the reverse process does not occur, i.e., removal of subunits VIa and VIb does not cause dissociation of the two functionally important, tightly bound cardiolipins. Nor does addition of exogenous cardiolipin result in reassociation of the two subunits with the remainder of the complex. We conclude that cardiolipin is not only essential for full electron transport activity, but also has an important structural role in stabilizing the association of subunits VIa and VIb within the remainder of the bovine heart enzyme.
To describe, implement, and test an efficient algorithm to obtain multipoint identity-by-descent (IBD) probabilities at arbitrary positions among marker loci for general pedigrees. Unlike existing programs, our algorithm can analyze data sets with large numbers of people and markers. The algorithm has been implemented in the SimWalk2 computer package.
Using a rigorous testing regimen containing five pedigrees of various sizes with realistic marker data, we compared several widely used IBD computation programs: Allegro, Aspex, GeneHunter, MapMaker/Sibs, Mendel, Sage, SimWalk2, and Solar.
The testing revealed a few discrepancies, particularly on consanguineous pedigrees, but overall excellent results in the deterministic multipoint packages. SimWalk2 was also found to be in good agreement with the deterministic multipoint programs, usually matching to two decimal places the kinship coefficient that ranges from 0 to 1. However, the packages based on single-point IBD estimation, while consistent with each other, often showed poor results, disagreeing with the multipoint kinship results by as much as 0.5.
Our testing has clearly shown that multipoint IBD estimation is much better than single-point estimation. In addition, our testing has validated our algorithm for estimating IBD probabilities at arbitrary positions on general pedigrees.
We investigated the interaction between cytochrome c oxidase and its substrate cytochrome c by catalyzing the covalent linkage of the two proteins to yield 1 : 1 covalent enzyme-substrate complexes under conditions of low ionic strength. In addition to the 'traditional' oxidized complex formed between oxidized cytochrome c and the oxidized enzyme we prepared complexes under steady-state reducing conditions. Whereas for the 'oxidized' complex cytochrome c became bound exclusively to subunit II of the enzyme, for the 'steady-state' complex cytochrome c became bound to subunit II and two low molecular mass subunits, most likely VIb and IV. For both complexes we investigated: (a) the ability of the covalently bound cytochrome c to relay electrons into the enzyme, and (b) the ability of the covalently bound enzyme to catalyze the oxidation of unbound (exogenous) ferrocytochrome c. Steady-state spectral analysis (400-630 nm) combined with stopped-flow studies, confirmed that the bound cytochrome c mediated the efficient transfer of electrons from the reducing agent ascorbate to the enzyme. In the case of the latter, the half life for the ascorbate reduction of the bound cytochrome c and that for the subsequent transfer of electrons to haem a were both < 5 ms. In contrast the covalent complexes, when reduced, were found to be totally unreactive towards oxidized cytochrome c oxidase confirming that the previously observed reduction of haem a within the complexes occurred via intramolecular rather than intermolecular electron transfer. Additionally, stopped-flow analysis at 550 nm showed that haem a within both covalent complexes catalyzed the oxidation of exogenous ferrocytochrome c: The second order rate constant for the traditional complex was 0.55x10(6) m(-1) x s(-1) while that for the steady-state was 0.27x10(6) m(-1) x s(-1). These values were approximately 25-50% of those observed for 1 : 1 electrostatic complexes of similar concentrations. These results combined with those of the ascorbate and the electrophoresis studies suggest that electrons are able to enter cytochrome c oxidase via two independent pathways. We propose that during enzyme turnover the enzyme cycles between two conformers, one with a substrate binding site at subunit II and the other along the interface of subunits II, IV and VIb. Structural analysis suggests that Glu112, Glu113, Glu114 and Asp125 of subunit IV and Glu40, Glu54, Glu78, Asp35, Asp49, Asp73 and Asp74 of subunit VIb are residues that might possibly be involved.
Detection of genotyping errors and integration of such errors in statistical analysis are relatively neglected topics, given their importance in gene mapping. A few inopportunely placed errors, if ignored, can tremendously affect evidence for linkage. The present study takes a fresh look at the calculation of pedigree likelihoods in the presence of genotyping error. To accommodate genotyping error, we present extensions to the Lander-Green-Kruglyak deterministic algorithm for small pedigrees and to the Markov-chain Monte Carlo stochastic algorithm for large pedigrees. These extensions can accommodate a variety of error models and refrain from simplifying assumptions, such as allowing, at most, one error per pedigree. In principle, almost any statistical genetic analysis can be performed taking errors into account, without actually correcting or deleting suspect genotypes. Three examples illustrate the possibilities. These examples make use of the full pedigree data, multiple linked markers, and a prior error model. The first example is the estimation of genotyping error rates from pedigree data. The second-and currently most useful-example is the computation of posterior mistyping probabilities. These probabilities cover both Mendelian-consistent and Mendelian-inconsistent errors. The third example is the selection of the true pedigree structure connecting a group of people from among several competing pedigree structures. Paternity testing and twin zygosity testing are typical applications.
This article describes a quick basic method adapted for the purification of mammalian mitochondria from different sources. The organelles obtained using this protocol are suitable for the investigation of biogenetic activities such as enzyme activity, mtDNA, mtRNA, mitochondrial protein synthesis, and mitochondrial tRNA aminoacylation. In addition, these mitochondria are capable of efficient protein import and the investigation of mtDNA/protein interactions by DNA footprinting is also possible.
Sperm motility is highly dependent on aerobic energy metabolism, of which the apparent rate-limiting step of the mitochondrial respiratory chain is catalyzed by cytochrome c oxidase (COX). COX is the only electron transport chain complex to display isoforms, consistent with its suggested rate-limiting role. Isoforms were previously described for four of the 13 subunits. We now report the discovery that COX subunit VIb displays a testes-specific isoform in human, bull, rat, and mouse (COX VIb-2). Analysis of a variety of rat and mouse tissues, including ovaries, demonstrates exclusive expression of VIb-2 in testes, whereas VIb-1 transcripts are absent in rodent testes, even at early developmental stages. In contrast, both isoforms are transcribed in human testes. In situ hybridizations with human, rat, and mouse testes sections reveal VIb-2 transcripts in all testicular cell types. Within the seminiferous tubules, VIb-1 shows stronger signals in the periphery than in the lumen. Previously, cytochrome c was the only component of the mitochondrial respiratory chain known to express a testes-specific isoform in mammals. COX subunit VIb connects the two COX monomers into the physiological dimeric form, and is the only COX subunit that, like cytochrome c, is solely located in the inter-membrane space. Significant differences between the isoform sequences, in particular changes in charged amino acids, suggest interactions with cytochrome c and sperm-specific energy requirements.
In the medical literature the term 'mitochondrial disorders' is to a large extent applied to the clinical syndromes associated with abnormalities of the common final pathway of mitochondrial energy metabolism, i.e. oxidative phosphorylation (OXPHOS). Faulty oxidative phosphorylation may be due to overall dysfunction of the respiratory chain, a heteromultimeric structure embedded in the inner mitochondrial membrane, or can be associated with single or multiple defects of the five complexes forming the respiratory chain itself. From the genetic standpoint, the respiratory chain is a unique structure of the inner mitochondrial membrane formed by means of the complementation of two separate genetic systems: the nuclear genome and the mitochondrial genome. The nuclear genome encodes the large majority of the protein subunits of the respiratory complexes and most of the mitochondrial DNA (mtDNA) replication and expression systems, whereas the mitochondrial genome encodes only 13 respiratory complex subunits, and some RNA components of the mitochondrial translational apparatus. Accordingly, mitochondrial disorders due to defects in OXPHOS include both mendelian-inherited and cytoplasmic-inherited diseases. This review describes human genetic diseases associated with mtDNA and nuclear DNA mutations leading to impaired OXPHOS.
Isolated complex I deficiency, the most frequent OXPHOS disorder in infants and children, is genetically heterogeneous. Mutations have been found in seven mitochondrial DNA (mtDNA) and eight nuclear DNA encoded subunits, respectively, but in most of the cases the genetic basis of the biochemical defect is unknown. We analyzed the entire mtDNA and 11 nuclear encoded complex I subunits in 23 isolated complex I-deficient children, classified into five clinical groups: Leigh syndrome, progressive leukoencephalopathy, neonatal cardiomyopathy, severe infantile lactic acidosis, and a miscellaneous group of unspecified encephalomyopathies. A genetic definition was reached in eight patients (35%). Mutations in mtDNA were found in six out of eight children with Leigh syndrome, indicating a prevalent association between this phenotype and abnormalities in ND genes. In two patients with leukoencephalopathy, homozygous mutations were detected in two different nuclear-encoded complex I genes, including a novel transition in NDUFS1 subunit. In addition to these, a child affected by mitochondrial encephalomyopathy had heterozygous mutations in NDUFA8 and NDUFS2 genes, while another child with neonatal cardiomyopathy had a complex rearrangement in a single NDUFS7 allele. The latter cases suggest the possibility of unconventional patterns of inheritance in complex I defects.
Mutations in the human TAZ gene are associated with Barth Syndrome, an often fatal X-linked disorder that presents with cardiomyopathy and neutropenia. The TAZ gene encodes Tafazzin, a putative phospholipid acyltranferase that is involved in the remodeling of cardiolipin, a phospholipid unique to the inner mitochondrial membrane. It has been shown that the disruption of the Tafazzin gene in yeast (Taz1) affects the assembly and stability of respiratory chain Complex IV and its supercomplex forms. However, the implications of these results for Barth Syndrome are restricted due to the additional presence of Complex I in humans that forms a supercomplex with Complexes III and IV. Here, we investigated the effects of Tafazzin, and hence cardiolipin deficiency in lymphoblasts from patients with Barth Syndrome, using blue-native polyacrylamide gel electrophoresis. Digitonin extraction revealed a more labile Complex I/III(2)/IV supercomplex in mitochondria from Barth Syndrome cells, with Complex IV dissociating more readily from the supercomplex. The interaction between Complexes I and III was also less stable, with decreased levels of the Complex I/III(2) supercomplex. Reduction of Complex I holoenzyme levels was observed also in the Barth Syndrome patients, with a corresponding decrease in steady-state subunit levels. We propose that the loss of mature cardiolipin species in Barth Syndrome results in unstable respiratory chain supercomplexes, thereby affecting Complex I biogenesis, respiratory activities and subsequent pathology.
The mitochondrial oxidative phosphorylation system is composed of five multiprotein complexes. The fourth complex of this system, cytochrome c oxidase (complex IV), consists of 13 subunits: 3 encoded by mitochondrial DNA and 10 encoded by the nuclear genome. Patients with an isolated complex IV deficiency frequently harbor mutations in nuclear genes encoding for proteins necessary for the assembly of the complex. Strikingly, until now, no mutations have been detected in the nuclear encoded structural subunits of complex IV in these patients. We report the results of a mutational analysis study in patients with isolated complex IV deficiency screened for mutations in all structural genes as well as assembly genes known to cause complex IV deficiency. Four patients carried mutations in the complex IV assembly gene SURF1. One patient harbored a mutation in the COX10 gene involved in heme A synthesis. Mutations in the 10 nuclear encoded structural genes were not present.
The whole structure of the 13-subunit oxi
- T Tsukihara
- H Aoyama
- E Yamashita
- T Tomizaki
- H Yamagu-Chi
- K Shinzawa-Itoh
- R Nakashima
- R Yaono
Tsukihara, T., Aoyama, H., Yamashita, E., Tomizaki, T., Yamagu-chi, H., Shinzawa-Itoh, K., Nakashima, R., Yaono, R., and Yosh-ikawa, S. (1996). The whole structure of the 13-subunit oxi-dized cytochrome c oxidase at 2.8 A. Science 272, 1136–1144.
Analysis (A) Family pedigree and haplotype analysis of the COX6B1 locus. Patients (#15, patient AK; #16, patient AW) are indicated by black symbols. (B) COX histoenzymatic staining of muscle biopsy. The reaction is diffusely low in the muscle from patient AW, compared with an age-matched control
- Genetic
- Clinical
Genetic, Clinical, and Biochemical Analysis (A) Family pedigree and haplotype analysis of the COX6B1 locus. Patients (#15, patient AK; #16, patient AW) are indicated by black symbols. (B) COX histoenzymatic staining of muscle biopsy. The reaction is diffusely low in the muscle from patient AW, compared with an age-matched control (inset).
Molecular Cloning: A Laboratory Manual (Cold Spring Harbor
- J Sambrook
Sambrook, J., and Russel, D.W. (2001). Molecular Cloning: A
Laboratory Manual (Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press).
- S L Williams
- I Valnot
- P Rustin
Williams, S.L., Valnot, I., Rustin, P., and Taanman, J.W. (2004).