Human Mitochondrial ATP Synthase Research Articles

Amelioration of Leigh syndrome induced by mouse blastocyst complementation with a mutant human mitochondrial ATP synthase 6

AbstractBackgroundMutation of the m. 8993 T > G ATP6 subunit of ATP synthase causes a maternally inherited Leigh Syndrome (LS), a rapidly fatal encephalomyelopathy in childhood called and related Neuropathy, Ataxia and Retinitis Pigmentosa (NARP) in adults. There is no cure, and relevant animal models are few. Here we describe a novel ATP6 mouse model of LS and preclinical efficacy of a gene therapy approach to neutralize the ATP6 mutation by competition with a mitochondrial targeted adeno‐associated virus serotype 9 (AAV9) vector containing wild‐type ATP6.MethodsLS transgenic mice with an ATP6 mutation were developed by microinjecting AAV2 modified by a COX8 mitochondrial targeting sequence (MTS) into mouse blastocysts to deliver the human ATP6 gene with a T > G mutation at position 8993 of the mitochondrial genome, responsible for clinical LS DNA, protein, and metabolic assays confirmed functional gene delivery and in depth physiological/clinical assessments supported germline transmission of an LS phenotype. Rescue of LS in this model was evaluated after intravenous injection of mito‐targeted AAV9 containing wild‐type ATP6 followed by reevaluation of phenotypes.ResultsLS transgenic mice expressed mutant ATP6 protein in multiple relevant tissues including brain, eye, liver. Mutant ATP6 incorporated into Complex V and conferred decreased ATP synthase efficiency. Mice exhibited hallmarks of the human disease with one or multiple systemic symptoms including early death, paralysis, hunching, vision loss and seizures over six generations. Necropsy revealed spongiform encephalopathy, retinal degeneration, and cardiomegaly. Rescue by mitochondrially directed AAV9 containing wild type ATP6 after disease onset conferred prolonged survival and reduced paralysis. When administered prior to disease onset all treated ATP6 mice survived with evidence of globally improved function.ConclusionsThe results are consistent with efficient delivery and expression of foreign genes in mitochondria by AAV vectors directed by MTS and support further development of such strategies via intravenous injection of mitochondrial targeted AAV9 to deliver corrective genes.

Inhibitors of F1F0-ATP synthase enzymes for the treatment of tuberculosis and cancer.

The spectacular success of the mycobacterial F1F0-ATP synthase inhibitor bedaquiline for the treatment of drug-resistant tuberculosis has generated wide interest in the development of other inhibitors of this enzyme. Work in this realm has included close analogues of bedaquiline with better safety profiles and 'bedaquiline-like'compounds, some of which show potent antibacterial activity in vitro although none have yet progressed to clinical trials. The search has lately extended to a range of new scaffolds as potential inhibitors, including squaramides, diaminoquinazolines, chloroquinolines, dihydropyrazolo[1,5-a]pyrazin-4-ones, thiazolidinediones, diaminopyrimidines and tetrahydroquinolines. Because of the ubiquitous expression of ATP synthase enzymes, there has also been interest in inhibitors of other bacterial ATP synthases, as well as inhibitors of human mitochondrial ATP synthase for cancer therapy. The latter encompass bothcomplex natural products and simpler small molecules. The review seeks to demonstrate the breadth of the structural types of molecules able to effectively inhibit the function of variants of this intriguing enzyme.

TMEM70 and TMEM242 help to assemble the rotor ring of human ATP synthase and interact with assembly factors for complex I

Human mitochondrial ATP synthase is a molecular machine with a rotary action bound in the inner organellar membranes. Turning of the rotor, driven by a proton motive force, provides energy to make ATP from ADP and phosphate. Among the 29 component proteins of 18 kinds, ATP6 and ATP8 are mitochondrial gene products, and the rest are nuclear gene products that are imported into the organelle. The ATP synthase is assembled from them via intermediate modules representing the main structural elements of the enzyme. One such module is the c8-ring, which provides the membrane sector of the enzyme's rotor, and its assembly is influenced by another transmembrane (TMEM) protein, TMEM70. We have shown that subunit c interacts with TMEM70 and another hitherto unidentified mitochondrial transmembrane protein, TMEM242. Deletion of TMEM242, similar to deletion of TMEM70, affects but does not completely eliminate the assembly of ATP synthase, and to a lesser degree the assembly of respiratory enzyme complexes I, III, and IV. Deletion of TMEM70 and TMEM242 together prevents assembly of ATP synthase and the impact on complex I is enhanced. Removal of TMEM242, but not of TMEM70, also affects the introduction of subunits ATP6, ATP8, j, and k into the enzyme. TMEM70 and TMEM242 interact with the mitochondrial complex I assembly (the MCIA) complex that supports assembly of the membrane arm of complex I. The interactions of TMEM70 and TMEM242 with MCIA could be part of either the assembly of ATP synthase and complex I or the regulation of their levels.

MitCHAP-60 and Hereditary Spastic Paraplegia SPG-13 Arise from an Inactive hsp60 Chaperonin that Fails to Fold the ATP Synthase \u03b2-Subunit

The human mitochondrial heat shock protein 60 (hsp60) is a tetradecameric chaperonin that folds proteins in the mitochondrial matrix. An hsp60 D3G mutation leads to MitCHAP-60, an early onset neurodegenerative disease while hsp60 V72I has been linked to SPG13, a form of hereditary spastic paraplegia. Previous studies have suggested that these mutations impair the protein folding activity of hsp60 complexes but the detailed mechanism by which these mutations lead the neuromuscular diseases remains unknown. It is known, is that the β-subunit of the human mitochondrial ATP synthase co-immunoprecipitates with hsp60 indicating that the β-subunit is likely a substrate for the chaperonin. Therefore, we hypothesized that hsp60 mutations cause misfolding of proteins that are critical for aerobic respiration. Negative-stain electron microscopy and DLS results suggest that the D3G and V72I complexes fall apart when treated with ATP or ADP and are therefore unable to fold denatured substrates such as α-lactalbumin, malate dehydrogenase (MDH), and the β-subunit of ATP synthase in in-vitro protein-folding assays. These data suggests that hsp60 plays a crucial role in folding important players in aerobic respiration such as the β-subunit of the ATP synthase. The hsp60 mutations D3G and V72I impair its ability to fold mitochondrial substrates leading to abnormal ATP synthesis and the development of the MitCHAP-60 and SPG13 neuromuscular degenerative disorders.

Plant-derived mitochondria-targeting cysteine-rich peptide modulates cellular bioenergetics

Mitochondria are attractive therapeutic targets for developing agents to delay age-related frailty and diseases. However, few promising leads have been identified from natural products. Previously, we identified roseltide rT1, a hyperstable 27-residue cysteine-rich peptide from Hibiscus sabdariffa, as a knottin-type neutrophil elastase inhibitor. Here, we show that roseltide rT1 is also a cell-penetrating, mitochondria-targeting peptide that increases ATP production. Results from flow cytometry, live-cell imaging, pulldown assays, and genetically-modified cell lines supported that roseltide rT1 enters cells via glycosaminoglycan-dependent endocytosis, and enters the mitochondria through TOM20, a mitochondrial protein import receptor. We further showed that roseltide rT1 increases cellular ATP production via mitochondrial membrane hyperpolarization. Using biotinylated roseltide rT1 for target identification and proteomic analysis, we showed that human mitochondrial membrane ATP synthase subunit O is an intramitochondrial target. Collectively, these data support our discovery that roseltide rT1 is a first-in-class mitochondria-targeting, cysteine-rich peptide with potentials to be developed into tools to further our understanding of mitochrondria-related diseases.

Assembly of human mitochondrial ATP synthase through two separate intermediates, F1-c-ring and b–e–g complex

Mitochondrial ATP synthase is a motor enzyme in which a central shaft rotates in the stator casings fixed with the peripheral stator stalk. When expression of d-subunit, a stator stalk component, was knocked-down, human cells could not form ATP synthase holocomplex and instead accumulated two subcomplexes, one containing a central rotor shaft plus catalytic subunits (F1-c-ring) and the other containing stator stalk components (“b–e–g” complex). F1-c-ring was also formed when expression of mitochondrial DNA-coded a-subunit and A6L was suppressed. Thus, the central rotor shaft and the stator stalk are formed separately and they assemble later. Similar assembly strategy has been known for ATP synthase of yeast and Escherichia coli and could be common to all organisms.

Mitochondrial ATP synthase: architecture, function and pathology.

Human mitochondrial (mt) ATP synthase, or complex V consists of two functional domains: F1, situated in the mitochondrial matrix, and Fo, located in the inner mitochondrial membrane. Complex V uses the energy created by the proton electrochemical gradient to phosphorylate ADP to ATP. This review covers the architecture, function and assembly of complex V. The role of complex V di-and oligomerization and its relation with mitochondrial morphology is discussed. Finally, pathology related to complex V deficiency and current therapeutic strategies are highlighted. Despite the huge progress in this research field over the past decades, questions remain to be answered regarding the structure of subunits, the function of the rotary nanomotor at a molecular level, and the human complex V assembly process. The elucidation of more nuclear genetic defects will guide physio(patho)logical studies, paving the way for future therapeutic interventions.

Selectivity of TMC207 towards Mycobacterial ATP Synthase Compared with That towards the Eukaryotic Homologue

The diarylquinoline TMC207 kills Mycobacterium tuberculosis by specifically inhibiting ATP synthase. We show here that human mitochondrial ATP synthase (50% inhibitory concentration [IC(50)] of >200 microM) displayed more than 20,000-fold lower sensitivity for TMC207 compared to that of mycobacterial ATP synthase (IC(50) of 10 nM). Also, oxygen consumption in mouse liver and bovine heart mitochondria showed very low sensitivity for TMC207. These results suggest that TMC207 may not elicit ATP synthesis-related toxicity in mammalian cells. ATP synthase, although highly conserved between prokaryotes and eukaryotes, may still qualify as an attractive antibiotic target.

A vertebrate RNA-binding protein Fox-1 regulates tissue-specific splicing via the pentanucleotide GCAUG.

Alternative splicing is one of the central mechanisms that regulate eukaryotic gene expression. Here we report a tissue-specific RNA-binding protein, Fox-1, which regulates alternative splicing in vertebrates. Fox-1 bound specifically to a pentanucleotide GCAUG in vitro. In zebrafish and mouse, fox-1 is expressed in heart and skeletal muscles. As candidates for muscle-specific targets of Fox-1, we considered two genes, the human mitochondrial ATP synthase gamma-subunit gene (F1gamma) and the rat alpha-actinin gene, because their primary transcripts contain several copies of GCAUG. In transfection experiments, Fox-1 induced muscle-specific exon skipping of the F1gamma gene via binding to GCAUG sequences upstream of the regulated exon. Fox-1 also regulated mutually exclusive splicing of the alpha-actinin gene, antagonizing the repressive effect of polypyrimidine tract-binding protein (PTB). It has been reported that GCAUG is essential for the alternative splicing regulation of several genes including fibronectin. We found that Fox-1 promoted inclusion of the fibronectin EIIIB exon. Thus, we conclude that Fox-1 plays key roles in both positive and negative regulation of tissue-specific splicing via GCAUG.

Isolation of a bi-directional promoter directing expression of the mouse GABPα and ATP synthase coupling factor 6 genes

The GA-binding protein (GABP) is a ubiquitous heteromeric transcription factor implicated in the regulation of several genes involved in mitochondrial energy metabolism including subunits of cytochrome c oxidase, ATP synthase, and mitochondrial transcription factor 1 (mtTF1). GABPα subunit binds the PEA3/Ets binding sites (EBS), while GABPβ contains a transcription activation domain and mediates αβ dimer and α 2β 2 tetramer formation essential for activation of transcription. Here we report the cloning of 2449 bp of the mouse (m) GABPα promoter region including 201 bp of the 5′ end of the published mGABPα cDNA sequence. Surprisingly, sequences homologous to the 5′UTR of mouse, rat and human mitochondrial ATP synthase coupling factor 6 (ATPsynCF6) cDNAs were found165–240 bp upstream of the mGABPα cDNA. A search of the non-redundant nucleotide database revealed a human genomic sequence derived from chromosome 21 (21q22) bearing significant homology to the mGABPα/ATPsynCF6 promoter region and encompassed the entire hGABPα and hATPsynCF6 genes. Primer extension analysis revealed multiple transcription start sites for both mGABPα and mATPsynCF6 mRNAs that mapped near the published cDNA 5′ ends. Sequence analysis identified several binding sites upstream of the GABPα cDNA sequence including sites for GABP (−86, −104, −169, −257, and −994), YY1 (−57), Sp1 (−242 and −226), and NRF1 (−5). No ‘TATA’ motif was identified near either the GABPα or ATPsynCF6 transcription start sites. The human and mouse promoters retain significant sequence identity including binding sites for several tissue-specific transcription factors. Transient transfection assays using Luciferase reporter constructs containing the intergenic region and flanking sequences confirmed that this region of DNA promotes transcription in both directions.

ETS transcription factors regulate the expression of the gene for the human mitochondrial ATP synthase beta-subunit.

Elements responsible for the transcriptional activity of the human ATP synthase beta-subunit (ATPsyn beta) gene promoter have been studied through transient expression in HepG2 hepatoma cells of a CAT gene connected with various 5'-deletion mutants of the 5'-flanking region. Promoter activity was mostly dependent upon a single CCAAT motif as well as a nearby Ets domain binding region. This last region contains two sites that bind Ets-related proteins present in liver nuclear extracts as well as recombinant purified Ets-1 protein. The ATPsyn beta promoter was trans-activated by Ets-1 and Ets-2 expression vectors, and this effect was lost when the Ets binding region was deleted. The Ets binding region of the ATPsyn beta promoter increased basal expression and conferred Ets-1- and Ets-2-dependent trans-activation to the herpes symplex thymidine kinase minimal promoter. A double-point mutation of the main Ets-binding site, which suppresses Ets binding, blocks Ets-dependent trans-activation. It is concluded that the gene for the mitochondrial ATPsyn beta is a target of transcriptional activation by members of the Ets family of transcription factors. It is suggested that Ets transcription factors may be involved in the enhanced expression of the ATPsyn beta gene in highly proliferating cells and in the coordinate transcription of nuclear genes for mitochondrial proteins.

Sequence analysis and mapping of a novel human mitochondrial ATP synthase subunit 9 cDNA (ATP5G3).

We describe the cloning, sequence analysis, and chromosomal mapping of a novel mitochondrial ATP synthase subunit 9cDNA, P3. Subunit 9 transports protons across the inner mitochondrial membrane to the F1-ATPase protruding on the matrix side, resulting in the generation of ATP. Sequence analysis of the P3 cDNA reveals only 80% identity with the human subunit 9 genes P1 and P2 in the DNA sequence encoding the mature peptide. However, this sequence predicts a mature protein identical to P1 and P2. The predicted sequence of the P3 leader peptide differs from the P1 and P2 leaders, but retains the "RFS" motif critical for mitochondrial import and maturation. The P3 gene (ATP5G3) maps to chromosome 2.

ATPF1 binding site, a positive cis-acting regulatory element of the mammalian ATP synthase alpha-subunit gene.

An analysis of the promoter of a bovine and human nuclear-encoded mitochondrial ATP synthase alpha-subunit gene (ATPA) revealed the presence of a positive control element. DNase I footprinting and electrophoretic mobility shift assays demonstrated that this cis-acting regulatory element contains a binding site for a protein present in human HeLa nuclei, termed ATP factor 1 (ATPF1). The ATPF1 binding site contains the sequence, CANNTG, a sequence identical to the recognition site for a family of transcription factors containing a basic region adjacent to a helix-loop-helix domain. Site-directed point mutations of the basic helix-loop-helix binding site demonstrated the critical role of this element for both binding of ATPF1 and also for transcriptional activation of the ATPA gene.

Gene structure of human mitochondrial ATP synthase gamma-subunit. Tissue specificity produced by alternative RNA splicing.

We completely sequenced the human gene for ATP synthase gamma-subunit, which was approximately 23 kilobases long and was composed of 10 exons. Exons 1 and 2 encoded the N-terminal presequence required for mitochondrial import, while exons 9 and 10 encoded the C-terminal portions of mature protein. Enzymatic amplification of human heart and liver cDNAs using the polymerase chain reaction revealed two mRNA transcripts that were predicted to encode two 30-kDa isoforms of the gamma-subunit, which differed by the addition of a single amino acid (Asp273) at the C terminus of the liver type isoform. These two mRNA transcripts of the heart (H) type and liver (L) type were generated by alternative splicing of an exon. The same alternative splicing event was observed in bovine tissue. In human tissues, the H type mRNA devoid of exon 9 was expressed specifically in the heart and skeletal muscle, which require rapid energy supply. The L type mRNA was expressed in the brain, liver, kidney etc. Both transcripts were expressed in the skin, intestine, stomach, and aorta. This tissue specificity of transcript heterogeneity suggests the distinct functional or regulatory roles of the gamma-subunit isoforms in the catalysis of ATP synthase. This is the first report on tissue-specific isoforms generated by alternative splicing in an energy transducing mitochondrial protein.

Amplification of the gene encoding the α-subunit of the mitochondrial ATP synthase complex in a human retinoblastoma cell line

A cDNA clone encoding the precursor of the α-subunit of the human mitochondrial ATP synthase (F1-ATPS) complex was isolated from a library prepared from the poly(A) +RNA present in a retinoblastoma (RB) cell line. Northern blot analysis of RNAs derived from a variety of transformed cell lines as well as from normal human fetal tissues indicated that RNA expression was significantly higher in two of the four RB cell lines analysed, Y79 (10- to 30-fold) and RB522A (3- to 8-fold), than in other cell lines or tissues. The increased mRNA level was apparently the result of gene amplification in Y79, but not in RB522A.

Antipeptide antibodies to the carboxy terminal and the DCCD binding region of the human mitochondrial ATP synthase β-subunit

Antibodies to defined epitopes on the human ATP synthase would provide a powerful tool in the definition of the subunit composition of the enzyme complex and in the characterization of any defect in its assembly in diseases associated with mitochondrial disorders. Antibodies have been thus raised against synthetic peptides, corresponding to two regions on the human ATP synthase β-subunit: the C-terminal region, and a region which includes the two dicyclohexylcarbodiimide (DCCD)-reactive glutamic acid residues suggested to be involved in the enzyme catalytic activity. The antibodies to the C-terminal peptide reacted with the ATP synthase β-subunit in ELISA, in Western immunoblotting and in immunohistochemical experiments, and had the ability to immunoprecipitate the enzyme complex. The antibodies to the DCCD-binding region peptide did not react to the ATP synthase β-subunit in its native configuration, although reacted well under Western immunoblotting conditions.

Antipeptide antibodies to the carboxy terminal and the DCCD binding region of the human mitochondrial ATP synthase β-subunit

Antipeptide antibodies to the carboxy terminal and the DCCD binding region of the human mitochondrial ATP synthase β-subunit

Molecular cloning of an import precursor of the δ-subunit of the human mitochondrial ATP synthase complex

Two types of cDNA clones encoding a precursor of the delta-subunit of the human mitochondrial F0F1 ATP synthase complex (EC 3.6.1.34) have been isolated from a human cDNA library. Both clones contain a 504 basepair open reading frame that encodes a polypeptide with a presequence 22 amino acids in length and a mature protein 146 residues in length. The difference between the two types of cDNA clones is the presence of a 296 basepair insert in the 3' untranslated region of the delta-subunit cDNA in one of the types.

Antipeptide antibodies to the carboxy terminal and the DCCD binding region of the human mitochondrial ATP synthase β-subunit

Antibodies to defined epitopes on the human ATP synthase would provide a powerful tool in the definition of the subunit composition of the enzyme complex and in the characterization of any defect in its assembly in diseases associated with mitochondrial disorders. Antibodies have been thus raised against synthetic peptides, corresponding to two regions on the human ATP synthase β-subunit: the C-terminal region, and a region which includes the two dicyclohexylcarbodiimide (DCCD)-reactive glutamic acid residues suggested to be involved in the enzyme catalytic activity. The antibodies to the C-terminal peptide reacted with the ATP synthase β-subunit in ELISA, in Western immunoblotting and in immunohistochemical experiments, and had the ability to immunoprecipitate the enzyme complex. The antibodies to the DCCD-binding region peptide did not react to the ATP synthase β-subunit in its native configuration, although reacted well under Western immunoblotting conditions.

Nucleotide sequence of a cDNA for the α subunit of human mitochondrial ATP synthase

A full length cDNA clone of the alpha subunit of mitochondrial ATP synthase (EC 3.6.1.34) has been isolated from a cDNA library prepared from LX-1 human tumor cells in the lambda-Zap vector. The clone is 1883 base pairs (bp) in length and contains a 1659 bp open reading frame encoding a polypeptide of 553 residues. The deduced amino acid sequence is highly homologous to ATP synthase from several other species.