Lancet study finds relationship between Redox Signaling molecules and mitochondria
Mitochondria are dynamic bioenergy organelles that maintain approximately 1500 proteins in the genome. Mutations in the mitochondrial or nuclear genome can disrupt cell metabolism and homogeneity. Mitochondrial diseases are the most common and severe type of hereditary diseases, which are characterized by clinical, biochemical and genetic heterogeneity, diagnostic disorders, and lack of disease-modifying therapies. With the wide application of high-throughput omics technology, mitochondrial biology and medicine have developed very well, and many new mitochondrial treatment methods have also been derived. New understanding of bioenergy and biosynthetic mitochondrial function has accelerated the genetic diagnosis of primary mitochondrial diseases and identified new mitochondrial pathological mechanisms and new targets for therapeutic intervention. As we enter the new era of mitochondrial medicine, omics technology will continue to reveal unresolved mitochondrial problems, paving the way for improving outcomes for patients with mitochondrial disease.
Mitochondrial function and disorders
Mitochondria are complex dynamic organelles that perform many functions related to cellular metabolism and homogeneous stability. Oxidative phosphorylation (OXPHOS) produces cellular energy that is a hallmark of mitochondria, but mitochondria also play a role in calcium homeostasis, caspase-dependent apoptosis initiation, cellular stress response, heme biosynthesis, sulfur metabolism and cytoplasmic protein degradation. effect. The unique feature of mitochondria is mitochondrial DNA (mtDNA), a small circular genome derived from the symbiotic evolutionary origin of organelles (encoding only 37 genes). MtDNA encodes 13 OXPHOS proteins; the remaining approximately 1500 mitochondrial proteome proteins are in the nuclear genome It is encoded and transferred to mitochondria by a complex system.
It has been reported that more than 350 genes from mitochondria and nuclear sources have been mutated, causing mitochondrial disease. These genetic diseases can be defined as mutations that cause OXPHOS dysfunction or other disorders of mitochondrial structure and function, including mutations in mitochondrial ultrastructure, abnormality of cofactors and vitamins, or impairment of other metabolic processes in the mitochondria. Carboxylic acid (TCA) cycle and pyruvate metabolism.
The mitochondrial disease shows an uneven phenotype and biochemical manifestations. This variation, coupled with an incomplete understanding of the pathophysiology of mitochondria, poses diagnostic challenges to mitochondrial diseases and lacks disease modification treatments. However, in recent years, high-throughput omics technologies-that is, high-throughput technologies capable of detecting differences in various molecular components in an organism (including genomics, transcriptomics, proteomics, metabolomics, and epigenomics) -And sophisticated bioinformatics tools have revealed new details of mitochondrial function and its contribution to cell health and disease. These new technologies have valuable value to several pillars of mitochondrial medicine, including elucidating basic aspects of mitochondrial structure and function, strengthening genetic diagnosis of mitochondrial diseases, and providing insights into new therapeutic goals for improving the efficacy of mitochondrial diseases.
Improve understanding of oxidative phosphorylation
Perhaps the most distinctive feature of mitochondria is that it is called the cell's "power station." The rationale behind this alternative name is to generate cellular energy through OXPHOS. The OXPHOS system is conserved from bacteria to higher eukaryotes. It consists of five polymerases, called complexes I to V, and two mobile electron carriers (Coenzyme Q10 [CoQ 10] and cytochrome c [cyt c]. ). Complex I (NADH: quinone oxidoreductase [CI]) pumps four protons from the mitochondrial membrane (IMM) into the intermembrane space (IMS) through NADH oxidation and CoQ 10 reduction. Complex II (succinate-coq oxidoreductase [CII]) transfers electrons from fad-dependent sources through reduction of CoQ 10; however, this enzyme cannot pump protons from imm. Complex III (ubiquinone-cytochrome c oxidoreductase [CIII]) redox CoQ 10, reduces Cytc, and releases four protons into the IMS. The final electron acceptor is molecular oxygen, which accepts four electrons in a reaction catalyzed by complex IV (cytochrome c oxidase (CIV) to form water molecules. This reaction is coupled to the transfer of protons to the IMS. These complexes The proton gradient generated by the activity of a substance produces a proton motive force, which is used by F1F0-ATP synthase (complex V) to phosphorylate ADP to ATP, which is the cell's main energy currency.
Mitochondria as gatekeepers of signal organelles and cellular homeostasis
A new concept in mitochondrial biology is the role of mitochondria as messenger organelles in constant communication with the nucleus and other subcellular compartments to titrate the generation of energy and metabolites to the specific needs and nutritional availability of cells. Details of retrograde signals from mitochondria to cytoplasm, including ROS, mitochondrial membrane potential, key intermediate metabolites, mitochondrial bioenergy (AMP / ATP) and redox (NAAD + / NADH) ratios, calcium flux on mitochondrial membrane, and GasoTurmim. At physiological concentrations, such as hydrogen sulfide, which promotes mitochondrial ROS production, although initially assumed to be harmful and contribute to mitochondria and cytopathology, regulation of cellular signal transduction and gene expression appears to be indispensable. OcLee, H2O2 and other ROS fine-tune mitochondrial morphology by altering fission balance (fragmented mitochondria) and fusion (long mitochondria). Elsewhere in the cell, mitochondrial H2O2 is involved in regulating cell survival, autophagy, and cell migration through regulatory kinase-driven pathways. At the organism level, it is important to note that ROS signals affect different cell types differently. All or part of several fusion metabolic pathways including TCA cycle, folate metabolism and sulfur metabolism.
Not only are the enzymes and metabolites in these pathways important for their respective metabolic functions, but these species also have pleiotropic effects on signalling molecules. In addition to generating reducing equivalents to the feed OXPHOS system, the TCA cycle has numerous compliments, including providing precursors for the biosynthesis of complex lipids, proteins, carbohydrates, and nucleotides. Signalling effects of cyclic intermediates on acetylcysteine and α-ketoglutarate affect histone acetylation and demethylation, respectively. Protein succinylation requires succinyl-CoA, and post-translational modifications have been proposed. There is a wide range of cellular effects in health and disease, including immune function and cancer. In addition, the alpha-ketoglutarate: succinic acid ratio has recently been shown to affect human stem cell differentiation. It should be noted that there is physiologically relevant evidence. F succinylation is still scarce. Impaired mitochondrial ATP synthesis is associated with an increase in the AMP: ATP ratio, leading to the activation of AMP-activated kinase (AMPK), which is a major cellular bioenergy sensor and is considered to be a shock source of nutrition-dependent signals. Identify AMPK phosphorylation targets, including the mitochondrial fission factor MFF, to connect nutritional sensing and mitochondrial dynamics. AMPK also alters the mitochondrial NAAD +: NADH redox ratio, resulting in sirtuin-mediated deacetylation of the primary transcriptional co-activator PGC1α. Enhance its gene expression and promote mitochondrial biosynthesis.
Application of genomics in mitochondrial diagnosis
Genomics: The earliest era of mitochondrial genomics began 30 years ago with sporadic large-scale mitochondrial DNA rearrangements in families with Leber's Hereditary Optic Neuropathy (LHON). These patients are accompanied by rough red fibre myopathy and Maternal hereditary mtDNA point mutations. The small size and known sequence of the mitochondrial genome have led to numerous reports of new disease-related mutations, spanning almost every base of mitochondrial DNA. During this period, it is clear that many cases of pediatric mitochondrial disease must be based on nuclear gene defects. Indirect evidence indicates that the incidence of blood relatives in infected families has increased and many patients do not have mtDNA mutations. Finding nuclear gene mutations that cause OXPHOS deficiency remains elusive until SDHA mutations in patients with Leigh syndrome caused by the lack of complex II are identified, the only OXPHOS complex encoded by a nuclear gene. Despite this breakthrough, mitochondrial disease is still resistant to genetic diagnosis in most cases, as the limitations of available methods are limited to candidate gene sequencing, with or without previous linkage analysis or (in the case of kinship families) homozygosity Drawing.
Turn to green omics: WES and WGS are powerful technologies, but genetic causes occur only in 25-50% of patients with mitochondrial disease. The reason for failure to diagnose 100% of cases is complex, but in determining de-novo mutations, 52 hidden splice site defects, copy number variants, insertion or deletion events, and mutations in deep introns or regulatory regions or refractory regions such as repeats. Parent-child triple sequencing can identify de novo mutations and is a strategy that is effectively used in the Decoding Development Disorder (DDD) and British Genome 100000 Genome (100K) projects. Recent studies have demonstrated the ability to sequence entire transcriptomes using RNA sequencing technology (RNAseq ) Prioritize candidate genes (such as those with reduced or even single allelic expression) and identify deep intron variants that affect splicing. RNAseq also helps explain the mutations identified by WGS. For example, RNAseq of a primary muscle RNA sample successfully identified a genetic defect in 21% of 50 patients with suspected hereditary myopathy, whose diagnosis has been resistant to WES and WGS. The study highlights unresolved cases The importance of studying clinically relevant organizations. However, since many mitochondrial disease genes are housekeeping genes, they are widely expressed, including in cultured skin fibroblasts. As a result, Kremer and his collaborators successfully used RNAseq in cultured skin fibroblasts to diagnose 10% of 48 patients with suspected mitochondrial disease, the latter of whom was undiagnosed after WES. RNAseq does present challenges and is subject to batches Impact and need strong methodology and filtering because it depends on the gene of interest expressed in the tissues being investigated, for genes not expressed in fibroblasts and genes that cannot be expressed in affected tissues (brain, heart) In this regard, reprogramming RNAseq of iPS cells may be a promising approach.
Proteomics: Another tool recently incorporated into mitochondrial diagnostic devices is quantitative proteomics. Progress is being made in cataloguing all about 1500 predicted human mitochondrial proteins, and the mitochondrial localization of the mitochondrial proteome has recently been studied in Saccharomyces cerevisiae and human cells. In addition, mass spectrometry-based cross-linking interaction group analysis has been used to study many interactions of mitochondrial proteins, and C17orf89 (NDUFAF8) has been identified as a novel complex I assembly factor mutated in patients with Leigh syndrome. All of this information is invaluable and may help to resolve 25-50% of undiagnosed cases after WES or WGS when identifying disease genes for new mitochondrial diseases.
Metabolomics: Biomarkers and therapeutic targets for the mitochondrial disease have been sought for decades. The first biochemical markers of mitochondrial diseases, such as lactic acid and pyruvate, have low sensitivity and low specificity. Mass spectrometry is a comprehensive and systematic analysis of thousands of small molecules in biological samples. It is the latest technology to find the ideal elusive biomarkers or drug targets for mitochondrial diseases. Metabolomics can be used to analyze the many downstream effects of mitochondrial dysfunction, including the consequences of oxidative stress, NAD / NADH redox imbalances, and energy deficiency worldwide.
New mitochondrial disease mechanism
Little is known about the supporting mechanisms of mitochondrial disease pathology. In recent years, however, the use of omics techniques to elucidate new pathophysiology mitochondrial mechanisms has led to exciting and often unexpected developments. The lack of bioenergy due to the ineffectiveness of OXPHOS is the starting point to explain the pathophysiology of mitochondrial diseases, especially for patients who have mutations in OXPHOS components. However, failure to detect ATP deficiency in vitro and in vivo has shown that energy deficiency cannot fully explain the pathophysiology of mitochondrial disease. Other putative mitochondrial pathological mechanisms include increased ROS production (or reduced antioxidant protection), loss of mitochondrial membrane potential, impaired mitochondrial calcium treatment,
Mitochondrial diseases may be caused by mutations in proteins with known effects, including OXPHOS structural subunits and assembly factors, mitochondrial DNA maintenance, mitochondrial translation, mitochondrial lipids, and mitochondrial dynamics. In contrast, dozens of mitochondrial disease mutations affect undetermined proteins. Elucidating the activity of these uncharacterized proteins provides valuable insights into mitochondrial pathophysiology and has been substantially promoted through non-targeted global analysis. Investigation of a new biotinylated affinity assay for mitochondrial protein-protein interactions enables functional understanding of the modified mitochondrial carrier protein SLC25A46, a mutation associated with Leigh syndrome and optic atrophy. These studies have implicated SLC25A46 in several physiological processes including mitochondrial dynamics, due to the mitochondrial hyper fused phenotype observed in patient cells. SLC25A46's recognition binding partners suggest additional roles, including as a part of the mitochondrial contact site and the 嵴 tissue system (MICOS), a protein complex responsible for connecting 嵴 to the IMM, and mitochondrial lipids through interaction with the endoplasm Metabolic stability (EMC), which is responsible for the transfer of phospholipids from the endoplasmic reticulum (the site of cellular phospholipid synthesis) to mitochondria. Defects in these processes help disrupt mitochondrial ultrastructure and alter the genetic and dysfunctional OXPHOS of mitochondrial DNA. Similar pathology and neuron phenotypes have been observed in patients with QIL1 MICOS subunit mutations
Genetic pathways for treating mitochondrial diseases can also be divided into two groups-targeting mtDNA, and those designed to correct nuclear gene defects. Due to the unique characteristics of mtDNA, that is, high copy number results in heterogeneity (coexistence of different proportions of mutant and wild-type mtDNA in different cells), unique genetic codes and unique maternal inheritance require different strategies.
Mitochondrial genome editing
For heterogeneous mtDNA mutations, genomic editing to selectively disrupt mutant sequences is an attractive option and has been pursued for more than 15 years using increasingly sophisticated tools. Preliminary proof-of-principle studies have shown that restriction endonucleases can selectively destroy mutated mtDNA, leaving the wild-type genome intact. Subsequent studies used zinc finger nucleases and mitochondrial TALENs to localize to mitochondria with restriction enzymes, initially as a cellular model of mitochondrial disease and more recently in whole animals. The technical application of CRISPR-Cas9 gene editing to mitochondrial DNA can become tricky because of the problem of RNA entering the mitochondria.