bims-mitdis Biomed News
on Mitochondrial disorders
Issue of 2022–10–16
thirty-two papers selected by
Catalina Vasilescu, University of Helsinki



  1. Comput Methods Biomech Biomed Engin. 2022 Oct 13. 1-13
      We describe a compartmental model of mitochondrial transport in axons, which we apply to compute mitochondrial age at different distances from the soma. The model predicts that at the tip of an axon that has a length of 1 cm, the average mitochondrial age is approximately 22 h. The mitochondria are youngest closest to the soma and their age scales approximately linearly with distance from the soma. To the best of the authors' knowledge, this is the first attempt to predict the spatial distribution of mitochondrial age within an axon. A sensitivity study of the mean age of mitochondria to various model parameters is also presented.
    Keywords:  Neurons; axonal transport; mathematical modeling; mean age; organelles
    DOI:  https://doi.org/10.1080/10255842.2022.2128784
  2. FEBS Lett. 2022 Oct 11.
      The compartmentation and distribution of metabolites between mitochondria and the rest of the cell is a key parameter of cell signalling and pathology. Here, we have developed a rapid fractionation procedure that enables us to take mouse heart and liver from in vivo and within ~ 30 seconds stabilise the distribution of metabolites between mitochondria and the cytosol by rapid cooling, homogenisation and dilution. This is followed by centrifugation of mitochondria through an oil layer to separate mitochondrial and cytosolic fractions for subsequent metabolic analysis. Using this procedure revealed the in vivo compartmentation of mitochondrial metabolites and will enable assessment of the distribution of metabolites between the cytosol and mitochondria during a range of situations in vivo.
    Keywords:  compartmentation; in vivo; ischemia; metabolites; mitochondria; rapid fractionation
    DOI:  https://doi.org/10.1002/1873-3468.14511
  3. J Biol Chem. 2022 Oct 06. pii: S0021-9258(22)01018-3. [Epub ahead of print] 102574
      Mitochondrial DNA (mtDNA) is present in multiple copies and phenotypic consequences of mtDNA mutations depend on the mutant load surpassing a specific threshold. Additionally, changes in mtDNA copy number can impact mitochondrial ATP production, resulting in disease. Therefore, the precise determination of mtDNA heteroplasmy and copy number is crucial to the study of mitochondrial diseases. However, current methods can be imprecise, and quantifying small changes in either heteroplasmy or copy number is challenging. We developed a new approach to measure mtDNA heteroplasmy using a single digital PCR (dPCR) probe. This method is based on the observation that fluorescent-labeled probes in dPCR exhibit different intensities depending on the presence of a single nucleotide change in the sequence bound by the probe. This finding allowed us to precisely and simultaneously determine mtDNA copy number and heteroplasmy levels using duplex dPCR. We tested this approach in two different models (human and mouse), which proved faster and more internally controlled when compared to other published methods routinely used in the mitochondrial genetics field. We believe this approach could be broadly applicable to the detection and quantification of other mixed genetic variations.
    DOI:  https://doi.org/10.1016/j.jbc.2022.102574
  4. Int J Mol Sci. 2022 Sep 27. pii: 11391. [Epub ahead of print]23(19):
      Mitochondria are the only organelles, along with the nucleus, that have their own DNA. Mitochondrial DNA (mtDNA) is a double-stranded circular molecule of ~16.5 kbp that can exist in multiple copies within the organelle. Both strands are translated and encode for 22 tRNAs, 2 rRNAs, and 13 proteins. mtDNA molecules are anchored to the inner mitochondrial membrane and, in association with proteins, form a structure called nucleoid, which exerts a structural and protective function. Indeed, mitochondria have evolved mechanisms necessary to protect their DNA from chemical and physical lesions such as DNA repair pathways similar to those present in the nucleus. However, there are mitochondria-specific mechanisms such as rapid mtDNA turnover, fission, fusion, and mitophagy. Nevertheless, mtDNA mutations may be abundant in somatic tissue due mainly to the proximity of the mtDNA to the oxidative phosphorylation (OXPHOS) system and, consequently, to the reactive oxygen species (ROS) formed during ATP production. In this review, we summarise the most common types of mtDNA lesions and mitochondria repair mechanisms. The second part of the review focuses on the physiological role of mtDNA damage in ageing and the effect of mtDNA mutations in neurodegenerative disorders such as Alzheimer's and Parkinson's disease. Considering the central role of mitochondria in maintaining cellular homeostasis, the analysis of mitochondrial function is a central point for developing personalised medicine.
    Keywords:  Alzheimer’s disease; DNA damage; DNA repair pathways; Parkinson’s disease; mitochondria; neurodegenerative diseases
    DOI:  https://doi.org/10.3390/ijms231911391
  5. Front Mol Biosci. 2022 ;9 910936
      The mitochondrion is an essential organelle enclosed by two membranes whose functionalities depend on their very specific protein and lipid compositions. Proteins from the outer mitochondrial membrane (OMM) are specialized in mitochondrial dynamics and mitophagy, whereas proteins of the inner mitochondrial membrane (IMM) have dedicated functions in cellular respiration and apoptosis. As for lipids, the OMM is enriched in glycerophosphatidyl choline but cardiolipin is exclusively found within the IMM. Though the lipid topology and distribution of the OMM and IMM are known since more than four decades, little is known about the interfacial and dynamic properties of the IMM and OMM lipid extracts. Here we build monolayers, supported bilayers and giant unilamellar vesicles (GUVs) of native OMM and IMM lipids extracts from porcine heart. Additionally, we perform a comparative analysis on the interfacial, phase immiscibility and mechanical properties of both types of extract. Our results show that IMM lipids form more expanded and softer membranes than OMM lipids, allowing a better understanding of the physicochemical and biophysical properties of mitochondrial membranes.
    Keywords:  FRAP; giant unilamellar vesicles (GUV); inner mitochondrial membrane; langmuir monolayers; micropipettes; outer mitochondrial membrane; supported lipid bilayers
    DOI:  https://doi.org/10.3389/fmolb.2022.910936
  6. Nat Biotechnol. 2022 Oct 13.
      Bacterial toxin DddA-derived cytosine base editors (DdCBEs)-composed of split DddAtox (a cytosine deaminase specific to double-stranded DNA), custom-designed TALE (transcription activator-like effector) DNA-binding proteins, and a uracil glycosylase inhibitor-enable mitochondrial DNA (mtDNA) editing in human cells, which may pave the way for therapeutic correction of pathogenic mtDNA mutations in patients. The utility of DdCBEs has been limited by off-target activity, which is probably caused by spontaneous assembly of the split DddAtox deaminase enzyme, independent of DNA-binding interactions. We engineered high-fidelity DddA-derived cytosine base editors (HiFi-DdCBEs) with minimal off-target activity by substituting alanine for amino acid residues at the interface between the split DddAtox halves. The resulting domains cannot form a functional deaminase without binding of their linked TALE proteins at adjacent sites on DNA. Whole mitochondrial genome sequencing shows that, unlike conventional DdCBEs, which induce hundreds of unwanted off-target C-to-T conversions in human mtDNA, HiFi-DdCBEs are highly efficient and precise, avoiding collateral off-target mutations, and as such, they will probably be desirable for therapeutic applications.
    DOI:  https://doi.org/10.1038/s41587-022-01486-w
  7. Genome Biol. 2022 Oct 12. 23(1): 211
      We present two methods for enhancing the efficiency of mitochondrial DNA (mtDNA) editing in mice with DddA-derived cytosine base editors (DdCBEs). First, we fused DdCBEs to a nuclear export signal (DdCBE-NES) to avoid off-target C-to-T conversions in the nuclear genome and improve editing efficiency in mtDNA. Second, mtDNA-targeted TALENs (mitoTALENs) are co-injected into mouse embryos to cleave unedited mtDNA. We generated a mouse model with the m.G12918A mutation in the MT-ND5 gene, associated with mitochondrial genetic disorders in humans. The mutant mice show hunched appearances, damaged mitochondria in kidney and brown adipose tissues, and hippocampal atrophy, resulting in premature death.
    Keywords:  DdCBE; Mitochondrial DNA editing; NES; mitoTALEN; mtDNA
    DOI:  https://doi.org/10.1186/s13059-022-02782-z
  8. J Mol Cell Cardiol. 2022 Oct 06. pii: S0022-2828(22)00531-4. [Epub ahead of print]173 73-74
      
    Keywords:  Cyclophilin D; Cyclosporin A; Ischemia/reperfusion injury; Mitochondrial permeability transition pore; Reactive oxygen species; Succinate
    DOI:  https://doi.org/10.1016/j.yjmcc.2022.09.006
  9. Bio Protoc. 2022 Sep 05. pii: e4498. [Epub ahead of print]12(17):
      Mitochondrial dysfunction is associated with perturbations in the cellular oxidative status, changes in energy production and metabolic rate, and the onset of pathological processes. Classic methods of assessing mitochondrial dysfunction rely on indirect measures, such as evaluating mitochondrial DNA copy numbers, or direct but more costly and skilled techniques, such as electron microscopy. The protocol presented here was recently implemented to evaluate mitochondrial dysfunction in response to insecticide exposure in Drosophila melanogaster larvae, and it relies on the use of a previously established MitoTimer mutant strain. MitoTimer is a genetically engineered mitochondrial protein that shows green fluorescence when newly synthetized, irreversibly turning into red as mitochondria age. The protocol described here allows for the easy and direct assessment of shifts in mitochondrial turnover, with tissue-specific accuracy. This protocol can be adapted to assess changes in mitochondrial turnover in response to drugs, rearing conditions, and/or mutations in larva, pupa, or adult fruit flies.
    Keywords:   Dissection ; Drosophila ; Fluorescence microscopy ; Insecticide ; Mitochondria ; Mitochondrial turnover ; Oxidative stress ; Tissue imaging
    DOI:  https://doi.org/10.21769/BioProtoc.4498
  10. Nucleic Acids Res. 2022 Oct 10. pii: gkac857. [Epub ahead of print]
      Genetic processes require the activity of multiple topoisomerases, essential enzymes that remove topological tension and intermolecular linkages in DNA. We have investigated the subcellular localisation and activity of the six human topoisomerases with a view to understanding the topological maintenance of human mitochondrial DNA. Our results indicate that mitochondria contain two topoisomerases, TOP1MT and TOP3A. Using molecular, genomic and biochemical methods we find that both proteins contribute to mtDNA replication, in addition to the decatenation role of TOP3A, and that TOP1MT is stimulated by mtSSB. Loss of TOP3A or TOP1MT also dysregulates mitochondrial gene expression, and both proteins promote transcription elongation in vitro. We find no evidence for TOP2 localisation to mitochondria, and TOP2B knockout does not affect mtDNA maintenance or expression. Our results suggest a division of labour between TOP3A and TOP1MT in mtDNA topology control that is required for the proper maintenance and expression of human mtDNA.
    DOI:  https://doi.org/10.1093/nar/gkac857
  11. Life Sci Alliance. 2023 Jan;pii: e202101305. [Epub ahead of print]6(1):
      In vertebrates, mitochondrial outer membrane fusion is mediated by two mitofusin paralogs, Mfn1 and Mfn2, conserved dynamin superfamily proteins. Here, we characterize a variant of mitofusin reported in patients with CMT2A where a serine is replaced with a proline (Mfn2-S350P and the equivalent in Mfn1, S329P). This serine is in a hinge domain (Hinge 2) that connects the globular GTPase domain to the adjacent extended helical bundle. We find that expression of this variant results in prolific and stable mitochondrial tethering that also blocks mitochondrial fusion by endogenous wild-type mitofusin. The formation of mitochondrial perinuclear clusters by this CMT2A variant requires normal GTPase domain function and formation of a mitofusin complex across two membranes. We propose that conformational dynamics mediated by Hinge 2 and regulated by GTP hydrolysis are disrupted by the substitution of proline at S329/S350 and this prevents progression from tethering to membrane fusion. Thus, our data are consistent with a model for mitofusin-mediated membrane fusion where Hinge 2 supports a power stroke to progress from the tethering complex to membrane fusion.
    DOI:  https://doi.org/10.26508/lsa.202101305
  12. EMBO J. 2022 Oct 10. e111115
      Mitochondria and peroxisomes are closely related metabolic organelles, both in terms of origin and in terms of function. Mitochondria and peroxisomes can also be turned over by autophagy, in processes termed mitophagy and pexophagy, respectively. However, despite their close relationship, it is not known if both organelles are turned over under similar conditions, and if so, how this might be coordinated molecularly. Here, we find that multiple selective autophagy pathways are activated upon iron chelation and show that mitophagy and pexophagy occur in a BNIP3L/NIX-dependent manner. We reveal that the outer mitochondrial membrane-anchored NIX protein, previously described as a mitophagy receptor, also independently localises to peroxisomes and drives pexophagy. We show this process happens in vivo, with mouse tissue that lacks NIX having a higher peroxisomal content. We further show that pexophagy is stimulated under the same physiological conditions that activate mitophagy, including cardiomyocyte and erythrocyte differentiation. Taken together, our work uncovers a dual role for NIX, not only in mitophagy but also in pexophagy, thus illustrating the interconnection between selective autophagy pathways.
    Keywords:  autophagy; mitochondria; mitophagy; peroxisomes; pexophagy
    DOI:  https://doi.org/10.15252/embj.2022111115
  13. N Engl J Med. 2022 Oct 13. 387(15): 1395-1403
      We describe the case of identical twin boys who presented with low body weight despite excessive caloric intake. An evaluation of their fibroblasts showed elevated oxygen consumption and decreased mitochondrial membrane potential. Exome analysis revealed a de novo heterozygous variant in ATP5F1B, which encodes the β subunit of mitochondrial ATP synthase (also called complex V). In yeast, mutations affecting the same region loosen coupling between the proton motive force and ATP synthesis, resulting in high rates of mitochondrial respiration. Expression of the mutant allele in human cell lines recapitulates this phenotype. These data support an autosomal dominant mitochondrial uncoupling syndrome with hypermetabolism. (Funded by the National Institutes of Health.).
    DOI:  https://doi.org/10.1056/NEJMoa2202949
  14. Nat Commun. 2022 Oct 13. 13(1): 6058
      Sustained muscle contraction occurs through interactions between actin and myosin filaments within sarcomeres and requires a constant supply of adenosine triphosphate (ATP) from nearby mitochondria. However, it remains unclear how different physical configurations between sarcomeres and mitochondria alter the energetic support for contractile function. Here, we show that sarcomere cross-sectional area (CSA) varies along its length in a cell type-dependent manner where the reduction in Z-disk CSA relative to the sarcomere center is closely coordinated with mitochondrial network configuration in flies, mice, and humans. Further, we find myosin filaments near the sarcomere periphery are curved relative to interior filaments with greater curvature for filaments near mitochondria compared to sarcoplasmic reticulum. Finally, we demonstrate variable myosin filament lattice spacing between filament ends and filament centers in a cell type-dependent manner. These data suggest both sarcomere structure and myofilament interactions are influenced by the location and orientation of mitochondria within muscle cells.
    DOI:  https://doi.org/10.1038/s41467-022-33678-y
  15. FEBS Lett. 2022 Oct 10.
      Mitochondria, organelles critical for energy production, modify their shape and location in response to developmental state and metabolic demands. Mitochondria are altered in diabetes, but the mechanistic basis is poorly defined, due to difficulties in assessing mitochondria within an intact organism. Here, we use in vivo imaging in transparent zebrafish larvae to demonstrate filamentous, interconnected mitochondrial networks within islet cells. Mitochondrial movements highly resemble what has been reported for human islet cells in vitro, showing conservation in behavior across species and cellular context. During islet development, mitochondrial content increases with emergence of cell motility, and mitochondria disperse within fine protrusions. Overall, this work presents quantitative analysis of mitochondria within their native environment and provides insights into mitochondrial behavior during organogenesis.
    Keywords:  Mitochondria; image analysis; in vivo imaging; islet; pancreas; zebrafish
    DOI:  https://doi.org/10.1002/1873-3468.14508
  16. Neurobiol Dis. 2022 Oct 05. pii: S0969-9961(22)00277-7. [Epub ahead of print]174 105885
      Mitochondrial dysfunction happens in both idiopathic (iPD) and LRRK2-related Parkinson's disease (LRRK2-PD). Nonetheless, previous studies suggested that a different type of mitochondrial pathology underlies the neurodegeneration in these two disorders. To further explore this hypothesis, we developed a novel multiplex digital PCR assay that allows the absolute quantification of cell-free mitochondrial DNA (cf-mtDNA) copy number and deletion ratio directly in cerebrospinal fluid (CSF) by simultaneously measuring two opposed regions of the mtDNA circular molecule, one of them in the commonly deleted major arc. The results confirmed that the content of cf-mtDNA in CSF was statistically significantly different between iPD and LRRK2-PD patients. Moreover, we found high cf-mtDNA deletion levels in CSF from patients with iPD, but not LRRK2-PD. The high cf-mtDNA deletion frequency in iPD was validated in an independent cohort. These results indicated that the content and deletion ratio of cf-mtDNA may differentiate iPD from LRRK2-PD, and provides further evidence of the different mitochondrial pathophysiology between these two forms of the disease.
    Keywords:  Digital PCR; LRRK2; Mitochondrial DNA; Mitochondrial DNA deletions; Parkinson's disease
    DOI:  https://doi.org/10.1016/j.nbd.2022.105885
  17. Int J Mol Sci. 2022 Sep 20. pii: 11002. [Epub ahead of print]23(19):
      Thymidine kinase (TK2) deficiency causes mitochondrial DNA depletion syndrome. We aimed to report the clinical, biochemical, genetic, histopathological, and ultrastructural features of a cohort of paediatric patients with TK2 deficiency. Mitochondrial DNA was isolated from muscle biopsies to assess depletions and deletions. The TK2 genes were sequenced using Sanger sequencing from genomic DNA. All muscle biopsies presented ragged red fibres (RRFs), and the prevalence was greater in younger ages, along with an increase in succinate dehydrogenase (SDH) activity and cytochrome c oxidase (COX)-negative fibres. An endomysial inflammatory infiltrate was observed in younger patients and was accompanied by an overexpression of major histocompatibility complex type I (MHC I). The immunofluorescence study for complex I and IV showed a greater number of fibres than those that were visualized by COX staining. In the ultrastructural analysis, we found three major types of mitochondrial alterations, consisting of concentrically arranged lamellar cristae, electrodense granules, and intramitochondrial vacuoles. The pathological features in the muscle showed substantial differences in the youngest patients when compared with those that had a later onset of the disease. Additional ultrastructural features are described in the muscle biopsy, such as sarcomeric de-structuration in the youngest patients with a more severe phenotype.
    Keywords:  TK2 deficiency; mitochondrial myopathies; muscle biopsy; paediatric patients; ragged red fibres; ultrastructural studies
    DOI:  https://doi.org/10.3390/ijms231911002
  18. Int J Mol Sci. 2022 Oct 09. pii: 11991. [Epub ahead of print]23(19):
      MOTS-c, a 16 amino acid mitochondrial derived peptide, is encoded from the 12S rRNA region of the mitochondrial genome. Under stress conditions, MOTS-c translocates to the nucleus where it regulates a wide range of genes in response to metabolic dysfunction. It is colocalized to mitochondria in various tissues and is found in plasma, but the levels decline with age. Since MOTS-c has important cellular functions as well as a possible hormonal role, it has been shown to have beneficial effects on age-related diseases including Diabetes, Cardiovascular diseases, Osteoporosis, postmenopausal obesity and Alzheimer. Aging is characterized by gradual loss of (mitochondrial) metabolic balance, decreased muscle homeostasis and eventual diminished physical capability, which potentially can be reversed with MOTS-c treatment. This review examines the latest findings on biological effects of MOTS-c as a nuclear regulatory peptide and focuses on the role of MOTS-c in aging and age-related disorders, including mechanisms of action and therapeutic potential.
    Keywords:  MOTS-c; age-related diseases; aging; mitochondrial derived peptides; mitochondrial dysfunction
    DOI:  https://doi.org/10.3390/ijms231911991
  19. FEBS J. 2022 Oct 14.
      Sarm1 is an evolutionary conserved innate immune adaptor protein that has emerged as a primary regulator of programmed axonal degeneration over the past decade. In vitro structural insights have revealed that although Sarm1 induces energy depletion by breaking down NAD+ , it is also allosterically inhibited by NAD+ . However, how NAD+ levels modulate the activation of intracellular Sarm1 has not been elucidated so far. This study focuses on understanding the events leading to Sarm1 activation in both neuronal and non-neuronal cells using the mitochondrial complex I inhibitor rotenone. Here we report the regulation of rotenone-induced cell death by loss of NAD+ that may act as a "biological trigger" of Sarm1 activation. Our study revealed that early loss of endogenous NAD+ levels arising due to PARP1 hyperactivation preceded Sarm1 induction following rotenone treatment. Interestingly, replenishing NAD+ levels by the PARP inhibitor, PJ34 restored mitochondrial complex I activity and also prevented subsequent Sarm1 activation in rotenone treated cells. These cellular data were further validated in Drosophila melanogaster where a significant reduction in rotenone mediated loss of locomotor abilities and reduced dSarm expression was observed in the flies following PARP inhibition. Taken together, these observations not only uncover a novel regulation of Sarm1 induction by endogenous NAD+ levels but also point towards an important understanding on how PARP inhibitors could be repurposed in the treatment of mitochondrial complex I deficiency disorders.
    Keywords:  Autophagy; Mitochondria; NAD+; PARP inhibitor; PJ34; Rotenone; Sarm1
    DOI:  https://doi.org/10.1111/febs.16652
  20. Nat Commun. 2022 Oct 13. 13(1): 6061
      Overexposure to manganese disrupts cellular energy metabolism across species, but the molecular mechanism underlying manganese toxicity remains enigmatic. Here, we report that excess cellular manganese selectively disrupts coenzyme Q (CoQ) biosynthesis, resulting in failure of mitochondrial bioenergetics. While respiratory chain complexes remain intact, the lack of CoQ as lipophilic electron carrier precludes oxidative phosphorylation and leads to premature cell and organismal death. At a molecular level, manganese overload causes mismetallation and proteolytic degradation of Coq7, a diiron hydroxylase that catalyzes the penultimate step in CoQ biosynthesis. Coq7 overexpression or supplementation with a CoQ headgroup analog that bypasses Coq7 function fully corrects electron transport, thus restoring respiration and viability. We uncover a unique sensitivity of a diiron enzyme to mismetallation and define the molecular mechanism for manganese-induced bioenergetic failure that is conserved across species.
    DOI:  https://doi.org/10.1038/s41467-022-33641-x
  21. Bioinformatics. 2022 Oct 13. pii: btac678. [Epub ahead of print]
       MOTIVATION: The advent of massive DNA sequencing technologies is producing a huge number of human single-nucleotide polymorphisms occurring in protein-coding regions and possibly changing their sequences. Discriminating harmful protein variations from neutral ones is one of the crucial challenges in precision medicine. Computational tools based on artificial intelligence provide models for protein sequence encoding, bypassing database searches for evolutionary information. We leverage the new encoding schemes for an efficient annotation of protein variants.
    RESULTS: E-SNPs&GO is a novel method that, given an input protein sequence and a single amino acid variation, can predict whether the variation is related to diseases or not. The proposed method adopts an input encoding completely based on protein language models and embedding techniques, specifically devised to encode protein sequences and GO functional annotations. We trained our model on a newly generated dataset of 101,146 human protein single amino acid variants in 13,661 proteins, derived from public resources. When tested on a blind set comprising 10,266 variants, our method well compares to recent approaches released in literature for the same task, reaching a Matthews Correlation Coefficient (MCC) score of 0.72. We propose E-SNPs&GO as a suitable, efficient and accurate large-scale annotator of protein variant datasets.
    AVAILABILITY: The method is available as a webserver at https://esnpsandgo.biocomp.unibo.it. Datasets and predictions are available at https://esnpsandgo.biocomp.unibo.it/datasets.
    SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
    DOI:  https://doi.org/10.1093/bioinformatics/btac678
  22. Methods Mol Biol. 2023 ;2553 417-439
      Computational cell metabolism models seek to provide metabolic explanations of cell behavior under different conditions or following genetic alterations, help in the optimization of in vitro cell growth environments, or predict cellular behavior in vivo and in vitro. In the extremes, mechanistic models can include highly detailed descriptions of a small number of metabolic reactions or an approximate representation of an entire metabolic network. To date, all mechanistic models have required details of individual metabolic reactions, either kinetic parameters or metabolic flux, as well as information about extracellular and intracellular metabolite concentrations. Despite the extensive efforts and the increasing availability of high-quality data, required in vivo data are not available for the majority of known metabolic reactions; thus, mechanistic models are based primarily on ex vivo kinetic measurements and limited flux information. Machine learning approaches provide an alternative for derivation of functional dependencies from existing data. The increasing availability of metabolomic and lipidomic data, with growing feature coverage as well as sample set size, is expected to provide new data options needed for derivation of machine learning models of cell metabolic processes. Moreover, machine learning analysis of longitudinal data can lead to predictive models of cell behaviors over time. Conversely, machine learning models trained on steady-state data can provide descriptive models for the comparison of metabolic states in different environments or disease conditions. Additionally, inclusion of metabolic network knowledge in these analyses can further help in the development of models with limited data.This chapter will explore the application of machine learning to the modeling of cell metabolism. We first provide a theoretical explanation of several machine learning and hybrid mechanistic machine learning methods currently being explored to model metabolism. Next, we introduce several avenues for improving these models with machine learning. Finally, we provide protocols for specific examples of the utilization of machine learning in the development of predictive cell metabolism models using metabolomic data. We describe data preprocessing, approaches for training of machine learning models for both descriptive and predictive models, and the utilization of these models in synthetic and systems biology. Detailed protocols provide a list of software tools and libraries used for these applications, step-by-step modeling protocols, troubleshooting, as well as an overview of existing limitations to these approaches.
    Keywords:  Hybrid modeling; Lipidomics, Flux analysis; Machine learning; Metabolism modeling; Metabolomics
    DOI:  https://doi.org/10.1007/978-1-0716-2617-7_18
  23. Proc Natl Acad Sci U S A. 2022 Oct 25. 119(43): e2207955119
      Oxygen plays a key role in supporting life on our planet. It is particularly important in higher eukaryotes where it boosts bioenergetics as a thermodynamically favorable terminal electron acceptor and has important roles in cell signaling and development. Many human diseases stem from either insufficient or excessive oxygen. Despite its fundamental importance, we lack methods with which to manipulate the supply of oxygen with high spatiotemporal resolution in cells and in organisms. Here, we introduce a genetic system, SupplemeNtal Oxygen Released from ChLorite (SNORCL), for on-demand local generation of molecular oxygen in living cells, by harnessing prokaryotic chlorite O2-lyase (Cld) enzymes that convert chlorite (ClO2-) into molecular oxygen (O2) and chloride (Cl-). We show that active Cld enzymes can be targeted to either the cytosol or mitochondria of human cells, and that coexpressing a chlorite transporter results in molecular oxygen production inside cells in response to externally added chlorite. This first-generation system allows fine temporal and spatial control of oxygen production, with immediate research applications. In the future, we anticipate that technologies based on SNORCL will have additional widespread applications in research, biotechnology, and medicine.
    Keywords:  Cld; SLC5A5; SNORCL; chlorite; oxygen
    DOI:  https://doi.org/10.1073/pnas.2207955119
  24. Int J Mol Sci. 2022 Sep 29. pii: 11536. [Epub ahead of print]23(19):
      CCG-1423 is a Rho A pathway inhibitor that has been reported to inhibit Rho/SRF-mediated transcriptional regulation. Serum response factor and its cofactors, which include ternary complex factors and myocardin-related transcription factors, regulate various cellular functions. In this study, we observed that CCG-1423 modulates the mitochondrial functions. The effect of this small molecule drug was determined by measuring mitochondrial function using an XFe96 Analyzer and an Oxygraph 2k (O2k) high-resolution respirometer. CCG-1423 treatment significantly reduced oxidative phosphorylation in a dose-dependent manner. However, CCG-1423 increased the glycolytic rate. We also observed that histone 4 at lysine-16 underwent hyperacetylation with the treatment of this drug. Immunolabeling with F-actin and MitoTracker revealed the alteration in the actin cytoskeleton and mitochondria. Taken together, our findings highlight a critical role of CCG-1423 in inhibiting the transcription of SRF/p49 and PGC-1α, β, resulting in the downregulation of mitochondrial genes, leading to the repression of mitochondrial oxidative phosphorylation and overall ATP reduction. This study provides a better understanding of the effects of CCG-1423 on mitochondria, which may be useful for the assessment of the potential clinical application of CCG-1423 and its derivatives.
    Keywords:  CCG-1423; acetylation; mitochondrial function; serum response factor
    DOI:  https://doi.org/10.3390/ijms231911536
  25. Methods Mol Biol. 2023 ;2553 285-323
      Protein interactions play a critical role in all biological processes, but experimental identification of protein interactions is a time- and resource-intensive process. The advances in next-generation sequencing and multi-omics technologies have greatly benefited large-scale predictions of protein interactions using machine learning methods. A wide range of tools have been developed to predict protein-protein, protein-nucleic acid, and protein-drug interactions. Here, we discuss the applications, methods, and challenges faced when employing the various prediction methods. We also briefly describe ways to overcome the challenges and prospective future developments in the field of protein interaction biology.
    Keywords:  Deep learning; Interaction; Machine learning; Neural networks; PPI; Protein networks
    DOI:  https://doi.org/10.1007/978-1-0716-2617-7_15
  26. Nat Commun. 2022 Oct 10. 13(1): 5948
      The steady-state localisation of proteins provides vital insight into their function. These localisations are context specific with proteins translocating between different subcellular niches upon perturbation of the subcellular environment. Differential localisation, that is a change in the steady-state subcellular location of a protein, provides a step towards mechanistic insight of subcellular protein dynamics. High-accuracy high-throughput mass spectrometry-based methods now exist to map the steady-state localisation and re-localisation of proteins. Here, we describe a principled Bayesian approach, BANDLE, that uses these data to compute the probability that a protein differentially localises upon cellular perturbation. Extensive simulation studies demonstrate that BANDLE reduces the number of both type I and type II errors compared to existing approaches. Application of BANDLE to several datasets recovers well-studied translocations. In an application to cytomegalovirus infection, we obtain insights into the rewiring of the host proteome. Integration of other high-throughput datasets allows us to provide the functional context of these data.
    DOI:  https://doi.org/10.1038/s41467-022-33570-9
  27. BMJ Open. 2022 10 10. 12(10): e061468
       INTRODUCTION: Despite the superior diagnostic performance of exome and genome sequencing compared with conventional genetic tests, evidence gaps related to clinical utility and cost effectiveness have limited their availability in routine clinical practice in many jurisdictions. To inform adoption and reimbursement policy, this protocol provides a chain of evidence approach to determining the diagnostic utility, clinical utility and cost-effectiveness of whole exome sequencing (WES) from seven medical genetic centres in two Canadian provinces.
    METHODS AND ANALYSIS: Using a multicentre observational cohort design, we will extract data specific to the pre-WES diagnostic pathway and 1-year post-WES medical management from electronic medical records for 650 patients with rare disease of suspected genetic aetiology who receive WES. The date from the clinical record will be linked to provincial administrative health database to capture healthcare resource use and estimate costs. Our analysis will: (1) define and describe diagnostic testing pathways that occur prior to WES among patients with rare disease, (2) determine the diagnostic utility of WES, characterised as the proportion of patients for whom causative DNA variants are identified, (3) determine the clinical utility of WES, characterised as a change in medical management triggered by WES results, (4) determine the pattern and cost of health service utilisation prior and 1 year following WES among patients who receive a diagnosis, do not receive a diagnosis, or receive an uncertain diagnosis and (5) estimate the cost-effectiveness of WES compared with conventional diagnostic testing pathways, measured by the incremental cost per additional patient diagnosed by WES using simulation modelling.
    ETHICS AND DISSEMINATION: This protocol was approved by Clinical Trials Ontario (CTO-1577) and research ethics boards at the University of Calgary (REB18-0744 and REB20-1449) and University of Alberta (Pro0009156). Findings will be disseminated through academic publications and policy reports.
    Keywords:  GENETICS; HEALTH ECONOMICS; Health policy
    DOI:  https://doi.org/10.1136/bmjopen-2022-061468
  28. Nat Commun. 2022 Oct 11. 13(1): 5989
      Mitochondrial ATP synthase forms stable dimers arranged into oligomeric assemblies that generate the inner-membrane curvature essential for efficient energy conversion. Here, we report cryo-EM structures of the intact ATP synthase dimer from Trypanosoma brucei in ten different rotational states. The model consists of 25 subunits, including nine lineage-specific, as well as 36 lipids. The rotary mechanism is influenced by the divergent peripheral stalk, conferring a greater conformational flexibility. Proton transfer in the lumenal half-channel occurs via a chain of five ordered water molecules. The dimerization interface is formed by subunit-g that is critical for interactions but not for the catalytic activity. Although overall dimer architecture varies among eukaryotes, we find that subunit-g together with subunit-e form an ancestral oligomerization motif, which is shared between the trypanosomal and mammalian lineages. Therefore, our data defines the subunit-g/e module as a structural component determining ATP synthase oligomeric assemblies.
    DOI:  https://doi.org/10.1038/s41467-022-33588-z
  29. Cell Discov. 2022 Oct 11. 8(1): 106
      Neonatal heart undergoes metabolic conversion and cell cycle arrest preparing for the increased workload during adulthood. Herein, we report that neonatal ketone body elevation is a critical regulatory factor for postnatal heart development. Through multiomics screening, we found that the expression of 3-hydroxy-3-methylglutaryl-CoA synthase 2 (HMGCS2), the rate-limiting enzyme of ketogenesis, was transiently induced by colostrum in the neonatal heart. Hmgcs2 knockout caused mitochondrial maturation defects. Meanwhile, postnatal heart development was compromised and cardiomyocytes reacquired proliferation capacity in Hmgcs2 knockout mice. Consequently, over 40% of newborn Hmgcs2 knockout mice died before weaning. The heart function of surviving Hmgcs2 knockout mice was also impaired, which could be rescued by ketone body supplementation during the suckling stage. Mechanistically, ketone body deficiency inhibited β-hydroxybutyrylation but enhanced acetylation of mitochondrial proteins, which might be responsible for the inhibition of the enzyme activity in mitochondria. These observations suggest that ketone body is critical for postnatal heart development through regulating mitochondrial maturation and metabolic reprogramming.
    DOI:  https://doi.org/10.1038/s41421-022-00447-6
  30. Nature. 2022 Oct 12.
      
    Keywords:  Neuroscience; Structural biology
    DOI:  https://doi.org/10.1038/d41586-022-03147-z
  31. Genet Mol Biol. 2022 ;pii: S1415-47572022000300104. [Epub ahead of print]45(3): e20220150
      Precision Medicine emerges from the genomic paradigm of health and disease. For precise molecular diagnoses of genetic diseases, we must analyze the Whole Exome (WES) or the Whole Genome (WGS). By not needing exon capture, WGS is more powerful to detect single nucleotide variants and copy number variants. In healthy individuals, we can observe monogenic highly penetrant variants, which may be causally responsible for diseases, and also susceptibility variants, associated with common polygenic diseases. But there is the major problem of penetrance. Thus, there is the question of whether it is worthwhile to perform WGS in all healthy individuals as a step towards Precision Medicine. The genetic architecture of disease is consistent with the fact that they are all polygenic. Moreover, ancestry adds another layer of complexity. We are now capable of obtaining Polygenic Risk Scores for all complex diseases using only data from new generation sequencing. Yet, review of available evidence does not at present favor the idea that WGS analyses are sufficiently developed to allow reliable predictions of the risk components for monogenic and polygenic hereditary diseases in healthy individuals. Probably, it is still better for WGS to remain reserved for the diagnosis of pathogenic variants of Mendelian diseases.
    DOI:  https://doi.org/10.1590/1678-4685-GMB-2022-0150