bims-mitdis Biomed News
on Mitochondrial disorders
Issue of 2022‒12‒18
48 papers selected by
Catalina Vasilescu
Helmholz Munich
  1. Genetic testing for mitochondrial disease: the United Kingdom best practice guidelines.
  2. An AAA-ATPase links mitochondrial division with DNA nucleoids.
  3. J-Domain Proteins Orchestrate the Multifunctionality of Hsp70s in Mitochondria: Insights from Mechanistic and Evolutionary Analyses.
  4. Cardiolipin, and not monolysocardiolipin, preferentially binds to the interface of complexes III and IV.
  5. Mitochondrial Dysfunction and Pharmacodynamics of Mitofusin Activation in Murine Charcot-Marie-Tooth Disease Type 2A.
  6. Exercise and mitochondrial remodeling to prevent age-related neurodegeneration.
  7. The BBSome regulates mitochondria dynamics and function.
  8. Oral nicotinamide riboside raises NAD+ and lowers biomarkers of neurodegenerative pathology in plasma extracellular vesicles enriched for neuronal origin.
  9. Changing faces of mitochondrial disease: autosomal recessive POLG disease mimicking myasthenia gravis and progressive supranuclear palsy.
  10. RNA granule-clustered mitochondrial aminoacyl-tRNA synthetases form multiple complexes with the potential to fine-tune tRNA aminoacylation.
  11. Importance of NAD+ Anabolism in Metabolic, Cardiovascular and Neurodegenerative Disorders.
  12. On the Mechanism of Sustained Mitochondrial Membrane Potential Without Functioning Complex IV.
  13. Thermoporter: a new regulatory mechanism for mitochondrial calcium uniporter activity.
  14. Mitochondrial form and function in hair cells.
  15. Implications of p53 in mitochondrial dysfunction and Parkinson's disease.
  16. A mitochondrial trigger for beige adipocyte development.
  17. Diversity of mitophagy pathways at a glance.
  18. Mitochondrial Permeability Transition.
  19. Cilia bent out of shape over dysfunctional astrocyte mitochondria.
  20. Mitochondria-endoplasmic reticulum contacts in sepsis-induced myocardial dysfunction.
  21. Mitochondrial dynamics proteins as emerging drug targets.
  22. NDP52 acts as a redox sensor in PINK1/Parkin-mediated mitophagy.
  23. Q-Flux: A method to assess hepatic mitochondrial succinate dehydrogenase, methylmalonyl-CoA mutase, and glutaminase fluxes in vivo.
  24. The mitochondrial UPR regulator ATF5 promotes intestinal barrier function via control of the satiety response.
  25. Transcriptomic analyses reveal neuronal specificity of Leigh syndrome associated genes.
  26. Human EXOG Possesses Strong AP Hydrolysis Activity: Implication on Mitochondrial DNA Base Excision Repair.
  27. Mechanisms of ion selectivity and throughput in the mitochondrial calcium uniporter.
  28. Mitochondrial dysfunction induces ALK5-SMAD2-mediated hypovascularization and arteriovenous malformations in mouse retinas.
  29. Methods to Assess the Role of PARPs in Regulating Mitochondrial Oxidative Function.
  30. TFAM's Contributions to mtDNA Replication and OXPHOS Biogenesis Are Genetically Separable.
  31. Protein import into peroxisomes occurs through a nuclear pore-like phase.
  32. Modulation of mitochondria by viral proteins.
  33. Cellular and subcellular optogenetic approaches towards neuroprotection and vision restoration.
  34. Biallelic variants in OGDH encoding oxoglutarate dehydrogenase lead to a neurodevelopmental disorder characterized by global developmental delay, movement disorder, and metabolic abnormalities.
  35. A novel homozygous CHMP1A variant arising from segmental uniparental disomy causes pontocerebellar hypoplasia type 8.
  36. Structural basis of the acyl-transfer mechanism of human GPAT1.
  37. Neuroimaging in Leber Hereditary Optic Neuropathy: State-of-the-art and future prospects.
  38. Deep Intronic FGF14 GAA Repeat Expansion in Late-Onset Cerebellar Ataxia.
  39. Identification and prediction of Parkinson's disease subtypes and progression using machine learning in two cohorts.
  40. Regulation of pre-mRNA splicing: roles in physiology and disease, and therapeutic prospects.
  41. Structural basis for Parkinson's disease-linked LRRK2's binding to microtubules.
  42. Third Generation Sequencing of Epigenetic DNA.
  43. An increase in mitochondrial TOM activates apoptosis to drive retinal neurodegeneration.
  44. Endosomal lipid signaling reshapes the endoplasmic reticulum to control mitochondrial function.
  45. Proteomics in Inherited Metabolic Disorders.
  46. Metabolic modulation of transcription: The role of one-carbon metabolism.
  47. Proteome-wide structural changes measured with limited proteolysis-mass spectrometry: an advanced protocol for high-throughput applications.
  48. Streamlined high-throughput cloning protocol to generate arrayed mutant libraries.
  1. Eur J Hum Genet. 2022 Dec 13.
    Genetic testing for mitochondrial disease: the United Kingdom best practice guidelines.
    Eleni Mavraki, Robyn Labrum, Kate Sergeant, Charlotte L Alston, Cathy Woodward, Conrad Smith, Charlotte V Y Knowles, Yogen Patel, Philip Hodsdon, Jack P Baines, Emma L Blakely, James Polke, Robert W Taylor, Carl Fratter.
      Primary mitochondrial disease describes a diverse group of neuro-metabolic disorders characterised by impaired oxidative phosphorylation. Diagnosis is challenging; >350 genes, both nuclear and mitochondrial DNA (mtDNA) encoded, are known to cause mitochondrial disease, leading to all possible inheritance patterns and further complicated by heteroplasmy of the multicopy mitochondrial genome. Technological advances, particularly next-generation sequencing, have driven a shift in diagnostic practice from 'biopsy first' to genome-wide analyses of blood and/or urine DNA. This has led to the need for a reference framework for laboratories involved in mitochondrial genetic testing to facilitate a consistent high-quality service. In the United Kingdom, consensus guidelines have been prepared by a working group of Clinical Scientists from the NHS Highly Specialised Service followed by national laboratory consultation. These guidelines summarise current recommended technologies and methodologies for the analysis of mtDNA and nuclear-encoded genes in patients with suspected mitochondrial disease. Genetic testing strategies for diagnosis, family testing and reproductive options including prenatal diagnosis are outlined. Importantly, recommendations for the minimum levels of mtDNA testing for the most common referral reasons are included, as well as guidance on appropriate referrals and information on the minimal appropriate gene content of panels when analysing nuclear mitochondrial genes. Finally, variant interpretation and recommendations for reporting of results are discussed, focussing particularly on the challenges of interpreting and reporting mtDNA variants.
    DOI:  https://doi.org/10.1038/s41431-022-01249-w
  2. Proc Natl Acad Sci U S A. 2022 Dec 20. 119(51): e2217949119
    An AAA-ATPase links mitochondrial division with DNA nucleoids.
    Nelson Yeung, Miho Iijima, Hiromi Sesaki.
      
    DOI:  https://doi.org/10.1073/pnas.2217949119
  3. Subcell Biochem. 2023 ;101 293-318
    J-Domain Proteins Orchestrate the Multifunctionality of Hsp70s in Mitochondria: Insights from Mechanistic and Evolutionary Analyses.
    Jaroslaw Marszalek, Elizabeth A Craig, Bartlomiej Tomiczek.
      Mitochondrial J-domain protein (JDP) co-chaperones orchestrate the function of their Hsp70 chaperone partner(s) in critical organellar processes that are essential for cell function. These include folding, refolding, and import of mitochondrial proteins, maintenance of mitochondrial DNA, and biogenesis of iron-sulfur cluster(s) (FeS), prosthetic groups needed for function of mitochondrial and cytosolic proteins. Consistent with the organelle's endosymbiotic origin, mitochondrial Hsp70 and the JDPs' functioning in protein folding and FeS biogenesis clearly descended from bacteria, while the origin of the JDP involved in protein import is less evident. Regardless of their origin, all mitochondrial JDP/Hsp70 systems evolved unique features that allowed them to perform mitochondria-specific functions. Their modes of functional diversification and specialization illustrate the versatility of JDP/Hsp70 systems and inform our understanding of system functioning in other cellular compartments.
    Keywords:  Co-chaperones; Iron-sulfur clusters; Mitochondrial matrix; Molecular chaperone; Protein evolution; Protein folding; Protein translocation
    DOI:  https://doi.org/10.1007/978-3-031-14740-1_10
  4. Chem Sci. 2022 Nov 23. 13(45): 13489-13498
    Cardiolipin, and not monolysocardiolipin, preferentially binds to the interface of complexes III and IV.
    Robin A Corey, Noah Harrison, Philllp J Stansfeld, Mark S P Sansom, Anna L Duncan.
      The mitochondrial electron transport chain comprises a series of protein complexes embedded in the inner mitochondrial membrane that generate a proton motive force via oxidative phosphorylation, ultimately generating ATP. These protein complexes can oligomerize to form larger structures called supercomplexes. Cardiolipin (CL), a conical lipid, unique within eukaryotes to the inner mitochondrial membrane, has proven essential in maintaining the stability and function of supercomplexes. Monolysocardiolipin (MLCL) is a CL variant that accumulates in people with Barth syndrome (BTHS). BTHS is caused by defects in CL biosynthesis and characterised by abnormal mitochondrial bioenergetics and destabilised supercomplexes. However, the mechanisms by which MLCL causes pathogenesis remain unclear. Here, multiscale molecular dynamics characterise the interactions of CL and MLCL with yeast and mammalian mitochondrial supercomplexes containing complex III (CIII) and complex IV (CIV). Coarse-grained simulations reveal that both CL and MLCL bind to sites at the interface between CIII and CIV of the supercomplex. Free energy perturbation calculations show that MLCL interaction is weaker than that of CL and suggest that interaction with CIV drives this difference. Atomistic contact analyses show that, although interaction with CIII is similar for CL and MLCL, CIV makes more contacts with CL than MLCL, demonstrating that CL is a more successful "glue" between the two complexes. Simulations of the human CIII2CIV supercomplex show that this interface site is maintained between species. Our study suggests that MLCL accumulation in people with BTHS disrupts supercomplex stability by formation of relatively weak interactions at the interface lipid binding site.
    DOI:  https://doi.org/10.1039/d2sc04072g
  5. J Pharmacol Exp Ther. 2022 Nov;383(2): 137-148
    Mitochondrial Dysfunction and Pharmacodynamics of Mitofusin Activation in Murine Charcot-Marie-Tooth Disease Type 2A.
    Antonietta Franco, Xiawei Dang, Lihong Zhang, Perry B Molinoff, Gerald W Dorn.
      Mitofusin (MFN) 1 and MFN2 are dynamin GTPase family mitochondrial proteins that mediate mitochondrial fusion requiring MFN conformational shifts, formation of macromolecular complexes on and between mitochondria, and GTP hydrolysis. Damaging MFN2 mutations cause an untreatable, largely pediatric progressive peripheral neuropathy, Charcot-Marie-Tooth (CMT) disease type 2A. We used small molecule allosteric mitofusin activators that promote MFN conformations favoring fusion to interrogate the effects of MFN2 conformation and GTPase activity on MFN2-mediated mitochondrial fusion and motility in vitro. We translated these findings in vivo by defining dose-dependent pharmacodynamic and disease-modifying effects of mitofusin activators in murine CMT2A. MFN2 catalytic GTPase activity and MFN2 conformational switching are essential for mitochondrial fusion, but the two processes are separate and dissociable. We report the first concentration-response relationships for mitofusin activators to stimulate mitochondrial transport through CMT2A neuronal axons, which is similar to their stimulation of mitochondrial fusion. In CMT2A mice, intermittent (daily short acting) and sustained (twice daily long acting) mitofusin activation were equally effective in reversing neuromuscular degeneration. Moreover, acute dose-dependent pharmacodynamic effects of mitofusin activators on mitochondrial transport through CMT2A neuronal axons anticipated those for long-term reversal of neurodegenerative phenotypes. A crossover study showed that CMT2A neuronal deficits recurred after mitofusin activators are discontinued, and revealed that CMT2A can be ameliorated by mitofusin activation even in old (>74 week) mice. These data add to our understanding of mitochondrial dysfunction induced by a CMT2A MFN2 GTPase mutation and provide additional information supporting the approach of pharmacological mitofusin activation in CMT2A. SIGNIFICANCE: This study interrogated the roles of MFN2 catalytic activity and allosteric activation on impaired mitochondrial fusion and neuronal transport as they impact an untreatable peripheral neuropathy caused by MFN2 mutations, Charcot-Marie-Tooth disease type 2A. The results mechanistically link mitochondrial fusion and motility to the relaxed MFN2 protein conformation and correction of mitochondrial abnormalities to in vivo reversal of neurodegeneration in murine CMT2A.
    DOI:  https://doi.org/10.1124/jpet.122.001332
  6. J Appl Physiol (1985). 2022 Dec 15.
    Exercise and mitochondrial remodeling to prevent age-related neurodegeneration.
    Colleen L O'Reilly, Benjamin F Miller, Tommy L Lewis.
      Healthy brain activity requires precise ion and energy management creating a strong reliance on mitochondrial function. Age-related neurodegeneration leads to a decline in mitochondrial function and increased oxidative stress, with associated declines in mitochondrial mass, respiration capacity, and respiration efficiency. The interdependent processes of mitochondrial protein turnover and mitochondrial dynamics, known together as mitochondrial remodeling, play essential roles in mitochondrial health and therefore brain function. This mini-review describes the role of mitochondria in neurodegeneration and brain health, current practices for assessing both aspects of mitochondrial remodeling, and how exercise mitigates the adverse effects of aging in the brain. Exercise training elicits functional adaptations to improve brain health, and current literature strongly suggests that mitochondrial remodeling plays a vital role in these positive adaptations. Despite substantial implications that the two aspects of mitochondrial remodeling are interdependent, very few investigations have simultaneously measured mitochondrial dynamics and protein synthesis. An improved understanding of the partnership between mitochondrial protein turnover and mitochondrial dynamics will provide a better understanding of their role in both brain health and disease, as well as how they induce protection following exercise.
    Keywords:  brain; exercise; mitochondria; mitochondria remodeling; neurodegeneration
    DOI:  https://doi.org/10.1152/japplphysiol.00611.2022
  7. Mol Metab. 2022 Dec 10. pii: S2212-8778(22)00223-X. [Epub ahead of print] 101654
    The BBSome regulates mitochondria dynamics and function.
    Deng-Fu Guo, Ronald A Merrill, Lan Qian, Ying Hsu, Qihong Zhang, Zhihong Lin, Daniel R Thedens, Yuriy M Usachev, Isabella Grumbach, Val C Sheffield, Stefan Strack, Kamal Rahmouni.
      OBJECTIVE: The essential role of mitochondria in regulation of metabolic function and other physiological processes has garnered enormous interest in understanding the mechanisms controlling the function of this organelle. We assessed the role of the BBSome, a protein complex composed of eight Bardet-Biedl syndrome (BBS) proteins, in the control of mitochondria dynamic and function.METHODS: We used a multidisciplinary approach that include CRISPR/Cas9 technology-mediated generation of a stable Bbs1 gene knockout hypothalamic N39 neuronal cell line. We also analyzed the phenotype of BBSome deficient mice in presence or absence of the gene encoding A-kinase anchoring protein 1 (AKAP1).
    RESULTS: Our data show that the BBSome play an important role in the regulation of mitochondria dynamics and function. Disruption of the BBSome cause mitochondria hyperfusion in cell lines, fibroblasts derived from patients as well as in hypothalamic neurons and brown adipocytes of mice. The morphological changes in mitochondria translate into functional abnormalities as indicated by the reduced oxygen consumption rate and altered mitochondrial distribution and calcium handling. Mechanistically, we demonstrate that the BBSome modulates the activity of dynamin-like protein 1 (DRP1), a key regulator of mitochondrial fission, by regulating its phosphorylation and translocation to the mitochondria. Notably, rescuing the decrease in DRP1 activity through deletion of one copy of the gene encoding AKAP1 was effective to normalize the defects in mitochondrial morphology and activity induced by BBSome deficiency. Importantly, this was associated with improvement in several of the phenotypes caused by loss of the BBSome such as the neuroanatomical abnormalities, metabolic alterations and obesity highlighting the importance of mitochondria defects in the pathophysiology of BBS.
    CONCLUSIONS: These findings demonstrate a critical role of the BBSome in the modulation of mitochondria function and point to mitochondrial defects as a key disease mechanism in BBS.
    Keywords:  Bardet-biedl syndrome proteins; Body weight; Leptin sensitivity; Mitochondria
    DOI:  https://doi.org/10.1016/j.molmet.2022.101654
  8. Aging Cell. 2022 Dec 14. e13754
    Oral nicotinamide riboside raises NAD+ and lowers biomarkers of neurodegenerative pathology in plasma extracellular vesicles enriched for neuronal origin.
    Michael Vreones, Maja Mustapic, Ruin Moaddel, Krishna A Pucha, Jacqueline Lovett, Douglas R Seals, Dimitrios Kapogiannis, Christopher R Martens.
      Declining nicotinamide adenine dinucleotide (NAD+ ) concentration in the brain during aging contributes to metabolic and cellular dysfunction and is implicated in the pathogenesis of aging-associated neurological disorders. Experimental therapies aimed at boosting brain NAD+ levels normalize several neurodegenerative phenotypes in animal models, motivating their clinical translation. Dietary intake of NAD+ precursors, such as nicotinamide riboside (NR), is a safe and effective avenue for augmenting NAD+ levels in peripheral tissues in humans, yet evidence supporting their ability to raise NAD+ levels in the brain or engage neurodegenerative disease pathways is lacking. Here, we studied biomarkers in plasma extracellular vesicles enriched for neuronal origin (NEVs) from 22 healthy older adults who participated in a randomized, placebo-controlled crossover trial (NCT02921659) of oral NR supplementation (500 mg, 2x /day, 6 weeks). We demonstrate that oral NR supplementation increases NAD+ levels in NEVs and decreases NEV levels of Aβ42, pJNK, and pERK1/2 (kinases involved in insulin resistance and neuroinflammatory pathways). In addition, changes in NAD(H) correlated with changes in canonical insulin-Akt signaling proteins and changes in pERK1/2 and pJNK. These findings support the ability of orally administered NR to augment neuronal NAD+ levels and modify biomarkers related to neurodegenerative pathology in humans. Furthermore, NEVs offer a new blood-based window into monitoring the physiologic response of NR in the brain.
    Keywords:  NAD+; NADH; extracellular vesicles; neurodegenerative disease
    DOI:  https://doi.org/10.1111/acel.13754
  9. BMJ Neurol Open. 2022 ;4(2): e000352
    Changing faces of mitochondrial disease: autosomal recessive POLG disease mimicking myasthenia gravis and progressive supranuclear palsy.
    Menatalla Elwan, Andrew M Schaefer, Kate Craig, Sila Hopton, Gavin Falkous, Emma L Blakely, Robert W Taylor, Naomi Warren.
      Background: Mitochondrial disorders are known to cause diverse neurological phenotypes which cause a diagnostic challenge to most neurologists. Pathogenic polymerase gamma (POLG) variants have been described as a cause of chronic progressive external ophthalmoplegia, which manifests with ptosis, horizontal and vertical eye movement restriction and myopathy. Autosomal dominant progressive external ophthalmoplegia is rarely associated with Parkinsonism responsive to levodopa.Methods: We report a case of a 58-year-old man who presented with an eye movement disorder then Parkinsonism who made his way through the myasthenia then the movement disorder clinic.
    Results: A diagnostic right tibialis anterior biopsy revealed classical hallmarks of mitochondrial disease, and genetic testing identified compound heterozygous pathogenic gene variants in the POLG gene. The patient was diagnosed with autosomal recessive POLG disease.
    Conclusions: It is important to maintain a high index of suspicion of pathogenic POLG variants in patients presenting with atypical Parkinsonism and ophthalmoplegia. Patients with POLG-related disease will usually have ptosis, and downgaze is typically preserved until late in the disease. Accurate diagnosis is essential for appropriate prognosis and genetic counselling.
    Keywords:  MITOCHONDRIAL DISORDERS; MYASTHENIA; SUPRANUCLEAR PALSY
    DOI:  https://doi.org/10.1136/bmjno-2022-000352
  10. Nucleic Acids Res. 2022 Dec 12. pii: gkac1141. [Epub ahead of print]
    RNA granule-clustered mitochondrial aminoacyl-tRNA synthetases form multiple complexes with the potential to fine-tune tRNA aminoacylation.
    Gui-Xin Peng, Xue-Ling Mao, Yating Cao, Shi-Ying Yao, Qing-Run Li, Xin Chen, En-Duo Wang, Xiao-Long Zhou.
      Mitochondrial RNA metabolism is suggested to occur in identified compartmentalized foci, i.e. mitochondrial RNA granules (MRGs). Mitochondrial aminoacyl-tRNA synthetases (mito aaRSs) catalyze tRNA charging and are key components in mitochondrial gene expression. Mutations of mito aaRSs are associated with various human disorders. However, the suborganelle distribution, interaction network and regulatory mechanism of mito aaRSs remain largely unknown. Here, we found that all mito aaRSs partly colocalize with MRG, and this colocalization is likely facilitated by tRNA-binding capacity. A fraction of human mitochondrial AlaRS (hmtAlaRS) and hmtSerRS formed a direct complex via interaction between catalytic domains in vivo. Aminoacylation activities of both hmtAlaRS and hmtSerRS were fine-tuned upon complex formation in vitro. We further established a full spectrum of interaction networks via immunoprecipitation and mass spectrometry for all mito aaRSs and discovered interactions between hmtSerRS and hmtAsnRS, between hmtSerRS and hmtTyrRS and between hmtThrRS and hmtArgRS. The activity of hmtTyrRS was also influenced by the presence of hmtSerRS. Notably, hmtSerRS utilized the same catalytic domain in mediating several interactions. Altogether, our results systematically analyzed the suborganelle localization and interaction network of mito aaRSs and discovered several mito aaRS-containing complexes, deepening our understanding of the functional and regulatory mechanisms of mito aaRSs.
    DOI:  https://doi.org/10.1093/nar/gkac1141
  11. Drugs Aging. 2022 Dec 13.
    Importance of NAD+ Anabolism in Metabolic, Cardiovascular and Neurodegenerative Disorders.
    Tessa Helman, Nady Braidy.
      The role of nicotinamide adenine dinucleotide (NAD+) in ageing has emerged as a critical factor in understanding links to a wide range of chronic diseases. Depletion of NAD+, a central redox cofactor and substrate of numerous metabolic enzymes, has been detected in many major age-related diseases. However, the mechanisms behind age-associated NAD+ decline remains poorly understood. Despite limited conclusive evidence, supplements aimed at increasing NAD+ levels are becoming increasingly popular. This review provides renewed insights regarding the clinical utility and benefits of NAD+ precursors, namely nicotinamide (NAM), nicotinic acid (NA), nicotinamide riboside (NR) and nicotinamide mononucleotide (NMN), in attenuating NAD+ decline and phenotypic characterization of age-related disorders, including metabolic, cardiovascular and neurodegenerative diseases. While it is anticipated that NAD+ precursors can play beneficial protective roles in several conditions, they vary in their ability to promote NAD+ anabolism with differing adverse effects. Careful evaluation of the role of NAD+, whether friend or foe in ageing, should be considered.
    DOI:  https://doi.org/10.1007/s40266-022-00989-0
  12. Adv Exp Med Biol. 2022 ;1395 367-372
    On the Mechanism of Sustained Mitochondrial Membrane Potential Without Functioning Complex IV.
    Eiji Takahashi, Yoshihisa Yamaoka.
      In intact mitochondria, the transport of electrons, respiration and generation of proton gradients across the inner membrane (proton motive force) are mutually coupled, according to Peter Mitchell's hypothesis on oxidative phosphorylation. Thus, the inhibition of electron transport at either respiratory complex III or IV in the electron transport chain leads to failure in producing proton motive force along with the abolition of respiration. Here, we determined the mitochondrial membrane potential (MMP), as a measure of proton motive force, and cellular respiration in various cultured cells and demonstrated that inhibition of complex IV by KCN abolished mitochondrial respiration while MMP was sustained. These results are unexpected and appear incompatible with Mitchell's chemiosmotic hypothesis.
    Keywords:  Electron transport; Mitchell’s chemiosmotic hypothesis; Mitochondria; Proton motive force
    DOI:  https://doi.org/10.1007/978-3-031-14190-4_60
  13. Trends Cell Biol. 2022 Dec 12. pii: S0962-8924(22)00259-8. [Epub ahead of print]
    Thermoporter: a new regulatory mechanism for mitochondrial calcium uniporter activity.
    Kaili Xue, Dongmei Wu, Yifu Qiu.
      The mitochondrial calcium uniporter (MCU) controls mitochondrial bioenergetics, and its activity varies greatly between tissues. Here, we highlight a recently identified MCU-EMRE-UCP1 complex, named thermoporter, in the adaptive thermogenesis of brown adipose tissue (BAT). The thermoporter enhances MCU activity to promote thermogenic metabolism, demonstrating a BAT-specific regulation for MCU activity.
    DOI:  https://doi.org/10.1016/j.tcb.2022.11.008
  14. Hear Res. 2022 Nov 25. pii: S0378-5955(22)00228-3. [Epub ahead of print]428 108660
    Mitochondrial form and function in hair cells.
    James D B O'Sullivan, Anwen Bullen, Zoë F Mann.
      Hair cells (HCs) are specialised sensory receptors residing in the neurosensory epithelia of inner ear sense organs. The precise morphological and physiological properties of HCs allow us to perceive sound and interact with the world around us. Mitochondria play a significant role in normal HC function and are also intricately involved in HC death. They generate ATP essential for sustaining the activity of ion pumps, Ca2+ transporters and the integrity of the stereociliary bundle during transduction as well as regulating cytosolic calcium homoeostasis during synaptic transmission. Advances in imaging techniques have allowed us to study mitochondrial populations throughout the HC, and how they interact with other organelles. These analyses have identified distinct mitochondrial populations between the apical and basolateral portions of the HC, in which mitochondrial morphology appears determined by the physiological processes in the different cellular compartments. Studies in HCs across species show that ototoxic agents, ageing and noise damage directly impact mitochondrial structure and function resulting in HC death. Deciphering the molecular mechanisms underlying this mitochondrial sensitivity, and how their morphology relates to their function during HC death, requires that we first understand this relationship in the context of normal HC function.
    Keywords:  Ca2+buffering; Development; Hearing loss; Metabolism; Mitochondria; Mitochondrial cristae; Synaptic transmission oxidative stress
    DOI:  https://doi.org/10.1016/j.heares.2022.108660
  15. Int J Neurosci. 2022 Dec 14. 1-22
    Implications of p53 in mitochondrial dysfunction and Parkinson's disease.
    Yi-Fang Wang, Ying-Di Wang, Song Gao, Wei Sun.
      Mitochondria are vital subcellular organelles for that maintain cellular function, and mitochondrial defect and impairment are primary causes of dopaminergic neuron degeneration in Parkinson's disease (PD). P53 is a multifunctional protein implicated in the regulation of diverse cellular processes via transcription-dependent and transcription-independent mechanisms. Increasing evidence has revealed that mitochondrial dysfunction-associated dopaminergic neuron degeneration is tightly regulated by p53 in PD pathogenesis. Neurodegenerative stress triggers p53 activation, which induces mitochondrial changes, including transmembrane permeability, reactive oxygen species production, Ca2+ overload, electron transport chain defects and other dynamic alterations, and these changes contribute to neurodegeneration and are linked closely with PD occurrence and development. P53 inhibition has been shown to attenuate mitochondrial dysfunction and protect dopaminergic neurons from degeneration under conditions of neurodegenerative stress, and thus, p53 appears to be a potential target for neuroprotective therapy. We review the contributions of p53 to mitochondrial changes leading to apoptosis and the subsequent degeneration of dopaminergic neurons to advance the understanding of the underlying molecular mechanisms and provide a potential therapeutic target for PD treatment.
    Keywords:  P53; Parkinson’s disease; mitochondrial dysfunction; neurodegeneration
    DOI:  https://doi.org/10.1080/00207454.2022.2158824
  16. Nat Rev Endocrinol. 2022 Dec 14.
    A mitochondrial trigger for beige adipocyte development.
    Shimona Starling.
      
    DOI:  https://doi.org/10.1038/s41574-022-00792-2
  17. J Cell Sci. 2022 Dec 01. pii: jcs259748. [Epub ahead of print]135(23):
    Diversity of mitophagy pathways at a glance.
    Ian G Ganley, Anne Simonsen.
      Mitochondria are crucial organelles that play a central role in various cell signaling and metabolic pathways. A healthy mitochondrial population is maintained through a series of quality control pathways and requires a fine-tuned balance between mitochondrial biogenesis and degradation. Defective targeting of dysfunctional mitochondria to lysosomes through mitophagy has been linked to several diseases, but the underlying mechanisms and the relative importance of distinct mitophagy pathways in vivo are largely unknown. In this Cell Science at a Glance and the accompanying poster, we describe our current understanding of how parts of, or whole, mitochondria are recognized by the autophagic machinery and targeted to lysosomes for degradation. We also discuss how this might be regulated under different physiological conditions to maintain mitochondrial and cellular health.
    Keywords:  BNIP3; HIF1; Mitochondria; Mitophagy; NIX; PINK1; Parkin; SLR; Selective autophagy
    DOI:  https://doi.org/10.1242/jcs.259748
  18. Cells. 2022 Nov 30. pii: 3866. [Epub ahead of print]11(23):
    Mitochondrial Permeability Transition.
    Paolo Bernardi, Evgeny Pavlov.
      The mitochondrial permeability transition (PT) is a phenomenon that can be broadly defined as an increase in the permeability of the mitochondrial inner membrane [...].
    DOI:  https://doi.org/10.3390/cells11233866
  19. J Cell Biol. 2023 Jan 02. pii: e202211123. [Epub ahead of print]222(1):
    Cilia bent out of shape over dysfunctional astrocyte mitochondria.
    Rachel M Bear, Tamara Caspary.
      Mitochondrial dysfunction in astrocytes drives neurodegenerative brain pathology. In this issue, Ignatenko et al. (2022. J. Cell. Biol.https://doi.org/10.1083/jcb.202203019) discover a novel connection between cilia and mitochondria in astrocytes, whereby mitochondrial dysfunction leads to abnormal cilia structure and a motile cilia program.
    DOI:  https://doi.org/10.1083/jcb.202211123
  20. Front Cell Dev Biol. 2022 ;10 1036225
    Mitochondria-endoplasmic reticulum contacts in sepsis-induced myocardial dysfunction.
    Tao Jiang, Qian Wang, Jiagao Lv, Li Lin.
      Mitochondrial and endoplasmic reticulum (ER) are important intracellular organelles. The sites that mitochondrial and ER are closely related in structure and function are called Mitochondria-ER contacts (MERCs). MERCs are involved in a variety of biological processes, including calcium signaling, lipid synthesis and transport, autophagy, mitochondrial dynamics, ER stress, and inflammation. Sepsis-induced myocardial dysfunction (SIMD) is a vital organ damage caused by sepsis, which is closely associated with mitochondrial and ER dysfunction. Growing evidence strongly supports the role of MERCs in the pathogenesis of SIMD. In this review, we summarize the biological functions of MERCs and the roles of MERCs proteins in SIMD.
    Keywords:  ER stress; autophagy; calcium signaling; inflammation; mitochondria-ER contacts; mitochondrial dynamics; sepsis-induced myocardial dysfunction
    DOI:  https://doi.org/10.3389/fcell.2022.1036225
  21. Trends Pharmacol Sci. 2022 Dec 07. pii: S0165-6147(22)00254-1. [Epub ahead of print]
    Mitochondrial dynamics proteins as emerging drug targets.
    Emmanouil Zacharioudakis, Evripidis Gavathiotis.
      The importance of mitochondrial dynamics, the physiological process of mitochondrial fusion and fission, in regulating diverse cellular functions and cellular fitness has been well established. Several pathologies are associated with aberrant mitochondrial fusion or fission that is often a consequence of deregulated mitochondrial dynamics proteins; however, pharmacological targeting of these proteins has been lacking and is challenged by complex molecular mechanisms. Recent studies have advanced our understanding in this area and have enabled rational drug design and chemical screening strategies. We provide an updated overview of the regulatory mechanisms of fusion and fission proteins, their structure-function relationships, and the discovery of pharmacological modulators demonstrating their therapeutic potential. These advances provide exciting opportunities for the development of prototype therapeutics for various diseases.
    Keywords:  activators; fission; fusion; inhibitors; mitochondria; mitochondrial dynamics; mitochondrial structure
    DOI:  https://doi.org/10.1016/j.tips.2022.11.004
  22. EMBO J. 2022 Dec 14. e111372
    NDP52 acts as a redox sensor in PINK1/Parkin-mediated mitophagy.
    Tetsushi Kataura, Elsje G Otten, Yoana Rabanal-Ruiz, Elias Adriaenssens, Francesca Urselli, Filippo Scialo, Lanyu Fan, Graham R Smith, William M Dawson, Xingxiang Chen, Wyatt W Yue, Agnieszka K Bronowska, Bernadette Carroll, Sascha Martens, Michael Lazarou, Viktor I Korolchuk.
      Mitophagy, the elimination of mitochondria via the autophagy-lysosome pathway, is essential for the maintenance of cellular homeostasis. The best characterised mitophagy pathway is mediated by stabilisation of the protein kinase PINK1 and recruitment of the ubiquitin ligase Parkin to damaged mitochondria. Ubiquitinated mitochondrial surface proteins are recognised by autophagy receptors including NDP52 which initiate the formation of an autophagic vesicle around the mitochondria. Damaged mitochondria also generate reactive oxygen species (ROS) which have been proposed to act as a signal for mitophagy, however the mechanism of ROS sensing is unknown. Here we found that oxidation of NDP52 is essential for the efficient PINK1/Parkin-dependent mitophagy. We identified redox-sensitive cysteine residues involved in disulphide bond formation and oligomerisation of NDP52 on damaged mitochondria. Oligomerisation of NDP52 facilitates the recruitment of autophagy machinery for rapid mitochondrial degradation. We propose that redox sensing by NDP52 allows mitophagy to function as a mechanism of oxidative stress response.
    Keywords:  NDP52; autophagy; mitophagy; p62; redox
    DOI:  https://doi.org/10.15252/embj.2022111372
  23. Cell Metab. 2022 Dec 08. pii: S1550-4131(22)00502-2. [Epub ahead of print]
    Q-Flux: A method to assess hepatic mitochondrial succinate dehydrogenase, methylmalonyl-CoA mutase, and glutaminase fluxes in vivo.
    Brandon T Hubbard, Traci E LaMoia, Leigh Goedeke, Rafael C Gaspar, Katrine D Galsgaard, Mario Kahn, Graeme F Mason, Gerald I Shulman.
      The mammalian succinate dehydrogenase (SDH) complex has recently been shown as capable of operating bidirectionally. Here, we develop a method (Q-Flux) capable of measuring absolute rates of both forward (VSDH(F)) and reverse (VSDH(R)) flux through SDH in vivo while also deconvoluting the amount of glucose derived from four discreet carbon sources in the liver. In validation studies, a mitochondrial uncoupler increased net SDH flux by >100% in awake rodents but also increased SDH cycling. During hyperglucagonemia, attenuated pyruvate cycling enhances phosphoenolpyruvate carboxykinase efficiency to drive increased gluconeogenesis, which is complemented by increased glutaminase (GLS) flux, methylmalonyl-CoA mutase (MUT) flux, and glycerol conversion to glucose. During hyperinsulinemic-euglycemic clamp, both pyruvate carboxylase and GLS are suppressed, while VSDH(R) is increased. Unstimulated MUT is a minor anaplerotic reaction but is readily induced by small amounts of propionate, which elicits glucagon-like metabolic rewiring. Taken together, Q-Flux yields a comprehensive picture of hepatic mitochondrial metabolism and should be broadly useful to researchers.
    Keywords:  anaplerosis; glucagon; glutaminase; insulin; mass spectrometry; metabolic flux analysis; methylmalonyl-CoA mutase; mitochondrial metabolism; propionate; succinate dehydrogenase
    DOI:  https://doi.org/10.1016/j.cmet.2022.11.011
  24. Cell Rep. 2022 Dec 13. pii: S2211-1247(22)01677-1. [Epub ahead of print]41(11): 111789
    The mitochondrial UPR regulator ATF5 promotes intestinal barrier function via control of the satiety response.
    Douja Chamseddine, Siraje A Mahmud, Aundrea K Westfall, Todd A Castoe, Rance E Berg, Mark W Pellegrino.
      Organisms use several strategies to mitigate mitochondrial stress, including the activation of the mitochondrial unfolded protein response (UPRmt). The UPRmt in Caenorhabditis elegans, regulated by the transcription factor ATFS-1, expands on this recovery program by inducing an antimicrobial response against pathogens that target mitochondrial function. Here, we show that the mammalian ortholog of ATFS-1, ATF5, protects the host during infection with enteric pathogens but, unexpectedly, by maintaining the integrity of the intestinal barrier. Intriguingly, ATF5 supports intestinal barrier function by promoting a satiety response that prevents obesity and associated hyperglycemia. This consequently averts dysregulated glucose metabolism that is detrimental to barrier function. Mechanistically, we show that intestinal ATF5 stimulates the satiety response by transcriptionally regulating the gastrointestinal peptide hormone cholecystokinin, which promotes the secretion of the hormone leptin. We propose that ATF5 protects the host from enteric pathogens by promoting intestinal barrier function through a satiety-response-mediated metabolic control mechanism.
    Keywords:  ATF5; CP: Metabolism; CP: Molecular biology; UPR(mt); cholecystokinin; colitis; epithelial barrier; host-pathogen interaction; hyperglycemia; leptin; mitochondria; satiety
    DOI:  https://doi.org/10.1016/j.celrep.2022.111789
  25. J Inherit Metab Dis. 2022 Dec 11.
    Transcriptomic analyses reveal neuronal specificity of Leigh syndrome associated genes.
    Azizia Wahedi, Chandika Soondram, Alan E Murphy, Nathan Skene, Shamima Rahman.
      Leigh syndrome is a rare inherited, complex, neurometabolic disorder with genetic and clinical heterogeneity. Features present in affected patients range from classical stepwise developmental regression to ataxia, seizures, tremor, and occasionally psychiatric manifestations. Currently, more than 100 monogenic causes of Leigh syndrome have been identified, yet, the pathophysiology remains unknown. Here, we sought to determine the cellular specificity within the brain of all genes currently associated with Leigh syndrome. Further, we aimed to investigate potential genetic commonalities between Leigh syndrome and other disorders with overlapping clinical features. Enrichment of our target genes within the brain was evaluated with co-expression (CoExp) network analyses constructed using existing UK Brain Expression Consortium data. To determine the cellular specificity of the Leigh associated genes, we employed expression weighted cell type enrichment (EWCE) analysis of single cell RNA-Seq data. Finally, CoExp network modules demonstrating enrichment of Leigh syndrome associated genes were then utilised for synaptic gene ontology analysis and heritability analysis. CoExp network analyses revealed that Leigh syndrome associated genes exhibit the highest levels of expression in brain regions most affected on MRI in affected patients. EWCE revealed significant enrichment of target genes in hippocampal and somatosensory pyramidal neurons and interneurons of the brain. Analysis of CoExp modules enriched with our target genes revealed preferential association with pre-synaptic structures. Heritability studies suggested some common enrichment between Leigh syndrome and Parkinson disease and epilepsy. Our findings suggest a primary mitochondrial dysfunction as the underlying basis of Leigh syndrome with associated genes primarily expressed in neuronal cells. This article is protected by copyright. All rights reserved.
    Keywords:  CoExp network; EWCE; Leigh syndrome spectrum; Primary mitochondrial disease; transcriptomics
    DOI:  https://doi.org/10.1002/jimd.12578
  26. J Am Chem Soc. 2022 Dec 14.
    Human EXOG Possesses Strong AP Hydrolysis Activity: Implication on Mitochondrial DNA Base Excision Repair.
    Michal R Szymanski, Anna Karlowicz, Geoffrey K Herrmann, Yana Cen, Y Whitney Yin.
      Most oxidative damage on mitochondrial DNA is corrected by the base excision repair (BER) pathway. However, the enzyme that catalyzes the rate-limiting reaction─deoxyribose phosphate (dRP) removal─in the multienzymatic reaction pathway has not been completely determined in mitochondria. Also unclear is how a logical order of enzymatic reactions is ensured. Here, we present structural and enzymatic studies showing that human mitochondrial EXOG (hEXOG) exhibits strong 5'-dRP removal ability. We show that, unlike the canonical dRP lyases that act on a single substrate, hEXOG functions on a variety of abasic sites, including 5'-dRP, its oxidized product deoxyribonolactone (dL), and the stable synthetic analogue tetrahydrofuran (THF). We determined crystal structures of hEXOG complexed with a THF-containing DNA and with a partial gapped DNA to 2.9 and 2.1 Å resolutions, respectively. The structures illustrate that hEXOG uses a controlled 5'-exonuclease activity to cleave the third phosphodiester bond away from the 5'-abasic site. This study provides a structural basis for hEXOG's broad spectrum of substrates. Further, we show that hEXOG can set the order of BER reactions by generating an ideal substrate for the subsequent reaction in BER and inhibit off-pathway reactions.
    DOI:  https://doi.org/10.1021/jacs.2c10558
  27. Sci Adv. 2022 Dec 16. 8(50): eade1516
    Mechanisms of ion selectivity and throughput in the mitochondrial calcium uniporter.
    Bryce D Delgado, Stephen B Long.
      The mitochondrial calcium uniporter, which regulates aerobic metabolism by catalyzing mitochondrial Ca2+ influx, is arguably the most selective ion channel known. The mechanisms for this exquisite Ca2+ selectivity have not been defined. Here, using a reconstituted system, we study the electrical properties of the channel's minimal Ca2+-conducting complex, MCU-EMRE, from Tribolium castaneum to probe ion selectivity mechanisms. The wild-type TcMCU-EMRE complex recapitulates hallmark electrophysiological properties of endogenous Uniporter channels. Through interrogation of pore-lining mutants, we find that a ring of glutamate residues, the "E-locus," serves as the channel's selectivity filter. Unexpectedly, a nearby "D-locus" at the mouth of the pore has diminutive influence on selectivity. Anomalous mole fraction effects indicate that multiple Ca2+ ions are accommodated within the E-locus. By facilitating ion-ion interactions, the E-locus engenders both exquisite Ca2+ selectivity and high ion throughput. Direct comparison with structural information yields the basis for selective Ca2+ conduction by the channel.
    DOI:  https://doi.org/10.1126/sciadv.ade1516
  28. Nat Commun. 2022 Dec 10. 13(1): 7637
    Mitochondrial dysfunction induces ALK5-SMAD2-mediated hypovascularization and arteriovenous malformations in mouse retinas.
    Haifeng Zhang, Busu Li, Qunhua Huang, Francesc López-Giráldez, Yoshiaki Tanaka, Qun Lin, Sameet Mehta, Guilin Wang, Morven Graham, Xinran Liu, In-Hyun Park, Anne Eichmann, Wang Min, Jenny Huanjiao Zhou.
      Although mitochondrial activity is critical for angiogenesis, its mechanism is not entirely clear. Here we show that mice with endothelial deficiency of any one of the three nuclear genes encoding for mitochondrial proteins, transcriptional factor (TFAM), respiratory complex IV component (COX10), or redox protein thioredoxin 2 (TRX2), exhibit retarded retinal vessel growth and arteriovenous malformations (AVM). Single-cell RNA-seq analyses indicate that retinal ECs from the three mutant mice have increased TGFβ signaling and altered gene expressions associated with vascular maturation and extracellular matrix, correlating with vascular malformation and increased basement membrane thickening in microvesels of mutant retinas. Mechanistic studies suggest that mitochondrial dysfunction from Tfam, Cox10, or Trx2 depletion induces a mitochondrial localization and MAPKs-mediated phosphorylation of SMAD2, leading to enhanced ALK5-SMAD2 signaling. Importantly, pharmacological blockade of ALK5 signaling or genetic deficiency of SMAD2 prevented retinal vessel growth retardation and AVM in all three mutant mice. Our studies uncover a novel mechanism whereby mitochondrial dysfunction via the ALK5-SMAD2 signaling induces retinal vascular malformations, and have therapeutic values for the alleviation of angiogenesis-associated human retinal diseases.
    DOI:  https://doi.org/10.1038/s41467-022-35262-w
  29. Methods Mol Biol. 2023 ;2609 227-249
    Methods to Assess the Role of PARPs in Regulating Mitochondrial Oxidative Function.
    Tünde Kovács, Boglárka Rauch, Edit Mikó, Péter Bai.
      PARP enzymes are involved in metabolic regulation and impact on a plethora of cellular metabolic pathways, among them, mitochondrial oxidative metabolism. The detrimental effects of PARP1 overactivation upon oxidative stress on mitochondrial oxidative metabolism was discovered in 1998. Since then, there was an enormous blooming in the understanding of the interplay between PARPs and mitochondria. Mitochondrial activity can be assessed by a comprehensive set of methods that we aim to introduce here.
    Keywords:  ARTD; Differentiation; Flow cytometry; Mitochondria; Mitochondrial fission; Mitochondrial fusion; Mitochondrial morphology; Oximetry; PARP; Seahorse extracellular flux analyzer; Substrate preference; Δψ
    DOI:  https://doi.org/10.1007/978-1-0716-2891-1_14
  30. Cells. 2022 Nov 24. pii: 3754. [Epub ahead of print]11(23):
    TFAM's Contributions to mtDNA Replication and OXPHOS Biogenesis Are Genetically Separable.
    Natalya Kozhukhar, Mikhail F Alexeyev.
      The ability of animal orthologs of human mitochondrial transcription factor A (hTFAM) to support the replication of human mitochondrial DNA (hmtDNA) does not follow a simple pattern of phylogenetic closeness or sequence similarity. In particular, TFAM from chickens (Gallus gallus, chTFAM), unlike TFAM from the "living fossil" fish coelacanth (Latimeria chalumnae), cannot support hmtDNA replication. Here, we implemented the recently developed GeneSwap approach for reverse genetic analysis of chTFAM to obtain insights into this apparent contradiction. By implementing limited "humanization" of chTFAM focused either on amino acid residues that make DNA contacts, or the ones with significant variances in side chains, we isolated two variants, Ch13 and Ch22. The former has a low mtDNA copy number (mtCN) but robust respiration. The converse is true of Ch22. Ch13 and Ch22 complement each other's deficiencies. Opposite directionalities of changes in mtCN and respiration were also observed in cells expressing frog TFAM. This led us to conclude that TFAM's contributions to mtDNA replication and respiratory chain biogenesis are genetically separable. We also present evidence that TFAM residues that make DNA contacts play the leading role in mtDNA replication. Finally, we present evidence for a novel mode of regulation of the respiratory chain biogenesis by regulating the supply of rRNA subunits.
    Keywords:  GeneSwap approach; TFAM; mtDNA instability; mtDNA replication; mtDNA transcription
    DOI:  https://doi.org/10.3390/cells11233754
  31. Science. 2022 Dec 16. 378(6625): eadf3971
    Protein import into peroxisomes occurs through a nuclear pore-like phase.
    Yuan Gao, Michael L Skowyra, Peiqiang Feng, Tom A Rapoport.
      Peroxisomes are ubiquitous organelles whose dysfunction causes fatal human diseases. Most peroxisomal proteins are imported from the cytosol in a folded state by the soluble receptor PEX5. How folded cargo crosses the membrane is unknown. Here, we show that peroxisomal import is similar to nuclear transport. The peroxisomal membrane protein PEX13 contains a conserved tyrosine (Y)- and glycine (G)-rich YG domain, which forms a selective phase resembling that formed by phenylalanine-glycine (FG) repeats within nuclear pores. PEX13 resides in the membrane in two orientations that oligomerize and suspend the YG meshwork within the lipid bilayer. Purified YG domains form hydrogels into which PEX5 selectively partitions, by using conserved aromatic amino acid motifs, bringing cargo along. The YG meshwork thus forms an aqueous conduit through which PEX5 delivers folded proteins into peroxisomes.
    DOI:  https://doi.org/10.1126/science.adf3971
  32. Life Sci. 2022 Dec 13. pii: S0024-3205(22)00971-7. [Epub ahead of print] 121271
    Modulation of mitochondria by viral proteins.
    Reshu Saxena, Priyanka Sharma, Sandeep Kumar, Niteshkumar Agrawal, Sumit Kumar Sharma, Amit Awasthi.
      Mitochondria are dynamic cellular organelles with diverse functions including energy production, calcium homeostasis, apoptosis, host innate immune signaling, and disease progression. Several viral proteins specifically target mitochondria to subvert host defense as mitochondria stand out as the most suitable target for the invading viruses. They have acquired the capability to control apoptosis, metabolic state, and evade immune responses in host cells, by targeting mitochondria. In this way, the viruses successfully allow the spread of viral progeny and thus the infection. Viruses employ their proteins to alter mitochondrial dynamics and their specific functions by a modulation of membrane potential, reactive oxygen species, calcium homeostasis, and mitochondrial bioenergetics to help them achieve a state of persistent infection. A better understanding of such viral proteins and their impact on mitochondrial forms and functions is the main focus of this review. We also attempt to emphasize the importance of exploring the role of mitochondria in the context of SARS-CoV2 pathogenesis and identify host-virus protein interactions.
    Keywords:  Mitochondria; SARS-CoV2; Viral proteins; Viruses
    DOI:  https://doi.org/10.1016/j.lfs.2022.121271
  33. Prog Retin Eye Res. 2022 Dec 08. pii: S1350-9462(22)00113-6. [Epub ahead of print] 101153
    Cellular and subcellular optogenetic approaches towards neuroprotection and vision restoration.
    Edward H Wood, Alexander Kreymerman, Tia Kowal, David Buickians, Yang Sun, Stephanie Muscat, Mark Mercola, Darius M Moshfeghi, Jeffrey L Goldberg.
      Optogenetics is defined as the combination of genetic and optical methods to induce or inhibit well-defined events in isolated cells, tissues, or animals. While optogenetics within ophthalmology has been primarily applied towards treating inherited retinal disease, there are a myriad of other applications that hold great promise for a variety of eye diseases including cellular regeneration, modulation of mitochondria and metabolism, regulation of intraocular pressure, and pain control. Supported by primary data from the authors' work with in vitro and in vivo applications, we introduce a novel approach to metabolic regulation, Opsins to Restore Cellular ATP (ORCA). We review the fundamental constructs for ophthalmic optogenetics, present current therapeutic approaches and clinical trials, and discuss the future of subcellular and signaling pathway applications for neuroprotection and vision restoration.
    Keywords:  Adeno-associated virus; Mitochondria; Optogenetics; Photoswitch; Retina; Subcellular
    DOI:  https://doi.org/10.1016/j.preteyeres.2022.101153
  34. Genet Med. 2022 Dec 14. pii: S1098-3600(22)01000-0. [Epub ahead of print]
    Biallelic variants in OGDH encoding oxoglutarate dehydrogenase lead to a neurodevelopmental disorder characterized by global developmental delay, movement disorder, and metabolic abnormalities.
    Ella F Whittle, Madison Chilian, Ehsan Ghayoor Karimiani, Helga Progri, Daniela Buhas, Melis Kose, Rebecca D Ganetzky, Mehran Beiraghi Toosi, Paria Najarzadeh Torbati, Reza Shervin Badv, Ivan Shelihan, Hui Yang, Houda Zghal Elloumi, Sukyeong Lee, Yalda Jamshidi, Alan M Pittman, Henry Houlden, Erika Ignatius, Shamima Rahman, Reza Maroofian, Wan Hee Yoon, Christopher J Carroll.
      PURPOSE: This study aimed to establish the genetic cause of a novel autosomal recessive neurodevelopmental disorder characterized by global developmental delay, movement disorder, and metabolic abnormalities.METHODS: We performed a detailed clinical characterization of 4 unrelated individuals from consanguineous families with a neurodevelopmental disorder. We used exome sequencing or targeted-exome sequencing, cosegregation, in silico protein modeling, and functional analyses of variants in HEK293 cells and Drosophila melanogaster, as well as in proband-derived fibroblast cells.
    RESULTS: In the 4 individuals, we identified 3 novel homozygous variants in oxoglutarate dehydrogenase (OGDH) (NM_002541.3), which encodes a subunit of the tricarboxylic acid cycle enzyme α-ketoglutarate dehydrogenase. In silico homology modeling predicts that c.566C>T:p.(Pro189Leu) and c.890C>A:p.(Ser297Tyr) variants interfere with the structure and function of OGDH. Fibroblasts from individual 1 showed that the p.(Ser297Tyr) variant led to a higher degradation rate of the OGDH protein. OGDH protein with p.(Pro189Leu) or p.(Ser297Tyr) variants in HEK293 cells showed significantly lower levels than the wild-type protein. Furthermore, we showed that expression of Drosophila Ogdh (dOgdh) carrying variants homologous to p.(Pro189Leu) or p.(Ser297Tyr), failed to rescue developmental lethality caused by loss of dOgdh. SpliceAI, a variant splice predictor, predicted that the c.935G>A:p.(Arg312Lys)/p.(Phe264_Arg312del) variant impacts splicing, which was confirmed through a mini-gene assay in HEK293 cells.
    CONCLUSION: We established that biallelic variants in OGDH cause a neurodevelopmental disorder with metabolic and movement abnormalities.
    Keywords:  Mitochondria; Neurodevelopmental disease; OGDH; Oxoglutarate dehydrogenase; α-ketoglutarate dehydrogenase deficiency
    DOI:  https://doi.org/10.1016/j.gim.2022.11.001
  35. J Hum Genet. 2022 Dec 13.
    A novel homozygous CHMP1A variant arising from segmental uniparental disomy causes pontocerebellar hypoplasia type 8.
    Masamune Sakamoto, Toshihide Shiiki, Shuji Matsui, Nobuhiko Okamoto, Eriko Koshimizu, Naomi Tsuchida, Yuri Uchiyama, Kohei Hamanaka, Atsushi Fujita, Satoko Miyatake, Kazuharu Misawa, Takeshi Mizuguchi, Naomichi Matsumoto.
      Pontocerebellar hypoplasia (PCH) is currently classified into 16 subgroups. Using mostly next-generation sequencing, pathogenic variants have been identified in as many as 24 PCH-associated genes. PCH type 8 (PCH8) is a rare heterogeneous disorder. Its clinical presentation includes severe development delay, increased muscle tone, microcephaly, and magnetic resonance imaging (MRI) abnormalities such as reduced cerebral white matter, a thin corpus callosum, and brainstem and cerebellar hypoplasia. To date, only two variants in the CHMP1A gene (MIM: 164010), NM_002768.5: c.88 C > T (p.Glu30*) and c.28-13 G > A, have been identified homozygously in seven patients with PCH8 from four families (MIM: 614961). CHMP1A is a subunit of the endosomal sorting complex required for transport III (ESCRT-III), which regulates the formation and release of extracellular vesicles. Biallelic CHMP1A loss of function impairs the ESCRT-III-mediated release of extracellular vesicles, which causes impaired progenitor proliferation in the developing brain. Herein, we report a patient with PCH8 who had a homozygous CHMP1A variant, c.122delA (p.Asn41Metfs*2), which arose from segmental uniparental disomy. Although our patient had similar MRI findings to those of previously reported patients, with no progression, we report some novel neurological and developmental findings that expand our knowledge of the clinical consequences associated with CHMP1A variants.
    DOI:  https://doi.org/10.1038/s10038-022-01098-x
  36. Nat Struct Mol Biol. 2022 Dec 15.
    Structural basis of the acyl-transfer mechanism of human GPAT1.
    Zachary Lee Johnson, Mark Ammirati, David Jonathan Wasilko, Jeanne S Chang, Stephen Noell, Timothy L Foley, Hyejin Yoon, Kathleen Smith, Shoh Asano, Katherine Hales, Min Wan, Qingyi Yang, Mary A Piotrowski, Kathleen A Farley, Tamara Gilbert, Lisa M Aschenbrenner, Kimberly F Fennell, Jason K Dutra, Mary Xu, Chunyang Guo, Alison E Varghese, Justin Bellenger, Alandra Quinn, Christopher W Am Ende, Graham M West, Matthew C Griffor, Donald Bennett, Matthew Calabrese, Claire M Steppan, Seungil Han, Huixian Wu.
      Glycerol-3-phosphate acyltransferase (GPAT)1 is a mitochondrial outer membrane protein that catalyzes the first step of de novo glycerolipid biosynthesis. Hepatic expression of GPAT1 is linked to liver fat accumulation and the severity of nonalcoholic fatty liver diseases. Here we present the cryo-EM structures of human GPAT1 in substrate analog-bound and product-bound states. The structures reveal an N-terminal acyltransferase domain that harbors important catalytic motifs and a tightly associated C-terminal domain that is critical for proper protein folding. Unexpectedly, GPAT1 has no transmembrane regions as previously proposed but instead associates with the membrane via an amphipathic surface patch and an N-terminal loop-helix region that contains a mitochondrial-targeting signal. Combined structural, computational and functional studies uncover a hydrophobic pathway within GPAT1 for lipid trafficking. The results presented herein lay a framework for rational inhibitor development for GPAT1.
    DOI:  https://doi.org/10.1038/s41594-022-00884-7
  37. Neuroimage Clin. 2022 ;pii: S2213-1582(22)00305-9. [Epub ahead of print]36 103240
    Neuroimaging in Leber Hereditary Optic Neuropathy: State-of-the-art and future prospects.
    Hugo T Chow-Wing-Bom, Martina F Callaghan, Junqing Wang, Shihui Wei, Frederic Dick, Patrick Yu-Wai-Man, Tessa M Dekker.
      Leber Hereditary Optic Neuropathy (LHON) is an inherited mitochondrial retinal disease that causes the degeneration of retinal ganglion cells and leads to drastic loss of visual function. In the last decades, there has been a growing interest in using Magnetic Resonance Imaging (MRI) to better understand mechanisms of LHON beyond the retina. This is partially due to the emergence of gene-therapies for retinal diseases, and the accompanying expanded need for reliably quantifying and monitoring visual processing and treatment efficiency in patient populations. This paper aims to draw a current picture of key findings in this field so far, the challenges of using neuroimaging methods in patients with LHON, and important open questions that MRI can help address about LHON disease mechanisms and prognoses, including how downstream visual brain regions are affected by the disease and treatment and why, and how scope for neural plasticity in these pathways may limit or facilitate recovery.
    Keywords:  DTI; LHON; MRI; Optic neuropathies; Optic pathways
    DOI:  https://doi.org/10.1016/j.nicl.2022.103240
  38. N Engl J Med. 2022 Dec 14.
    Deep Intronic FGF14 GAA Repeat Expansion in Late-Onset Cerebellar Ataxia.
    David Pellerin, Matt C Danzi, Carlo Wilke, Mathilde Renaud, Sarah Fazal, Marie-Josée Dicaire, Carolin K Scriba, Catherine Ashton, Christopher Yanick, Danique Beijer, Adriana Rebelo, Clarissa Rocca, Zane Jaunmuktane, Joshua A Sonnen, Roxanne Larivière, David Genís, Laura Molina Porcel, Karine Choquet, Rawan Sakalla, Sylvie Provost, Rebecca Robertson, Xavier Allard-Chamard, Martine Tétreault, Sarah J Reiling, Sara Nagy, Vikas Nishadham, Meera Purushottam, Seena Vengalil, Mainak Bardhan, Atchayaram Nalini, Zhongbo Chen, Jean Mathieu, Rami Massie, Colin H Chalk, Anne-Louise Lafontaine, François Evoy, Marie-France Rioux, Jiannis Ragoussis, Kym M Boycott, Marie-Pierre Dubé, Antoine Duquette, Henry Houlden, Gianina Ravenscroft, Nigel G Laing, Phillipa J Lamont, Mario A Saporta, Rebecca Schüle, Ludger Schöls, Roberta La Piana, Matthis Synofzik, Stephan Zuchner, Bernard Brais.
      BACKGROUND: The late-onset cerebellar ataxias (LOCAs) have largely resisted molecular diagnosis.METHODS: We sequenced the genomes of six persons with autosomal dominant LOCA who were members of three French Canadian families and identified a candidate pathogenic repeat expansion. We then tested for association between the repeat expansion and disease in two independent case-control series - one French Canadian (66 patients and 209 controls) and the other German (228 patients and 199 controls). We also genotyped the repeat in 20 Australian and 31 Indian index patients. We assayed gene and protein expression in two postmortem cerebellum specimens and two induced pluripotent stem-cell (iPSC)-derived motor-neuron cell lines.
    RESULTS: In the six French Canadian patients, we identified a GAA repeat expansion deep in the first intron of FGF14, which encodes fibroblast growth factor 14. Cosegregation of the repeat expansion with disease in the families supported a pathogenic threshold of at least 250 GAA repeats ([GAA]≥250). There was significant association between FGF14 (GAA)≥250 expansions and LOCA in the French Canadian series (odds ratio, 105.60; 95% confidence interval [CI], 31.09 to 334.20; P<0.001) and in the German series (odds ratio, 8.76; 95% CI, 3.45 to 20.84; P<0.001). The repeat expansion was present in 61%, 18%, 15%, and 10% of French Canadian, German, Australian, and Indian index patients, respectively. In total, we identified 128 patients with LOCA who carried an FGF14 (GAA)≥250 expansion. Postmortem cerebellum specimens and iPSC-derived motor neurons from patients showed reduced expression of FGF14 RNA and protein.
    CONCLUSIONS: A dominantly inherited deep intronic GAA repeat expansion in FGF14 was found to be associated with LOCA. (Funded by Fondation Groupe Monaco and others.).
    DOI:  https://doi.org/10.1056/NEJMoa2207406
  39. NPJ Parkinsons Dis. 2022 Dec 16. 8(1): 172
    Identification and prediction of Parkinson's disease subtypes and progression using machine learning in two cohorts.
    Anant Dadu, Vipul Satone, Rachneet Kaur, Sayed Hadi Hashemi, Hampton Leonard, Hirotaka Iwaki, Mary B Makarious, Kimberley J Billingsley, Sara Bandres-Ciga, Lana J Sargent, Alastair J Noyce, Ali Daneshmand, Cornelis Blauwendraat, Ken Marek, Sonja W Scholz, Andrew B Singleton, Mike A Nalls, Roy H Campbell, Faraz Faghri.
      The clinical manifestations of Parkinson's disease (PD) are characterized by heterogeneity in age at onset, disease duration, rate of progression, and the constellation of motor versus non-motor features. There is an unmet need for the characterization of distinct disease subtypes as well as improved, individualized predictions of the disease course. We used unsupervised and supervised machine learning methods on comprehensive, longitudinal clinical data from the Parkinson's Disease Progression Marker Initiative (n = 294 cases) to identify patient subtypes and to predict disease progression. The resulting models were validated in an independent, clinically well-characterized cohort from the Parkinson's Disease Biomarker Program (n = 263 cases). Our analysis distinguished three distinct disease subtypes with highly predictable progression rates, corresponding to slow, moderate, and fast disease progression. We achieved highly accurate projections of disease progression 5 years after initial diagnosis with an average area under the curve (AUC) of 0.92 (95% CI: 0.95 ± 0.01) for the slower progressing group (PDvec1), 0.87 ± 0.03 for moderate progressors, and 0.95 ± 0.02 for the fast-progressing group (PDvec3). We identified serum neurofilament light as a significant indicator of fast disease progression among other key biomarkers of interest. We replicated these findings in an independent cohort, released the analytical code, and developed models in an open science manner. Our data-driven study provides insights to deconstruct PD heterogeneity. This approach could have immediate implications for clinical trials by improving the detection of significant clinical outcomes. We anticipate that machine learning models will improve patient counseling, clinical trial design, and ultimately individualized patient care.
    DOI:  https://doi.org/10.1038/s41531-022-00439-z
  40. Nat Rev Genet. 2022 Dec 16.
    Regulation of pre-mRNA splicing: roles in physiology and disease, and therapeutic prospects.
    Malgorzata Ewa Rogalska, Claudia Vivori, Juan Valcárcel.
      The removal of introns from mRNA precursors and its regulation by alternative splicing are key for eukaryotic gene expression and cellular function, as evidenced by the numerous pathologies induced or modified by splicing alterations. Major recent advances have been made in understanding the structures and functions of the splicing machinery, in the description and classification of physiological and pathological isoforms and in the development of the first therapies for genetic diseases based on modulation of splicing. Here, we review this progress and discuss important remaining challenges, including predicting splice sites from genomic sequences, understanding the variety of molecular mechanisms and logic of splicing regulation, and harnessing this knowledge for probing gene function and disease aetiology and for the design of novel therapeutic approaches.
    DOI:  https://doi.org/10.1038/s41576-022-00556-8
  41. Nat Struct Mol Biol. 2022 Dec;29(12): 1196-1207
    Structural basis for Parkinson's disease-linked LRRK2's binding to microtubules.
    David M Snead, Mariusz Matyszewski, Andrea M Dickey, Yu Xuan Lin, Andres E Leschziner, Samara L Reck-Peterson.
      Leucine-rich repeat kinase 2 (LRRK2) is one of the most commonly mutated genes in familial Parkinson's disease (PD). Under some circumstances, LRRK2 co-localizes with microtubules in cells, an association enhanced by PD mutations. We report a cryo-EM structure of the catalytic half of LRRK2, containing its kinase, in a closed conformation, and GTPase domains, bound to microtubules. We also report a structure of the catalytic half of LRRK1, which is closely related to LRRK2 but is not linked to PD. Although LRRK1's structure is similar to that of LRRK2, we find that LRRK1 does not interact with microtubules. Guided by these structures, we identify amino acids in LRRK2's GTPase that mediate microtubule binding; mutating them disrupts microtubule binding in vitro and in cells, without affecting LRRK2's kinase activity. Our results have implications for the design of therapeutic LRRK2 kinase inhibitors.
    DOI:  https://doi.org/10.1038/s41594-022-00863-y
  42. Angew Chem Int Ed Engl. 2022 Dec 16.
    Third Generation Sequencing of Epigenetic DNA.
    Bethany Searle, Markus Müller, Thomas Carell, Andrew Kellett.
      The discovery of epigenetic bases has revolutionised the understanding of disease and development. Among the most studied epigenetic marks are cytosines covalently modified at the 5 position. In order to gain insight into their biological significance, the ability to determine their spatiotemporal distribution within the genome is essential. Techniques for sequencing on 'Next Generation' platforms often involve harsh chemical treatments leading to sample degradation. Third generation sequencing promises to further revolutionise the field by providing long reads, enabling coverage of highly repetitive regions of the genome or structural variants considered unmappable by next generation sequencing technology. While the ability of third generation platforms to directly detect epigenetic modifications is continuously improving, at present chemical or enzymatic derivatisation presents the most convenient means of enhancing reliability. This review presents techniques available for the detection of cytosine modifications on third generation platforms.
    Keywords:  epigenetics * DNA methylation * nanopore * sequence determination * SMRT
    DOI:  https://doi.org/10.1002/anie.202215704
  43. Sci Rep. 2022 Dec 14. 12(1): 21634
    An increase in mitochondrial TOM activates apoptosis to drive retinal neurodegeneration.
    Agalya Periasamy, Naomi Mitchell, Olga Zaytseva, Arjun S Chahal, Jiamin Zhao, Peter M Colman, Leonie M Quinn, Jacqueline M Gulbis.
      Intronic polymorphic TOMM40 variants increasing TOMM40 mRNA expression are strongly correlated to late onset Alzheimer's Disease. The gene product, hTomm40, encoded in the APOE gene cluster, is a core component of TOM, the translocase that imports nascent proteins across the mitochondrial outer membrane. We used Drosophila melanogaster eyes as an in vivo model to investigate the relationship between elevated Tom40 (the Drosophila homologue of hTomm40) expression and neurodegeneration. Here we provide evidence that an overabundance of Tom40 in mitochondria invokes caspase-dependent cell death in a dose-dependent manner, leading to degeneration of the primarily neuronal eye tissue. Degeneration is contingent on the availability of co-assembling TOM components, indicating that an increase in assembled TOM is the factor that triggers apoptosis and degeneration in a neural setting. Eye death is not contingent on inner membrane translocase components, suggesting it is unlikely to be a direct consequence of impaired import. Another effect of heightened Tom40 expression is upregulation and co-association of a mitochondrial oxidative stress biomarker, DmHsp22, implicated in extension of lifespan, providing new insight into the balance between cell survival and death. Activation of regulated death pathways, culminating in eye degeneration, suggests a possible causal route from TOMM40 polymorphisms to neurodegenerative disease.
    DOI:  https://doi.org/10.1038/s41598-022-23280-z
  44. Science. 2022 Dec 16. 378(6625): eabq5209
    Endosomal lipid signaling reshapes the endoplasmic reticulum to control mitochondrial function.
    Wonyul Jang, Dmytro Puchkov, Paula Samsó, YongTian Liang, Michal Nadler-Holly, Stephan J Sigrist, Ulrich Kintscher, Fan Liu, Kamel Mamchaoui, Vincent Mouly, Volker Haucke.
      Cells respond to fluctuating nutrient supply by adaptive changes in organelle dynamics and in metabolism. How such changes are orchestrated on a cell-wide scale is unknown. We show that endosomal signaling lipid turnover by MTM1, a phosphatidylinositol 3-phosphate [PI(3)P] 3-phosphatase mutated in X-linked centronuclear myopathy in humans, controls mitochondrial morphology and function by reshaping the endoplasmic reticulum (ER). Starvation-induced endosomal recruitment of MTM1 impairs PI(3)P-dependent contact formation between tubular ER membranes and early endosomes, resulting in the conversion of ER tubules into sheets, the inhibition of mitochondrial fission, and sustained oxidative metabolism. Our results unravel an important role for early endosomal lipid signaling in controlling ER shape and, thereby, mitochondrial form and function to enable cells to adapt to fluctuating nutrient environments.
    DOI:  https://doi.org/10.1126/science.abq5209
  45. Int J Mol Sci. 2022 Nov 25. pii: 14744. [Epub ahead of print]23(23):
    Proteomics in Inherited Metabolic Disorders.
    Maria Del Pilar Chantada-Vázquez, Susana B Bravo, Sofía Barbosa-Gouveia, José V Alvarez, María L Couce.
      Inherited metabolic disorders (IMD) are rare medical conditions caused by genetic defects that interfere with the body's metabolism. The clinical phenotype is highly variable and can present at any age, although it more often manifests in childhood. The number of treatable IMDs has increased in recent years, making early diagnosis and a better understanding of the natural history of the disease more important than ever. In this review, we discuss the main challenges faced in applying proteomics to the study of IMDs, and the key advances achieved in this field using tandem mass spectrometry (MS/MS). This technology enables the analysis of large numbers of proteins in different body fluids (serum, plasma, urine, saliva, tears) with a single analysis of each sample, and can even be applied to dried samples. MS/MS has thus emerged as the tool of choice for proteome characterization and has provided new insights into many diseases and biological systems. In the last 10 years, sequential window acquisition of all theoretical fragmentation spectra mass spectrometry (SWATH-MS) has emerged as an accurate, high-resolution technique for the identification and quantification of proteins differentially expressed between healthy controls and IMD patients. Proteomics is a particularly promising approach to help obtain more information on rare genetic diseases, including identification of biomarkers to aid early diagnosis and better understanding of the underlying pathophysiology to guide the development of new therapies. Here, we summarize new and emerging proteomic technologies and discuss current uses and limitations of this approach to identify and quantify proteins. Moreover, we describe the use of proteomics to identify the mechanisms regulating complex IMD phenotypes; an area of research essential to better understand these rare disorders and many other human diseases.
    Keywords:  biomarkers; enzyme replacement therapy; inborn errors of metabolism; lysosomal disorders; proteomics
    DOI:  https://doi.org/10.3390/ijms232314744
  46. Cell Chem Biol. 2022 Dec 01. pii: S2451-9456(22)00415-9. [Epub ahead of print]
    Metabolic modulation of transcription: The role of one-carbon metabolism.
    Jung-Ming G Lin, Savvas Kourtis, Ritobrata Ghose, Natalia Pardo Lorente, Stefan Kubicek, Sara Sdelci.
      While it is well known that expression levels of metabolic enzymes regulate the metabolic state of the cell, there is mounting evidence that the converse is also true, that metabolite levels themselves can modulate gene expression via epigenetic modifications and transcriptional regulation. Here we focus on the one-carbon metabolic pathway, which provides the essential building blocks of many classes of biomolecules, including purine nucleotides, thymidylate, serine, and methionine. We review the epigenetic roles of one-carbon metabolic enzymes and their associated metabolites and introduce an interactive computational resource that places enzyme essentiality in the context of metabolic pathway topology. Therefore, we briefly discuss examples of metabolic condensates and higher-order complexes of metabolic enzymes downstream of one-carbon metabolism. We speculate that they may be required to the formation of transcriptional condensates and gene expression control. Finally, we discuss new ways to exploit metabolic pathway compartmentalization to selectively target these enzymes in cancer.
    Keywords:  cancer; chromatin; epigenetics; folate metabolism; metabolic condensates; nuclear condensates; nuclear metabolism; nucleotides; one-carbon metabolism; phase separation; purinergic signaling; transcription regulation; transcriptional condensates
    DOI:  https://doi.org/10.1016/j.chembiol.2022.11.009
  47. Nat Protoc. 2022 Dec 16.
    Proteome-wide structural changes measured with limited proteolysis-mass spectrometry: an advanced protocol for high-throughput applications.
    Liliana Malinovska, Valentina Cappelletti, Devon Kohler, Ilaria Piazza, Tsung-Heng Tsai, Monika Pepelnjak, Patrick Stalder, Christian Dörig, Fabian Sesterhenn, Franziska Elsässer, Lucie Kralickova, Nigel Beaton, Lukas Reiter, Natalie de Souza, Olga Vitek, Paola Picotti.
      Proteins regulate biological processes by changing their structure or abundance to accomplish a specific function. In response to a perturbation, protein structure may be altered by various molecular events, such as post-translational modifications, protein-protein interactions, aggregation, allostery or binding to other molecules. The ability to probe these structural changes in thousands of proteins simultaneously in cells or tissues can provide valuable information about the functional state of biological processes and pathways. Here, we present an updated protocol for LiP-MS, a proteomics technique combining limited proteolysis with mass spectrometry, to detect protein structural alterations in complex backgrounds and on a proteome-wide scale. In LiP-MS, proteins undergo a brief proteolysis in native conditions followed by complete digestion in denaturing conditions, to generate structurally informative proteolytic fragments that are analyzed by mass spectrometry. We describe advances in the throughput and robustness of the LiP-MS workflow and implementation of data-independent acquisition-based mass spectrometry, which together achieve high reproducibility and sensitivity, even on large sample sizes. We introduce MSstatsLiP, an R package dedicated to the analysis of LiP-MS data for the identification of structurally altered peptides and differentially abundant proteins. The experimental procedures take 3 d, mass spectrometric measurement time and data processing depend on sample number and statistical analysis typically requires ~1 d. These improvements expand the adaptability of LiP-MS and enable wide use in functional proteomics and translational applications.
    DOI:  https://doi.org/10.1038/s41596-022-00771-x
  48. STAR Protoc. 2022 Dec 13. pii: S2666-1667(22)00810-3. [Epub ahead of print]4(1): 101930
    Streamlined high-throughput cloning protocol to generate arrayed mutant libraries.
    Kerry T Sun, Tark S Patel, Justin Kim, Helen S H Tang, Ghazaleh Eskandari-Sedighi, Haresh Sureshkumar, Dean Schieve, S A Mok.
      Large-scale, site-directed mutagenesis enables rapid characterization of the biochemical and biological properties of proteins. Here, we present a cost-effective and adaptable cloning pipeline to generate arrayed gene libraries for a construct of interest. We detail steps to use an open access web app to automate the design of mutagenesis primers optimized for our cloning protocols in a 96-well plate format. The protocol allows most molecular biology labs to clone 96 mutants (from PCR to sequence ready plasmid) in 3 days.
    Keywords:  Genetics; High Throughput Screening; Molecular Biology; Sequencing
    DOI:  https://doi.org/10.1016/j.xpro.2022.101930
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