bims-mitdyn 2021-01-24 papers

bims-mitdyn

Biomed News

on Mitochondrial dynamics: mechanisms

Issue of 2021‒01‒24
twenty papers selected by
Edmond Chan
Queen’s University, School of Medicine

Emr1 regulates the number of foci of the endoplasmic reticulum-mitochondria encounter structure complex.
UPRmt scales mitochondrial network expansion with protein synthesis via mitochondrial import in Caenorhabditis elegans.
MOTS-c is an exercise-induced mitochondrial-encoded regulator of age-dependent physical decline and muscle homeostasis.
BNIP3L/NIX-mediated mitophagy protects against glucocorticoid-induced synapse defects.
The TIM22 complex mediates the import of Sideroflexins and is required for efficient mitochondrial one-carbon metabolism.
Proteomic and metabolomic advances uncover biomarkers of mitochondrial disease pathophysiology and severity.
Impaired complex I repair causes recessive Leber's hereditary optic neuropathy.
Circulating markers of NADH-reductive stress correlate with mitochondrial disease severity.
BNIP3-dependent mitophagy promotes cytosolic localization of LC3B and metabolic homeostasis in the liver.
Autophagy restricts mitochondrial DNA damage-induced release of ENDOG (endonuclease G) to regulate genome stability.
Atg43, a novel autophagy-related protein, serves as a mitophagy receptor to bridge mitochondria with phagophores in fission yeast.
Visualizing, quantifying, and manipulating mitochondrial DNA in vivo.
AggreCount: an unbiased image analysis tool for identifying and quantifying cellular aggregates in a spatially defined manner.
Structure, mechanism, and regulation of mitochondrial DNA transcription initiation.
Nrf2 is activated by disruption of mitochondrial thiol homeostasis but not by enhanced mitochondrial superoxide production.
Inhibition of mitochondrial oxidative metabolism attenuates EMCV replication and protects β-cells from virally mediated lysis.
PDE5 inhibition rescues mitochondrial dysfunction and angiogenic responses induced by Akt3 inhibition by promotion of PRC expression.
ERAD deficiency promotes mitochondrial dysfunction and transcriptional rewiring in human hepatic cells.
Stop codon read-through of mammalian MTCH2 leading to an unstable isoform regulates mitochondrial membrane potential.
Listeria monocytogenes upregulates mitochondrial calcium signalling to inhibit LC3-associated phagocytosis as a survival strategy.

Nat Commun. 2021 Jan 22. 12(1): 521

Emr1 regulates the number of foci of the endoplasmic reticulum-mitochondria encounter structure complex.

Faiz Rasul, Fan Zheng, Fenfen Dong, Jiajia He, Ling Liu, Wenyue Liu, Javairia Yousuf Cheema, Wenfan Wei, Chuanhai Fu.

The endoplasmic reticulum-mitochondria encounter structure (ERMES) complex creates contact sites between the endoplasmic reticulum and mitochondria, playing crucial roles in interorganelle communication, mitochondrial fission, mtDNA inheritance, lipid transfer, and autophagy. The mechanism regulating the number of ERMES foci within the cell remains unclear. Here, we demonstrate that the mitochondrial membrane protein Emr1 contributes to regulating the number of ERMES foci. We show that the absence of Emr1 significantly decreases the number of ERMES foci. Moreover, we find that Emr1 interacts with the ERMES core component Mdm12 and colocalizes with Mdm12 on mitochondria. Similar to ERMES mutant cells, cells lacking Emr1 display defective mitochondrial morphology and impaired mitochondrial segregation, which can be rescued by an artificial tether capable of linking the endoplasmic reticulum and mitochondria. We further demonstrate that the cytoplasmic region of Emr1 is required for regulating the number of ERMES foci. This work thus reveals a crucial regulatory protein necessary for ERMES functions and provides mechanistic insights into understanding the dynamic regulation of endoplasmic reticulum-mitochondria communication.

DOI: https://doi.org/10.1038/s41467-020-20866-x
Nat Commun. 2021 01 20. 12(1): 479

UPRmt scales mitochondrial network expansion with protein synthesis via mitochondrial import in Caenorhabditis elegans.

Tomer Shpilka, YunGuang Du, Qiyuan Yang, Andrew Melber, Nandhitha Uma Naresh, Joshua Lavelle, Sookyung Kim, Pengpeng Liu, Hilla Weidberg, Rui Li, Jun Yu, Lihua Julie Zhu, Lara Strittmatter, Cole M Haynes.

As organisms develop, individual cells generate mitochondria to fulfill physiological requirements. However, it remains unknown how mitochondrial network expansion is scaled to cell growth. The mitochondrial unfolded protein response (UPRmt) is a signaling pathway mediated by the transcription factor ATFS-1 which harbors a mitochondrial targeting sequence (MTS). Here, using the model organism Caenorhabditis elegans we demonstrate that ATFS-1 mediates an adaptable mitochondrial network expansion program that is active throughout normal development. Mitochondrial network expansion requires the relatively inefficient MTS in ATFS-1, which allows the transcription factor to be responsive to parameters that impact protein import capacity of the mitochondrial network. Increasing the strength of the ATFS-1 MTS impairs UPRmt activity by increasing accumulation within mitochondria. Manipulations of TORC1 activity increase or decrease ATFS-1 activity in a manner that correlates with protein synthesis. Lastly, expression of mitochondrial-targeted GFP is sufficient to expand the muscle cell mitochondrial network in an ATFS-1-dependent manner. We propose that mitochondrial network expansion during development is an emergent property of the synthesis of highly expressed mitochondrial proteins that exclude ATFS-1 from mitochondrial import, causing UPRmt activation.

DOI: https://doi.org/10.1038/s41467-020-20784-y
Nat Commun. 2021 01 20. 12(1): 470

MOTS-c is an exercise-induced mitochondrial-encoded regulator of age-dependent physical decline and muscle homeostasis.

Joseph C Reynolds, Rochelle W Lai, Jonathan S T Woodhead, James H Joly, Cameron J Mitchell, David Cameron-Smith, Ryan Lu, Pinchas Cohen, Nicholas A Graham, Bérénice A Benayoun, Troy L Merry, Changhan Lee.

Healthy aging can be promoted by enhanced metabolic fitness and physical capacity. Mitochondria are chief metabolic organelles with strong implications in aging that also coordinate broad physiological functions, in part, using peptides that are encoded within their independent genome. However, mitochondrial-encoded factors that actively regulate aging are unknown. Here, we report that mitochondrial-encoded MOTS-c can significantly enhance physical performance in young (2 mo.), middle-age (12 mo.), and old (22 mo.) mice. MOTS-c can regulate (i) nuclear genes, including those related to metabolism and proteostasis, (ii) skeletal muscle metabolism, and (iii) myoblast adaptation to metabolic stress. We provide evidence that late-life (23.5 mo.) initiated intermittent MOTS-c treatment (3x/week) can increase physical capacity and healthspan in mice. In humans, exercise induces endogenous MOTS-c expression in skeletal muscle and in circulation. Our data indicate that aging is regulated by genes encoded in both of our co-evolved mitochondrial and nuclear genomes.

DOI: https://doi.org/10.1038/s41467-020-20790-0
Nat Commun. 2021 01 20. 12(1): 487

BNIP3L/NIX-mediated mitophagy protects against glucocorticoid-induced synapse defects.

Gee Euhn Choi, Hyun Jik Lee, Chang Woo Chae, Ji Hyeon Cho, Young Hyun Jung, Jun Sung Kim, Seo Yihl Kim, Jae Ryong Lim, Ho Jae Han.

Stress-induced glucocorticoids disturb mitochondrial bioenergetics and dynamics; however, instead of being removed via mitophagy, the damaged mitochondria accumulate. Therefore, we investigate the role of glucocorticoids in mitophagy inhibition and subsequent synaptic defects in hippocampal neurons, SH-SY5Y cells, and ICR mice. First, we observe that glucocorticoids decrease both synaptic density and vesicle recycling due to suppressed mitophagy. Screening data reveal that glucocorticoids downregulate BNIP3-like (BNIP3L)/NIX, resulting in the reduced mitochondrial respiration function and synaptic density. Notably, we find that glucocorticoids direct the glucocorticoid receptor to bind directly to the PGC1α promoter, downregulating its expression and nuclear translocation. PGC1α downregulation selectively decreases NIX-dependent mitophagy. Consistent with these results, NIX enhancer pre-treatment of a corticosterone-exposed mouse elevates mitophagy and synaptic density in hippocampus, improving the outcome of a spatial memory task. In conclusion, glucocorticoids inhibit mitophagy via downregulating NIX and that NIX activation represents a potential target for restoring synapse function.

DOI: https://doi.org/10.1038/s41467-020-20679-y
Mol Biol Cell. 2021 Jan 21. mbcE20060390

The TIM22 complex mediates the import of Sideroflexins and is required for efficient mitochondrial one-carbon metabolism.

Thomas D Jackson, Daniella H Hock, Kenji M Fujihara, Catherine S Palmer, Ann E Frazier, Yau C Low, Yilin Kang, Ching-Seng Ang, Nicholas J Clemons, David R Thorburn, David A Stroud, Diana Stojanovski.

Acylglycerol Kinase (AGK) is a mitochondrial lipid kinase that contributes to protein biogenesis as a subunit of the TIM22 complex at the inner mitochondrial membrane. Mutations in AGK cause Sengers syndrome, an autosomal recessive condition characterized by congenital cataracts, hypertrophic cardiomyopathy, skeletal myopathy and lactic acidosis. We mapped the proteomic changes in Sengers patient fibroblasts and AGKKO cell lines to understand the effects of AGK dysfunction on mitochondria. This uncovered downregulation of a number of proteins at the inner mitochondrial membrane, including many SLC25 carrier family proteins, which are predicted substrates of the complex. We also observed downregulation of SFXN proteins, which contain five transmembrane domains, and show that they represent a novel class of TIM22 complex substrate. Perturbed biogenesis of SFXN proteins in cells lacking AGK reduces the proliferative capabilities of these cells in the absence of exogenous serine, suggesting that dysregulation of one carbon metabolism is a molecular feature in the biology of Sengers syndrome.

DOI: https://doi.org/10.1091/mbc.E20-06-0390
J Clin Invest. 2021 Jan 19. pii: 145158. [Epub ahead of print]131(2):

Proteomic and metabolomic advances uncover biomarkers of mitochondrial disease pathophysiology and severity.

Marjan Gucek, Michael N Sack.

Advancing proteomic and metabolomic technologies that integrate curated omic databases have crossed a threshold to enable their clinical utility. In this issue of the JCI, Sharma et al. exploit emerging technologies to evaluate whether biomarkers identified in the mitochondrial encephalomyopathy lactic acidosis and stroke-like episodes (MELAS) syndrome could refine disease characterization, uncover pathways to monitor therapeutic efficacy, and/or delineate disease-modifying targets. The authors analyzed blood and urine samples from patients with this genetic mitochondrial disease and elucidated proteins and metabolites related to NADH-reductive stress. These circulating biomarkers have intriguing clinical potential that implicate disease pathophysiology and may prove important biomarkers for the future management of MELAS.

DOI: https://doi.org/10.1172/JCI145158
J Clin Invest. 2021 Jan 19. pii: 138267. [Epub ahead of print]

Impaired complex I repair causes recessive Leber's hereditary optic neuropathy.

Sarah L Stenton, Natalia L Sheremet, Claudia B Catarino, Natalia Andreeva, Zahra Assouline, Piero Barboni, Ortal Barel, Riccardo Berutti, Igor O Bychkov, Leonardo Caporali, Mariantonietta Capristo, Michele Carbonelli, Maria Lucia Cascavilla, Peter Charbel Issa, Peter Freisinger, Sylvie Gerber, Daniele Ghezzi, Elisabeth Graf, Juliana Heidler, Maja Hempel, Elise Heon, Yulia S Itkis, Elisheva Javasky, Josseline Kaplan, Robert Kopajtich, Cornelia Kornblum, Reka Kovacs-Nagy, Tatiana Krylova, Wolfram S Kunz, Chiara La Morgia, Costanza Lamperti, Christina Ludwig, Pedro F Malacarne, Alessandra Maresca, Johannes A Mayr, Jana Meisterknecht, Tatiana Nevinitsyna, Flavia Palombo, Ben Pode-Shakked, Maria S Shmelkova, Tim M Strom, Francesca Tagliavini, Michal Tzadok, Amelie T van der Ven, Catherine Vignal-Clermont, Matias Wagner, Ekaterina Zakharova, Nino Zhorzholadze, Jen-Michel Rozet, Valerio Carelli, Polina Tsygankova, Thomas Klopstock, Ilka Wittig, Holger Prokisch.

  Leber's hereditary optic neuropathy (LHON) is the most frequent mitochondrial disease and was the first to be genetically defined by a point mutation in the mitochondrial DNA (mtDNA). A molecular diagnosis is reached in up to 95%, the vast majority of which are accounted for by three mutations within mitochondrial complex I (CI) subunit encoding genes in the mtDNA (mtLHON). Here, we resolve the enigma of LHON in the absence of pathogenic mtDNA mutations. We describe biallelic mutations in a nuclear encoded gene, DNAJC30, in 33 unsolved patients from 29 families and establish an autosomal recessive mode of inheritance for LHON (arLHON), which to date has been a prime example of a maternally inherited disorder. Remarkably, all hallmarks of mtLHON are recapitulated, including incomplete penetrance, male predominance, and significant idebenone responsivity. Moreover, by tracking protein turnover in patient-derived cell lines and a DNAJC30-knock-out cellular model, we measure reduced turnover of specific CI N-module subunits and a resultant impairment of CI function. This demonstrates DNAJC30 is to be a chaperone protein needed for the efficient exchange of CI subunits exposed to reactive oxygen species and integral to a mitochondrial CI repair mechanism, thereby providing the first example of a disease resulting from impaired exchange of assembled respiratory chain subunits.

Keywords:  Genetic diseases; Genetics; Neuroscience

DOI:  https://doi.org/10.1172/JCI138267
J Clin Invest. 2021 01 19. pii: 136055. [Epub ahead of print]131(2):

Circulating markers of NADH-reductive stress correlate with mitochondrial disease severity.

Rohit Sharma, Bryn Reinstadler, Kristin Engelstad, Owen S Skinner, Erin Stackowitz, Ronald G Haller, Clary B Clish, Kerry Pierce, Melissa A Walker, Robert Fryer, Devin Oglesbee, Xiangling Mao, Dikoma C Shungu, Ashok Khatri, Michio Hirano, Darryl C De Vivo, Vamsi K Mootha.

  Mitochondrial disorders represent a large collection of rare syndromes that are difficult to manage both because we do not fully understand biochemical pathogenesis and because we currently lack facile markers of severity. The m.3243A>G variant is the most common heteroplasmic mitochondrial DNA mutation and underlies a spectrum of diseases, notably mitochondrial encephalomyopathy lactic acidosis and stroke-like episodes (MELAS). To identify robust circulating markers of m.3243A>G disease, we first performed discovery proteomics, targeted metabolomics, and untargeted metabolomics on plasma from a deeply phenotyped cohort (102 patients, 32 controls). In a validation phase, we measured concentrations of prioritized metabolites in an independent cohort using distinct methods. We validated 20 analytes (1 protein, 19 metabolites) that distinguish patients with MELAS from controls. The collection includes classic (lactate, alanine) and more recently identified (GDF-15, α-hydroxybutyrate) mitochondrial markers. By mining untargeted mass-spectra we uncovered 3 less well-studied metabolite families: N-lactoyl-amino acids, β-hydroxy acylcarnitines, and β-hydroxy fatty acids. Many of these 20 analytes correlate strongly with established measures of severity, including Karnofsky status, and mechanistically, nearly all markers are attributable to an elevated NADH/NAD+ ratio, or NADH-reductive stress. Our work defines a panel of organelle function tests related to NADH-reductive stress that should enable classification and monitoring of mitochondrial disease.

Keywords:  Genetics; Intermediary metabolism; Metabolism; Mitochondria; Monogenic diseases; RET; HS6ST1; sE-selectin; integrated stress response; creatine; pyruvate; 2-hydroxybutyrate; alpha-hydroxybutyrate; lactoyl-amino acids; hydroxy-fatty acids; hydroxy-acylcarnitines

DOI:  https://doi.org/10.1172/JCI136055
Autophagy. 2021 Jan 17.

BNIP3-dependent mitophagy promotes cytosolic localization of LC3B and metabolic homeostasis in the liver.

Maya Z Springer, Logan P Poole, Lauren E Drake, Althea Bock-Hughes, Michelle L Boland, Alexandra G Smith, John Hart, Aparajita H Chourasia, Ivan Liu, Grazyna Bozek, Kay F Macleod.

  Mitophagy formed the basis of the original description of autophagy by Christian de Duve when he demonstrated that GCG (glucagon) induced macroautophagic/autophagic turnover of mitochondria in the liver. However, the molecular basis of liver-specific activation of mitophagy by GCG, or its significance for metabolic stress responses in the liver is not understood. Here we show that BNIP3 is required for GCG-induced mitophagy in the liver through interaction with processed LC3B; an interaction that is also necessary to localize LC3B out of the nucleus to cytosolic mitophagosomes in response to nutrient deprivation. Loss of BNIP3-dependent mitophagy caused excess mitochondria to accumulate in the liver, disrupting metabolic zonation within the liver parenchyma, with expansion of zone 1 metabolism at the expense of zone 3 metabolism. These results identify BNIP3 as a regulator of metabolic homeostasis in the liver through its effect on mitophagy and mitochondrial mass distribution.

Keywords:  BNIP3; LC3B; glucagon; hepatocyte; liver zonation; mitophagy; nutrient deprivation

DOI:  https://doi.org/10.1080/15548627.2021.1877469
Autophagy. 2021 Jan 19. 1-17

Autophagy restricts mitochondrial DNA damage-induced release of ENDOG (endonuclease G) to regulate genome stability.

Tung Chao, Hsueh-Tzu Shih, Shih-Chin Hsu, Pei-Jer Chen, Yu-Shan Fan, Yung-Ming Jeng, Zhao-Qing Shen, Ting-Fen Tsai, Zee-Fen Chang.

  Genotoxic insult causes nuclear and mitochondrial DNA damages with macroautophagy/autophagy induction. The role of mitochondrial DNA (mtDNA) damage in the requirement of autophagy for nuclear DNA (nDNA) stability is unclear. Using site-specific DNA damage approaches, we show that specific nDNA damage alone does not require autophagy for repair unless in the presence of mtDNA damage. We provide evidence that after IR exposure-induced mtDNA and nDNA damages, autophagy suppression causes non-apoptotic mitochondrial permeability, by which mitochondrial ENDOG (endonuclease G) is released and translocated to nuclei to sustain nDNA damage in a TET (tet methylcytosine dioxygenase)-dependent manner. Furthermore, blocking lysosome function is sufficient to increase the amount of mtDNA leakage to the cytosol, accompanied by ENDOG-free mitochondrial puncta formation with concurrent ENDOG nuclear accumulation. We proposed that autophagy eliminates the mitochondria specified by mtDNA damage-driven mitochondrial permeability to prevent ENDOG-mediated genome instability. Finally, we showed that HBx, a hepatitis B viral protein capable of suppressing autophagy, also causes mitochondrial permeability-dependent ENDOG mis-localization in nuclei and is linked to hepatitis B virus (HBV)-mediated hepatocellular carcinoma development. Abbreviations: 3-MA: 3-methyladenine; 5-hmC: 5-hydroxymethylcytosine; ACTB: actin beta; ATG5: autophagy related 5; ATM: ATM serine/threonine kinase; DFFB/CAD: DNA fragmentation factor subunit beta; cmtDNA: cytosolic mitochondrial DNA; ConA: concanamycin A; CQ: chloroquine; CsA: cyclosporin A; Dox: doxycycline; DSB: double-strand break; ENDOG: endonuclease G; GFP: green fluorescent protein; Gy: gray; H2AX: H2A.X variant histone; HBV: hepatitis B virus; HBx: hepatitis B virus X protein; HCC: hepatocellular carcinoma; I-PpoI: intron-encoded endonuclease; IR: ionizing radiation; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MOMP: mitochondrial outer membrane permeability; mPTP: mitochondrial permeability transition pore; mtDNA: mitochondrial DNA; nDNA: nuclear DNA; 4-OHT: 4-hydroxytamoxifen; rDNA: ribosomal DNA; ROS: reactive oxygen species; SQSTM1/p62: sequestosome 1; TET: tet methylcytosine dioxygenase; TFAM: transcription factor A, mitochondrial; TOMM20: translocase of outer mitochondrial membrane 20; VDAC: voltage dependent anion channel.

Keywords:  Autophagy; TET; endonuclease G; genome instability; mitochondrial DNA; mitochondrial permeability

DOI:  https://doi.org/10.1080/15548627.2021.1874209
Autophagy. 2021 Jan 21. 1-2

Atg43, a novel autophagy-related protein, serves as a mitophagy receptor to bridge mitochondria with phagophores in fission yeast.

Tomoyuki Fukuda, Tomotake Kanki.

  Mitophagy is a selective type of autophagy in which damaged or unnecessary mitochondria are sequestered by double-membranous structures called phagophores and delivered to vacuoles/lysosomes for degradation. The molecular mechanisms underlying mitophagy have been studied extensively in budding yeast and mammalian cells. To gain more diverse insights, our recent study identified Atg43 as a mitophagy receptor in the fission yeast Schizosaccharomyces pombe. Atg43 is localized on the mitochondrial outer membrane through the Mim1-Mim2 complex and binds to Atg8, a ubiquitin-like protein conjugated to phagophore membranes. Artificial tethering of Atg8 to mitochondria can bypass the requirement of Atg43 for mitophagy, suggesting that the main role of Atg43 in mitophagy is to stabilize phagophore expansion on mitochondria by interacting with Atg8. Atg43 shares no sequence similarity with mitophagy receptors in other organisms and has a mitophagy-independent function, raising the possibility that Atg43 has acquired the mitophagic function by convergent evolution.

Keywords:  Atg43; Atg8; MIM complex; autophagy; mitochondria; mitophagy; selective autophagy; yeast

DOI:  https://doi.org/10.1080/15548627.2021.1874662
J Biol Chem. 2020 Dec 18. pii: S0021-9258(17)50642-0. [Epub ahead of print]295(51): 17588-17601

Visualizing, quantifying, and manipulating mitochondrial DNA in vivo.

David L Prole, Patrick F Chinnery, Nick S Jones.

  Mitochondrial DNA (mtDNA) encodes proteins and RNAs that support the functions of mitochondria and thereby numerous physiological processes. Mutations of mtDNA can cause mitochondrial diseases and are implicated in aging. The mtDNA within cells is organized into nucleoids within the mitochondrial matrix, but how mtDNA nucleoids are formed and regulated within cells remains incompletely resolved. Visualization of mtDNA within cells is a powerful means by which mechanistic insight can be gained. Manipulation of the amount and sequence of mtDNA within cells is important experimentally and for developing therapeutic interventions to treat mitochondrial disease. This review details recent developments and opportunities for improvements in the experimental tools and techniques that can be used to visualize, quantify, and manipulate the properties of mtDNA within cells.

Keywords:  aging; gene editing; microscopy; mitochondria; mitochondrial DNA (mtDNA); mitochondrial disease; mitophagy

DOI:  https://doi.org/10.1074/jbc.REV120.015101
J Biol Chem. 2020 Dec 18. pii: S0021-9258(17)50648-1. [Epub ahead of print]295(51): 17672-17683

AggreCount: an unbiased image analysis tool for identifying and quantifying cellular aggregates in a spatially defined manner.

Jacob Aaron Klickstein, Sirisha Mukkavalli, Malavika Raman.

  Protein quality control is maintained by a number of integrated cellular pathways that monitor the folding and functionality of the cellular proteome. Defects in these pathways lead to the accumulation of misfolded or faulty proteins that may become insoluble and aggregate over time. Protein aggregates significantly contribute to the development of a number of human diseases such as amyotrophic lateral sclerosis, Huntington's disease, and Alzheimer's disease. In vitro, imaging-based, cellular studies have defined key biomolecular components that recognize and clear aggregates; however, no unifying method is available to quantify cellular aggregates, limiting our ability to reproducibly and accurately quantify these structures. Here we describe an ImageJ macro called AggreCount to identify and measure protein aggregates in cells. AggreCount is designed to be intuitive, easy to use, and customizable for different types of aggregates observed in cells. Minimal experience in coding is required to utilize the script. Based on a user-defined image, AggreCount will report a number of metrics: (i) total number of cellular aggregates, (ii) percentage of cells with aggregates, (iii) aggregates per cell, (iv) area of aggregates, and (v) localization of aggregates (cytosol, perinuclear, or nuclear). A data table of aggregate information on a per cell basis, as well as a summary table, is provided for further data analysis. We demonstrate the versatility of AggreCount by analyzing a number of different cellular aggregates including aggresomes, stress granules, and inclusion bodies caused by huntingtin polyglutamine expansion.

Keywords:  Huntington disease; ImageJ; aggregate; aggregation; aggresome; amyotrophic lateral sclerosis (ALS) (Lou Gehrig disease); image-based analysis; inclusion body; microscopic imaging; misfolded protein; p97/valosin-containing protein; polyQ inclusion body; polyubiquitin chain; protein aggregation; protein misfolding p97/valosin-containing protein; protein quality control; proteostasis; stress granule; ubiquitin

DOI:  https://doi.org/10.1074/jbc.RA120.015398
J Biol Chem. 2020 Dec 25. pii: S0021-9258(17)50708-5. [Epub ahead of print]295(52): 18406-18425

Structure, mechanism, and regulation of mitochondrial DNA transcription initiation.

Urmimala Basu, Alicia M Bostwick, Kalyan Das, Kristin E Dittenhafer-Reed, Smita S Patel.

  Mitochondria are specialized compartments that produce requisite ATP to fuel cellular functions and serve as centers of metabolite processing, cellular signaling, and apoptosis. To accomplish these roles, mitochondria rely on the genetic information in their small genome (mitochondrial DNA) and the nucleus. A growing appreciation for mitochondria's role in a myriad of human diseases, including inherited genetic disorders, degenerative diseases, inflammation, and cancer, has fueled the study of biochemical mechanisms that control mitochondrial function. The mitochondrial transcriptional machinery is different from nuclear machinery. The in vitro re-constituted transcriptional complexes of Saccharomyces cerevisiae (yeast) and humans, aided with high-resolution structures and biochemical characterizations, have provided a deeper understanding of the mechanism and regulation of mitochondrial DNA transcription. In this review, we will discuss recent advances in the structure and mechanism of mitochondrial transcription initiation. We will follow up with recent discoveries and formative findings regarding the regulatory events that control mitochondrial DNA transcription, focusing on those involved in cross-talk between the mitochondria and nucleus.

Keywords:  DNA transcription; RNA polymerase; enzyme mechanism; enzyme structure; human mitochondrial RNA polymerase; mitochondria; mitochondrial DNA (mtDNA); mitochondrial DNA transcription; mitochondrial gene regulation; structure-function; transcription; transcription initiation factors; transcription regulation; yeast mitochondrial RNA polymerase

DOI:  https://doi.org/10.1074/jbc.REV120.011202
J Biol Chem. 2020 Dec 13. pii: S0021-9258(20)00163-5. [Epub ahead of print]296 100169

Nrf2 is activated by disruption of mitochondrial thiol homeostasis but not by enhanced mitochondrial superoxide production.

Filip Cvetko, Stuart T Caldwell, Maureen Higgins, Takafumi Suzuki, Masayuki Yamamoto, Hiran A Prag, Richard C Hartley, Albena T Dinkova-Kostova, Michael P Murphy.

  The transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) regulates the expression of genes involved in antioxidant defenses to modulate fundamental cellular processes such as mitochondrial function and GSH metabolism. Previous reports proposed that mitochondrial reactive oxygen species production and disruption of the GSH pool activate the Nrf2 pathway, suggesting that Nrf2 senses mitochondrial redox signals and/or oxidative damage and signals to the nucleus to respond appropriately. However, until now, it has not been possible to disentangle the overlapping effects of mitochondrial superoxide/hydrogen peroxide production as a redox signal from changes to mitochondrial thiol homeostasis on Nrf2. Recently, we developed mitochondria-targeted reagents that can independently induce mitochondrial superoxide and hydrogen peroxide production mitoParaquat (MitoPQ) or selectively disrupt mitochondrial thiol homeostasis MitoChlorodinitrobenzoic acid (MitoCDNB). Using these reagents, here we have determined how enhanced generation of mitochondrial superoxide and hydrogen peroxide or disruption of mitochondrial thiol homeostasis affects activation of the Nrf2 system in cells, which was assessed by the Nrf2 protein level, nuclear translocation, and expression of its target genes. We found that selective disruption of the mitochondrial GSH pool and inhibition of its thioredoxin system by MitoCDNB led to Nrf2 activation, whereas using MitoPQ to enhance the production of mitochondrial superoxide and hydrogen peroxide alone did not. We further showed that Nrf2 activation by MitoCDNB requires cysteine sensors of Kelch-like ECH-associated protein 1 (Keap1). These findings provide important information on how disruption to mitochondrial redox homeostasis is sensed in the cytoplasm and signaled to the nucleus.

Keywords:  MitoCDNB; MitoPQ; Nrf2; energy metabolism; reactive oxygen species (ROS); redox signaling; thiol oxidation

DOI:  https://doi.org/10.1074/jbc.RA120.016551
J Biol Chem. 2020 Dec 04. pii: S0021-9258(17)50482-2. [Epub ahead of print]295(49): 16655-16664

Inhibition of mitochondrial oxidative metabolism attenuates EMCV replication and protects β-cells from virally mediated lysis.

Joshua D Stafford, Zachary R Shaheen, Chay Teng Yeo, John A Corbett.

  Viral infection is one environmental factor that may contribute to the initiation of pancreatic β-cell destruction during the development of autoimmune diabetes. Picornaviruses, such as encephalomyocarditis virus (EMCV), induce a pro-inflammatory response in islets leading to local production of cytokines, such as IL-1, by resident islet leukocytes. Furthermore, IL-1 is known to stimulate β-cell expression of iNOS and production of the free radical nitric oxide. The purpose of this study was to determine whether nitric oxide contributes to the β-cell response to viral infection. We show that nitric oxide protects β-cells against virally mediated lysis by limiting EMCV replication. This protection requires low micromolar, or iNOS-derived, levels of nitric oxide. At these concentrations nitric oxide inhibits the Krebs enzyme aconitase and complex IV of the electron transport chain. Like nitric oxide, pharmacological inhibition of mitochondrial oxidative metabolism attenuates EMCV-mediated β-cell lysis by inhibiting viral replication. These findings provide novel evidence that cytokine signaling in β-cells functions to limit viral replication and subsequent β-cell lysis by attenuating mitochondrial oxidative metabolism in a nitric oxide-dependent manner.

Keywords:  B-cell; EMCV; autoimmune diabetes; diabetes; innate immunity; mitochondrial metabolism; nitric oxide; plus-stranded RNA virus; β-cells

DOI:  https://doi.org/10.1074/jbc.RA120.014851
J Biol Chem. 2020 Dec 25. pii: S0021-9258(17)50684-5. [Epub ahead of print]295(52): 18091-18104

PDE5 inhibition rescues mitochondrial dysfunction and angiogenic responses induced by Akt3 inhibition by promotion of PRC expression.

Daniel G Corum, Dorea P Jenkins, James A Heslop, Lacey M Tallent, Gyda C Beeson, Jeremy L Barth, Rick G Schnellmann, Robin C Muise-Helmericks.

  Akt3 regulates mitochondrial content in endothelial cells through the inhibition of PGC-1α nuclear localization and is also required for angiogenesis. However, whether there is a direct link between mitochondrial function and angiogenesis is unknown. Here we show that Akt3 depletion in primary endothelial cells results in decreased uncoupled oxygen consumption, increased fission, decreased membrane potential, and increased expression of the mitochondria-specific protein chaperones, HSP60 and HSP10, suggesting that Akt3 is required for mitochondrial homeostasis. Direct inhibition of mitochondrial homeostasis by the model oxidant paraquat results in decreased angiogenesis, showing a direct link between angiogenesis and mitochondrial function. Next, in exploring functional links to PGC-1α, the master regulator of mitochondrial biogenesis, we searched for compounds that induce this process. We found that, sildenafil, a phosphodiesterase 5 inhibitor, induced mitochondrial biogenesis as measured by increased uncoupled oxygen consumption, mitochondrial DNA content, and voltage-dependent anion channel protein expression. Sildenafil rescued the effects on mitochondria by Akt3 depletion or pharmacological inhibition and promoted angiogenesis, further supporting that mitochondrial homeostasis is required for angiogenesis. Sildenafil also induces the expression of PGC-1 family member PRC and can compensate for PGC-1α activity during mitochondrial stress by an Akt3-independent mechanism. The induction of PRC by sildenafil depends upon cAMP and the transcription factor CREB. Thus, PRC can functionally substitute during Akt3 depletion for absent PGC-1α activity to restore mitochondrial homeostasis and promote angiogenesis. These findings show that mitochondrial homeostasis as controlled by the PGC family of transcriptional activators is required for angiogenic responses.

Keywords:  Akt PKB; Akt3; Akt3/PKBγ; PGC-1α; PPARGC1A; PRC; angiogenesis; endothelial cells; mitochondria; peroxisome proliferator–activated receptor γ coactivator-1 α; phosphodiesterases

DOI:  https://doi.org/10.1074/jbc.RA120.013716
J Biol Chem. 2020 Dec 04. pii: S0021-9258(17)50489-5. [Epub ahead of print]295(49): 16743-16753

ERAD deficiency promotes mitochondrial dysfunction and transcriptional rewiring in human hepatic cells.

Qingqing Liu, Xiaoqin Yang, Guangyu Long, Yabing Hu, Zhenglong Gu, Yves R Boisclair, Qiaoming Long.

  Mitochondrial dysfunction is associated with a variety of human diseases including neurodegeneration, diabetes, nonalcohol fatty liver disease (NAFLD), and cancer, but its underlying causes are incompletely understood. Using the human hepatic cell line HepG2 as a model, we show here that endoplasmic reticulum-associated degradation (ERAD), an ER protein quality control process, is critically required for mitochondrial function in mammalian cells. Pharmacological inhibition or genetic ablation of key proteins involved in ERAD increased cell death under both basal conditions and in response to proinflammatory cytokines, a situation frequently found in NAFLD. Decreased viability of ERAD-deficient HepG2 cells was traced to impaired mitochondrial functions including reduced ATP production, enhanced reactive oxygen species (ROS) accumulation, and increased mitochondrial outer membrane permeability. Transcriptome profiling revealed widespread down-regulation of genes underpinning mitochondrial functions, and up-regulation of genes associated with tumor growth and aggression. These results highlight a critical role for ERAD in maintaining mitochondrial functional and structural integrity and raise the possibility of improving cellular and organismal mitochondrial function via enhancing cellular ERAD capacity.

Keywords:  ERAD; ROS; SEL1L; calcium; cell death; cytochrome c; endoplasmic reticulum; endoplasmic reticulum stress (ER stress); endoplasmic reticulum-associated protein degradation (ERAD); hepatocyte death; liver; mitochondria; mitochondrial disease; mitochondrial permeability transition (MPT)

DOI:  https://doi.org/10.1074/jbc.RA120.013987
J Biol Chem. 2020 Dec 11. pii: S0021-9258(17)50596-7. [Epub ahead of print]295(50): 17009-17026

Stop codon read-through of mammalian MTCH2 leading to an unstable isoform regulates mitochondrial membrane potential.

Lekha E Manjunath, Anumeha Singh, Sarthak Sahoo, Ashutosh Mishra, Jinsha Padmarajan, Chaithanya G Basavaraju, Sandeep M Eswarappa.

  Stop codon read-through (SCR) is a process of continuation of translation beyond a stop codon. This phenomenon, which occurs only in certain mRNAs under specific conditions, leads to a longer isoform with properties different from that of the canonical isoform. MTCH2, which encodes a mitochondrial protein that regulates mitochondrial metabolism, was selected as a potential read-through candidate based on evolutionary conservation observed in the proximal region of its 3' UTR. Here, we demonstrate translational read-through across two evolutionarily conserved, in-frame stop codons of MTCH2 using luminescence- and fluorescence-based assays, and by analyzing ribosome-profiling and mass spectrometry (MS) data. This phenomenon generates two isoforms, MTCH2x and MTCH2xx (single- and double-SCR products, respectively), in addition to the canonical isoform MTCH2, from the same mRNA. Our experiments revealed that a cis-acting 12-nucleotide sequence in the proximal 3' UTR of MTCH2 is the necessary signal for SCR. Functional characterization showed that MTCH2 and MTCH2x were localized to mitochondria with a long t1/2 (>36 h). However, MTCH2xx was found predominantly in the cytoplasm. This mislocalization and its unique C terminus led to increased degradation, as shown by greatly reduced t1/2 (<1 h). MTCH2 read-through-deficient cells, generated using CRISPR-Cas9, showed increased MTCH2 expression and, consistent with this, decreased mitochondrial membrane potential. Thus, double-SCR of MTCH2 regulates its own expression levels contributing toward the maintenance of normal mitochondrial membrane potential.

Keywords:  MTCH2; codon; mRNA; mitochondria; mitochondrial membrane potential; protein degradation; ribosome; stop; stop codon; translation control; translational read-through

DOI:  https://doi.org/10.1074/jbc.RA120.014253
Nat Microbiol. 2021 Jan 18.

Listeria monocytogenes upregulates mitochondrial calcium signalling to inhibit LC3-associated phagocytosis as a survival strategy.

Tianliang Li, Ligang Kong, Xinghui Li, Sijin Wu, Kuldeep S Attri, Yan Li, Weipeng Gong, Bao Zhao, Lupeng Li, Laura E Herring, John M Asara, Lei Xu, Xiaobo Luo, Yu L Lei, Qin Ma, Stephanie Seveau, John S Gunn, Xiaolin Cheng, Pankaj K Singh, Douglas R Green, Haibo Wang, Haitao Wen.

Mitochondria are believed to have originated ~2.5 billion years ago. As well as energy generation in cells, mitochondria have a role in defence against bacterial pathogens. Despite profound changes in mitochondrial morphology and functions following bacterial challenge, whether intracellular bacteria can hijack mitochondria to promote their survival remains elusive. We report that Listeria monocytogenes-an intracellular bacterial pathogen-suppresses LC3-associated phagocytosis (LAP) by modulation of mitochondrial Ca2+ (mtCa2+) signalling in order to survive inside cells. Invasion of macrophages by L. monocytogenes induced mtCa2+ uptake through the mtCa2+ uniporter (MCU), which in turn increased acetyl-coenzyme A (acetyl-CoA) production by pyruvate dehydrogenase. Acetylation of the LAP effector Rubicon with acetyl-CoA decreased LAP formation. Genetic ablation of MCU attenuated intracellular bacterial growth due to increased LAP formation. Our data show that modulation of mtCa2+ signalling can increase bacterial survival inside cells, and highlight the importance of mitochondrial metabolism in host-microbial interactions.

DOI: https://doi.org/10.1038/s41564-020-00843-2