bims-minimp 2022-03-20 papers

on Mitochondria, innate immunity, proteostasis

Issue of 2022–03–20
24 papers selected by
Hanna Salmonowicz, International Institute of Molecular Mechanisms and Machines of the Polish Academy of Sciences

Parallel kinase pathways stimulate actin polymerization at depolarized mitochondria.
Disruption of Mitochondrial Quality Control Genes Promotes Caspase-Resistant Cell Survival Following Apoptotic Stimuli.
Applying Sodium Carbonate Extraction Mass Spectrometry to Investigate Defects in the Mitochondrial Respiratory Chain.
SPOTting stress at the mitochondrial outer membrane.
Acute pH alterations do not impact cardiac mitochondrial respiration in naked mole-rats or mice.
Mitochondrial transport and metabolism of the vitamin B-derived cofactors thiamine pyrophosphate, coenzyme A, FAD and NAD+ , and related diseases: A review.
Phosphorylation and acetylation of mitochondrial transcription factor A promote transcription processivity without compromising initiation or DNA compaction.
Lon upregulation contributes to cisplatin resistance by triggering NCLX-mediated mitochondrial Ca2+ release in cancer cells.
Correction: Cytoplasmic Irradiation Results in Mitochondrial Dysfunction and DRP1-Dependent Mitochondrial Fission.
POLG mutations lead to abnormal mitochondrial remodeling during neural differentiation of human pluripotent stem cells via SIRT3/AMPK pathway inhibition.
Regulation of the urea cycle by CPS1 O-GlcNAcylation in response to dietary restriction and aging.
Orally-active, clinically-translatable senolytics restore α-Klotho in mice and humans.
Activation of the UPR sensor ATF6α is regulated by its redox-dependent dimerization and ER retention by ERp18.
Mitochondrial depolarization after acute ethanol treatment drives mitophagy in living mice.
Mitochondrial uncoupling attenuates sarcopenic obesity by enhancing skeletal muscle mitophagy and quality control.
Mitochondrial complex IV defects induce metabolic and signaling perturbations that expose potential vulnerabilities in HCT116 cells.
Abnormal mitochondria in Down syndrome iPSC-derived GABAergic interneurons and organoids.
A role for cell polarity in lifespan and mitochondrial quality control in the budding yeast Saccharomyces cerevisiae.
Expression analysis and function of mitochondrial genome-encoded microRNAs.
BMAL1 moonlighting as a gatekeeper for LINE1 repression and cellular senescence in primates.
Identification of the Highly Active, Species Cross-Reactive Complex I Inhibitor BAY-179.
Inhibition of unfolded protein response prevents post-anesthesia neuronal hyperactivity and synapse loss in aged mice.
Structure of the BAK-activating antibody 7D10 bound to BAK reveals an unexpected role for the α1-α2 loop in BAK activation.
Quality control of protein complex assembly by the ubiquitin-proteasome system.

Curr Biol. 2022 Mar 08. pii: S0960-9822(22)00328-1. [Epub ahead of print]

Parallel kinase pathways stimulate actin polymerization at depolarized mitochondria.

Tak Shun Fung, Rajarshi Chakrabarti, Jana Kollasser, Klemens Rottner, Theresia E B Stradal, Frieda Kage, Henry N Higgs.

  Mitochondrial damage (MtD) represents a dramatic change in cellular homeostasis, necessitating metabolic changes and stimulating mitophagy. One rapid response to MtD is a rapid peri-mitochondrial actin polymerization termed ADA (acute damage-induced actin). The activation mechanism for ADA is unknown. Here, we use mitochondrial depolarization or the complex I inhibitor metformin to induce ADA. We show that two parallel signaling pathways are required for ADA. In one pathway, increased cytosolic calcium in turn activates PKC-β, Rac, WAVE regulatory complex, and Arp2/3 complex. In the other pathway, a drop in cellular ATP in turn activates AMPK (through LKB1), Cdc42, and FMNL formins. We also identify putative guanine nucleotide exchange factors for Rac and Cdc42, Trio and Fgd1, respectively, whose phosphorylation states increase upon mitochondrial depolarization and whose suppression inhibits ADA. The depolarization-induced calcium increase is dependent on the mitochondrial sodium-calcium exchanger NCLX, suggesting initial mitochondrial calcium efflux. We also show that ADA inhibition results in enhanced mitochondrial shape changes upon mitochondrial depolarization, suggesting that ADA inhibits these shape changes. These depolarization-induced shape changes are not fragmentation but a circularization of the inner mitochondrial membrane, which is dependent on the inner mitochondrial membrane protease Oma1. ADA inhibition increases the proteolytic processing of an Oma1 substrate, the dynamin GTPase Opa1. These results show that ADA requires the combined action of the Arp2/3 complex and formin proteins to polymerize a network of actin filaments around mitochondria and that the ADA network inhibits the rapid mitochondrial shape changes that occur upon mitochondrial depolarization.

Keywords:  AMPK; Arp2/3 complex; CCCP; FMNL formins; OMA1; OPA1; PKCβ; actin; calcium; mitochondrial depolarization

DOI:  https://doi.org/10.1016/j.cub.2022.02.058
J Biol Chem. 2022 Mar 15. pii: S0021-9258(22)00275-7. [Epub ahead of print] 101835

Disruption of Mitochondrial Quality Control Genes Promotes Caspase-Resistant Cell Survival Following Apoptotic Stimuli.

Yulia Kushnareva, Vivian Moraes, Julian Suess, Bjoern Peters, Donald D Newmeyer, Tomomi Kuwana.

  In cells undergoing cell-intrinsic apoptosis, mitochondrial outer membrane permeabilization (MOMP) typically marks an irreversible step in the cell death process. However, in some cases a subpopulation of treated cells can exhibit a sublethal response, termed "minority MOMP". In this phenomenon, the affected cells survive, despite a low level of caspase activation and subsequent limited activation of the endonuclease CAD (DFFB). Consequently, these cells can experience DNA damage, increasing the probability of oncogenesis. However, little is known about the minority MOMP response. To discover genes that affect the MOMP response in individual cells, we conducted an imaging-based phenotypic siRNA screen. We identified multiple candidate genes whose downregulation increased the heterogeneity of MOMP within single cells, among which were genes related to mitochondrial dynamics and mitophagy that participate in the mitochondrial quality control (MQC) system. Furthermore, to test the hypothesis that functional MQC is important for reducing the frequency of minority MOMP, we developed an assay to measure the clonogenic survival of caspase-engaged cells. We found that cells deficient in various MQC genes were indeed prone to aberrant post-MOMP survival. Our data highlight the important role of proteins involved in mitochondrial dynamics and mitophagy in preventing apoptotic dysregulation and oncogenesis.

Keywords:  apoptosis; mitochondrial dynamics; mitochondrial heterogeneity; mitochondrial outer membrane permeabilization; mitochondrial quality control; mitophagy; oncogenesis; siRNA screen

DOI:  https://doi.org/10.1016/j.jbc.2022.101835
Front Cell Dev Biol. 2022 ;10 786268

Applying Sodium Carbonate Extraction Mass Spectrometry to Investigate Defects in the Mitochondrial Respiratory Chain.

David R L Robinson, Daniella H Hock, Linden Muellner-Wong, Roopasingam Kugapreethan, Boris Reljic, Elliot E Surgenor, Carlos H M Rodrigues, Nikeisha J Caruana, David A Stroud.

  Mitochondria are complex organelles containing 13 proteins encoded by mitochondrial DNA and over 1,000 proteins encoded on nuclear DNA. Many mitochondrial proteins are associated with the inner or outer mitochondrial membranes, either peripherally or as integral membrane proteins, while others reside in either of the two soluble mitochondrial compartments, the mitochondrial matrix and the intermembrane space. The biogenesis of the five complexes of the oxidative phosphorylation system are exemplars of this complexity. These large multi-subunit complexes are comprised of more than 80 proteins with both membrane integral and peripheral associations and require soluble, membrane integral and peripherally associated assembly factor proteins for their biogenesis. Mutations causing human mitochondrial disease can lead to defective complex assembly due to the loss or altered function of the affected protein and subsequent destabilization of its interactors. Here we couple sodium carbonate extraction with quantitative mass spectrometry (SCE-MS) to track changes in the membrane association of the mitochondrial proteome across multiple human knockout cell lines. In addition to identifying the membrane association status of over 840 human mitochondrial proteins, we show how SCE-MS can be used to understand the impacts of defective complex assembly on protein solubility, giving insights into how specific subunits and sub-complexes become destabilized.

Keywords:  OXPHOS (oxidative phosphorylation); carbonate extraction; membrane protein; mitochondria; proteomic analyses; respiratory chain assembly

DOI:  https://doi.org/10.3389/fcell.2022.786268
Mol Cell. 2022 Mar 17. pii: S1097-2765(22)00168-X. [Epub ahead of print]82(6): 1086-1088

SPOTting stress at the mitochondrial outer membrane.

Max-Hinderk Schuler, Adam L Hughes.

Li et al. (2022) discover that Toxoplasma infection triggers remodeling of the mitochondrial outer membrane through generation of a mitochondrial subdomain termed "structure positive for outer mitochondrial membrane" (SPOT).

DOI: https://doi.org/10.1016/j.molcel.2022.02.030
Comp Biochem Physiol A Mol Integr Physiol. 2022 Mar 09. pii: S1095-6433(22)00043-5. [Epub ahead of print]268 111185

Acute pH alterations do not impact cardiac mitochondrial respiration in naked mole-rats or mice.

Kenny W Huynh, Soulene Sabir, Hang Cheng, Matthew E Pamenter.

  Energetically demanding conditions such as hypoxia and exercise favour anaerobic metabolism (glycolysis), which leads to acidification of the cellular milieu from ATP hydrolysis and accumulation of the anaerobic end-product, lactate. Cellular acidification may damage mitochondrial proteins and/or alter the H+ gradient across the mitochondrial inner membrane, which may in turn impact mitochondrial respiration and thus aerobic ATP production. Naked mole-rats are among the most hypoxia-tolerant mammals, and putatively experience intermittent environmental and systemic hypoxia while resting and exercising in their underground burrows. Previous studies in naked mole-rat brain, heart, and skeletal muscle mitochondria have demonstrated adaptations that favour improved efficiency in hypoxic conditions; however, the impact of cellular acidification on mitochondrial function has not been explored. We hypothesized that, relative to hypoxia-intolerant mice, naked mole-rat cardiac mitochondrial respiration is less sensitive to cellular pH changes. To test this, we used high-resolution respirometry to measure mitochondrial respiration by permeabilized cardiac muscle fibres from naked mole-rats and mice exposed in vitro to a pH range from 6.6 to 7.6. Surprisingly, we found that acute pH changes do not impact cardiac mitochondrial respiration or compromise mitochondrial integrity in either species. Our results suggest that acute alterations of cellular pH have minimal impact on cardiac mitochondrial respiration.

Keywords:  Acidity; Electron transport system; Mitochondrial integrity; alkaline; heart; oxidative phosphorylation

DOI:  https://doi.org/10.1016/j.cbpa.2022.111185
IUBMB Life. 2022 Mar 18.

Mitochondrial transport and metabolism of the vitamin B-derived cofactors thiamine pyrophosphate, coenzyme A, FAD and NAD+ , and related diseases: A review.

Ferdinando Palmieri, Magnus Monné, Giuseppe Fiermonte, Luigi Palmieri.

  Multiple mitochondrial matrix enzymes playing key roles in metabolism require cofactors for their action. Due to the high impermeability of the mitochondrial inner membrane, these cofactors need to be synthesized within the mitochondria or be imported, themselves or one of their precursors, into the organelles. Transporters belonging to the protein family of mitochondrial carriers have been identified to transport the coenzymes: thiamine pyrophosphate, coenzyme A, FAD and NAD+ , which are all structurally similar to nucleotides and derived from different B-vitamins. These mitochondrial cofactors bind more or less tightly to their enzymes and, after having been involved in a specific reaction step, are regenerated, spontaneously or by other enzymes, to return to their active form, ready for the next catalysis round. Disease-causing mutations in the mitochondrial cofactor carrier genes compromise not only the transport reaction but also the activity of all mitochondrial enzymes using that particular cofactor and the metabolic pathways in which the cofactor-dependent enzymes are involved. The mitochondrial transport, metabolism and diseases of the cofactors thiamine pyrophosphate, coenzyme A, FAD and NAD+ are the focus of this review.

Keywords:  coenzyme; coenzyme A; flavin adenine dinucleotide; mitochondria; mitochondrial carrier family SLC25; nicotinamide adenine dinucleotide; thiamine pyrophosphate

DOI:  https://doi.org/10.1002/iub.2612
J Biol Chem. 2022 Mar 09. pii: S0021-9258(22)00255-1. [Epub ahead of print] 101815

Phosphorylation and acetylation of mitochondrial transcription factor A promote transcription processivity without compromising initiation or DNA compaction.

Sean D Reardon, Tatiana V Mishanina.

  Mitochondrial transcription factor A (TFAM) plays important roles in mitochondrial DNA (mtDNA) compaction, transcription initiation, and in the regulation of processes like transcription and replication processivity. It is possible that TFAM is locally regulated within the mitochondrial matrix via such mechanisms like phosphorylation by protein kinase A (PKA) and non-enzymatic acetylation by acetyl-CoA. Here we demonstrate that DNA-bound TFAM is less susceptible to these modifications. We confirmed using electrophoretic mobility shift assays that phosphorylated or acetylated TFAM compacted circular double-stranded DNA just as well as unmodified TFAM and provide an in-depth analysis of acetylated sites on TFAM. We show that both modifications of TFAM increase the processivity of mitochondrial RNA polymerase during transcription through TFAM-imposed barriers on DNA, but that TFAM bearing either modification retains its full activity in transcription initiation. We conclude that TFAM phosphorylation by PKA and non-enzymatic acetylation by acetyl-CoA are unlikely to occur at the mtDNA and that modified free TFAM retains its vital functionalities like compaction and transcription initiation while enhancing transcription processivity.

Keywords:  Acetylation; DNA compaction; Mitochondrial transcription; Phosphorylation

DOI:  https://doi.org/10.1016/j.jbc.2022.101815
Cell Death Dis. 2022 Mar 16. 13(3): 241

Lon upregulation contributes to cisplatin resistance by triggering NCLX-mediated mitochondrial Ca2+ release in cancer cells.

Vidhya Tangeda, Yu Kang Lo, Ananth Ponneri Babuharisankar, Han-Yu Chou, Cheng-Liang Kuo, Yung-Hsi Kao, Alan Yueh-Luen Lee, Jang-Yang Chang.

Mitochondria are the major organelles in sensing cellular stress and inducing the response for cell survival. Mitochondrial Lon has been identified as an important stress protein involved in regulating proliferation, metastasis, and apoptosis in cancer cells. However, the mechanism of retrograde signaling by Lon on mitochondrial DNA (mtDNA) damage remains to be elucidated. Here we report the role of Lon in the response to cisplatin-induced mtDNA damage and oxidative stress, which confers cancer cells on cisplatin resistance via modulating calcium levels in mitochondria and cytosol. First, we found that cisplatin treatment on oral cancer cells caused oxidative damage of mtDNA and induced Lon expression. Lon overexpression in cancer cells decreased while Lon knockdown sensitized the cytotoxicity towards cisplatin treatment. We further identified that cisplatin-induced Lon activates the PYK2-SRC-STAT3 pathway to stimulate Bcl-2 and IL-6 expression, leading to the cytotoxicity resistance to cisplatin. Intriguingly, we found that activation of this pathway is through an increase of intracellular calcium (Ca2+) via NCLX, a mitochondrial Na+/Ca2+ exchanger. We then verified that NCLX expression is dependent on Lon levels; Lon interacts with and activates NCLX activity. NCLX inhibition increased the level of mitochondrial calcium and sensitized the cytotoxicity to cisplatin in vitro and in vivo. In summary, mitochondrial Lon-induced cisplatin resistance is mediated by calcium release into cytosol through NCLX, which activates calcium-dependent PYK2-SRC-STAT3-IL-6 pathway. Thus, our work uncovers the novel retrograde signaling by mitochondrial Lon on resistance to cisplatin-induced mtDNA stress, indicating the potential use of Lon and NCLX inhibitors for better clinical outcomes in chemoresistant cancer patients.

DOI: https://doi.org/10.1038/s41419-022-04668-1
Cancer Res. 2022 Mar 15. 82(6): 1154

Correction: Cytoplasmic Irradiation Results in Mitochondrial Dysfunction and DRP1-Dependent Mitochondrial Fission.

Bo Zhang, Mercy M Davidson, Hongning Zhou, Chunxin Wang, Winsome F Walker, Tom K Hei.

DOI: https://doi.org/10.1158/0008-5472.CAN-21-4330
Cell Cycle. 2022 Mar 17. 1-16

POLG mutations lead to abnormal mitochondrial remodeling during neural differentiation of human pluripotent stem cells via SIRT3/AMPK pathway inhibition.

Anbin Chen, Cecilie Katrin Kristiansen, Lena Elise Høyland, Mathias Ziegler, Jian Wang, Gareth John Sullivan, Xingang Li, Laurence A Bindoff, Kristina Xiao Liang.

  We showed previously that POLG mutations cause major changes in mitochondrial function, including loss of mitochondrial respiratory chain (MRC) complex I, mitochondrial DNA (mtDNA) depletion and an abnormal NAD+/NADH ratio in both neural stem cells (NSCs) and astrocytes differentiated from induced pluripotent stem cells (iPSCs). In the current study, we looked at mitochondrial remodeling as stem cells transit pluripotency and during differentiation from NSCs to both dopaminergic (DA) neurons and astrocytes comparing the process in POLG-mutated and control stem cells. We saw that mitochondrial membrane potential (MMP), mitochondrial volume, ATP production and reactive oxygen species (ROS) changed in similar ways in POLG and control NSCs, but mtDNA replication, MRC complex I and NAD+ metabolism failed to remodel normally. In DA neurons differentiated from NSCs, we saw that POLG mutations caused failure to increase MMP and ATP production and blunted the increase in mtDNA and complex I. Interestingly, mitochondrial remodeling during astrocyte differentiation from NSCs was similar in both POLG-mutated and control NSCs. Further, we showed downregulation of the SIRT3/AMPK pathways in POLG-mutated cells, suggesting that POLG mutations lead to abnormal mitochondrial remodeling in early neural development due to the downregulation of these pathways. [Figure: see text].

Keywords:  DA neurons; Mitochondrial remodeling; NSCs; POLG; astrocytes; iPSCs

DOI:  https://doi.org/10.1080/15384101.2022.2044136
J Mol Cell Biol. 2022 Mar 14. pii: mjac016. [Epub ahead of print]

Regulation of the urea cycle by CPS1 O-GlcNAcylation in response to dietary restriction and aging.

Jing Wu, Jiayu Liu, Kalina Lapenta, Reina Desrouleaux, Min-Dian Li, Xiaoyong Yang.

  O-linked N-acetyl-glucosamine glycosylation (O-GlcNAcylation) of intracellular proteins is a dynamic process broadly implicated in age-related disease, yet it remains uncharacterized whether and how O-GlcNAcylation contributes to the natural aging process. O-GlcNAc transferase (OGT) and the opposing enzyme O-GlcNAcase (OGA) control this nutrient-sensing protein modification in cells. Here, we show that global O-GlcNAc levels are increased in multiple tissues of aged mice. In aged liver, carbamoyl phosphate synthetase 1 (CPS1) is among the most heavily O-GlcNAcylated proteins. CPS1 O-GlcNAcylation is reversed by calorie restriction and is sensitive to genetic and pharmacological manipulations of the O-GlcNAc pathway. High glucose stimulates CPS1 O-GlcNAcylation and inhibits CPS1 activity. Liver-specific deletion of OGT potentiates CPS1 activity and renders CPS1 irresponsive to further stimulation by a prolonged fasting. Our results identify CPS1 O-GlcNAcylation as a key nutrient-sensing regulatory step in the urea cycle during aging and dietary restriction, implying a role for mitochondrial O-GlcNAcylation in nutritional regulation of longevity.

Keywords:   O-GlcNAcylation; ageing; calorie restriction; carbamoyl-phosphate synthetase 1; dietary restriction; post-translational modification; urea cycle

DOI:  https://doi.org/10.1093/jmcb/mjac016
EBioMedicine. 2022 Mar 12. pii: S2352-3964(22)00096-2. [Epub ahead of print] 103912

Orally-active, clinically-translatable senolytics restore α-Klotho in mice and humans.

Yi Zhu, Larissa G P Langhi Prata, Erin O Wissler Gerdes, Jair Machado Espindola Netto, Tamar Pirtskhalava, Nino Giorgadze, Utkarsh Tripathi, Christina L Inman, Kurt O Johnson, Ailing Xue, Allyson K Palmer, Tingjun Chen, Kalli Schaefer, Jamie N Justice, Anoop M Nambiar, Nicolas Musi, Stephen B Kritchevsky, Jun Chen, Sundeep Khosla, Diana Jurk, Marissa J Schafer, Tamar Tchkonia, James L Kirkland.

   BACKGROUND: α-Klotho is a geroprotective protein that can attenuate or alleviate deleterious changes with ageing and disease. Declines in α-Klotho play a role in the pathophysiology of multiple diseases and age-related phenotypes. Pre-clinical evidence suggests that boosting α-Klotho holds therapeutic potential. However, readily clinically-translatable, practical strategies for increasing α-Klotho are not at hand. Here, we report that orally-active, clinically-translatable senolytics can increase α-Klotho in mice and humans.
METHODS: We examined α-Klotho expression in three different human primary cell types co-cultured with conditioned medium (CM) from senescent or non-senescent cells with or without neutralizing antibodies. We assessed α-Klotho expression in aged, obese, and senescent cell-transplanted mice treated with vehicle or senolytics. We assayed urinary α-Klotho in patients with idiopathic pulmonary fibrosis (IPF) who were treated with the senolytic drug combination, Dasatinib plus Quercetin (D+Q).
FINDINGS: We found exposure to the senescent cell secretome reduces α-Klotho in multiple nonsenescent human cell types. This was partially prevented by neutralizing antibodies against the senescence-associated secretory phenotype (SASP) factors, activin A and Interleukin 1α (IL-1α). Consistent with senescent cells' being a cause of decreased α-Klotho, transplanting senescent cells into younger mice reduced brain and urine α-Klotho. Selectively removing senescent cells genetically or pharmacologically increased α-Klotho in urine, kidney, and brain of mice with increased senescent cell burden, including naturally-aged, diet-induced obese (DIO), or senescent cell-transplanted mice. D+Q increased α-Klotho in urine of patients with IPF, a disease linked to cellular senescence.
INTERPRETATION: Senescent cells cause reduced α-Klotho, partially due to their production of activin A and IL-1α. Targeting senescent cells boosts α-Klotho in mice and humans. Thus, clearing senescent cells restores α-Klotho, potentially opening a novel, translationally-feasible avenue for developing orally-active small molecule, α-Klotho-enhancing clinical interventions. Furthermore, urinary α-Klotho may prove to be a useful test for following treatments in senolytic clinical trials.
FUNDING: This work was supported by National Institute of Health grants AG013925 (J.L.K.), AG062413 (J.L.K., S.K.), AG044271 (N.M.), AG013319 (N.M.), and the Translational Geroscience Network (AG061456: J.L.K., T.T., N.M., S.B.K., S.K.), Robert and Arlene Kogod (J.L.K.), the Connor Group (J.L.K.), Robert J. and Theresa W. Ryan (J.L.K.), and the Noaber Foundation (J.L.K.). The previous IPF clinical trial was supported by the Claude D. Pepper Older Americans Independence Centers at WFSM (AG021332: J.N.J., S.B.K.), UTHSCA (AG044271: A.M.N.), and the Translational Geroscience Network.

Keywords:  Cellular senescence; Senolytics; α-Klotho

DOI:  https://doi.org/10.1016/j.ebiom.2022.103912
Proc Natl Acad Sci U S A. 2022 Mar 22. 119(12): e2122657119

Activation of the UPR sensor ATF6α is regulated by its redox-dependent dimerization and ER retention by ERp18.

Ojore Benedict Valentine Oka, Arvin Shedrach Pierre, Marie Anne Pringle, Wanida Tungkum, Zhenbo Cao, Bethany Fleming, Neil John Bulleid.

  SignificanceMembrane and secretory proteins are synthesized in the endoplasmic reticulum (ER). Perturbations to ER function disrupts protein folding, causing misfolded proteins to accumulate, a condition known as ER stress. Cells adapt to stress by activating the unfolded protein response (UPR), which ultimately restores proteostasis. A key player in the UPR response is ATF6α, which requires release from ER retention and modulation of its redox status during activation. Here, we report that ER stress promotes formation of a specific ATF6α dimer, which is preferentially trafficked to the Golgi for processing. We show that ERp18 regulates ATF6α by mitigating its dimerization and trafficking to the Golgi and identify redox-dependent oligomerization of ATF6α as a key mechanism regulating its function during the UPR.

Keywords:  ATF6; ER stress; proteostasis; unfolded protein response

DOI:  https://doi.org/10.1073/pnas.2122657119
Autophagy. 2022 Mar 16. 1-15

Mitochondrial depolarization after acute ethanol treatment drives mitophagy in living mice.

Devadoss J Samuvel, Li Li, Yasodha Krishnasamy, Monika Gooz, Kenji Takemoto, Patrick M Woster, John J Lemasters, Zhi Zhong.

  Ethanol increases hepatic mitophagy driven by unknown mechanisms. Type 1 mitophagy sequesters polarized mitochondria for nutrient recovery and cytoplasmic remodeling. In Type 2, mitochondrial depolarization (mtDepo) initiates mitophagy to remove the damaged organelles. Previously, we showed that acute ethanol administration produces reversible hepatic mtDepo. Here, we tested the hypothesis that ethanol-induced mtDepo initiates Type 2 mitophagy. GFP-LC3 transgenic mice were gavaged with ethanol (2-6 g/kg) with and without pre-treatment with agents that decrease or increase mtDepo-Alda-1, tacrolimus, or disulfiram. Without ethanol, virtually all hepatocytes contained polarized mitochondria with infrequent autophagic GFP-LC3 puncta visualized by intravital microscopy. At ~4 h after ethanol treatment, mtDepo occurred in an all-or-none fashion within individual hepatocytes, which increased dose dependently. GFP-LC3 puncta increased in parallel, predominantly in hepatocytes with mtDepo. Mitochondrial PINK1 and PRKN/parkin also increased. After covalent labeling of mitochondria with MitoTracker Red (MTR), GFP-LC3 puncta encircled MTR-labeled mitochondria after ethanol treatment, directly demonstrating mitophagy. GFP-LC3 puncta did not associate with fat droplets visualized with BODIPY558/568, indicating that increased autophagy was not due to lipophagy. Before ethanol administration, rhodamine-dextran (RhDex)-labeled lysosomes showed little association with GFP-LC3. After ethanol treatment, TFEB (transcription factor EB) translocated to nuclei, and lysosomal mass increased. Many GFP-LC3 puncta merged with RhDex-labeled lysosomes, showing autophagosomal processing into lysosomes. After ethanol treatment, disulfiram increased, whereas Alda-1 and tacrolimus decreased mtDepo, and mitophagy changed proportionately. In conclusion, mtDepo after acute ethanol treatment induces mitophagic sequestration and subsequent lysosomal processing.Abbreviations : AcAld, acetaldehyde; ADH, alcohol dehydrogenase; ALDH, aldehyde dehydrogenase; ALD, alcoholic liver disease; Alda-1, N-(1,3-benzodioxol-5-ylmethyl)-2,6-dichlorobenzamide; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GFP, green fluorescent protein; LAMP1, lysosomal-associated membrane protein 1; LMNB1, lamin B1; MAA, malondialdehyde-acetaldehyde adducts; MAP1LC3/LC3, microtubule-associated protein 1 light chain 3; MPT, mitochondrial permeability transition; mtDAMPS, mitochondrial damage-associated molecular patterns; mtDepo, mitochondrial depolarization; mtDNA, mitochondrial DNA; MTR, MitoTracker Red; PI, propidium iodide; PINK1, PTEN induced putative kinase 1; PRKN, parkin; RhDex, rhodamine dextran; TFEB, transcription factor EB; Tg, transgenic; TMRM, tetramethylrhodamine methylester; TOMM20, translocase of outer mitochondrial membrane 20; VDAC, voltage-dependent anion channel.

Keywords:  Acetaldehyde; Alda-1; alcoholic liver disease; mitochondrial depolarization; mitophagy; tacrolimus

DOI:  https://doi.org/10.1080/15548627.2022.2046457
J Cachexia Sarcopenia Muscle. 2022 Mar 19.

Mitochondrial uncoupling attenuates sarcopenic obesity by enhancing skeletal muscle mitophagy and quality control.

Wagner S Dantas, Elizabeth R M Zunica, Elizabeth C Heintz, Bolormaa Vandanmagsar, Z Elizabeth Floyd, Yongmei Yu, Hisashi Fujioka, Charles L Hoppel, Kathryn P Belmont, Christopher L Axelrod, John P Kirwan.

   BACKGROUND: Sarcopenic obesity is a highly prevalent disease with poor survival and ineffective medical interventions. Mitochondrial dysfunction is purported to be central in the pathogenesis of sarcopenic obesity by impairing both organelle biogenesis and quality control. We have previously identified that a mitochondrial-targeted furazano[3,4-b]pyrazine named BAM15 is orally available and selectively lowers respiratory coupling efficiency and protects against diet-induced obesity in mice. Here, we tested the hypothesis that mitochondrial uncoupling simultaneously attenuates loss of muscle function and weight gain in a mouse model of sarcopenic obesity.
METHODS: Eighty-week-old male C57BL/6J mice with obesity were randomized to 10 weeks of high fat diet (CTRL) or BAM15 (BAM15; 0.1% w/w in high fat diet) treatment. Body weight and food intake were measured weekly. Body composition, muscle function, energy expenditure, locomotor activity, and glucose tolerance were determined after treatment. Skeletal muscle was harvested and evaluated for histology, gene expression, protein signalling, and mitochondrial structure and function.
RESULTS: BAM15 decreased body weight (54.0 ± 2.0 vs. 42.3 ± 1.3 g, P < 0.001) which was attributable to increased energy expenditure (10.1 ± 0.1 vs. 11.3 ± 0.4 kcal/day, P < 0.001). BAM15 increased muscle mass (52.7 ± 0.4 vs. 59.4 ± 1.0%, P < 0.001), strength (91.1 ± 1.3 vs. 124.9 ± 1.2 g, P < 0.0001), and locomotor activity (347.0 ± 14.4 vs. 432.7 ± 32.0 m, P < 0.001). Improvements in physical function were mediated in part by reductions in skeletal muscle inflammation (interleukin 6 and gp130, both P < 0.05), enhanced mitochondrial function, and improved endoplasmic reticulum homeostasis. Specifically, BAM15 activated mitochondrial quality control (PINK1-ubiquitin binding and LC3II, P < 0.01), increased mitochondrial activity (citrate synthase and complex II activity, all P < 0.05), restricted endoplasmic reticulum (ER) misfolding (decreased oligomer A11 insoluble/soluble ratio, P < 0.0001) while limiting ER stress (decreased PERK signalling, P < 0.0001), apoptotic signalling (decreased cytochrome C release and Caspase-3/9 activation, all P < 0.001), and muscle protein degradation (decreased 14-kDa actin fragment insoluble/soluble ratio, P < 0.001).
CONCLUSIONS: Mitochondrial uncoupling by agents such as BAM15 may mitigate age-related decline in muscle mass and function by molecular and cellular bioenergetic adaptations that confer protection against sarcopenic obesity.

Keywords:  Ageing; BAM15; Bioenergetics; Mitochondrial uncoupling; Obesity; Sarcopenia

DOI:  https://doi.org/10.1002/jcsm.12982
FEBS Open Bio. 2022 Mar 18.

Mitochondrial complex IV defects induce metabolic and signaling perturbations that expose potential vulnerabilities in HCT116 cells.

Oro Uchenunu, Alexander V Zhdanov, Phillipe Hutton, Predrag Jovanovic, Ye Wang, Dmitry E Andreev, Laura Hulea, David J Papadopoli, Daina Avizonis, Pavel V Baranov, Michael N Pollak, Dmitri B Papkovsky, Ivan Topisirovic.

  Mutations in genes encoding cytochrome c oxidase (COX; mitochondrial complex IV) subunits and assembly factors (e.g., SCO1, SCO2, COA6) are linked to severe metabolic syndromes. Notwithstanding that SCO2 is under transcriptional control of tumour suppressor p53, the role of mitochondrial complex IV dysfunction in cancer metabolism remains obscure. Herein, we demonstrate that the loss of SCO2 in HCT116 colorectal cancer cells leads to significant metabolic and signaling perturbations. Specifically, abrogation of SCO2 increased NAD+ regenerating reactions and decreased glucose oxidation through citric acid cycle while enhancing pyruvate carboxylation. This was accompanied by a reduction in amino acid levels and the accumulation of lipid droplets. In addition, SCO2 loss resulted in hyperactivation of the IGF1R/AKT axis with paradoxical downregulation of mTOR signaling which was accompanied by increased AMPK activity. Accordingly, abrogation of SCO2 expression appears to increase the sensitivity of cells to IGF1R and AKT, but not mTOR inhibitors. Finally, the loss of SCO2 was associated with reduced proliferation and enhanced migration of HCT116 cells. Collectively, herein we describe potential adaptive signaling and metabolic perturbations triggered by mitochondrial complex IV dysfunction.

Keywords:  AKT; AMPK; SCO2; cytochrome C oxidase; mTOR; metabolism; mitochondrial dysfunction

DOI:  https://doi.org/10.1002/2211-5463.13398
Biochim Biophys Acta Mol Basis Dis. 2022 Mar 14. pii: S0925-4439(22)00051-5. [Epub ahead of print] 166388

Abnormal mitochondria in Down syndrome iPSC-derived GABAergic interneurons and organoids.

Lei Xu, Hai-Qin Huo, Kai-Qin Lu, Xiao-Yan Tang, Yuan Hong, Xiao Han, Zi-Xing Fu, Kai-Heng Fang, Min Xu, Xing Guo, Yan Liu.

  Down syndrome (DS) is caused by trisomy 21, and it is characterized by developmental brain disorders and neurological dysfunction. Clinical studies and basic research have revealed that defects in mitochondrial function contribute to the pathogenesis of DS. However, the underlying mechanisms of mitochondrial dysfunction in DS remain unclear. In this study, we first generated GABAergic interneurons and medial ganglionic eminence (MGE) organoids from DS patients and control induced pluripotent stem cells. The mitochondria were abnormally clustered in the perinuclear region of GABA neurons and cell in MGE organoids from DS patients, which exhibited impaired mitochondrial function as assessed by seahorse oxidative phosphorylation assay. Inhibition of the DSCAM-PAK1 pathway by gene editing or treatment with a small molecule corrected mitochondrial perinuclear aggregation in cells from DS patients. Therefore, our study provides insight into the potential mechanism of mitochondrial dysfunction in DS.

Keywords:  Down syndrome; GABAergic interneurons; MGE organoids; Mitochondria; iPSC

DOI:  https://doi.org/10.1016/j.bbadis.2022.166388
iScience. 2022 Mar 18. 25(3): 103957

A role for cell polarity in lifespan and mitochondrial quality control in the budding yeast Saccharomyces cerevisiae.

Emily J Yang, Wolfgang M Pernice, Liza A Pon.

  Babies are born young, largely independent of the age of their mothers. Mother-daughter age asymmetry in yeast is achieved, in part, by inheritance of higher-functioning mitochondria by buds and retention of some high-functioning mitochondria in mother cells. The mitochondrial F box protein, Mfb1p, tethers mitochondria at both poles in a cell cycle-regulated manner: it localizes to and anchors mitochondria at the mother cell tip throughout the cell cycle and at the bud tip before cytokinesis. Here, we report that cell polarity and polarized localization of Mfb1p decline with age in Saccharomyces cerevisiae. Moreover, deletion of genes (BUD1, BUD2, and BUD5) that mediate symmetry breaking during establishment of cell polarity and asymmetric yeast cell division cause depolarized Mfb1p localization and defects in mitochondrial distribution and quality control. Our results support a role for the polarity machinery in lifespan through modulating Mfb1 function in asymmetric inheritance of mitochondria during yeast cell division.

Keywords:  Biological sciences; Cell biology; Genetics; Molecular biology

DOI:  https://doi.org/10.1016/j.isci.2022.103957
J Cell Sci. 2022 Mar 17. pii: jcs.258937. [Epub ahead of print]

Expression analysis and function of mitochondrial genome-encoded microRNAs.

Raviprasad Kuthethur, Vaibhav Shukla, Sandeep Mallya, Divya Adiga, Shama Prasada Kabekkodu, Lingadakai Ramachandra, Prakash Saxena Pu, Kapaettu Satyamoorthy, Sanjiban Chakrabarty.

  MicroRNAs play a significant role in nuclear and mitochondrial anterograde and retrograde signaling. Most of the miRNAs found inside mitochondria are nuclear genome encoded, with few mitochondrial genome encoded non-coding RNAs have been reported. In this study, we have identified 13 mitochondrial genome-encoded microRNAs (mitomiRs), which were differentially expressed in breast cancer cell lines (MCF-7, MDA-MB-468, and MDA-MB-231), non-malignant breast epithelial cell line (MCF-10A), and normal and breast cancer tissue specimens. We found that mitochondrial DNA depletion and inhibition of mitochondrial transcription leads to reduced expression of mitomiRs in breast cancer cells. MitomiRs physically interact with Ago2, an RNA-induced silencing complex (RISC) protein, in the cytoplasm and inside mitochondria. MitomiRs regulate the expression of both nuclear and mitochondrial transcripts in breast cancer cells. We showed that mitomiR-5 targets PPARGC1A and regulates mtDNA copy number in breast cancer cells. MitomiRs identified in the present study may be a promising tool for expression and functional analysis in patients with a defective mitochondrial phenotype, including cancer and metabolic syndromes.

Keywords:   PPARGC1A ; MicroRNAs; Mitochondria; mitomiRs; mtDNA copy number

DOI:  https://doi.org/10.1242/jcs.258937
Nucleic Acids Res. 2022 Mar 14. pii: gkac146. [Epub ahead of print]

BMAL1 moonlighting as a gatekeeper for LINE1 repression and cellular senescence in primates.

Chuqian Liang, Qiong Ke, Zunpeng Liu, Jie Ren, Weiqi Zhang, Jianli Hu, Zehua Wang, Hong Chen, Kai Xia, Xingqiang Lai, Qiaoran Wang, Kuan Yang, Wei Li, Zeming Wu, Chao Wang, Haoteng Yan, Xiaoyu Jiang, Zhejun Ji, Miyang Ma, Xiao Long, Si Wang, Huating Wang, Hao Sun, Juan Carlos Izpisua Belmonte, Jing Qu, Andy Peng Xiang, Guang-Hui Liu.

Aging in humans is intricately linked with alterations in circadian rhythms concomitant with physiological decline and stem cell exhaustion. However, whether the circadian machinery directly regulates stem cell aging, especially in primates, remains poorly understood. In this study, we found that deficiency of BMAL1, the only non-redundant circadian clock component, results in an accelerated aging phenotype in both human and cynomolgus monkey mesenchymal progenitor cells (MPCs). Unexpectedly, this phenotype was mainly attributed to a transcription-independent role of BMAL1 in stabilizing heterochromatin and thus preventing activation of the LINE1-cGAS-STING pathway. In senescent primate MPCs, we observed decreased capacity of BMAL1 to bind to LINE1 and synergistic activation of LINE1 expression. Likewise, in the skin and muscle tissues from the BMAL1-deficient cynomolgus monkey, we observed destabilized heterochromatin and aberrant LINE1 transcription. Altogether, these findings uncovered a noncanonical role of BMAL1 in stabilizing heterochromatin to inactivate LINE1 that drives aging in primate cells.

DOI: https://doi.org/10.1093/nar/gkac146
ACS Med Chem Lett. 2022 Mar 10. 13(3): 348-357

Identification of the Highly Active, Species Cross-Reactive Complex I Inhibitor BAY-179.

Jeffrey Mowat, Alexander H M Ehrmann, Sven Christian, Carolyn Sperl, Stephan Menz, Judith Günther, Roman C Hillig, Marcus Bauser, Wolfgang Schwede.

Mitochondria are key regulators of energy supply and cell death. Generation of ATP within mitochondria occurs through oxidative phosphorylation (OXPHOS), a process which utilizes the four complexes (complex I-IV) of the electron transport chain and ATP synthase. Certain oncogenic mutations (e.g., LKB1 or mIDH) can further enhance the reliance of cancer cells on OXPHOS for their energetic requirements, rendering cells sensitive to complex I inhibition and highlighting the potential value of complex I as a therapeutic target. Herein, we describe the discovery of a potent, selective, and species cross-reactive complex I inhibitor. A high-throughput screen of the Bayer compound library followed by hit triaging and initial hit-to-lead activities led to a lead structure which was further optimized in a comprehensive lead optimization campaign. Focusing on balancing potency and metabolic stability, this program resulted in the identification of BAY-179, an excellent in vivo suitable tool with which to probe the biological relevance of complex I inhibition in cancer indications.

DOI: https://doi.org/10.1021/acsmedchemlett.1c00666
Aging Cell. 2022 Mar 17. e13592

Inhibition of unfolded protein response prevents post-anesthesia neuronal hyperactivity and synapse loss in aged mice.

Kai Chen, Qiuping Hu, Riya Gupta, Jessie Stephens, Zhongcong Xie, Guang Yang.

  Delirium is the most common postoperative complication in older patients after prolonged anesthesia and surgery and is associated with accelerated cognitive decline and dementia. The neuronal pathogenesis of postoperative delirium is largely unknown. The unfolded protein response (UPR) is an adaptive reaction of cells to perturbations in endoplasmic reticulum function. Dysregulation of UPR has been implicated in a variety of diseases including Alzheimer's disease and related dementias. However, whether UPR plays a role in anesthesia-induced cognitive impairment remains unexplored. By performing in vivo calcium imaging in the mouse frontal cortex, we showed that exposure of aged mice to the inhalational anesthetic sevoflurane for 2 hours resulted in a marked elevation of neuronal activity during recovery, which lasted for at least 24 hours after the end of exposure. Concomitantly, sevoflurane anesthesia caused a prolonged increase in phosphorylation of PERK and eIF2α, the markers of UPR activation. Genetic deletion or pharmacological inhibition of PERK prevented neuronal hyperactivity and memory impairment induced by sevoflurane. Moreover, we showed that PERK suppression also reversed various molecular and synaptic changes induced by sevoflurane anesthesia, including alterations of synaptic NMDA receptors, tau protein phosphorylation, and dendritic spine loss. Together, these findings suggest that sevoflurane anesthesia causes abnormal UPR in the aged brain, which contributes to neuronal hyperactivity, synapse loss and cognitive decline in aged mice.

Keywords:  delirium; dendritic spine; neuronal activity; sevoflurane; synapse; unfolded protein response

DOI:  https://doi.org/10.1111/acel.13592
Cell Death Differ. 2022 Mar 12.

Structure of the BAK-activating antibody 7D10 bound to BAK reveals an unexpected role for the α1-α2 loop in BAK activation.

Adeline Y Robin, Michelle S Miller, Sweta Iyer, Melissa X Shi, Ahmad Z Wardak, Daisy Lio, Nicholas A Smith, Brian J Smith, Richard W Birkinshaw, Peter E Czabotar, Ruth M Kluck, Peter M Colman.

Pro-apoptotic BAK and BAX are activated by BH3-only proteins to permeabilise the outer mitochondrial membrane. The antibody 7D10 also activates BAK on mitochondria and its epitope has previously been mapped to BAK residues in the loop connecting helices α1 and α2 of BAK. A crystal structure of the complex between the Fv fragment of 7D10 and the BAK mutant L100A suggests a possible mechanism of activation involving the α1-α2 loop residue M60. M60 mutants of BAK have reduced stability and elevated sensitivity to activation by BID, illustrating that M60, through its contacts with residues in helices α1, α5 and α6, is a linchpin stabilising the inert, monomeric structure of BAK. Our data demonstrate that BAK's α1-α2 loop is not a passive covalent connector between secondary structure elements, but a direct restraint on BAK's activation.

DOI: https://doi.org/10.1038/s41418-022-00961-w
Trends Cell Biol. 2022 Mar 14. pii: S0962-8924(22)00038-1. [Epub ahead of print]

Quality control of protein complex assembly by the ubiquitin-proteasome system.

Carlos Pla-Prats, Nicolas H Thomä.

  The majority of human proteins operate as multimeric complexes with defined compositions and distinct architectures. How the assembly of these complexes is surveyed and how defective complexes are recognized is just beginning to emerge. In eukaryotes, over 600 E3 ubiquitin ligases form part of the ubiquitin-proteasome system (UPS) which detects structural characteristics in its target proteins and selectively induces their degradation. The UPS has recently been shown to oversee key quality control steps during the assembly of protein complexes. We review recent findings on how E3 ubiquitin ligases regulate protein complex assembly and highlight unanswered questions relating to their mechanism of action.

Keywords:  AQC; UPS; assembly; protein complex; quality control; ubiquitin

DOI:  https://doi.org/10.1016/j.tcb.2022.02.005