bims-mitdyn Biomed News
on Mitochondrial dynamics: mechanisms
Issue of 2023‒05‒07
twelve papers selected by
Edmond Chan
Queen’s University, School of Medicine
  1. Molecular basis of translation termination at noncanonical stop codons in human mitochondria.
  2. A DddA ortholog-based and transactivator-assisted nuclear and mitochondrial cytosine base editors with expanded target compatibility.
  3. The mitochondrial unfolded protein response regulates hippocampal neural stem cell aging.
  4. Inter-organelle cross-talk supports acetyl-coenzyme A homeostasis and lipogenesis under metabolic stress.
  5. Acetylcarnitine shuttling links mitochondrial metabolism to histone acetylation and lipogenesis.
  6. Methionine restriction constrains lipoylation and activates mitochondria for nitrogenic synthesis of amino acids.
  7. CLUH functions as negative regulator of inflammation in human macrophages and determines ulcerative colitis pathogenesis.
  8. Mitochondrial protein import clogging as a mechanism of disease.
  9. Noncanonical role of singleminded-2s in mitochondrial respiratory chain formation in breast cancer.
  10. Direct tests of cytochrome c and c1 functions in the electron transport chain of malaria parasites.
  11. MICU1 occludes the mitochondrial calcium uniporter in divalent-free conditions.
  12. PARKIN UBIQUITINATION OF KINDLIN-2 ENABLES MITOCHONDRIA-ASSOCIATED METASTASIS SUPPRESSION.
  1. Science. 2023 May 05. 380(6644): 531-536
    Molecular basis of translation termination at noncanonical stop codons in human mitochondria.
    Martin Saurer, Marc Leibundgut, Hima Priyanka Nadimpalli, Alain Scaiola, Tanja Schönhut, Richard G Lee, Stefan J Siira, Oliver Rackham, René Dreos, Tea Lenarčič, Eva Kummer, David Gatfield, Aleksandra Filipovska, Nenad Ban.
      The genetic code that specifies the identity of amino acids incorporated into proteins during protein synthesis is almost universally conserved. Mitochondrial genomes feature deviations from the standard genetic code, including the reassignment of two arginine codons to stop codons. The protein required for translation termination at these noncanonical stop codons to release the newly synthesized polypeptides is not currently known. In this study, we used gene editing and ribosomal profiling in combination with cryo-electron microscopy to establish that mitochondrial release factor 1 (mtRF1) detects noncanonical stop codons in human mitochondria by a previously unknown mechanism of codon recognition. We discovered that binding of mtRF1 to the decoding center of the ribosome stabilizes a highly unusual conformation in the messenger RNA in which the ribosomal RNA participates in specific recognition of the noncanonical stop codons.
    DOI:  https://doi.org/10.1126/science.adf9890
  2. Mol Cell. 2023 Apr 30. pii: S1097-2765(23)00283-6. [Epub ahead of print]
    A DddA ortholog-based and transactivator-assisted nuclear and mitochondrial cytosine base editors with expanded target compatibility.
    Junfan Guo, Wenxia Yu, Min Li, Hongyu Chen, Jie Liu, Xiaowen Xue, Jianxiang Lin, Shisheng Huang, Wenjie Shu, Xingxu Huang, Zhen Liu, Shengqi Wang, Yunbo Qiao.
      Bacterial double-stranded DNA (dsDNA) cytosine deaminase DddAtox-derived cytosine base editor (DdCBE) and its evolved variant, DddA11, guided by transcription-activator-like effector (TALE) proteins, enable mitochondrial DNA (mtDNA) editing at TC or HC (H = A, C, or T) sequence contexts, while it remains relatively unattainable for GC targets. Here, we identified a dsDNA deaminase originated from a Roseburia intestinalis interbacterial toxin (riDddAtox) and generated CRISPR-mediated nuclear DdCBEs (crDdCBEs) and mitochondrial CBEs (mitoCBEs) using split riDddAtox, which catalyzed C-to-T editing at both HC and GC targets in nuclear and mitochondrial genes. Moreover, transactivator (VP64, P65, or Rta) fusion to the tail of DddAtox- or riDddAtox-mediated crDdCBEs and mitoCBEs substantially improved nuclear and mtDNA editing efficiencies by up to 3.5- and 1.7-fold, respectively. We also used riDddAtox-based and Rta-assisted mitoCBE to efficiently stimulate disease-associated mtDNA mutations in cultured cells and in mouse embryos with conversion frequencies of up to 58% at non-TC targets.
    Keywords:  DdCBE; Roseburia intestinalis; mitoCBE; mitochondrial DNA; mtDNA; non-TC; nuclear DNA; transactivator
    DOI:  https://doi.org/10.1016/j.molcel.2023.04.012
  3. Cell Metab. 2023 Apr 28. pii: S1550-4131(23)00139-0. [Epub ahead of print]
    The mitochondrial unfolded protein response regulates hippocampal neural stem cell aging.
    Chih-Ling Wang, Rika Ohkubo, Wei-Chieh Mu, Wei Chen, Jiang Lan Fan, Zehan Song, Ayane Maruichi, Peter H Sudmant, Angela O Pisco, Dena B Dubal, Na Ji, Danica Chen.
      Aging results in a decline in neural stem cells (NSCs), neurogenesis, and cognitive function, and evidence is emerging to demonstrate disrupted adult neurogenesis in the hippocampus of patients with several neurodegenerative disorders. Here, single-cell RNA sequencing of the dentate gyrus of young and old mice shows that the mitochondrial protein folding stress is prominent in activated NSCs/neural progenitors (NPCs) among the neurogenic niche, and it increases with aging accompanying dysregulated cell cycle and mitochondrial activity in activated NSCs/NPCs in the dentate gyrus. Increasing mitochondrial protein folding stress results in compromised NSC maintenance and reduced neurogenesis in the dentate gyrus, neural hyperactivity, and impaired cognitive function. Reducing mitochondrial protein folding stress in the dentate gyrus of old mice improves neurogenesis and cognitive function. These results establish the mitochondrial protein folding stress as a driver of NSC aging and suggest approaches to improve aging-associated cognitive decline.
    Keywords:  SIRT1; SIRT2; SIRT3; SIRT6; SIRT7; cognitive aging; mitochondrial unfolded protein response; neural stem cell aging; sirtuin; stem cell aging
    DOI:  https://doi.org/10.1016/j.cmet.2023.04.012
  4. Sci Adv. 2023 May 03. 9(18): eadf0138
    Inter-organelle cross-talk supports acetyl-coenzyme A homeostasis and lipogenesis under metabolic stress.
    Ramya S Kuna, Avi Kumar, Karl A Wessendorf-Rodriguez, Hector Galvez, Courtney R Green, Grace H McGregor, Thekla Cordes, Reuben J Shaw, Robert U Svensson, Christian M Metallo.
      Proliferating cells rely on acetyl-CoA to support membrane biogenesis and acetylation. Several organelle-specific pathways are available for provision of acetyl-CoA as nutrient availability fluctuates, so understanding how cells maintain acetyl-CoA homeostasis under such stresses is critically important. To this end, we applied 13C isotope tracing cell lines deficient in these mitochondrial [ATP-citrate lyase (ACLY)]-, cytosolic [acetyl-CoA synthetase (ACSS2)]-, and peroxisomal [peroxisomal biogenesis factor 5 (PEX5)]-dependent pathways. ACLY knockout in multiple cell lines reduced fatty acid synthesis and increased reliance on extracellular lipids or acetate. Knockout of both ACLY and ACSS2 (DKO) severely stunted but did not entirely block proliferation, suggesting that alternate pathways can support acetyl-CoA homeostasis. Metabolic tracing and PEX5 knockout studies link peroxisomal oxidation of exogenous lipids as a major source of acetyl-CoA for lipogenesis and histone acetylation in cells lacking ACLY, highlighting a role for inter-organelle cross-talk in supporting cell survival in response to nutrient fluctuations.
    DOI:  https://doi.org/10.1126/sciadv.adf0138
  5. Sci Adv. 2023 May 03. 9(18): eadf0115
    Acetylcarnitine shuttling links mitochondrial metabolism to histone acetylation and lipogenesis.
    Luke T Izzo, Sophie Trefely, Christina Demetriadou, Jack M Drummond, Takuya Mizukami, Nina Kuprasertkul, Aimee T Farria, Phuong T T Nguyen, Nivitha Murali, Lauren Reich, Daniel S Kantner, Joshua Shaffer, Hayley Affronti, Alessandro Carrer, Andrew Andrews, Brian C Capell, Nathaniel W Snyder, Kathryn E Wellen.
      The metabolite acetyl-CoA is necessary for both lipid synthesis in the cytosol and histone acetylation in the nucleus. The two canonical precursors to acetyl-CoA in the nuclear-cytoplasmic compartment are citrate and acetate, which are processed to acetyl-CoA by ATP-citrate lyase (ACLY) and acyl-CoA synthetase short-chain 2 (ACSS2), respectively. It is unclear whether other substantial routes to nuclear-cytosolic acetyl-CoA exist. To investigate this, we generated cancer cell lines lacking both ACLY and ACSS2 [double knockout (DKO) cells]. Using stable isotope tracing, we show that both glucose and fatty acids contribute to acetyl-CoA pools and histone acetylation in DKO cells and that acetylcarnitine shuttling can transfer two-carbon units from mitochondria to cytosol. Further, in the absence of ACLY, glucose can feed fatty acid synthesis in a carnitine responsive and carnitine acetyltransferase (CrAT)-dependent manner. The data define acetylcarnitine as an ACLY- and ACSS2-independent precursor to nuclear-cytosolic acetyl-CoA that can support acetylation, fatty acid synthesis, and cell growth.
    DOI:  https://doi.org/10.1126/sciadv.adf0115
  6. Nat Commun. 2023 May 02. 14(1): 2504
    Methionine restriction constrains lipoylation and activates mitochondria for nitrogenic synthesis of amino acids.
    Wen Fang, Liu Jiang, Yibing Zhu, Sen Yang, Hong Qiu, Jiou Cheng, Qingxi Liang, Zong-Cai Tu, Cunqi Ye.
      Methionine restriction (MR) provides metabolic benefits in many organisms. However, mechanisms underlying the MR-induced effect remain incompletely understood. Here, we show in the budding yeast S. cerevisiae that MR relays a signal of S-adenosylmethionine (SAM) deprivation to adapt bioenergetic mitochondria to nitrogenic anabolism. In particular, decreases in cellular SAM constrain lipoate metabolism and protein lipoylation required for the operation of the tricarboxylic acid (TCA) cycle in the mitochondria, leading to incomplete glucose oxidation with an exit of acetyl-CoA and α-ketoglutarate from the TCA cycle to the syntheses of amino acids, such as arginine and leucine. This mitochondrial response achieves a trade-off between energy metabolism and nitrogenic anabolism, which serves as an effector mechanism promoting cell survival under MR.
    DOI:  https://doi.org/10.1038/s41467-023-38289-9
  7. JCI Insight. 2023 May 04. pii: e161096. [Epub ahead of print]
    CLUH functions as negative regulator of inflammation in human macrophages and determines ulcerative colitis pathogenesis.
    Shaziya Khan, Desh Raj, Shikha Sahu, Anam Naseer, Nishakumari C Singh, Sunaina Kumari, Sharmeen Ishteyaque, Jyotsna Sharma, Promila Lakra, Madhav Nilakanth Mugale, Arun Kumar Trivedi, Mrigank Srivastava, Tulika Chandra, Vivek Bhosale, Manoj Kumar Barthwal, Shashi Kumar Gupta, Kalyan Mitra, Aamir Nazir, Uday C Ghoshal, Amit Lahiri.
      Altered mitochondrial function without a well-defined cause has been documented in the patients with ulcerative colitis (UC). In our efforts to understand UC pathogenesis, we observed reduced expression of clustered mitochondrial homologue, CLUH, only in the active UC tissues compared to the unaffected areas from the same patient and healthy controls. Stimulation with bacterial toll like receptor (TLR) ligands similarly reduced CLUH expression in the human primary macrophages. Further, CLUH negatively regulated secretion of pro-inflammatory cytokines IL6, TNF-α and rendered a pro-inflammatory niche in the TLR stimulated macrophage. CLUH was further found to bind to mitochondrial fission protein DRP-1 and also regulated DRP-1 transcription in the human macrophages. In the TLR ligand stimulated macrophages, absence of CLUH led to enhanced DRP-1 availability for mitochondrial fission and smaller dysfunctional mitochondrial pool was observed. Mechanistically, this fissioned mitochondrial pool in turn enhanced mitochondrial ROS production, reduced mitophagy and lysosomal function in the CLUH knockout macrophages. Remarkably, our studies in the mice model of colitis with CLUH knockdown displayed exacerbated disease pathology. Taken together, this is the first report signifying the role of CLUH in UC pathogenesis, by means of regulating inflammation via maintaining mitochondrial-lysosomal functions in the human macrophages and intestinal mucosa.
    Keywords:  Autoimmune diseases; Gastroenterology
    DOI:  https://doi.org/10.1172/jci.insight.161096
  8. Elife. 2023 May 02. pii: e84330. [Epub ahead of print]12
    Mitochondrial protein import clogging as a mechanism of disease.
    Liam P Coyne, Xiaowen Wang, Jiyao Song, Ebbing de Jong, Karin Schneider, Paul T Massa, Frank A Middleton, Thomas Becker, Xin Jie Chen.
      Mitochondrial biogenesis requires the import of >1,000 mitochondrial preproteins from the cytosol. Most studies on mitochondrial protein import are focused on the core import machinery. Whether and how the biophysical properties of substrate preproteins affect overall import efficiency is underexplored. Here, we show that protein traffic into mitochondria can be disrupted by amino acid substitutions in a single substrate preprotein. Pathogenic missense mutations in ADP/ATP translocase 1 (ANT1), and its yeast homolog Aac2, cause the protein to accumulate along the protein import pathway, thereby obstructing general protein translocation into mitochondria. This impairs mitochondrial respiration, cytosolic proteostasis and cell viability independent of ANT1's nucleotide transport activity. The mutations act synergistically, as double mutant Aac2/ANT1 cause severe clogging primarily at the Translocase of the Outer Membrane (TOM) complex. This confers extreme toxicity in yeast. In mice, expression of a super-clogger ANT1 variant led to neurodegeneration and an age-dependent dominant myopathy that phenocopy ANT1-induced human disease, suggesting clogging as a mechanism of disease. More broadly, this work implies the existence of uncharacterized amino acid requirements for mitochondrial carrier proteins to avoid clogging and subsequent disease.
    Keywords:  S. cerevisiae; biochemistry; chemical biology; mouse
    DOI:  https://doi.org/10.7554/eLife.84330
  9. Exp Mol Med. 2023 May 01.
    Noncanonical role of singleminded-2s in mitochondrial respiratory chain formation in breast cancer.
    Steven W Wall, Lilia Sanchez, Kelly Scribner Tuttle, Scott J Pearson, Shivatheja Soma, Garhett L Wyatt, Hannah N Carter, Ramsey M Jenschke, Lin Tan, Sara A Martinez, Philip L Lorenzi, Vishal M Gohil, Monique Rijnkels, Weston W Porter.
      Dysregulation of cellular metabolism is a hallmark of breast cancer progression and is associated with metastasis and therapeutic resistance. Here, we show that the breast tumor suppressor gene SIM2 promotes mitochondrial oxidative phosphorylation (OXPHOS) using breast cancer cell line models. Mechanistically, we found that SIM2s functions not as a transcription factor but localizes to mitochondria and directly interacts with the mitochondrial respiratory chain (MRC) to facilitate functional supercomplex (SC) formation. Loss of SIM2s expression disrupts SC formation through destabilization of MRC Complex III, leading to inhibition of electron transport, although Complex I (CI) activity is retained. A metabolomic analysis showed that knockout of SIM2s leads to a compensatory increase in ATP production through glycolysis and accelerated glutamine-driven TCA cycle production of NADH, creating a favorable environment for high cell proliferation. Our findings indicate that SIM2s is a novel stabilizing factor required for SC assembly, providing insight into the impact of the MRC on metabolic adaptation and breast cancer progression.
    DOI:  https://doi.org/10.1038/s12276-023-00996-0
  10. Proc Natl Acad Sci U S A. 2023 05 09. 120(19): e2301047120
    Direct tests of cytochrome c and c1 functions in the electron transport chain of malaria parasites.
    Tanya J Espino-Sanchez, Henry Wienkers, Rebecca G Marvin, Shai-Anne Nalder, Aldo E García-Guerrero, Peter E VanNatta, Yasaman Jami-Alahmadi, Amanda Mixon Blackwell, Frank G Whitby, James A Wohlschlegel, Matthew T Kieber-Emmons, Christopher P Hill, Paul A Sigala.
      The mitochondrial electron transport chain (ETC) of Plasmodium malaria parasites is a major antimalarial drug target, but critical cytochrome (cyt) functions remain unstudied and enigmatic. Parasites express two distinct cyt c homologs (c and c-2) with unusually sparse sequence identity and uncertain fitness contributions. P. falciparum cyt c-2 is the most divergent eukaryotic cyt c homolog currently known and has sequence features predicted to be incompatible with canonical ETC function. We tagged both cyt c homologs and the related cyt c1 for inducible knockdown. Translational repression of cyt c and cyt c1 was lethal to parasites, which died from ETC dysfunction and impaired ubiquinone recycling. In contrast, cyt c-2 knockdown or knockout had little impact on blood-stage growth, indicating that parasites rely fully on the more conserved cyt c for ETC function. Biochemical and structural studies revealed that both cyt c and c-2 are hemylated by holocytochrome c synthase, but UV-vis absorbance and EPR spectra strongly suggest that cyt c-2 has an unusually open active site in which heme is stably coordinated by only a single axial amino acid ligand and can bind exogenous small molecules. These studies provide a direct dissection of cytochrome functions in the ETC of malaria parasites and identify a highly divergent Plasmodium cytochrome c with molecular adaptations that defy a conserved role in eukaryotic evolution.
    Keywords:  Plasmodium; cytochrome; electron transport chain; malaria; mitochondria
    DOI:  https://doi.org/10.1073/pnas.2301047120
  11. Proc Natl Acad Sci U S A. 2023 May 09. 120(19): e2218999120
    MICU1 occludes the mitochondrial calcium uniporter in divalent-free conditions.
    Macarena Rodríguez-Prados, Elena Berezhnaya, Maria Teresa Castromonte, Sergio L Menezes-Filho, Melanie Paillard, György Hajnóczky.
      Mitochondrial Ca2+ uptake is mediated by the mitochondrial uniporter complex (mtCU) that includes a tetramer of the pore-forming subunit, MCU, a scaffold protein, EMRE, and the EF-hand regulatory subunit, MICU1 either homodimerized or heterodimerized with MICU2/3. MICU1 has been proposed to regulate Ca2+ uptake via the mtCU by physically occluding the pore and preventing Ca2+ flux at resting cytoplasmic [Ca2+] (free calcium concentration) and to increase Ca2+ flux at high [Ca2+] due to cooperative activation of MICUs EF-hands. However, mtCU and MICU1 functioning when its EF-hands are unoccupied by Ca2+ is poorly studied due to technical limitations. To overcome this barrier, we have studied the mtCU in divalent-free conditions by assessing the Ru265-sensitive Na+ influx using fluorescence-based measurement of mitochondrial matrix [Na+] (free sodium concentration) rise and the ensuing depolarization and swelling. We show an increase in all these measures of Na+ uptake in MICU1KO cells as compared to wild-type (WT) and rescued MICU1KO HEK cells. However, mitochondria in WT cells and MICU1 stable-rescued cells still allowed some Ru265-sensitive Na+ influx that was prevented by MICU1 in excess upon acute overexpression. Thus, MICU1 restricts the cation flux across the mtCU in the absence of Ca2+, but even in cells with high endogenous MICU1 expression such as HEK, some mtCU seem to lack MICU1-dependent gating. We also show rearrangement of the mtCU and altered number of functional channels in MICU1KO and different rescues, and loss of MICU1 during mitoplast preparation, that together might have obscured the pore-blocking function of MICU1 in divalent-free conditions in previous studies.
    Keywords:  EMRE; MICU1; Na+; mitochondrial calcium uniporter; mitoplast
    DOI:  https://doi.org/10.1073/pnas.2218999120
  12. J Biol Chem. 2023 May 02. pii: S0021-9258(23)01802-1. [Epub ahead of print] 104774
    PARKIN UBIQUITINATION OF KINDLIN-2 ENABLES MITOCHONDRIA-ASSOCIATED METASTASIS SUPPRESSION.
    Minjeong Yeon, Irene Bertolini, Ekta Agarwal, Jagadish C Ghosh, Hsin-Yao Tang, David W Speicher, Frederick Keeney, Khalid Sossey-Alaoui, Elzbieta Pluskota, Katarzyna Bialkowska, Edward F Plow, Lucia R Languino, Emmanuel Skordalakes, M Cecilia Caino, Dario C Altieri.
      Mitochondria are signaling organelles implicated in cancer, but the mechanisms are elusive. Here, we show that Parkin, an E3 ubiquitin ligase altered in Parkinson's Disease (PD), forms a complex with the regulator of cell motility, Kindlin-2 (K2) at mitochondria of tumor cells. In turn, Parkin ubiquitinates Lys581 and Lys582 using Lys48 linkages, resulting in proteasomal degradation of K2 and shortened half-life from ∼5 h to ∼1.5 h. Loss of K2 inhibits focal adhesion turnover and β1 integrin activation, impairs membrane lamellipodia size and frequency, and inhibits mitochondrial dynamics, altogether suppressing tumor cell-ECM interactions, migration, and invasion. Conversely, Parkin does not affect tumor cell proliferation, cell cycle transitions or apoptosis. Expression of a Parkin ubiquitination-resistant K2 Lys581Ala/Lys582Ala double mutant is sufficient to restore membrane lamellipodia dynamics, correct mitochondrial fusion/fission, and preserve single-cell migration and invasion. In a 3D model of mammary gland developmental morphogenesis, impaired K2 ubiquitination drives multiple oncogenic traits of EMT, increased cell proliferation, reduced apoptosis and disrupted basal-apical polarity. Therefore, deregulated K2 is a potent oncogene and its ubiquitination by Parkin enables mitochondria-associated metastasis suppression.
    Keywords:  Kindlin-2; Parkin; metastasis suppression; tumor cell motility; ubiquitination
    DOI:  https://doi.org/10.1016/j.jbc.2023.104774
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