bims-nenemi Biomed News
on Neuroinflammation, neurodegeneration and mitochondria
Issue of 2023–11–05
thirteen papers selected by
Marco Tigano, Thomas Jefferson University
  1. An optimized high-yield protocol for expression and purification of monomeric full-length BAX protein.
  2. TBK1 is ubiquitinated by TRIM5α to assemble mitophagy machinery.
  3. Mitochondrial antiviral signaling protein: a potential therapeutic target in renal disease.
  4. Thiol reductive stress activates the hypoxia response pathway.
  5. A Kinase Interacting Protein 1 regulates mitochondrial protein levels in energy metabolism and promotes mitochondrial turnover after exercise.
  6. Guanine quadruplexes mediate mitochondrial RNA polymerase pausing.
  7. Autoregulatory control of mitochondrial glutathione homeostasis.
  8. Remodeling the cellular stress response for enhanced genetic code expansion in mammalian cells.
  9. Twinkle-Catalyzed Toehold-Mediated DNA Strand Displacement Reaction.
  10. Activated Drp1 Initiates the Formation of Endoplasmic Reticulum-Mitochondrial Contacts via Shrm4-Mediated Actin Bundling.
  11. Methane-Rich Saline Suppresses ER-Mitochondria Contact and Activation of the NLRP3 Inflammasome by Regulating the PERK Signaling Pathway to Ameliorate Intestinal Ischemia‒Reperfusion Injury.
  12. Correction: Independent regulation of mitochondrial DNA quantity and quality in Caenorhabditis elegans primordial germ cells.
  13. Neo-functionalization in Saccharomyces cerevisiae: a novel Nrg1-Rtg3 chimeric transcriptional modulator is essential to maintain mitochondrial DNA integrity.
  1. bioRxiv. 2023 Oct 19. pii: 2023.10.16.562589. [Epub ahead of print]
    An optimized high-yield protocol for expression and purification of monomeric full-length BAX protein.
    Yiyang Chen, Jesse D Gelles, Jarvier N Mohammed, Jerry Edward Chipuk.
      Diverse developmental signals and pro-death stresses converge on regulation of the mitochondrial pathway of apoptosis. BAX, a pro-apoptotic BCL-2 effector, directly forms proteolipid pores in the outer mitochondrial member to activate the mitochondrial pathway of apoptosis. BAX is a viable pharmacological target for various human diseases, and increasing efforts have been made to study the molecular regulation of BAX and identify small molecules selectively targeting BAX. However, generating large quantities of monomeric and functionally-competent BAX has been challenging due to its aggregation-prone nature. Additionally, there is a lack of detailed and instructional protocols available for investigators who are not already familiar with recombinant BAX production. Here, we present a comprehensive high-yield protocol for expressing, purifying, and storing functional recombinant BAX protein. We utilize an intein-tagged BAX construct and employ a two-step chromatography strategy to capture and purify BAX, and provide example standard assays to observe BAX activation. We also highlight best practices for handling and storing BAX to effectively preserve its quality, shelf-life, and function.
    DOI:  https://doi.org/10.1101/2023.10.16.562589
  2. bioRxiv. 2023 Oct 20. pii: 2023.10.19.563195. [Epub ahead of print]
    TBK1 is ubiquitinated by TRIM5α to assemble mitophagy machinery.
    Bhaskar Saha, Hallvard Olsvik, Geneva L Williams, Seeun Oh, Gry Evjen, Eva Sjøttem, Michael A Mandell.
      Ubiquitination of mitochondrial proteins provides a basis for the downstream recruitment of mitophagy machinery, yet whether ubiquitination of the machinery itself contributes to mitophagy is unknown. Here, we show that K63-linked polyubiquitination of the key mitophagy regulator TBK1 is essential for its mitophagy functions. This modification is catalyzed by the ubiquitin ligase TRIM5α. Mitochondrial damage triggers TRIM5α's auto-ubiquitination and its interaction with ubiquitin-binding autophagy adaptors including NDP52, optineurin, and NBR1. Autophagy adaptors, along with TRIM27, enable TRIM5α to engage with TBK1. TRIM5α with intact ubiquitination function is required for the proper accumulation of active TBK1 on damaged mitochondria in Parkin-dependent and Parkin-independent mitophagy pathways. Additionally, we show that TRIM5α can directly recruit autophagy initiation machinery to damaged mitochondria. Our data support a model in which TRIM5α provides a self-amplifying, mitochondria-localized, ubiquitin-based, assembly platform for TBK1 and mitophagy adaptors that is ultimately required to recruit the core autophagy machinery.
    DOI:  https://doi.org/10.1101/2023.10.19.563195
  3. Front Immunol. 2023 ;14 1266461
    Mitochondrial antiviral signaling protein: a potential therapeutic target in renal disease.
    Meng Wu, Zhiyin Pei, Guangfeng Long, Hongbing Chen, Zhanjun Jia, Weiwei Xia.
      Mitochondrial antiviral signaling protein (MAVS) is a key innate immune adaptor on the outer mitochondrial membrane that acts as a switch in the immune signal transduction response to viral infections. Some studies have reported that MAVS mediates NF-κB and type I interferon signaling during viral infection and is also required for optimal NLRP3 inflammasome activity. Recent studies have reported that MAVS is involved in various cancers, systemic lupus erythematosus, kidney diseases, and cardiovascular diseases. Herein, we summarize the structure, activation, pathophysiological roles, and MAVS-based therapies for renal diseases. This review provides novel insights into MAVS's role and therapeutic potential in the pathogenesis of renal diseases.
    Keywords:  MAVS; NLRP3; inflammation; innate immune; renal disease
    DOI:  https://doi.org/10.3389/fimmu.2023.1266461
  4. EMBO J. 2023 Oct 02. e114093
    Thiol reductive stress activates the hypoxia response pathway.
    Ravi, Ajay Kumar, Shalmoli Bhattacharyya, Jogender Singh.
      Owing to their capability to disrupt the oxidative protein folding environment in the endoplasmic reticulum (ER), thiol antioxidants, such as dithiothreitol (DTT), are used as ER-specific stressors. We recently showed that thiol antioxidants modulate the methionine-homocysteine cycle by upregulating an S-adenosylmethionine-dependent methyltransferase, rips-1, in Caenorhabditis elegans. However, the changes in cellular physiology induced by thiol stress that modulate the methionine-homocysteine cycle remain uncharacterized. Here, using forward genetic screens in C. elegans, we discover that thiol stress enhances rips-1 expression via the hypoxia response pathway. We demonstrate that thiol stress activates the hypoxia response pathway. The activation of the hypoxia response pathway by thiol stress is conserved in human cells. The hypoxia response pathway enhances thiol toxicity via rips-1 expression and confers protection against thiol toxicity via rips-1-independent mechanisms. Finally, we show that DTT might activate the hypoxia response pathway by producing hydrogen sulfide. Our studies reveal an intriguing interaction between thiol-mediated reductive stress and the hypoxia response pathway and challenge the current model that thiol antioxidant DTT disrupts only the ER milieu in the cell.
    Keywords:  C. elegans; endoplasmic reticulum; hif‐1; hypoxia; reductive stress
    DOI:  https://doi.org/10.15252/embj.2023114093
  5. Sci Rep. 2023 Nov 01. 13(1): 18822
    A Kinase Interacting Protein 1 regulates mitochondrial protein levels in energy metabolism and promotes mitochondrial turnover after exercise.
    Kirsten T Nijholt, Pablo I Sánchez-Aguilera, Belend Mahmoud, Albert Gerding, Justina C Wolters, Anouk H G Wolters, Ben N G Giepmans, Herman H W Silljé, Rudolf A de Boer, Barbara M Bakker, B Daan Westenbrink.
      A Kinase Interacting Protein 1 (AKIP1) is a signalling adaptor that promotes mitochondrial respiration and attenuates mitochondrial oxidative stress in cultured cardiomyocytes. We sought to determine whether AKIP1 influences mitochondrial function and the mitochondrial adaptation in response to exercise in vivo. We assessed mitochondrial respiratory capacity, as well as electron microscopy and mitochondrial targeted-proteomics in hearts from mice with cardiomyocyte-specific overexpression of AKIP1 (AKIP1-TG) and their wild type (WT) littermates. These parameters were also assessed after four weeks of voluntary wheel running. In contrast to our previous in vitro study, respiratory capacity measured as state 3 respiration on palmitoyl carnitine was significantly lower in AKIP1-TG compared to WT mice, whereas state 3 respiration on pyruvate remained unaltered. Similar findings were observed for maximal respiration, after addition of FCCP. Mitochondrial DNA damage and oxidative stress markers were not elevated in AKIP1-TG mice and gross mitochondrial morphology was similar. Mitochondrial targeted-proteomics did reveal reductions in mitochondrial proteins involved in energy metabolism. Exercise performance was comparable between genotypes, whereas exercise-induced cardiac hypertrophy was significantly increased in AKIP1-TG mice. After exercise, mitochondrial state 3 respiration on pyruvate substrates was significantly lower in AKIP1-TG compared with WT mice, while respiration on palmitoyl carnitine was not further decreased. This was associated with increased mitochondrial fission on electron microscopy, and the activation of pathways associated with mitochondrial fission and mitophagy. This study suggests that AKIP1 regulates the mitochondrial proteome involved in energy metabolism and promotes mitochondrial turnover after exercise. Future studies are required to unravel the mechanistic underpinnings and whether the mitochondrial changes are required for the AKIP1-induced physiological cardiac growth.
    DOI:  https://doi.org/10.1038/s41598-023-45961-z
  6. bioRxiv. 2023 Oct 19. pii: 2023.10.17.562821. [Epub ahead of print]
    Guanine quadruplexes mediate mitochondrial RNA polymerase pausing.
    Ryan Snyder, Don Delker, Joshua T Burdick, Vivian G Cheung, Jason A Watts.
      The information content within nucleic acids extends beyond the primary sequence to include secondary structures with functional roles in cells. Guanine-rich sequences form structures called guanine quadruplexes (G4) that result from non-canonical base pairing between guanine residues. These stable structures are enriched in gene promoters and have been correlated with the locations of RNA polymerase II pausing (Pol II). While promoter-proximal RNA polymerase pausing regulates gene expression, the effects of guanine quadruplexes on gene transcription have been less clear. We determined the pattern of mitochondrial RNA polymerase (mtRNAP) pausing in human fibroblasts and found that it pauses over 400 times on the mitochondrial genome. We identified quadruplexes as a mediator of mtRNAP pausing and show that stabilization of quadruplexes impeded transcription by mtRNAP. Gene products encoded by the mitochondrial genome are required for oxidative phosphorylation and the decreased transcription by mtRNAP resulted in lower expression of mitochondrial genes and significantly reduced ATP generation. Energy from mitochondria is essential for transport function in renal epithelia, and impeded mitochondrial transcription inhibits transport function in renal proximal tubule cells. These results link formation of guanine quadruplex structures to regulation of mtRNAP elongation and mitochondrial function.
    DOI:  https://doi.org/10.1101/2023.10.17.562821
  7. Science. 2023 Nov 02. eadf4154
    Autoregulatory control of mitochondrial glutathione homeostasis.
    Yuyang Liu, Shanshan Liu, Anju Tomar, Frederick S Yen, Gokhan Unlu, Nathalie Ropek, Ross A Weber, Ying Wang, Artem Khan, Mark Gad, Junhui Peng, Erdem Terzi, Hanan Alwaseem, Alexandra E Pagano, Søren Heissel, Henrik Molina, Benjamin Allwein, Timothy C Kenny, Richard L Possemato, Li Zhao, Richard K Hite, Ekaterina V Vinogradova, Sheref S Mansy, Kıvanç Birsoy.
      Mitochondria must maintain adequate amounts of metabolites for protective and biosynthetic functions. However, how mitochondria sense the abundance of metabolites and regulate metabolic homeostasis is not well understood. We focused on glutathione (GSH), a critical redox metabolite in mitochondria and identified a feedback mechanism that controls its abundance through the mitochondrial GSH transporter, SLC25A39. Under physiological conditions, SLC25A39 is rapidly degraded by a mitochondrial protease, AFG3L2. Depletion of GSH dissociates AFG3L2 from SLC25A39, causing a compensatory increase in mitochondrial GSH uptake. Genetic and proteomic analysis identified a putative iron-sulfur cluster in the matrix-facing loop of SLC25A39 to be essential for this regulation, coupling mitochondrial iron homeostasis to GSH import. Altogether, our work revealed a paradigm for the autoregulatory control of metabolic homeostasis in organelles.
    DOI:  https://doi.org/10.1126/science.adf4154
  8. Nat Commun. 2023 Oct 31. 14(1): 6931
    Remodeling the cellular stress response for enhanced genetic code expansion in mammalian cells.
    Mikhail E Sushkin, Christine Koehler, Edward A Lemke.
      Genetic code expansion (GCE) reprograms the translational machinery to site-specifically incorporate noncanonical amino acids (ncAAs) into a selected protein. The efficiency of GCE in mammalian cells might be compromised by cellular stress responses, among which, the protein kinase R(PKR)-dependent eIF2α phosphorylation pathway can reduce translation rates. Here we test several strategies to engineer the eIF2α pathway and boost the rate of translation and show that such interventions increase GCE efficiency in mammalian cells. In particular, addition of the N-terminal PKR fragment (1-174) provides a substantial enhancement in cytoplasmic GCE and also in GCE realized by OTOs (orthogonally translating designer organelles), which built on the principle of 2D phase separation to enable mRNA-selective ncAA incorporation. Our study demonstrates an approach for improving the efficiency of GCE and provides a means by which the power of designer organelles can be further optimized to tune protein translation.
    DOI:  https://doi.org/10.1038/s41467-023-42689-2
  9. J Am Chem Soc. 2023 Nov 02.
    Twinkle-Catalyzed Toehold-Mediated DNA Strand Displacement Reaction.
    Anupam Singh, Gayatri Patel, Smita S Patel.
      Strand exchange between homologous nucleic acid sequences is the basis for cellular DNA repair, recombination, and genome editing technologies. Specialized enzymes catalyze cellular strand exchange; however, the reaction occurs spontaneously when a single-stranded DNA toehold can dock the invader strand on the target DNA to initiate strand exchange through branch migration. Due to its precise response, the spontaneous toehold-mediated strand displacement (TMSD) reaction is widely employed in DNA nanotechnology. However, enzyme-free TMSD suffers from slow rates, resulting in slow response times. Here, we show that human mitochondrial DNA helicase Twinkle can accelerate TMSD up to 6000-fold. Mechanistic studies indicate that Twinkle accelerates TMSD by catalyzing the docking step, which typically limits spontaneous reactions. The catalysis occurs without ATP, and Twinkle-catalyzed TMSD rates remain sensitive to base-pair mismatches. The simple catalysis, tunability, and speed improvement of the catalyzed TMSD can be leveraged in nanotechnology, requiring sensitive detection and faster response times.
    DOI:  https://doi.org/10.1021/jacs.3c04970
  10. Adv Sci (Weinh). 2023 Nov 01. e2304885
    Activated Drp1 Initiates the Formation of Endoplasmic Reticulum-Mitochondrial Contacts via Shrm4-Mediated Actin Bundling.
    Chenyang Duan, Ruixue Liu, Lei Kuang, Zisen Zhang, Dongyao Hou, Danyang Zheng, Xinming Xiang, He Huang, Liangming Liu, Tao Li.
      Excessive mitochondrial fission following ischemia and hypoxia relies on the formation of contacts between the endoplasmic reticulum and mitochondria (ER-Mito); however, the specific mechanisms behind this process remain unclear. Confocal microscopy and time course recording are used to investigate how ischemia and hypoxia affect the activation of dynamin-related protein 1 (Drp1), a protein central to mitochondrial dynamics, ER-Mito interactions, and the consequences of modifying the expression of Drp1, shroom (Shrm) 4, and inverted formin (INF) 2 on ER-Mito contact establishment. Both Drp1 activation and ER-Mito contact initiation cause excessive mitochondrial fission and dysfunction under ischemic-hypoxic conditions. The activated form of Drp1 aids in ER-Mito contact initiation by recruiting Shrm4 and promoting actin bundling between the ER and mitochondria. This process relies on the structural interplay between INF2 and scattered F-actin on the ER. This study uncovers new roles of cytoplasmic Drp1, providing valuable insights for devising strategies to manage mitochondrial imbalances in the context of ischemic-hypoxic injury.
    Keywords:  Drp1; ER-Mito contact; actin bundling; mitochondrial fission; shrm4
    DOI:  https://doi.org/10.1002/advs.202304885
  11. Inflammation. 2023 Oct 29.
    Methane-Rich Saline Suppresses ER-Mitochondria Contact and Activation of the NLRP3 Inflammasome by Regulating the PERK Signaling Pathway to Ameliorate Intestinal Ischemia‒Reperfusion Injury.
    Zeyu Li, Ben Wang, Lifei Tian, Bobo Zheng, Xu Zhao, Ruiting Liu.
      Intestinal ischemia‒reperfusion (I/R) injury is a common pathological process in patients undergoing gastrointestinal surgery, leading to local intestinal damage and increased microvascular permeability, eventually causing extraintestinal multiple organ dysfunction or sepsis. The NLRP3-mediated inflammatory response is associated with I/R injury. Methane saline (MS) has anti-pyroptosis properties. This study aims to explore the protective effect of MS on intestinal I/R injury and its potential mechanisms. After MS pretreatment, the in vivo model was established by temporarily clipping the mouse superior mesentery artery with a noninvasive vascular clamp, and the in vitro model was established by OGD/R on Caco-2 cells. The results of HE and TUNEL staining showed intestinal barrier damage after I/R injury, which was consistent with the IHC staining results of tight junction proteins. Moreover, the expression of the NLRP3 signaling pathway was increased after I/R injury, and inhibition of NLRP3 activation reduced Caco-2 cell injury, indicating that NLRP3-mediated pyroptosis was one of the main forms of cell death after I/R injury. Subsequently, we found that MS treatment ameliorated intestinal barrier function after I/R injury by suppressing NLRP3-mediated pyroptosis. MS treatment also reduced mitochondria-associated membrane (MAM) formation, which was considered to be a platform for activation of the NLRP3 inflammasome. Importantly, MS reduced ER stress, which was related to the PERK signaling pathway. Knocking down PERK, a key protein involved in ER stress and MAM formation, reversed the protective effect of MS, indicating that MS suppressed NLRP3 by reducing ER stress and MAM formation. In conclusion, we believe that MS suppresses MAMs and activation of the NLRP3 inflammasome by regulating the PERK signaling pathway to ameliorate intestinal I/R injury.
    Keywords:  Inflammasome; Intestinal ischemia‒reperfusion injury; Methane saline; Mitochondria-associated membranes
    DOI:  https://doi.org/10.1007/s10753-023-01916-0
  12. Elife. 2023 Nov 01. pii: e93941. [Epub ahead of print]12
    Correction: Independent regulation of mitochondrial DNA quantity and quality in Caenorhabditis elegans primordial germ cells.
    Aaron Z A Schwartz, Nikita Tsyba, Yusuff Abdu, Maulik R Patel, Jeremy Nance.
      
    Keywords:  C. elegans; cell biology; developmental biology
    DOI:  https://doi.org/10.7554/eLife.93941
  13. R Soc Open Sci. 2023 Nov;10(11): 231209
    Neo-functionalization in Saccharomyces cerevisiae: a novel Nrg1-Rtg3 chimeric transcriptional modulator is essential to maintain mitochondrial DNA integrity.
    Carlos Campero-Basaldua, James González, Janeth Alejandra García, Edgar Ramírez, Hugo Hernández, Beatriz Aguirre, Nayeli Torres-Ramírez, Dariel Márquez, Norma Silvia Sánchez, Nicolás Gómez-Hernández, Ana Lilia Torres-Machorro, Lina Riego-Ruiz, Claudio Scazzocchio, Alicia González.
      In Saccharomyces cerevisiae, the transcriptional repressor Nrg1 (Negative Regulator of Glucose-repressed genes) and the β-Zip transcription factor Rtg3 (ReTroGrade regulation) mediate glucose repression and signalling from the mitochondria to the nucleus, respectively. Here, we show a novel function of these two proteins, in which alanine promotes the formation of a chimeric Nrg1/Rtg3 regulator that represses the ALT2 gene (encoding an alanine transaminase paralog of unknown function). An NRG1/NRG2 paralogous pair, resulting from a post-wide genome small-scale duplication event, is present in the Saccharomyces genus. Neo-functionalization of only one paralog resulted in the ability of Nrg1 to interact with Rtg3. Both nrg1Δ and rtg3Δ single mutant strains were unable to use ethanol and showed a typical petite (small) phenotype on glucose. Neither of the wild-type genes complemented the petite phenotype, suggesting irreversible mitochondrial DNA damage in these mutants. Neither nrg1Δ nor rtg3Δ mutant strains expressed genes encoded by any of the five polycistronic units transcribed from mitochondrial DNA in S. cerevisiae. This, and the direct measurement of the mitochondrial DNA gene complement, confirmed that irreversible damage of the mitochondrial DNA occurred in both mutant strains, which is consistent with the essential role of the chimeric Nrg1/Rtg3 regulator in mitochondrial DNA maintenance.
    Keywords:  mitochondrial genes; neo-functionalization; respiratory metabolism; transcriptional coregulators
    DOI:  https://doi.org/10.1098/rsos.231209
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