bims-mikwok 2022-11-27 papers

bims-mikwok

Biomed News

on Mitochondrial quality control

Issue of 2022‒11‒27
sixteen papers selected by
Avinash N. Mukkala
University of Toronto

SELECTIVE AND REVERSIBLE DISRUPTION OF MITOCHONDRIAL INNER MEMBRANE PROTEIN COMPLEXES BY LIPOPHILIC CATIONS.
Drp1 and the cytoskeleton: mechanistic nexus in mitochondrial division.
Short-term hypoxia triggers ROS and SAFB mediated nuclear matrix and mRNA splicing remodeling.
FACS-mediated isolation of native autophagic vesicles.
Mito-SiPE is a sequence-independent and PCR-free mtDNA enrichment method for accurate ultra-deep mitochondrial sequencing.
TraB, a Novel Plant ER-Mitochondrial Contact Site Protein Functions as a Mitophagy Receptor in Plants.
Mitochondrial permeability transition pore-dependent necrosis.
Autophagy promotes cell survival by maintaining NAD levels.
1,5-disubstituted-1,2,3-triazoles counteract mitochondrial dysfunction acting on F1FO-ATPase in models of cardiovascular diseases.
Real-time simultaneous imaging of temporal alterations in cytoplasmic and mitochondrial redox in single cells during cell division and cell death.
Mechanical characterization of isolated mitochondria under conditions of oxidative stress.
Apelin-13 facilitates mitochondria homeostasis via mitophagy to prevent against airway oxidative injury in asthma.
The ULK complex-LRRK1 axis regulates Parkin-mediated mitophagy via Rab7 Ser-72 phosphorylation.
Understanding the Role of Yeast Yme1 in Mitochondrial Function Using Biochemical and Proteomics Analyses.
Compact zinc finger base editors that edit mitochondrial or nuclear DNA in vitro and in vivo.
The phosphorylation of AMPKβ1 is critical for increasing autophagy and maintaining mitochondrial homeostasis in response to fatty acids.

Mitochondrion. 2022 Nov 16. pii: S1567-7249(22)00103-9. [Epub ahead of print]

SELECTIVE AND REVERSIBLE DISRUPTION OF MITOCHONDRIAL INNER MEMBRANE PROTEIN COMPLEXES BY LIPOPHILIC CATIONS.

Anezka Kafkova, Lisa Tilokani, Filip Trčka, Veronika Šrámková, Marie Vancová, Tomáš Bílý, Jana Nebesařová, Julien Prudent, Jan Trnka.

  Triphenylphosphonium (TPP) derivatives are commonly used to target chemical into mitochondria. We show that alkyl-TPP cause reversible, dose- and hydrophobicity-dependent alterations of mitochondrial morphology and function and a selective decrease of mitochondrial inner membrane proteins including subunits of the respiratory chain complexes, as well as components of the mitochondrial calcium uniporter complex. The treatment with alkyl-TPP resulted in the cleavage of the pro-fusion and cristae organisation regulator Optic atrophy-1. The structural and functional effects of alkyl-TPP were found to be reversible and not merely due to loss of membrane potential. A similar effect was observed with the mitochondria-targeted antioxidant MitoQ.

Keywords:  MitoQ; inner mitochondrial membrane; lipophilic cations; mitochondria; mitochondrial dynamics; respiratory chain

DOI:  https://doi.org/10.1016/j.mito.2022.11.006
Curr Opin Physiol. 2022 Oct;pii: 100574. [Epub ahead of print]29

Drp1 and the cytoskeleton: mechanistic nexus in mitochondrial division.

Jason A Mears, Rajesh Ramachandran.

  Dynamin-related protein 1 (Drp1), the master regulator of mitochondrial division (MD), interacts with the cytoskeletal elements, namely filamentous actin (F-actin), microtubules (MT), and septins that coincidentally converge at MD sites. However, the mechanistic contributions of these critical elements to, and their cooperativity in, MD remain poorly characterized. Emergent data indicate that the cytoskeleton plays combinatorial modulator, mediator, and effector roles in MD by 'priming' and 'channeling' Drp1 for mechanoenzymatic membrane remodeling. In this brief review, we will outline our current understanding of Drp1-cytoskeleton interactions, focusing on recent progress in the field and a plausible 'diffusion barrier' role for the cytoskeleton in MD.

Keywords:  Drp1; Mitochondria; actin; fission; microtubules; septins

DOI:  https://doi.org/10.1016/j.cophys.2022.100574
Redox Biol. 2022 Nov 17. pii: S2213-2317(22)00317-2. [Epub ahead of print]58 102545

Short-term hypoxia triggers ROS and SAFB mediated nuclear matrix and mRNA splicing remodeling.

Chrysa Taze, Sotiria Drakouli, Martina Samiotaki, George Panayotou, George Simos, Eleni Georgatsou, Ilias Mylonis.

  The cellular response to hypoxia, in addition to HIF-dependent transcriptional reprogramming, also involves less characterized transcription-independent processes, such as alternative splicing of the VEGFA transcript leading to the production of the proangiogenic VEGF form. We now show that this event depends on reorganization of the splicing machinery, triggered after short-term hypoxia by ROS production and intranuclear redistribution of the nucleoskeletal proteins SAFB1/2. Exposure to low oxygen causes fast dissociation of SAFB1/2 from the nuclear matrix, which is reversible, inhibited by antioxidant treatment, and also observed under normoxia when the mitochondrial electron transport chain is blocked. This is accompanied by altered interactions between SAFB1/2 and the splicing machinery, translocation of kinase SRPK1 to the cytoplasm, and dephosphorylation of RS-splicing factors. Depletion of SAFB1/2 under normoxia phenocopies the hypoxic and ROS-mediated switch in VEGF mRNA splicing. These data suggest that ROS-dependent remodeling of the nuclear architecture can promote production of splicing variants that facilitate adaptation to hypoxia.

Keywords:  Hypoxia; Nuclear matrix; SAFB; Splicing; VEGFA

DOI:  https://doi.org/10.1016/j.redox.2022.102545
Autophagy. 2022 Nov 23.

FACS-mediated isolation of native autophagic vesicles.

Daniel Schmitt, Süleyman Bozkurt, Pascale Henning-Domres, Heike Huesmann, Stefan Eimer, Laura Bindila, Christian Behrends, Emily Boyle, Florian Wilfling, Georg Tascher, Christian Münch, Christian Behl, Andreas Kern.

  Autophagosome isolation enables the thorough investigation of structural components and engulfed materials. Recently, we introduced a novel antibody-based FACS-mediated method for isolation of native macroautophagic/autophagic vesicles and confirmed the quality of the preparations. We performed phospholipidomic and proteomic analyses to characterize autophagic vesicle-associated phospholipids and protein cargoes under different autophagy conditions. Lipidomic analyses identified phosphoglycerides and sphingomyelins within autophagic vesicles and revealed that the lipid composition was unaffected by different rates of autophagosome formation. Proteomic analyses identified more than 4500 potential autophagy substrates and showed that in comparison to autophagic vesicles isolated under basal autophagy conditions, starvation only marginally affected the cargo profile. Proteasome inhibition, however, resulted in the enhanced degradation of ubiquitin-proteasome system components. Taken together, the novel isolation method enriched large quantities of autophagic vesicles and enabled detailed analyses of their lipid and cargo composition.

Keywords:  autophagic vesicles; autophagy; cargo profiling; lipid profiling; vesicle isolation

DOI:  https://doi.org/10.1080/15548627.2022.2151188
Commun Biol. 2022 Nov 19. 5(1): 1269

Mito-SiPE is a sequence-independent and PCR-free mtDNA enrichment method for accurate ultra-deep mitochondrial sequencing.

Darren J Walsh, David J Bernard, Faith Pangilinan, Madison Esposito, Denise Harold, Anne Parle-McDermott, Lawrence C Brody.

The analysis of somatic variation in the mitochondrial genome requires deep sequencing of mitochondrial DNA. This is ordinarily achieved by selective enrichment methods, such as PCR amplification or probe hybridization. These methods can introduce bias and are prone to contamination by nuclear-mitochondrial sequences (NUMTs), elements that can introduce artefacts into heteroplasmy analysis. We isolated intact mitochondria using differential centrifugation and alkaline lysis and subjected purified mitochondrial DNA to a sequence-independent and PCR-free method to obtain ultra-deep (>80,000X) sequencing coverage of the mitochondrial genome. This methodology avoids false-heteroplasmy calls that occur when long-range PCR amplification is used for mitochondrial DNA enrichment. Previously published methods employing mitochondrial DNA purification did not measure mitochondrial DNA enrichment or utilise high coverage short-read sequencing. Here, we describe a protocol that yields mitochondrial DNA and have quantified the increased level of mitochondrial DNA post-enrichment in 7 different mouse tissues. This method will enable researchers to identify changes in low frequency heteroplasmy without introducing PCR biases or NUMT contamination that are incorrectly identified as heteroplasmy when long-range PCR is used.

DOI: https://doi.org/10.1038/s42003-022-04182-2
Autophagy. 2022 Nov 24.

TraB, a Novel Plant ER-Mitochondrial Contact Site Protein Functions as a Mitophagy Receptor in Plants.

Patrick Duckney, Chengyang Li, Patrick J Hussey, Pengwei Wang.

  Autophagic degradation of mitochondria (known as mitophagy) is known to occur in all eukaryotes, and is important for the turnover of damaged mitochondria and recycling of nutrients during starvation. Targeting of mitochondria for autophagic degradation is regulated by recognition of mitochondrial-localized mitophagy receptors by the autophagy adaptor protein, ATG8, which regulates the formation of phagophore membranes to encapsulate mitochondrial cargo. Mitophagy receptor proteins have been well characterized in animals and yeast; however, proteins that function as mitophagy receptors in plants have not been discovered until now. We have recently characterized the plant TraB-family proteins AT1G05270/TRB1 and AT2G32340/TRB2, as novel mitophagy receptors, elucidating novel mechanisms of mitophagy in plants.

Keywords:  Arabidopsis; ER-mitochondrial contact sites; TraB-family proteins; VAP27; mitophagy; mitophagy receptor

DOI:  https://doi.org/10.1080/15548627.2022.2151190
J Mol Cell Cardiol. 2022 Nov 18. pii: S0022-2828(22)00563-6. [Epub ahead of print]

Mitochondrial permeability transition pore-dependent necrosis.

Dexter J Robichaux, Mikako Harata, Elizabeth Murphy, Jason Karch.

  Mitochondrial permeability transition pore (mPTP)-dependent necrotic cell death is a form of necrotic cell death that is driven by mitochondrial dysfunction by the opening of the mPTP and is triggered by increases in matrix levels of Ca2+ and reactive oxygen species. This form of cell death has been implicated in ischemic injuries of the heart and brain as well as numerous degenerative diseases in the brain and skeletal muscle. This review focuses on the molecular triggers and regulators of mPTP-dependent necrosis in the context of myocardial ischemia reperfusion injury. Research over the past 50 years has led to the identity of regulators and putative pore-forming components of the mPTP. Finally, downstream consequences of activation of the mPTP as well as ongoing questions and areas of research are discussed. These questions pose a particular interest as targeting the mPTP could potentially represent an efficacious therapeutic strategy to reduce infarct size following an ischemic event.

Keywords:  ANT; ATP synthase; BAK; BAX; Calcium; CypD; Ischemia reperfusion; MPTP; Mitochondria; Mitochondrial dysfunction; Necrosis; Permeability transition; ROS

DOI:  https://doi.org/10.1016/j.yjmcc.2022.11.003
Dev Cell. 2022 Nov 21. pii: S1534-5807(22)00760-2. [Epub ahead of print]57(22): 2584-2598.e11

Autophagy promotes cell survival by maintaining NAD levels.

Tetsushi Kataura, Lucia Sedlackova, Elsje G Otten, Ruchika Kumari, David Shapira, Filippo Scialo, Rhoda Stefanatos, Kei-Ichi Ishikawa, George Kelly, Elena Seranova, Congxin Sun, Dorothea Maetzel, Niall Kenneth, Sergey Trushin, Tong Zhang, Eugenia Trushina, Charles C Bascom, Ryan Tasseff, Robert J Isfort, John E Oblong, Satomi Miwa, Michael Lazarou, Rudolf Jaenisch, Masaya Imoto, Shinji Saiki, Manolis Papamichos-Chronakis, Ravi Manjithaya, Oliver D K Maddocks, Alberto Sanz, Sovan Sarkar, Viktor I Korolchuk.

  Autophagy is an essential catabolic process that promotes the clearance of surplus or damaged intracellular components. Loss of autophagy in age-related human pathologies contributes to tissue degeneration through a poorly understood mechanism. Here, we identify an evolutionarily conserved role of autophagy from yeast to humans in the preservation of nicotinamide adenine dinucleotide (NAD) levels, which are critical for cell survival. In respiring mouse fibroblasts with autophagy deficiency, loss of mitochondrial quality control was found to trigger hyperactivation of stress responses mediated by NADases of PARP and Sirtuin families. Uncontrolled depletion of the NAD(H) pool by these enzymes ultimately contributed to mitochondrial membrane depolarization and cell death. Pharmacological and genetic interventions targeting several key elements of this cascade improved the survival of autophagy-deficient yeast, mouse fibroblasts, and human neurons. Our study provides a mechanistic link between autophagy and NAD metabolism and identifies targets for interventions in human diseases associated with autophagic, lysosomal, and mitochondrial dysfunction.

Keywords:  DNA damage; NAD; PARP; Sirtuins; ageing; autophagy; metabolism; mitochondria; mitophagy

DOI:  https://doi.org/10.1016/j.devcel.2022.10.008
Pharmacol Res. 2022 Nov 18. pii: S1043-6618(22)00507-2. [Epub ahead of print] 106561

1,5-disubstituted-1,2,3-triazoles counteract mitochondrial dysfunction acting on F1FO-ATPase in models of cardiovascular diseases.

Cristina Algieri, Chiara Bernardini, Saverio Marchi, Maurizio Forte, Matteo Antonio Tallarida, Franca Bianchi, Debora La Mantia, Vincenzo Algieri, Rosita Stanzione, Maria Cotugno, Paola Costanzo, Fabiana Trombetti, Loredana Maiuolo, Monica Forni, Antonio De Nino, Flavio Di Nonno, Sebastiano Sciarretta, Massimo Volpe, Speranza Rubattu, Salvatore Nesci.

  The compromised viability and function of cardiovascular cells are rescued by small molecules of triazole derivatives (Tzs), identified as 3a and 3b, by preventing mitochondrial dysfunction. The oxidative phosphorylation improves the respiratory control rate in the presence of Tzs independently of the substrates that energize the mitochondria. The F1FO-ATPase, the main candidate in mitochondrial permeability transition pore (mPTP) formation, is the biological target of Tzs and hydrophilic F1 domain of the enzyme is depicted as the binding region of Tzs. The protective effect of Tz molecules on isolated mitochondria was corroborated by immortalized cardiomyocytes results. Indeed, mPTP opening was attenuated in response to ionomycin. Consequently, increased mitochondrial roundness and reduction of both length and interconnections between mitochondria. In in-vitro and ex-vivo models of cardiovascular pathologies (i.e., hypoxia-reoxygenation and hypertension) were used to evaluate the Tzs cardioprotective action. Key parameters of porcine aortic endothelial cells (pAECs) oxidative metabolism and cell viability were not affected by Tzs. However, in the presence of either 1μM 3a or 0.5μM 3b the impaired cell metabolism of pAECs injured by hypoxia-reoxygenation was restored to control respiratory profile. Moreover, endothelial cells isolated from SHRSP exposed to high-salt treatment rescued the Complex I activity and the endothelial capability to form vessel-like tubes and vascular function in presence of Tzs. As a result, the specific biochemical mechanism of Tzs to block Ca2+-activated F1FO-ATPase protected cell viability and preserved the pAECs bioenergetic metabolism upon hypoxia-reoxygenation injury. Moreover, SHRSP improved vascular dysfunction in response to a high-salt treatment.

Keywords:  F(1)F(O)-ATPase; Mitochondria; binding sites; cardiovascular diseases; permeability transition pore; triazoles

DOI:  https://doi.org/10.1016/j.phrs.2022.106561
Free Radic Biol Med. 2022 Nov 22. pii: S0891-5849(22)00994-7. [Epub ahead of print]

Real-time simultaneous imaging of temporal alterations in cytoplasmic and mitochondrial redox in single cells during cell division and cell death.

Aneesh Chandrasekharan, Shankara Narayanan Varadarajan, Asha Lekshmi, T R Santhoshkumar.

  Cytosolic and organelle redox are highly interrelated, and their alterations play critical roles in both physiological and pathological cell states. This highly regulated process is crucial in life-death decisions of cells. Among organelles, the mitochondrion is the major source of intracellular-ROS and contributes to oxidation damage-induced cell death. Increase in cytosolic-redox and mitochondrial-redox is evident in cells undergoing diverse forms of cell death, such as apoptosis, necrosis, and necroptosis. The hierarchical profiling of redox signaling at the cytosol and mitochondria in a single cell is important to understand the relative contribution of each species in the initiation and shaping of cell death. Here, we demonstrate the potential application of ratiometric redox GFP (roGFP) and intensity-based redox-sensitive RFP (rxRFP) targeted to mitochondria in revealing both rapid and slow progressing changes in redox during cell division and in cells undergoing multiple modes of cell death. To generate imaging quality signal, single-cell clones stably expressing both roGFP at the cytosol and rxRFP probes targeted to mitochondria were generated. The cells provided sufficient temporal resolution with imaging-ready signal for the real-time visualization of rapidly progressing redox alterations at the cytosol and mitochondria. The long-time imaging of the cells revealed that a moderate increase in cytosolic ROS marks the division stage. Similarly, distinct forms of cell death trigger a unique and temporally regulated redox change at the cytosol and mitochondria, suggesting the potential utility of the sensor cells to dissect the nature of cell death pathways induced by specific forms of stress.

Keywords:  Cell death; Cytosolic redox; Fluorescent probes; Mitochondrial redox; Redox imaging

DOI:  https://doi.org/10.1016/j.freeradbiomed.2022.11.031
Biomicrofluidics. 2022 Dec;16(6): 064101

Mechanical characterization of isolated mitochondria under conditions of oxidative stress.

Yesaswini Komaragiri, Muzaffar H Panhwar, Bob Fregin, Gayatri Jagirdar, Carmen Wolke, Stefanie Spiegler, Oliver Otto.

Mechanical properties have been proven to be a pivotal parameter to enhance our understanding of living systems. While research during the last decades focused on cells and tissues, little is known about the role of organelle mechanics in cell function. Here, mitochondria are of specific interest due to their involvement in numerous physiological and pathological processes, e.g., in the production and homeostasis of reactive oxygen species (ROS). Using real-time fluorescence and deformability cytometry, we present a microfluidic technology that is capable to determine the mechanical properties of individual mitochondria at a throughput exceeding 100 organelles per second. Our data on several thousands of viable mitochondria isolated from rat C6 glial cells yield a homogenous population with a median deformation that scales with the applied hydrodynamic stress. In two proof-of-principle studies, we investigated the impact of exogenously and endogenously produced ROS on mitochondria mechanics. Exposing C6 cells to hydrogen peroxide (H2O2) triggers superoxide production and leads to a reduction in mitochondria size while deformation is increased. In a second study, we focused on the knockout of tafazzin, which has been associated with impaired remodeling of the mitochondrial membrane and elevated levels of ROS. Interestingly, our results reveal the same mechanical alterations as observed after the exposure to H2O2, which points to a unified biophysical mechanism of how mitochondria respond to the presence of oxidative stress. In summary, we introduce high-throughput mechanical phenotyping into the field of organelle biology with potential applications for understanding sub-cellular dynamics that have not been accessible before.

DOI: https://doi.org/10.1063/5.0111581
Mol Immunol. 2022 Nov 17. pii: S0161-5890(22)00472-2. [Epub ahead of print]153 1-9

Apelin-13 facilitates mitochondria homeostasis via mitophagy to prevent against airway oxidative injury in asthma.

Meixuan Liu, Yunxuan Zhang, Lin Dong, Zhongliang Guo.

  Oxidative stress is a major mediator in the pathogenesis of allergens-induced asthma. Mitochondria damage and dysfunction is considered to be closely related with oxidative stress. Apelin-13 is a novel multifunctional protein with anti-inflammatory and anti-oxidative properties in neuroinflammation and ischemia-reperfusion injury. However, its role in mitochondria homeostasis under asthma-associated airway oxidative injury and the potential mechanisms have not been elucidated. A murine model of asthma was established by house dust mite (HDM) allergen sensitization and challenge. The mice were received Apelin-13 protein through intraperitoneal administration before HDM challenge. Airway inflammation, histopathological changes and oxidative stress were examined. The regulatory effects of Apelin-13 on mitochondria function were evaluated using airway epithelial BEAS-2B cells, including mitochondria membrane potential (MMP), mitophagy and the possible signaling pathway. The HDM-challenged mice group exhibited robust inflammation and apoptosis in airway epithelium compared to the control group. The airway impairment in asthmatic mice was partly lessened after Apelin-13 administration. Meanwhile, protein expressions of mitophagy-related markers PINK1, Parkin, Tomm20 and LC3 were significantly increased in the lungs of Apelin-13-treated asthmatic mice. In vitro, Apelin-13 treatment significantly improved MMP levels and reduced ROS production in BEAS-2B cells exposed to HDM, accompanied with the increase of cell viability. Furthermore, Apelin-13 was found to promote the activation of PINK1/Parkin signaling in BEAS-2B cells, thereby increasing mitophagy activity and facilitating mitochondria homeostasis. These results demonstrate that Apelin-13 acts as a regulator of mitochondria homeostasis by driving mitophagy to protect against HDM allergen-induced airway oxidative injury. Apelin-13 may serve as a promising protective agent for treating asthma.

Keywords:  Airway injury; Apelin-13; Asthma; Mitochondria homeostasis; Mitophagy; Oxidative stress

DOI:  https://doi.org/10.1016/j.molimm.2022.11.012
J Cell Sci. 2022 Nov 21. pii: jcs.260395. [Epub ahead of print]

The ULK complex-LRRK1 axis regulates Parkin-mediated mitophagy via Rab7 Ser-72 phosphorylation.

Keitaro Fujita, Shin Kedashiro, Takuya Yagi, Naoki Hisamoto, Kunihiro Matsumoto, Hiroshi Hanafusa.

  Mitophagy, a type of selective autophagy, specifically targets damaged mitochondria. The ULK complex regulates Parkin-mediated mitophagy, but the mechanism through which the ULK complex initiates mitophagosome formation remains unknown. The Rab7 GTPase is a key initiator of mitophagosome formation, and Ser-72 phosphorylation of Rab7 is important for this process. We have previously identified LRRK1 as a protein kinase responsible for Rab7 Ser-72 phosphorylation. In this study, we investigated the role of LRRK1 in mitophagy. We showed that LRRK1 functions downstream of ULK1/ULK2 in Parkin-mediated mitophagy. Furthermore, we demonstrated that ectopic targeting of active LRRK1 to mitochondria is sufficient to induce the Ser-72 phosphorylation of Rab7, circumventing the requirement for ATG13, a component of the ULK complex. Thus, the ULK complex recruits LRRK1 to mitochondria by interacting with ATG13 to initiate mitophagosome formation. This study highlights the crucial role of the ULK complex-LRRK1 axis in the regulation of Parkin-mediated mitophagy.

Keywords:  LRRK1; Mitophagy; Rab7

DOI:  https://doi.org/10.1242/jcs.260395
Int J Mol Sci. 2022 Nov 08. pii: 13694. [Epub ahead of print]23(22):

Understanding the Role of Yeast Yme1 in Mitochondrial Function Using Biochemical and Proteomics Analyses.

Kwan Ting Kan, Michael G Nelson, Chris M Grant, Simon J Hubbard, Hui Lu.

  Mitochondrial i-AAA proteinase Yme1 is a multifunctional protein that plays important roles in maintaining mitochondrial protein homeostasis and regulating biogenesis and function of mitochondrial proteins. However, due to the complex interplay of mitochondria and the multifunctional nature of Yme1, how Yme1 affects mitochondrial function and protein homeostasis is still poorly understood. In this study, we investigated how YME1 deletion affects yeast Saccharomyces cerevisiae growth, chronological life span, mitochondrial protein homeostasis and function, with a focus on the mitochondrial oxidative phosphorylation (OXPHOS) complexes. Our results show that whilst the YME1 deleted cells grow poorly under respiratory conditions, they grow similar to wild-type yeast under fermentative conditions. However, the chronological life span is impaired, indicating that Yme1 plays a key role in longevity. Using highly enriched mitochondrial extract and proteomic analysis, we show that the abundances of many mitochondrial proteins are altered by YME1 deletion. Several components of the respiratory chain complexes II, III, IV and V were significantly decreased, suggesting that Yme1 plays an important role in maintaining the level and function of complexes II-V. This result was confirmed using blue native-PAGE and in-solution-based enzyme activity assays. Taken together, this study shows that Yme1 plays an important role in the chronological life span and mitochondrial protein homeostasis and has deciphered its function in maintaining the activity of mitochondrial OXPHOS complexes.

Keywords:  AAA proteinase; OXPHOS complex; mitochondrial function; mitochondrial proteomics

DOI:  https://doi.org/10.3390/ijms232213694
Nat Commun. 2022 Nov 23. 13(1): 7204

Compact zinc finger base editors that edit mitochondrial or nuclear DNA in vitro and in vivo.

Julian C W Willis, Pedro Silva-Pinheiro, Lily Widdup, Michal Minczuk, David R Liu.

DddA-derived cytosine base editors (DdCBEs) use programmable DNA-binding TALE repeat arrays, rather than CRISPR proteins, a split double-stranded DNA cytidine deaminase (DddA), and a uracil glycosylase inhibitor to mediate C•G-to-T•A editing in nuclear and organelle DNA. Here we report the development of zinc finger DdCBEs (ZF-DdCBEs) and the improvement of their editing performance through engineering their architectures, defining improved ZF scaffolds, and installing DddA activity-enhancing mutations. We engineer variants with improved DNA specificity by integrating four strategies to reduce off-target editing. We use optimized ZF-DdCBEs to install or correct disease-associated mutations in mitochondria and in the nucleus. Leveraging their small size, we use a single AAV9 to deliver into heart, liver, and skeletal muscle in post-natal mice ZF-DdCBEs that efficiently install disease-associated mutations. While off-target editing of ZF-DdCBEs is likely too high for therapeutic applications, these findings demonstrate a compact, all-protein base editing research tool for precise editing of organelle or nuclear DNA without double-strand DNA breaks.

DOI: https://doi.org/10.1038/s41467-022-34784-7
Proc Natl Acad Sci U S A. 2022 Nov 29. 119(48): e2119824119

The phosphorylation of AMPKβ1 is critical for increasing autophagy and maintaining mitochondrial homeostasis in response to fatty acids.

Eric M Desjardins, Brennan K Smith, Emily A Day, Serge Ducommun, Matthew J Sanders, Joshua P Nederveen, Rebecca J Ford, Stephen L Pinkosky, Logan K Townsend, Robert M Gutgesell, Rachel Lu, Kei Sakamoto, Gregory R Steinberg.

  Fatty acids are vital for the survival of eukaryotes, but when present in excess can have deleterious consequences. The AMP-activated protein kinase (AMPK) is an important regulator of multiple branches of metabolism. Studies in purified enzyme preparations and cultured cells have shown that AMPK is allosterically activated by small molecules as well as fatty acyl-CoAs through a mechanism involving Ser108 within the regulatory AMPK β1 isoform. However, the in vivo physiological significance of this residue has not been evaluated. In the current study, we generated mice with a targeted germline knock-in (KI) mutation of AMPKβ1 Ser108 to Ala (S108A-KI), which renders the site phospho-deficient. S108A-KI mice had reduced AMPK activity (50 to 75%) in the liver but not in the skeletal muscle. On a chow diet, S108A-KI mice had impairments in exogenous lipid-induced fatty acid oxidation. Studies in mice fed a high-fat diet found that S108A-KI mice had a tendency for greater glucose intolerance and elevated liver triglycerides. Consistent with increased liver triglycerides, livers of S108A-KI mice had reductions in mitochondrial content and respiration that were accompanied by enlarged mitochondria, suggestive of impairments in mitophagy. Subsequent studies in primary hepatocytes found that S108A-KI mice had reductions in palmitate- stimulated Cpt1a and Ppargc1a mRNA, ULK1 phosphorylation and autophagic/mitophagic flux. These data demonstrate an important physiological role of AMPKβ1 Ser108 phosphorylation in promoting fatty acid oxidation, mitochondrial biogenesis and autophagy under conditions of high lipid availability. As both ketogenic diets and intermittent fasting increase circulating free fatty acid levels, AMPK activity, mitochondrial biogenesis, and mitophagy, these data suggest a potential unifying mechanism which may be important in mediating these effects.

Keywords:  AMPK; NAFLD; autophagy; fat oxidation; mitochondria

DOI:  https://doi.org/10.1073/pnas.2119824119