bims-mitdyn 2022-07-24 papers

bims-mitdyn

Biomed News

on Mitochondrial dynamics: mechanisms

Issue of 2022–07–24
twelve papers selected by
Edmond Chan, Queen’s University, School of Medicine

Oocytes maintain ROS-free mitochondrial metabolism by suppressing complex I.
Crystal structure of human NADK2 reveals a dimeric organization and active site occlusion by lysine acetylation.
Mitochondrial genome undergoes de novo DNA methylation that protects mtDNA against oxidative damage during the peri-implantation window.
TFEB induces mitochondrial itaconate synthesis to suppress bacterial growth in macrophages.
The ER protein Creld regulates ER-mitochondria contact dynamics and respiratory complex 1 activity.
AIFM1 is a component of the mitochondrial disulfide relay that drives complex I assembly through efficient import of NDUFS5.
Mitochondria dysfunction in Charcot Marie Tooth 2B Peripheral Sensory Neuropathy.
Pathological mitophagy disrupts mitochondrial homeostasis in Leber's hereditary optic neuropathy.
The oxidoreductase CLIC4 is required to maintain mitochondrial function and resistance to exogenous oxidants in breast cancer cells.
Optogenetic Studies of Mitochondria.
An overview of the molecular mechanisms of mitophagy in yeast.
G6PD-mediated increase in de novo NADP+ biosynthesis promotes antioxidant defense and tumor metastasis.

Nature. 2022 Jul 20.

Oocytes maintain ROS-free mitochondrial metabolism by suppressing complex I.

Aida Rodríguez-Nuevo, Ariadna Torres-Sanchez, Juan M Duran, Cristian De Guirior, Maria Angeles Martínez-Zamora, Elvan Böke.

Oocytes form before birth and remain viable for several decades before fertilization1. Although poor oocyte quality accounts for most female fertility problems, little is known about how oocytes maintain cellular fitness, or why their quality eventually declines with age2. Reactive oxygen species (ROS) produced as by-products of mitochondrial activity are associated with lower rates of fertilization and embryo survival3-5. Yet, how healthy oocytes balance essential mitochondrial activity with the production of ROS is unknown. Here we show that oocytes evade ROS by remodelling the mitochondrial electron transport chain through elimination of complex I. Combining live-cell imaging and proteomics in human and Xenopus oocytes, we find that early oocytes exhibit greatly reduced levels of complex I. This is accompanied by a highly active mitochondrial unfolded protein response, which is indicative of an imbalanced electron transport chain. Biochemical and functional assays confirm that complex I is neither assembled nor active in early oocytes. Thus, we report a physiological cell type without complex I in animals. Our findings also clarify why patients with complex-I-related hereditary mitochondrial diseases do not experience subfertility. Complex I suppression represents an evolutionarily conserved strategy that allows longevity while maintaining biological activity in long-lived oocytes.

DOI: https://doi.org/10.1038/s41586-022-04979-5
Mol Cell. 2022 Jul 13. pii: S1097-2765(22)00609-8. [Epub ahead of print]

Crystal structure of human NADK2 reveals a dimeric organization and active site occlusion by lysine acetylation.

Charline Mary, Mona Hoseini Soflaee, Rushendhiran Kesavan, Muriel Gelin, Harrison Brown, G Zacharias, Thomas P Mathews, Andrew Lemoff, Corinne Lionne, Gilles Labesse, Gerta Hoxhaj.

  NAD+ kinases (NADKs) are metabolite kinases that phosphorylate NAD+ molecules to make NADP+, a limiting substrate for the generation of reducing power NADPH. NADK2 sustains mitochondrial NADPH production that enables proline biosynthesis and antioxidant defense. However, its molecular architecture and mechanistic regulation remain undescribed. Here, we report the crystal structure of human NADK2, revealing a substrate-driven mode of activation. We find that NADK2 presents an unexpected dimeric organization instead of the typical tetrameric assemblage observed for other NADKs. A specific extended segment (aa 325-365) is crucial for NADK2 dimerization and activity. Moreover, we characterize numerous acetylation events, including those on Lys76 and Lys304, which reside near the active site and inhibit NADK2 activity without disrupting dimerization, thereby reducing mitochondrial NADP(H) production, proline synthesis, and cell growth. These findings reveal important molecular insight into the structure and regulation of a vital enzyme in mitochondrial NADPH and proline metabolism.

Keywords:  NAD kinases; NADK2; NADPH metabolism; crystal structure; mitochondrial metabolism; post-translational modifications; proline metabolism

DOI:  https://doi.org/10.1016/j.molcel.2022.06.026
Proc Natl Acad Sci U S A. 2022 Jul 26. 119(30): e2201168119

Mitochondrial genome undergoes de novo DNA methylation that protects mtDNA against oxidative damage during the peri-implantation window.

Yuan Yue, Likun Ren, Chao Zhang, Kai Miao, Kun Tan, Qianying Yang, Yupei Hu, Guangyin Xi, Gang Luo, Mingyao Yang, Jingyu Zhang, Zhuocheng Hou, Lei An, Jianhui Tian.

  Mitochondrial remodeling during the peri-implantation stage is the hallmark event essential for normal embryogenesis. Among the changes, enhanced oxidative phosphorylation is critical for supporting high energy demands of postimplantation embryos, but increases mitochondrial oxidative stress, which in turn threatens mitochondrial DNA (mtDNA) stability. However, how mitochondria protect their own histone-lacking mtDNA, during this stage remains unclear. Concurrently, the mitochondrial genome gain DNA methylation by this stage. Its spatiotemporal coincidence with enhanced mitochondrial stress led us to ask if mtDNA methylation has a role in maintaining mitochondrial genome stability. Herein, we report that mitochondrial genome undergoes de novo mtDNA methylation that can protect mtDNA against enhanced oxidative damage during the peri-implantation window. Mitochondrial genome gains extensive mtDNA methylation during transition from blastocysts to postimplantation embryos, thus establishing relatively hypermethylated mtDNA from hypomethylated state in blastocysts. Mechanistic study revealed that DNA methyltransferase 3A (DNMT3A) and DNMT3B enter mitochondria during this process and bind to mtDNA, via their unique mitochondrial targeting sequences. Importantly, loss- and gain-of-function analyses indicated that DNMT3A and DNMT3B are responsible for catalyzing de novo mtDNA methylation, in a synergistic manner. Finally, we proved, in vivo and in vitro, that increased mtDNA methylation functions to protect mitochondrial genome against mtDNA damage induced by increased mitochondrial oxidative stress. Together, we reveal mtDNA methylation dynamics and its underlying mechanism during the critical developmental window. We also provide the functional link between mitochondrial epigenetic remodeling and metabolic changes, which reveals a role for nuclear-mitochondrial crosstalk in establishing mitoepigenetics and maintaining mitochondrial homeostasis.

Keywords:  DNMT3A/3B; de novo DNA methylation; mitochondrial DNA; mitochondrial oxidative damage; peri-implantation

DOI:  https://doi.org/10.1073/pnas.2201168119
Nat Metab. 2022 Jul 21.

TFEB induces mitochondrial itaconate synthesis to suppress bacterial growth in macrophages.

Ev-Marie Schuster, Maximilian W Epple, Katharina M Glaser, Michael Mihlan, Kerstin Lucht, Julia A Zimmermann, Anna Bremser, Aikaterini Polyzou, Nadine Obier, Nina Cabezas-Wallscheid, Eirini Trompouki, Andrea Ballabio, Jörg Vogel, Joerg M Buescher, Alexander J Westermann, Angelika S Rambold.

Successful elimination of bacteria in phagocytes occurs in the phago-lysosomal system, but also depends on mitochondrial pathways. Yet, how these two organelle systems communicate is largely unknown. Here we identify the lysosomal biogenesis factor transcription factor EB (TFEB) as regulator for phago-lysosome-mitochondria crosstalk in macrophages. By combining cellular imaging and metabolic profiling, we find that TFEB activation, in response to bacterial stimuli, promotes the transcription of aconitate decarboxylase (Acod1, Irg1) and synthesis of its product itaconate, a mitochondrial metabolite with antimicrobial activity. Activation of the TFEB-Irg1-itaconate signalling axis reduces the survival of the intravacuolar pathogen Salmonella enterica serovar Typhimurium. TFEB-driven itaconate is subsequently transferred via the Irg1-Rab32-BLOC3 system into the Salmonella-containing vacuole, thereby exposing the pathogen to elevated itaconate levels. By activating itaconate production, TFEB selectively restricts proliferating Salmonella, a bacterial subpopulation that normally escapes macrophage control, which contrasts TFEB's role in autophagy-mediated pathogen degradation. Together, our data define a TFEB-driven metabolic pathway between phago-lysosomes and mitochondria that restrains Salmonella Typhimurium burden in macrophages in vitro and in vivo.

DOI: https://doi.org/10.1038/s42255-022-00605-w
Sci Adv. 2022 Jul 22. 8(29): eabo0155

The ER protein Creld regulates ER-mitochondria contact dynamics and respiratory complex 1 activity.

Marie Paradis, Nicole Kucharowski, Gabriela Edwards Faret, Santiago José Maya Palacios, Christian Meyer, Birgit Stümpges, Isabell Jamitzky, Julia Kalinowski, Christoph Thiele, Reinhard Bauer, Achim Paululat, Julia Sellin, Margret Helene Bülow.

Dynamic contacts are formed between endoplasmic reticulum (ER) and mitochondria that enable the exchange of calcium and phospholipids. Disturbed contacts between ER and mitochondria impair mitochondrial dynamics and are a molecular hallmark of Parkinson's disease, which is also characterized by impaired complex I activity and dopaminergic neuron degeneration. Here, we analyzed the role of cysteine-rich with EGF-like domain (Creld), a poorly characterized risk gene for Parkinson's disease, in the regulation of mitochondrial dynamics and function. We found that loss of Creld leads to mitochondrial hyperfusion and reduced ROS signaling in Drosophila melanogaster, Xenopus tropicalis, and human cells. Creld fly mutants show differences in ER-mitochondria contacts and reduced respiratory complex I activity. The resulting low-hydrogen peroxide levels are linked to disturbed neuronal activity and lead to impaired locomotion, but not neurodegeneration, in Creld mutants. We conclude that Creld regulates ER-mitochondria communication and thereby hydrogen peroxide formation, which is required for normal neuron function.

DOI: https://doi.org/10.1126/sciadv.abo0155
EMBO J. 2022 Jul 20. e110784

AIFM1 is a component of the mitochondrial disulfide relay that drives complex I assembly through efficient import of NDUFS5.

Silja Lucia Salscheider, Sarah Gerlich, Alfredo Cabrera-Orefice, Esra Peker, Robin Alexander Rothemann, Lena Maria Murschall, Yannik Finger, Karolina Szczepanowska, Zeinab Alsadat Ahmadi, Sergio Guerrero-Castillo, Alican Erdogan, Mark Becker, Muna Ali, Markus Habich, Carmelina Petrungaro, Nele Burdina, Guenter Schwarz, Merlin Klußmann, Ines Neundorf, David A Stroud, Michael T Ryan, Aleksandra Trifunovic, Ulrich Brandt, Jan Riemer.

  The mitochondrial intermembrane space protein AIFM1 has been reported to mediate the import of MIA40/CHCHD4, which forms the import receptor in the mitochondrial disulfide relay. Here, we demonstrate that AIFM1 and MIA40/CHCHD4 cooperate beyond this MIA40/CHCHD4 import. We show that AIFM1 and MIA40/CHCHD4 form a stable long-lived complex in vitro, in different cell lines, and in tissues. In HEK293 cells lacking AIFM1, levels of MIA40 are unchanged, but the protein is present in the monomeric form. Monomeric MIA40 neither efficiently interacts with nor mediates the import of specific substrates. The import defect is especially severe for NDUFS5, a subunit of complex I of the respiratory chain. As a consequence, NDUFS5 accumulates in the cytosol and undergoes rapid proteasomal degradation. Lack of mitochondrial NDUFS5 in turn results in stalling of complex I assembly. Collectively, we demonstrate that AIFM1 serves two overlapping functions: importing MIA40/CHCHD4 and constituting an integral part of the disulfide relay that ensures efficient interaction of MIA40/CHCHD4 with specific substrates.

Keywords:  AIFM1; MIA40-CHCHD4; NDUFS5; complex I; mitochondrial disulfide relay

DOI:  https://doi.org/10.15252/embj.2022110784
Commun Biol. 2022 Jul 18. 5(1): 717

Mitochondria dysfunction in Charcot Marie Tooth 2B Peripheral Sensory Neuropathy.

Yingli Gu, Flora Guerra, Mingzheng Hu, Alexander Pope, Kijung Sung, Wanlin Yang, Simone Jetha, Thomas A Shoff, Tessanya Gunatilake, Owen Dahlkamp, Linda Zhixia Shi, Fiore Manganelli, Maria Nolano, Yue Zhou, Jianqing Ding, Cecilia Bucci, Chengbiao Wu.

Rab7 GTPase regulates mitochondrial morphology and function. Missense mutation(s) of Rab7 underlies the pathogenesis of Charcot Marie Tooth 2B (CMT2B) peripheral neuropathy. Herein, we investigate how mitochondrial morphology and function are impacted by the CMT2B associated Rab7V162M mutation. In contrast to recent studies of using heterologous overexpression systems, our results demonstrate significant mitochondrial fragmentation in both human CMT2B patient fibroblasts and CMT2B embryonic fibroblasts (MEFs). Primary cultured E18 dorsal root ganglion (DRG) sensory neurons also show mitochondrial fragmentation and altered axonal mitochondrial movement. In addition, we demonstrate that inhibitors to either the mitochondrial fission protein Drp1 or to the nucleotide binding to Rab7 normalize the mitochondrial deficits in both MEFs and E18 cultured DRG neurons. Our study reveals, for the first time, that expression of CMT2B Rab7 mutation at the physiological level enhances Drp1 activity to promote mitochondrial fission, potentially underlying selective vulnerability of peripheral sensory neurons in CMT2B pathogenesis.

DOI: https://doi.org/10.1038/s42003-022-03632-1
Cell Rep. 2022 Jul 19. pii: S2211-1247(22)00930-5. [Epub ahead of print]40(3): 111124

Pathological mitophagy disrupts mitochondrial homeostasis in Leber's hereditary optic neuropathy.

Alberto Danese, Simone Patergnani, Alessandra Maresca, Camille Peron, Andrea Raimondi, Leonardo Caporali, Saverio Marchi, Chiara La Morgia, Valentina Del Dotto, Claudia Zanna, Angelo Iannielli, Alice Segnali, Ivano Di Meo, Andrea Cavaliere, Magdalena Lebiedzinska-Arciszewska, Mariusz R Wieckowski, Andrea Martinuzzi, Milton N Moraes-Filho, Solange R Salomao, Adriana Berezovsky, Rubens Belfort, Christopher Buser, Fred N Ross-Cisneros, Alfredo A Sadun, Carlo Tacchetti, Vania Broccoli, Carlotta Giorgi, Valeria Tiranti, Valerio Carelli, Paolo Pinton.

  Leber's hereditary optic neuropathy (LHON), a disease associated with a mitochondrial DNA mutation, is characterized by blindness due to degeneration of retinal ganglion cells (RGCs) and their axons, which form the optic nerve. We show that a sustained pathological autophagy and compartment-specific mitophagy activity affects LHON patient-derived cells and cybrids, as well as induced pluripotent-stem-cell-derived neurons. This is variably counterbalanced by compensatory mitobiogenesis. The aberrant quality control disrupts mitochondrial homeostasis as reflected by defective bioenergetics and excessive reactive oxygen species production, a stress phenotype that ultimately challenges cell viability by increasing the rate of apoptosis. We counteract this pathological mechanism by using autophagy regulators (clozapine and chloroquine) and redox modulators (idebenone), as well as genetically activating mitochondrial biogenesis (PGC1-α overexpression). This study substantially advances our understanding of LHON pathophysiology, providing an integrated paradigm for pathogenesis of mitochondrial diseases and druggable targets for therapy.

Keywords:  CP: Neuroscience; LHON; autophagy; cybrids; iPSCs; mitochondria; mitophagy; mtDNA; optic nerve; retinal ganglion cells; therapy

DOI:  https://doi.org/10.1016/j.celrep.2022.111124
J Biol Chem. 2022 Jul 18. pii: S0021-9258(22)00717-7. [Epub ahead of print] 102275

The oxidoreductase CLIC4 is required to maintain mitochondrial function and resistance to exogenous oxidants in breast cancer cells.

Heba Al Khamici, Vanesa C Sanchez, Hualong Yan, Christophe Cataisson, Aleksandra M Michalowski, Howard H Yang, Luowei Li, Maxwell P Lee, Jing Huang, Stuart H Yuspa.

  The Chloride Intracellular Channel-4 (CLIC4) is one of six highly conserved proteins in the CLIC family that share high structural homology with glutathione-S-transferase (GST)-omega in the GST superfamily. While CLIC4 is a multifunctional protein that resides in multiple cellular compartments, the discovery of its enzymatic glutaredoxin-like activity in vitro suggested that it could function as an antioxidant. Here, we found that deleting CLIC4 from murine 6DT1 breast tumor cells using CRISPR enhanced the accumulation of reactive oxygen species (ROS) and sensitized cells to apoptosis in response to H2O2 as a ROS-inducing agent. In intact cells, H2O2 increased the expression of both CLIC4 mRNA and protein. In addition, increased superoxide production in 6DT1 cells lacking CLIC4 was associated with mitochondrial hyperactivity including increased mitochondrial membrane potential and mitochondrial organelle enlargement. In the absence of CLIC4, however, H2O2-induced apoptosis was associated with low expression and degradation of the anti-apoptotic mitochondrial protein Bcl2 and the negative regulator of mitochondrial ROS, UCP2. Furthermore, transcriptomic profiling of H2O2-treated control and CLIC4-null cells revealed up-regulation of genes associated with ROS-induced apoptosis and down-regulation of genes that sustain mitochondrial functions. Accordingly, tumors that formed from transplantation of CLIC4 deficient 6DT1 cells were highly necrotic. These results highlight a critical role for CLIC4 in maintaining redox-homeostasis and mitochondrial functions in 6DT1 cells. Our findings also raise the possibility of targeting CLIC4 to increase cancer cell sensitivity to chemotherapeutic drugs that are based on elevating ROS in cancer cells.

Keywords:  Antioxidant; CLIC4; Cancer cells; Cancer redox; H(2)O(2)-induced apoptosis; Mitochondrial ROS

DOI:  https://doi.org/10.1016/j.jbc.2022.102275
Methods Mol Biol. 2022 ;2501 311-324

Optogenetic Studies of Mitochondria.

Kai Chen, Patrick Ernst, Xiaoguang Margaret Liu, Lufang Zhou.

  While optogenetic approaches have been widely used for remote control of cell membrane excitability and intracellular signaling pathways, their application in mitochondrial study has been limited, largely due to the challenge of effectively and specifically expressing heterologous light-gated rhodopsin channels in the mitochondria. Here, we describe the methods for expressing functional channelrhodopsin 2 (ChR2) proteins in the mitochondrial inner membrane with an unusually long mitochondrial leading sequence and characterizing optogenetic-mediated mitochondrial membrane potential (ΔΨm) depolarization. We then illustrate how this next-generation optogenetic approach can be used to study the effect of ΔΨm on mitochondrial functions such as mitophagy, programed cell death, and preconditioning-mediated cytoprotection. We anticipate that this innovative technology will enable new insights into the mechanisms by which changes in ΔΨm differentially impacts mitochondrial and cellular functions.

Keywords:  Apoptosis; Mitochondria; Mitochondrial protein import; Mitophagy; Optogenetics; Preconditioning

DOI:  https://doi.org/10.1007/978-1-0716-2329-9_15
Biochim Biophys Acta Gen Subj. 2022 Jul 13. pii: S0304-4165(22)00121-0. [Epub ahead of print] 130203

An overview of the molecular mechanisms of mitophagy in yeast.

Ramona Schuster, Koji Okamoto.

  Autophagy-dependent selective degradation of excess or damaged mitochondria, termed mitophagy, is a tightly regulated process necessary for mitochondrial quality and quantity control. Mitochondria are highly dynamic and major sites for vital cellular processes such as ATP and iron‑sulfur cluster biogenesis. Due to their pivotal roles for immunity, apoptosis, and aging, the maintenance of mitochondrial function is of utmost importance for cellular homeostasis. In yeast, mitophagy is mediated by the receptor protein Atg32 that is localized to the outer mitochondrial membrane. Upon mitophagy induction, Atg32 expression is transcriptionally upregulated, which leads to its accumulation on the mitochondrial surface and to recruitment of the autophagic machinery via its direct interaction with Atg11 and Atg8. Importantly, post-translational modifications such as phosphorylation further fine-tune the mitophagic response. This review summarizes the current knowledge about mitophagy in yeast and its connection with mitochondrial dynamics and the ubiquitin-proteasome system.

Keywords:  Atg11; Atg32; Atg8; Autophagy; Mitochondria; Yeast

DOI:  https://doi.org/10.1016/j.bbagen.2022.130203
Sci Adv. 2022 Jul 22. 8(29): eabo0404

G6PD-mediated increase in de novo NADP+ biosynthesis promotes antioxidant defense and tumor metastasis.

Yang Zhang, Yi Xu, Wenyun Lu, Jinyang Li, Sixiang Yu, Eric J Brown, Ben Z Stanger, Joshua D Rabinowitz, Xiaolu Yang.

Metastasizing cancer cells are able to withstand high levels of oxidative stress through mechanisms that are poorly understood. Here, we show that under various oxidative stress conditions, pancreatic cancer cells markedly expand NADPH and NADP+ pools. This expansion is due to up-regulation of glucose-6-phosphate dehydrogenase (G6PD), which stimulates the cytoplasmic nicotinamide adenine dinucleotide kinase (NADK1) to produce NADP+ while converting NADP+ to NADPH. G6PD is activated by the transcription factor TAp73, which is, in turn, regulated by two pathways. Nuclear factor-erythroid 2 p45-related factor-2 suppresses expression of the ubiquitin ligase PIRH2, stabilizing the TAp73 protein. Checkpoint kinases 1/2 and E2F1 induce expression of the TAp73 gene. Levels of G6PD and its upstream activators are elevated in metastatic pancreatic cancer. Knocking down G6PD impedes pancreatic cancer metastasis, whereas forced G6PD expression promotes it. These findings reveal an intracellular network that maintains redox homeostasis through G6PD-mediated increase in de novo NADP+ biosynthesis, which may be co-opted by tumor cells to enable metastasis.

DOI: https://doi.org/10.1126/sciadv.abo0404