bims-mikwok 2020-12-13 papers

bims-mikwok

Biomed News

on Mitochondrial quality control

Issue of 2020‒12‒13
twelve papers selected by
Avinash N. Mukkala, University of Toronto

Aberrant mitochondrial morphology and function associated with impaired mitophagy and DNM1L-MAPK/ERK signaling are found in aged mutant Parkinsonian LRRK2R1441G mice.
Deficiency in E3 Ubiquitin Ligase Parkin Exacerbates Chronic Alcohol Intake-Induced Cardiomyopathy through an Ambra1-Dependent Mechanism.
Downregulation of the zinc transporter SLC39A13 (ZIP13) is responsible for the activation of CaMKII at reperfusion and leads to myocardial ischemia/reperfusion injury in mouse hearts.
Altered glucose metabolism and cell function in keloid fibroblasts under hypoxia.
Molecular Nature and Physiological Role of the Mitochondrial Calcium Uniporter Channel.
Cryo-EM Structures Reveal Transcription Initiation Steps by Yeast Mitochondrial RNA Polymerase.
Nrf2 is activated by disruption of mitochondrial thiol homeostasis but not by enhanced mitochondrial superoxide production.
Mycobacterial origin protein Rv0674 localizes into mitochondria, interacts with D-loop and regulates OXPHOS for intracellular persistence of Mycobacterium tuberculosis.
TGR5 deficiency aggravates hepatic ischemic/reperfusion injury via inhibiting SIRT3/FOXO3/HIF-1ɑ pathway.
Mechanisms of EMRE-Dependent MCU Opening in the Mitochondrial Calcium Uniporter Complex.
Reconstitution of mammalian mitochondrial translation system capable of correct initiation and long polypeptide synthesis from leaderless mRNA.
Human GTPBP5 is involved in the late stage of mitoribosome large subunit assembly.

Autophagy. 2020 Dec 10. 1-25

Aberrant mitochondrial morphology and function associated with impaired mitophagy and DNM1L-MAPK/ERK signaling are found in aged mutant Parkinsonian LRRK2R1441G mice.

Huifang Liu, Philip Wing-Lok Ho, Chi-Ting Leung, Shirley Yin-Yu Pang, Eunice Eun Seo Chang, Zoe Yuen-Kiu Choi, Michelle Hiu-Wai Kung, David Boyer Ramsden, Shu-Leong Ho.

  Mitochondrial dysfunction causes energy deficiency and nigrostriatal neurodegeneration which is integral to the pathogenesis of Parkinson disease (PD). Clearance of defective mitochondria involves fission and ubiquitin-dependent degradation via mitophagy to maintain energy homeostasis. We hypothesize that LRRK2 (leucine-rich repeat kinase 2) mutation disrupts mitochondrial turnover causing accumulation of defective mitochondria in aging brain. We found more ubiquitinated mitochondria with aberrant morphology associated with impaired function in aged (but not young) LRRK2R1441G knockin mutant mouse striatum compared to wild-type (WT) controls. LRRK2R1441G mutant mouse embryonic fibroblasts (MEFs) exhibited reduced MAP1LC3/LC3 activation indicating impaired macroautophagy/autophagy. Mutant MEFs under FCCP-induced (mitochondrial uncoupler) stress showed increased LC3-aggregates demonstrating impaired mitophagy. Using a novel flow cytometry assay to quantify mitophagic rates in MEFs expressing photoactivatable mito-PAmCherry, we found significantly slower mitochondria clearance in mutant cells. Specific LRRK2 kinase inhibition using GNE-7915 did not alleviate impaired mitochondrial clearance suggesting a lack of direct relationship to increased kinase activity alone. DNM1L/Drp1 knockdown in MEFs slowed mitochondrial clearance indicating that DNM1L is a prerequisite for mitophagy. DNM1L knockdown in slowing mitochondrial clearance was less pronounced in mutant MEFs, indicating preexisting impaired DNM1L activation. DNM1L knockdown disrupted mitochondrial network which was more evident in mutant MEFs. DNM1L-Ser616 and MAPK/ERK phosphorylation which mediate mitochondrial fission and downstream mitophagic processes was apparent in WT using FCCP-induced stress but not mutant MEFs, despite similar total MAPK/ERK and DNM1L levels. In conclusion, aberrant mitochondria morphology and dysfunction associated with impaired mitophagy and DNM1L-MAPK/ERK signaling are found in mutant LRRK2 MEFs and mouse brain. Abbreviations: ATP: adenosine triphosphate; BAX: BCL2-associated X protein; CDK1: cyclin-dependent kinase 1; CDK5: cyclin-dependent kinase 5; CQ: chloroquine; CSF: cerebrospinal fluid; DNM1L/DRP1: dynamin 1-like; ELISA: enzyme-linked immunosorbent assay; FACS: fluorescence-activated cell sorting; FCCP: carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; LAMP2A: lysosomal-associated membrane protein 2A; LRRK2: leucine-rich repeat kinase 2; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MAPK1/ERK2: mitogen-activated protein kinase 1; MEF: mouse embryonic fibroblast; MFN1: mitofusin 1; MMP: mitochondrial membrane potential; PAmCherry: photoactivatable-mCherry; PD: Parkinson disease; PINK1: PTEN induced putative kinase 1; PRKN/PARKIN: parkin RBR E3 ubiquitin protein ligase; RAB10: RAB10, member RAS oncogene family; RAF: v-raf-leukemia oncogene; SNCA: synuclein, alpha; TEM: transmission electron microscopy; VDAC: voltage-dependent anion channel; WT: wild type; SQSTM1/p62: sequestosome 1.

Keywords:  Aging; Dnm1l/DRP1; SQSTM1/p62; knockin mice; macroautophagy; mitochondria dysfunction; mitochondrial fission; mitophagy; parkinson disease; ubiquitination

DOI:  https://doi.org/10.1080/15548627.2020.1850008
Br J Pharmacol. 2020 Dec 10.

Deficiency in E3 Ubiquitin Ligase Parkin Exacerbates Chronic Alcohol Intake-Induced Cardiomyopathy through an Ambra1-Dependent Mechanism.

Mingjie Yang, Shuyi Wang, Shouzhi Fu, Ne N Wu, Xihui Xu, Shiqun Sun, Yingmei Zhang, Jun Ren.

  BACKGROUND AND PURPOSE: Chronic alcohol consumption contributes to contractile dysfunction and unfavorable geometric changes in myocardium, accompanied with altered autophagy and disturbed mitochondrial homeostasis. The E3 ubiquitin ligase Parkin encoded by PARK2 gene maintains a fundamental role in regulating mitophagy and mitochondrial homeostasis although little is known for its role in the etiology of alcoholic cardiomyopathy.EXPERIMENTAL APPROACH: This study aimed to discern the impact of Parkin deletion in chronic alcohol-evoked cardiotoxicity. Following alcohol (4%) or control diet intake for 8 weeks, adult male wild-type (WT) and PARK2 knockout (Parkin-/- ) mice were examined using echocardiography. Cardiomyocyte mechanical properties, morphology of myocardium and mitochondrial damage were also evaluated. Besides, autophagy and mitophagy levels were assessed by LC3B and GFP-LC3 puncta, whereas lysosome-dependent autophagic flux was scrutinized using GFP-mRFP-LC3 puncta and Bafilomycin A1 treatment.
KEY RESULTS: Our data indicated that chronic alcohol exposure provoked unfavorable geometric changes in myocardium, including enlarged left ventricles, increased cell size, worsened interstitial fibrosis and mitochondrial swelling. Moreover, alcohol intake led to mitochondrial dysfunction and cardiac contractile defects, such as decreased ejection fraction, fractional shortening, peak shortening, velocity of shortening and relengthening, and prolonged relengthening duration, the effects of which were further exacerbated by Parkin knockout. Chronic alcohol exposure provoked autophagy and PINK1/Parkin-mediated mitophagy without affecting lysosome-dependent autophagic flux, the effects of which were diminished by Parkin ablation. Parkin adenovirus infection in neonatal rat cardiomyocytes further increased autophagy and protected against alcohol-induced myocardial injury, the effect of which was negated by Ambra1 (Autophagy and Beclin1 regulator 1) silencing by siRNA. Immunofluorescence staining and co-immunoprecipitation assay denoted evident interaction between Parkin and Ambra1.
CONCLUSIONS AND IMPLICATIONS: In conclusion, our results revealed that Parkin is obligatory to the cardiac homeostasis in alcohol challenge, likely due to promotion of autophagy/mitophagy and maintenance of mitochondrial integrity through its interaction with Ambra1.

Keywords:  Ambra1; Parkin; alcohol; autophagy; cardiac function; mitophagy

DOI:  https://doi.org/10.1111/bph.15340
J Mol Cell Cardiol. 2020 Dec 08. pii: S0022-2828(20)30342-4. [Epub ahead of print]

Downregulation of the zinc transporter SLC39A13 (ZIP13) is responsible for the activation of CaMKII at reperfusion and leads to myocardial ischemia/reperfusion injury in mouse hearts.

Jie Wang, Xinxin Cheng, Huanhuan Zhao, Qing Yang, Zhelong Xu.

  While Zn2+ dyshomeostasis is known to contribute to ischemia/reperfusion (I/R) injury, the roles of zinc transporters that are responsible for Zn2+ homeostasis in the pathogenesis of I/R injury remain to be addressed. This study reports that ZIP13 (SLC39A13), a zinc transporter, plays a role in myocardial I/R injury by modulating the Ca2+ signaling pathway rather than by regulating Zn2+ transport. ZIP13 is downregulated upon reperfusion in mouse hearts or in H9c2 cells at reoxygenation. Ca2+ but not Zn2+ was responsible for ZIP13 downregulation, implying that ZIP13 may play a role in I/R injury through the Ca2+ signaling pathway. In line with our assumption, knockout of ZIP13 resulted in phosphorylation (Thr287) of Ca2+-calmodulin-dependent protein kinase (CaMKII), indicating that downregulation of ZIP13 leads to CaMKII activation. Further studies showed that the heart-specific knockout of ZIP13 enhanced I/R-induced CaMKII phosphorylation in mouse hearts. In contrast, overexpression of ZIP13 suppressed I/R-induced CaMKII phosphorylation. Moreover, the heart-specific knockout of ZIP13 exacerbated myocardial infarction in mouse hearts subjected to I/R, whereas overexpression of ZIP13 reduced infarct size. In addition, knockout of ZIP13 induced increases of mitochondrial Ca2+, ROS, mitochondrial swelling, decrease in the mitochondrial respiration control rate (RCR), and dissipation of mitochondrial membrane potential (ΔΨm) in a CaMKII-dependent manner. These data suggest that downregulation of ZIP13 at reperfusion contributes to myocardial I/R injury through activation of CaMKII and the mitochondrial death pathway.

Keywords:  Ca(2+); CaMKII; Ischemia/reperfusion; Mitochondrial death pathway; ZIP13

DOI:  https://doi.org/10.1016/j.yjmcc.2020.12.002
Redox Biol. 2020 Nov 28. pii: S2213-2317(20)31020-X. [Epub ahead of print]38 101815

Altered glucose metabolism and cell function in keloid fibroblasts under hypoxia.

Qifei Wang, Pu Wang, Zelian Qin, Xin Yang, Bailin Pan, Fangfei Nie, Hongsen Bi.

  Keloids exhibit metabolic reprogramming including enhanced glycolysis and attenuated oxidative phosphorylation. Hypoxia induces a series of protective responses in mammalian cells. However, the metabolic phenotype of keloid fibroblasts under hypoxic conditions remains to be elucidated. The present study aimed to investigate glycolytic activity, mitochondrial function and morphology, and the HIF1α and PI3K/AKT signaling pathways in keloid fibroblasts (KFB) under hypoxic conditions. Our results showed that hypoxia promoted proliferation, migration invasion and collagen synthesis and inhibited apoptosis in KFB. The mRNA levels, protein expressions and enzyme activities of glycolytic enzymes in KFB were higher than those in normal skin fibroblasts (NFB) under normoxia. Moreover, hypoxia remarkedly upregulated glycolysis in KFB. Decreased activities of mitochondrial complexes and abnormal mitochondria were detected in KFB under normoxic conditions and the damage was aggravated by hypoxia. An intracellular metabolic profile assay suggested hypoxia increased glycolytic parameters except glycolytic reserve but inhibited the key parameters of mitochondrial function apart from H+ leak. Protein levels of HIF1α and phosphorylation levels of the PI3K/AKT signaling pathway were upregulated in the context of 3% oxygen. Enhanced total reactive oxygen species (ROS), mitochondrial ROS (mitoROS) and antioxidant activities of KFB were observed in response to hypoxia. Additionally, autophagy was induced by hypoxia. Our data collectively demonstrated potentiated glycolysis and attenuated mitochondrial function under hypoxia, indicating that altered glucose metabolism regulated by hypoxia could be a therapeutic target for keloids.

Keywords:  Autophagy; Cell metabolism; Fibroblasts; Hypoxia; Keloid; Redox homeostasis

DOI:  https://doi.org/10.1016/j.redox.2020.101815
Am J Physiol Cell Physiol. 2020 Dec 09.

Molecular Nature and Physiological Role of the Mitochondrial Calcium Uniporter Channel.

B Rita Alevriadou, Akshar Patel, Megan Noble, Sagnika Ghosh, Vishal M Gohil, Peter B Stathopulos, Muniswamy Madesh.

  Calcium (Ca2+) signaling is critical for cell function and cell survival. Mitochondria play a major role in regulating the intracellular Ca2+ concentration ([Ca2+]i). Mitochondrial Ca2+ uptake is an important determinant of cell fate and governs respiration, mitophagy/autophagy, and mitochondrial pathway of apoptosis. Mitochondrial Ca2+ uptake occurs via the mitochondrial Ca2+ uniporter (MCU) complex. This review summarizes the current knowledge on the function of MCU complex, regulation of MCU channel, and the role of MCU in Ca2+ homeostasis and human disease pathogenesis. The channel core consists of four MCU subunits and EMRE. Regulatory proteins that interact with them include mitochondrial Ca2+ uptake 1/2 (MICU1/2), MCU dominant negative beta subunit (MCUb), MCU regu-lator 1 (MCUR1) and solute carrier 25A23 (SLC25A23). In addition to these proteins, cardiolipin, a mito-chondrial mem-brane-specific phospholipid, has been shown to interact with the channel core. The dynamic interplay between the core and regu-latory proteins modulates MCU channel activity after sensing local changes in [Ca2+]i, reactive oxygen species, and other environmental factors. Here, we highlight the structural details of the human MCU heteromeric assemblies and their known roles in regulating mitochondrial Ca2+ homeostasis. MCU dysfunction has been shown to alter mitochondrial Ca2+ dynamics, in turn eliciting cell apoptosis. Changes in mitochondrial Ca2+ uptake have been implicated in pathological con-ditions af-fecting multiple organs, including the heart, skeletal muscle, and brain. However, our structural and functional knowledge of this vital protein complex remains incomplete and under-standing the precise role for MCU-mediated mito-chondrial Ca2+ signaling in disease requires further research ef-forts.

Keywords:  Calcium; Channel; MCU; mitochondria; uniporter

DOI:  https://doi.org/10.1152/ajpcell.00502.2020
Mol Cell. 2020 Nov 25. pii: S1097-2765(20)30794-2. [Epub ahead of print]

Cryo-EM Structures Reveal Transcription Initiation Steps by Yeast Mitochondrial RNA Polymerase.

Brent De Wijngaert, Shemaila Sultana, Anupam Singh, Chhaya Dharia, Hans Vanbuel, Jiayu Shen, Daniel Vasilchuk, Sergio E Martinez, Eaazhisai Kandiah, Smita S Patel, Kalyan Das.

  Mitochondrial RNA polymerase (mtRNAP) is crucial in cellular energy production, yet understanding of mitochondrial DNA transcription initiation lags that of bacterial and nuclear DNA transcription. We report structures of two transcription initiation intermediate states of yeast mtRNAP that explain promoter melting, template alignment, DNA scrunching, abortive synthesis, and transition into elongation. In the partially melted initiation complex (PmIC), transcription factor MTF1 makes base-specific interactions with flipped non-template (NT) nucleotides "AAGT" at -4 to -1 positions of the DNA promoter. In the initiation complex (IC), the template in the expanded 7-mer bubble positions the RNA and NTP analog UTPαS, while NT scrunches into an NT loop. The scrunched NT loop is stabilized by the centrally positioned MTF1 C-tail. The IC and PmIC states coexist in solution, revealing a dynamic equilibrium between two functional states. Frequent scrunching/unscruching transitions and the imminent steric clashes of the inflating NT loop and growing RNA:DNA with the C-tail explain abortive synthesis and transition into elongation.

Keywords:  MTF1; POLRMT; RNA polymerase; RNAP; RPO41; TFB2M; mitochondria; transcription factor

DOI:  https://doi.org/10.1016/j.molcel.2020.11.016
J Biol Chem. 2020 Dec 09. pii: jbc.RA120.016551. [Epub ahead of print]

Nrf2 is activated by disruption of mitochondrial thiol homeostasis but not by enhanced mitochondrial superoxide production.

Filip Cvetko, Stuart T Caldwell, Maureen Higgins, Takafumi Suzuki, Masayuki Yamamoto, Hiran A Prag, Richard C Hartley, Albena Dinkova-Kostova, Michael P Murphy.

  The transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) regulates the expression of genes involved in antioxidant defenses to modulate fundamental cellular processes such as mitochondrial function and glutathione metabolism. Previous reports proposed that mitochondrial ROS production and disruption of the glutathione pool activate the Nrf2 pathway, suggesting that Nrf2 senses mitochondrial redox signals and/or oxidative damage and signals to the nucleus to respond appropriately. However, until now it has not been possible to disentangle the overlapping effects of mitochondrial superoxide/ hydrogen peroxide production as a redox signal from changes to mitochondrial thiol homeostasis on Nrf2. Recently, we developed mitochondria-targeted reagents that can independently induce mitochondrial superoxide and hydrogen peroxide production (MitoPQ), or selectively disrupt mitochondrial thiol homeostasis (MitoCDNB). Using these reagents, here we have determined how enhanced generation of mitochondrial superoxide and hydrogen peroxide, or disruption of mitochondrial thiol homeostasis affect activation of the Nrf2 system in cells, which was assessed by Nrf2 protein level, nuclear translocation and expression of its target genes. We found that selective disruption of the mitochondrial glutathione pool and inhibition of its thioredoxin system by MitoCDNB led to Nrf2 activation, while using MitoPQ to enhance production of mitochondrial superoxide and hydrogen peroxide alone did not. We further showed that Nrf2 activation by MitoCDNB requires cysteine sensors of Kelch-like ECH-associated protein 1 (Keap1). These findings provide important information on how disruption to mitochondrial redox homeostasis is sensed in the cytoplasm and signaled to the nucleus.

Keywords:  Nuclear factor 2 (erythroid-derived 2-like factor) (NFE2L2) (Nrf2); mitochondria; reactive oxygen species (ROS); superoxide ion; thiol

DOI:  https://doi.org/10.1074/jbc.RA120.016551
Mitochondrion. 2020 Dec 03. pii: S1567-7249(20)30221-X. [Epub ahead of print]

Mycobacterial origin protein Rv0674 localizes into mitochondria, interacts with D-loop and regulates OXPHOS for intracellular persistence of Mycobacterium tuberculosis.

Rikesh Kumar Dubey, Ekta Dhamija, Alok Kumar Mishra, Dheeraj Soam, Shivraj Mohanrao Yabaji, Kanchan Srivastava, Kishore K Srivastava.

  Mycobacterium tuberculosis (Mtb) employs diverse strategies to survive inside the host macrophages. In this study, we have identified a conserved hypothetical protein of Mtb; Rv0674, which is present in the mitochondria of the host cell. The genetic knock-out of rv0674 (Mtb-KO) showed increased growth of Mtb. The intracellular infection with recombinant Mycobacterium smegmatis (MSMEG) expressing Rv0674 (MS_Rv0674), established that the protein is involved in promoting the apoptotic cell death of the macrophage. To investigate the mechanism incurred in mitochondria, we observed that the protein physically interacts with the control region (D-loop) of the mitochondrial DNA (LSP and HSP promoters of the loop) of the macrophages and facilitates the increased expression of mRNA in all the complexes of mitochondrial encoded OXPHOS subunits. The changes in OXPHOS levels corroborated with the ATP synthesis, mitochondrial membrane potential and superoxide production. The infection with MS_Rv0674 confirmed the role of this protein in effecting the intracellular infection. The fluorescent and confocal microscopy confirmed that the protein is localized in the mitochondria of infected macrophages and in the cells of BAL of TB patients. Together these findings indicate towards the novel function of the protein which is unlike to the earlier established mechanisms of mycobacterial physiology.

Keywords:  Apoptosis; Intracellular survival; Mitochondria; Mycobacteria; OXPHOS; Rv0674

DOI:  https://doi.org/10.1016/j.mito.2020.11.014
Cell Death Discov. 2020 Nov 01. 6(1): 116

TGR5 deficiency aggravates hepatic ischemic/reperfusion injury via inhibiting SIRT3/FOXO3/HIF-1ɑ pathway.

Qi Wang, Song Wei, Lei Li, Jiannan Qiu, Shun Zhou, Chengyu Shi, Yong Shi, Haoming Zhou, Ling Lu.

Ischemia/reperfusion (I/R) injury is responsible for liver injury during hepatic resection and liver transplantation. The plasma membrane-bound G protein-coupled bile acid receptor (TGR5) could regulate immune response in multiple liver diseases. Nevertheless, the underlying role of TGR5 in hepatic I/R injury remains largely unknown. This study aimed to investigate the potential mechanism of TGR5 in hepatic I/R injury. Wild-type (WT) and TGR5 knockout (TGR5KO) mice were used to perform hepatic I/R, and macrophages were isolated from mice for in vitro experiments. The results demonstrated that knockout of TGR5 in mice significantly exacerbated liver injury and inflammatory response. TGR5KO mice infused with WT macrophages showed relieved liver injury. Further study revealed that TGR5 knockout inhibited sirtuin 3 (SIRT3) and forkhead box O3 (FOXO3) expression. In vitro experiments indicated that SIRT3 inhibited acetylation, ubiquitination and degradation of FOXO3. FOXO3 inhibited HIF-1α transcription by binding to its promoter. TGR5 knockout inhibited SIRT3 expression, thus promoted the acetylation, ubiquitination, and degradation of FOXO3, which resulted in increased HIF-1α transcription and macrophages proinflammatory response. Collectively, TGR5 plays a critical protective role in hepatic I/R injury. FOXO3 deacetylation mediated by SIRT3 can attenuate hepatic I/R injury. TGR5 deficiency aggravates hepatic I/R injury via inhibiting SIRT3/FOXO3/HIF-1α pathway.

DOI: https://doi.org/10.1038/s41420-020-00347-2
Cell Rep. 2020 Dec 08. pii: S2211-1247(20)31475-3. [Epub ahead of print]33(10): 108486

Mechanisms of EMRE-Dependent MCU Opening in the Mitochondrial Calcium Uniporter Complex.

Anna M Van Keuren, Chen-Wei Tsai, Enrique Balderas, Madison X Rodriguez, Dipayan Chaudhuri, Ming-Feng Tsai.

  The mitochondrial calcium uniporter is a multi-subunit Ca2+-activated Ca2+ channel, made up of the pore-forming MCU protein, a metazoan-specific EMRE subunit, and MICU1/MICU2, which mediate Ca2+ activation. It has been established that metazoan MCU requires EMRE binding to conduct Ca2+, but how EMRE promotes MCU opening remains unclear. Here, we demonstrate that EMRE controls MCU activity via its transmembrane helix, while using an N-terminal PKP motif to strengthen binding with MCU. Opening of MCU requires hydrophobic interactions mediated by MCU residues near the pore's luminal end. Enhancing these interactions by single mutation allows human MCU to transport Ca2+ without EMRE. We further show that EMRE may facilitate MCU opening by stabilizing the open state in a conserved MCU gating mechanism, present also in non-metazoan MCU homologs. These results provide insights into the evolution of the uniporter machinery and elucidate the mechanism underlying the physiologically crucial EMRE-dependent MCU activation process.

Keywords:  calcium channels; calcium signaling; membrane transport; mitochondrial calcium

DOI:  https://doi.org/10.1016/j.celrep.2020.108486
Nucleic Acids Res. 2020 Dec 09. pii: gkaa1165. [Epub ahead of print]

Reconstitution of mammalian mitochondrial translation system capable of correct initiation and long polypeptide synthesis from leaderless mRNA.

Muhoon Lee, Noriko Matsunaga, Shiori Akabane, Ippei Yasuda, Takuya Ueda, Nono Takeuchi-Tomita.

Mammalian mitochondria have their own dedicated protein synthesis system, which produces 13 essential subunits of the oxidative phosphorylation complexes. We have reconstituted an in vitro translation system from mammalian mitochondria, utilizing purified recombinant mitochondrial translation factors, 55S ribosomes from pig liver mitochondria, and a tRNA mixture from either Escherichia coli or yeast. The system is capable of translating leaderless mRNAs encoding model proteins (DHFR and nanoLuciferase) or some mtDNA-encoded proteins. We show that a leaderless mRNA, encoding nanoLuciferase, is faithfully initiated without the need for any auxiliary factors other than IF-2mt and IF-3mt. We found that the ribosome-dependent GTPase activities of both the translocase EF-G1mt and the recycling factor EF-G2mt are insensitive to fusidic acid (FA), the translation inhibitor that targets bacterial EF-G homologs, and consequently the system is resistant to FA. Moreover, we demonstrate that a polyproline sequence in the protein causes 55S mitochondrial ribosome stalling, yielding ribosome nascent chain complexes. Analyses of the effects of the Mg concentration on the polyproline-mediated ribosome stalling suggested the unique regulation of peptide elongation by the mitoribosome. This system will be useful for analyzing the mechanism of translation initiation, and the interactions between the nascent peptide chain and the mitochondrial ribosome.

DOI: https://doi.org/10.1093/nar/gkaa1165
Nucleic Acids Res. 2020 Dec 07. pii: gkaa1131. [Epub ahead of print]

Human GTPBP5 is involved in the late stage of mitoribosome large subunit assembly.

Miriam Cipullo, Sarah F Pearce, Isabel G Lopez Sanchez, Shreekara Gopalakrishna, Annika Krüger, Florian Schober, Jakob D Busch, Xinping Li, Anna Wredenberg, Ilian Atanassov, Joanna Rorbach.

Human mitoribosomes are macromolecular complexes essential for translation of 11 mitochondrial mRNAs. The large and the small mitoribosomal subunits undergo a multistep maturation process that requires the involvement of several factors. Among these factors, GTP-binding proteins (GTPBPs) play an important role as GTP hydrolysis can provide energy throughout the assembly stages. In bacteria, many GTPBPs are needed for the maturation of ribosome subunits and, of particular interest for this study, ObgE has been shown to assist in the 50S subunit assembly. Here, we characterize the role of a related human Obg-family member, GTPBP5. We show that GTPBP5 interacts specifically with the large mitoribosomal subunit (mt-LSU) proteins and several late-stage mitoribosome assembly factors, including MTERF4:NSUN4 complex, MRM2 methyltransferase, MALSU1 and MTG1. Interestingly, we find that interaction of GTPBP5 with the mt-LSU is compromised in the presence of a non-hydrolysable analogue of GTP, implying a different mechanism of action of this protein in contrast to that of other Obg-family GTPBPs. GTPBP5 ablation leads to severe impairment in the oxidative phosphorylation system, concurrent with a decrease in mitochondrial translation and reduced monosome formation. Overall, our data indicate an important role of GTPBP5 in mitochondrial function and suggest its involvement in the late-stage of mt-LSU maturation.

DOI: https://doi.org/10.1093/nar/gkaa1131