The oxidoreductase PYROXD1 uses NAD(P)+ as an antioxidant to sustain tRNA ligase activity in pre-tRNA splicing and unfolded protein response

نویسندگان

چکیده

•The RNA ligase RTCB is susceptible to copper-dependent oxidative inactivation•The flavoprotein PYROXD1 uses NAD(P)+ safeguard mammalian against oxidation•Depletion of impairs pre-tRNA splicing and the unfolded protein response•Myopathy-causing genetic variants fail preserve activity The tRNA complex (tRNA-LC) splices precursor tRNAs (pre-tRNA), Xbp1-mRNA during response (UPR). In aerobic conditions, a cysteine residue bound two metal ions in its ancient, catalytic subunit could make tRNA-LC inactivation. Here, we confirm this hypothesis reveal co-evolutionary association between PYROXD1, conserved essential oxidoreductase. We that preserves UPR. binds presence NAD(P)H converts RTCB-bound into NAD(P)+, typical co-enzyme. However, here acts as an antioxidant protects from inactivation, which dependent on copper ions. Genetic cause human myopathies only partially support activity. Thus, establish oxidation-sensitive metalloenzyme, safeguarded by through unexpected redox mechanism. Post-transcriptional processing molecules guarantees proper expression genome. During pre-mRNA splicing, spliceosome removes introns joins adjacent exons generate mature, functional mRNAs (Will Lührmann, 2011Will C.L. Lührmann R. Spliceosome structure function.Cold Spring Harb. Perspect. Biol. 2011; 3: a003707Crossref PubMed Scopus (935) Google Scholar). A spliceosome-independent, non-canonical pathway occurs subset (pre-tRNAs) (Phizicky Hopper, 2015Phizicky E.M. Hopper A.K. processing, modification, subcellular dynamics: past, present, future.RNA. 2015; 21: 483-485Crossref (26) Scholar; Schmidt Matera, 2020Schmidt C.A. Matera A.G. introns: presence, purpose.Wiley Interdiscip. Rev. RNA. 2020; 11: e1583Crossref (18) Scholar) mRNA encoding XBP1, most transcription factor required for (UPR) (Walter Ron, 2011Walter P. Ron D. response: stress homeostatic regulation.Science. 334: 1081-1086Crossref (3654) Since all pre-tRNAs decoding GTA, TAT, CAA codons contain introns, life (Gogakos et al., 2017Gogakos T. Brown M. Garzia A. Meyer C. Hafner Tuschl Characterizing Expression Processing Precursor Mature Human Hydro-tRNAseq PAR-CLIP.Cell Rep. 2017; 20: 1463-1475Abstract Full Text PDF (100) Non-canonical entails distinct enzymatic reactions: endonucleolytic cleavage single intron (Peebles 1979Peebles Ogden R.C. Knapp G. Abelson J. Splicing yeast precursors: two-stage reaction.Cell. 1979; 18: 27-35Abstract (89) Yoshida 2001Yoshida H. Matsui Yamamoto Okada Mori K. XBP1 induced ATF6 spliced IRE1 ER produce highly active factor.Cell. 2001; 107: 881-891Abstract (2870) ligation exon halves. Both reaction catalyzed (Jurkin 2014Jurkin Henkel Nielsen A.F. Minnich Popow Kaufmann Heindl Hoffmann Busslinger Martinez mediates controls antibody secretion plasma cells.EMBO 2014; 33: 2922-2936Crossref (112) Kosmaczewski 2014Kosmaczewski S.G. Edwards T.J. Han S.M. Eckwahl M.J. B.I. Peach S. Hesselberth J.R. Wolin S.L. Hammarlund RtcB component metazoan response.EMBO 15: 1278-1285Crossref (99) Lu 2014Lu Y. Liang F.X. Wang X. synthetic biology approach identifies UPR RtcB.Mol. Cell. 55: 758-770Abstract (142) 2011Popow Englert Weitzer Schleiffer Mierzwa B. Mechtler Trowitzsch Will Söll HSPC117 complex.Science. 331: 760-764Crossref (153) cofactor Archease, guanosine triphosphate (GTP) enable multiple cycles (Popow 2014Popow Jurkin Analysis orthologous groups reveals archease DDX1 factors.Nature. 511: 104-107Crossref (66) RTCB, enzyme present domains life. anaerobic archaeon Pyrococcus horikoshii, binuclear metalloenzyme whose site contains coordinated (Desai 2013Desai K.K. Bingman Phillips Jr., G.N. Raines R.T. Structures noncanonical mechanism histidine guanylylation.Biochemistry. 2013; 52: 2518-2525Crossref (32) 2012Englert Xia Nakamura Tanavde V. Yao Eom S.H. Konigsberg W.H. Structural mechanistic insights guanylylation RNA-splicing joining 3?-terminal phosphate 5?-OH.Proc. Natl. Acad. Sci. USA. 2012; 109: 15235-15240Crossref (38) Such sites are often sensitive inactivation under they can encounter both molecular oxygen reduced reactive species; oxidized crystal aerobically purified archaeal RCTB (Banerjee 2021Banerjee Goldgur Shuman Structure 3?-PO4/5?-OH with 5?-OH oligonucleotide.RNA. 2021; https://doi.org/10.1261/rna.078692.121Crossref (5) Scholar), well accumulation halves oxidatively stressed cells (Hanada 2013Hanada Mair Bernreuther Wainger B.J. Ichida Hanada Orthofer Cronin S.J. Komnenovic al.CLP1 links metabolism progressive motor-neuron loss.Nature. 495: 474-480Crossref (159) suggests may be case RTCB. To ensure catalysis, regulation, stability, metalloenzymes organisms have evolved protective adaptations (Imlay 2019Imlay J.A. Sethu Rohaun S.K. Evolutionary enzymes tolerate stress.Free Radic. Med. 2019; 140: 4-13Crossref (23) Adaptive mechanisms enabling function organisms, including humans, remain unknown. within tRNA-LC, inactivated stress. oxidoreductase has co-evolved sustain mouse. contrary established cellular role prevents ion-mediated our vitro reconstituted system. protect not been reported. Finally, causing display decreased oxidation. identify factors potentially providing protection maintaining Eukarya, searched proteins same species distribution subunits proteomes This analysis, using clusters groups, revealed some (e.g., plants, fungi) replaced non-homologous Trl1 (Greer 1983Greer Peebles Gegenheimer Mechanism action splicing.Cell. 1983; 32: 537-546Abstract (178) while others animals) it appears pyridine nucleotide-disulfide domain-containing 1 (PYROXD1; Uniprot: Q8WU10), putative (Figure 1A). essential, cytosolic protein, early-onset myopathy (N155S Q372H) (O’Grady 2016O’Grady G.L. Best H.A. Sztal T.E. Schartner Sanjuan-Vazquez Donkervoort Abath Neto O. Sutton R.B. Ilkovski Romero N.B. al.Variants Oxidoreductase Cause Early-Onset Myopathy Internalized Nuclei Myofibrillar Disorganization.Am. Hum. Genet. 2016; 99: 1086-1105Abstract (29) adult-onset limb-girdle-type muscular dystrophy Y354C) (Sainio 2019Sainio M.T. Välipakka Rinaldi Lapatto Paetau Ojanen Brilhante Jokela Huovinen Auranen al.Recessive mutations dystrophy.J. Neurol. 266: 353-360Crossref (8) humans. homologous textbook oxidoreductases, NADH dehydrogenases oxidases (Figures 1B S1A). Decreased respiration PYROXD1-deficient (Lornage 2019Lornage Balbueno I. Biancalana Willis Echaniz-Laguna Scheidecker Quinlivan Fardeau Malfatti E. al.Clinical, histological, characterization PYROXD1-related myopathy.Acta Neuropathol. Commun. 7: 138Crossref ability complement loss glutathione-reductase suggest also displays test whether activity, silenced interference (RNAi). depletion abrogated 1C) cell extracts S1B) impaired 1D). resulted fragments derived intron-containing, tyrosine-GTA 1E), accumulate upon S1C) levels catalysis were affected S1D), conclude cells, absence PYROXD1. found deletion embryonically lethal mice. analyze vivo, generated tamoxifen-inducible knockout (KO) heart S1E). Mice survived 30 days after tamoxifen injection S1F), time observed weakening cardiac ejection volume S1G), increased weight S1H), muscle fibrosis S1I), resulting fatal failure. Deleting Pyroxd1 gene led tyrosine-tRNA 1F), confirming interaction vivo. Our results show vivo depends characterize at biochemical level, produced recombinant Escherichia coli homogeneity affinity capture gel filtration S2A S2B). loaded prosthetic group flavin adenine dinucleotide (FAD) 2A S2C) coenzymes NADPH 2B S2D), converted NADP+ NAD+, respectively. N155S variant S2E), rate ?25-fold lower 2C indicating oxidation physiological function. Reduced re-oxidized O2 turnover S2F), but limited remains hinders access 2D; see Figure S3 detailed presentation underlying data). Model substrates containing disulfides substitute reaction, incorrectly named (Q8WU10) disulfide reductase S3). 3.2 Å resolution (see Table structural statistics) architecture three domains: NAD(P)(H)-binding domain C-terminal domain, connected presumably non-structured loop ambiguous electron density, FAD-binding FAD 3A 3B ). overall generally when compared 3C). substrate-binding motifs homologs displaced away 3C S4A) no known motifs, line lack ring covered peptide stretch I131-D133 indole W376 3B). replacement alanine S2E) oxidase ?4-fold showing further limits FAD. model NADP(H)-bound state, combined all-atom simulation approaches ensemble docking S5 model). predicts N155 involved positioning 3D) formed conformational change nucleotide binding S4B); explains reductive 2C).Table 1Table statisticsData statisticsSpace groupP3112a, b, c (Å)101.23, 101.23, 143.45Resolution20–3.2 (3.28–3.2)Rmeas (%)11.0 (186.4)CC½99.8 (56.1)I/?(I)13.58 (1.24)No. unique reflections (anomalous)26,957 (1,997)Completeness (%)99.3 (99.7)Redundancy5.5 (5.7)Refinement statisticsResolution20–3.2 (3.314–3.2)No. (non-anomalous)14,003 (1,393)Completeness (%)99.21 (99.64)Rwork/Rfree (%)0.217/0.234No. atoms Protein3,327 Ligand53B-factor Protein123.90 Ligand103.60Rmsd Bond lengths (Å)0.003 angles (°)0.56Data refinement statistics related FAD, deposited PDB: 6ZK7. Open table new tab Data Recombinant exists predominantly monomer 3E). H410 monomeric occupies similar geometric position residues dimeric, S4C), (H410A) S2E S2F). makes among family members (Argyrou Blanchard, 2004Argyrou Blanchard J.S. Flavoprotein reductases: advances chemistry function.Prog. Nucleic Acid Res. Mol. 2004; 78: 89-142Crossref (165) S4D S4E) without need homodimerize S6 evidence claim). While insect below). maintains rather than promotes protecting 4A) investigate indeed stress, treated HeLa increasing concentrations stressors menadione H2O2, monitored assay. was specifically abrogated, since endonuclease (TSEN) complex, remained intact 4B S7A). Using interstrand ligation, northern blot demonstrated dose S7A–S7E), reversible removal stressing agent 4C), rescued N-acetylcysteine (NAC), boosted even treatment oxidants (i.e., background stress) 4D). These indicate sensitivity used reversible, malleable regulation. reconstitute vitro, incubated either S7F) or S7G) proteins, Archease H2O2. H2O2 cases 4E). Prior S7H). confirmed original alone tRNA-LC. reducing TCEP prevented S7I). Co-incubation 4F) S7J) dimedone, labels sulfenic acid situ, uncovered correlation cysteine(s) slightly higher dimedone signal, speculate system significant extent driven sulfinic sulfonic acid. Under acids helicase 4F), fine-tune regulation Oxidative thus vitro. relatively high lacking sensitizing factors. hypothesized toward depend least one ion site. chelator imidazole purification, reasoned fully native metals trace buffer. this, pre-treated buffer ion-chelating resin Chelex 100, followed supplementation series regulatory metals, Mg largely where had removed pre-treatment 100 4G). Supplementation nanomolar Cu2+, other ions, restored 4H). Cu2+ sensitized near-equimolar concentrations, did affect S7K; corresponding quantification panel). Accumulation dimedone-labeled incubation similarly S7L). conditions system, amounts free Cu critical stability. tested physical mediate Co-immunoprecipitation interact, NAD(P)(H) 5A), effect mimicked ATP, GTP, ubiquinone-1. nucleotides equally capable interaction, NAD+ less efficient 5A). Mass spectrometry analysis co-immunoprecipitates expressing FLAG-PYROXD1 detected main interactors addition 5B) S8A S8B) extracts. reproduced these findings pulling down StrepII-GFP-RTCB 5C); here, binding. apparent NAD(P)+-mediated 5A occur due partial conversion NAD(P)H. interacts mainly potential nicotinamide dinucleotides added Addition made more resistant 6A S9A), suggesting alters properties. Incubation streptavidin magnetic beads coated biotinylated enabled abolished NADPH, NADP+, NADH, competitors S9B). like NADP(H) NAD(H). triggers Fenton reactions chemical systems, production (ROS) direct (Rowley Halliwell, 1985Rowley D.A. Halliwell Formation hydroxyl radicals salts.J. Inorg. Biochem. 1985; 23: 103-108Crossref (30) mode protector sensitizer, shown reconstitution experiments, context. effects included bathocuproinedisulfonic (BCS), intermediate Cu+, BCS turned nucleotides, plays crucial modulation 6B). finally composed Cu. 6C). pronounced disease 6C), oxidizes 2C, 3D, S2D, When Q372H (causing myopathy) H410A (designed based structure) proportionally impairment their activities S2F S9C). Similarly, efficiently dimeric form S4D, S4E, S9D). Heat-denatured S9E). addition, prevent, rescue, upstream cascade S9F). data demonstrate oxidizing environments. archaic preserved those co-evolution taken place; reflected modern-day context codependency cancer lines (DepMap: PYROXD1). Consistent proposed prone behaves local generator ROS, such HO? (hydroxyl) HOO? (hydroperoxyl) radicals, 6D). RNA-enzyme-metabolite (REM) concept (Hentze Preiss, 2010Hentze M.W. Preiss REM phase regulation.Trends 2010; 35: 423-426Abstract (81) RNA, GTP (Englert accommodate NAD(P)H, act suppressor stimulator, respectively, Cu-mediated 6A). believe NAD(P)(H), consequence nature allowed turn hazardous inactivating controlled process regulate tightly kinetics, molecule per double-edged sword: ensures protecting, site, produces undesirable vicinity, illustrated gain-of-function W376A, fails inhibits S9G). must operate narrow range, evidenced failure loss- Furthermore, bind dissociates generation 5C), precisely avoid would lead besides O2, additional, far unknown acceptor exist, suppress idea consistent PYROXD1’s complementing glutathione Donker

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ژورنال

عنوان ژورنال: Molecular Cell

سال: 2021

ISSN: ['1097-4164', '1097-2765']

DOI: https://doi.org/10.1016/j.molcel.2021.04.007