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Functional Reconstruction of a Eukaryotic-Like E1/E2/(RING) E3 Ubiquitylation Cascade from an Uncultured Archaeon

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Functional Reconstruction of a Eukaryotic-Like E1/E2/(RING) E3 Ubiquitylation Cascade from an Uncultured Archaeon ARTICLE DOI: 10.1038/s41467-017-01162-7 OPEN Functional reconstruction of a eukaryotic-like E1/E2/(RING) E3 ubiquitylation cascade from an uncultured archaeon Rory Hennell James1, Eva F. Caceres2, Alex Escasinas3, Haya Alhasan3, Julie A. Howard4, Michael J. Deery4, Thijs J.G. Ettema 2 & Nicholas P. Robinson 3 The covalent modification of protein substrates by ubiquitin regulates a diverse range of critical biological functions. Although it has been established that ubiquitin-like modifiers evolved from prokaryotic sulphur transfer proteins it is less clear how complex eukaryotic ubiquitylation system arose and diversified from these prokaryotic antecedents. The dis- covery of ubiquitin, E1-like, E2-like and small-RING finger (srfp) protein components in the Aigarchaeota and the Asgard archaea superphyla has provided a substantive step toward addressing this evolutionary question. Encoded in operons, these components are likely representative of the progenitor apparatus that founded the modern eukaryotic ubiquitin modification systems. Here we report that these proteins from the archaeon Candidatus ‘Caldiarchaeum subterraneum’ operate together as a bona fide ubiquitin modification system, mediating a sequential ubiquitylation cascade reminiscent of the eukaryotic process. Our observations support the hypothesis that complex eukaryotic ubiquitylation signalling path- ways have developed from compact systems originally inherited from an archaeal ancestor. 1 Department of Biochemistry, The University of Cambridge, Cambridge, CB2 1GA, UK. 2 Department of Cell and Molecular Biology, Science for Life Laboratory, Uppsala University, Uppsala, 751 24, Sweden. 3 Division of Biomedical and Life Sciences, Faculty of Health and Medicine, Lancaster University, Lancaster, LA1 4YG, UK. 4 Department of Biochemistry and Cambridge Systems Biology Centre, Cambridge Centre for Proteomics, The University of Cambridge, Cambridge, CB2 1GA, UK. Correspondence and requests for materials should be addressed to N.P.R. (email: [email protected]) NATURE COMMUNICATIONS | 8: 1120 | DOI: 10.1038/s41467-017-01162-7 | www.nature.com/naturecommunications 1 ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-01162-7 he complex ubiquitylation systems of eukaryotes orches- complex descendants has garnered considerable interest of late. Ttrate diverse regulatory cell signalling pathways that play Indeed, it has been hypothesised that the acquisition of a primitive instrumental roles in maintaining cellular viability. It is well prokaryotic ubiquitylation system may have been a prerequisite to documented that the attachment of the ubiquitin small modifier is permit the endosymbiotic event central to eukaryogenesis9. Fur- central to proteasomal degradation pathways, transcriptional thermore, the diversification of this protein modification system control, DNA repair, cell cycle regulation and a plethora of other has been shown to be concomitant with the increasing cellular – – regulatory pathways1 8. How these ubiquitylation systems were complexity during eukaryotic evolution10 14. acquired by the earliest eukaryotes and then subsequently devel- The canonical eukaryotic ubiquitylation process involves three oped into the sophisticated apparatus employed by the most key biochemical steps, operating in a sequential cascade, which a Aigarchaeota archaeon SCGC AAA471 F17 ASPD01000002.1_68 Aigarchaeota archaeon SCGC AAA471 G05 ASLV01000001.1_69 Aigarchaeota archaeon SCGC AAA471 J08 ASLX01000006.1_25 Thaumarchaeota archaeon JGI OTU 1 AWOE01000006.1_7 Aigarchaeota archaeon SCGC AAA471 D15 AQTB01000052.1_49 Thaumarchaeota archaeon JGI OTU 2 AWOD01000002.1_49 Aigarchaeota archaeon SCGC AAA471 E14 AQTA01000028.1_2 Thaumarchaeota archaeon JGI OTU 3 AWOC01000031.1_2 Cluster I Aigarchaeota archaeon JGI 0000001 A7 ASLY01000016.1_7 Caldiarchaeum subterraneum BAJ51327.1 Candidatus Heimdallarchaeota archaeon AB_125 OLS31900.1 Candidatus Heimdallarchaeota archaeon LC_2 OLS25722.1 Candidatus Heimdallarchaeota archaeon LC_3 OLS27773.1 Candidatus Heimdallarchaeota archaeon LC_3 OLS27494.1 Candidatus Odinarchaeota archaeon LCB_4 OLS18183.1 Candidatus Heimdallarchaeota archaeon AB_125 OLS32357.1 Candidatus Heimdallarchaeota archaeon LC_2 OLS28180.1 Candidatus Heimdallarchaeota archaeon LC_3 OLS27108.1 Cluster II Candidatus Lokiarchaeota archaeon CR_4 OLS16232.1 Lokiarchaeum sp. GC14 _75 KKK43157.1 1 kb Ubiquitin-domain protein - IPR029071, IPR000626 Putative E1-like - IPR000594 Putative E2-like ubiquiting conjugating enzyme - IPR000608 Putative E3-like - IPR013083 Putative deubiquitinating enzyme - IPR000555 Ubiquitin domain - CD Search Predicted ubiquitin / Ubl domain - Phyre2 Search Other CSUB_C1473 ubq E2l E1l srfp rpn11 CSUB_ CSUB_ CSUB_C1476 CSUB_ C1474 C1475 C1477 Fig. 1 Operonic arrangement of the putative archaeal ubiquitylation apparatus. a Gene clusters of the putative ubiquitin-like protein modifier system identified in archaeal species: the ubiquitin, E1-like, E2-like and small RING finger protein (srfp) components are coloured as indicated in the key. The Cluster I and II division is described in Supplementary Fig. 1, which provides a more comprehensive analysis showing an additional Cluster III. See Methods and Supplementary Table 2 for more details on the E1-like C-terminal ubiquitin-like (UBL) domain identification. (boxed inset): Operonic arrangement of the genes encoding the components of the C. subterraneum ubiquitylation cascade investigated in this study. The Rpn11/JAMM domain metalloprotease homologue is juxtaposed to the operon and transcribed right to left, whereas the ubiquitylation operon is transcribed left to right 2 NATURE COMMUNICATIONS | 8: 1120 | DOI: 10.1038/s41467-017-01162-7 | www.nature.com/naturecommunications NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-01162-7 ARTICLE ab c –++ – + – – + ATP – + – + ATP Coomassie ––++ ––++ E2 +++E2+ – + Rpn11 ++++++++ E1 +++E1+ ––––++++ Ubq-CLV + + – – Ubq-GG + + Ubq-FL ++++–––– Ubq-FL ––++Ubq-FL 55 kDa E1~Ubq E1~Ubq E1 E1 35 kDa E2~Ubq E2~Ubq 25 kDa Rpn11 20 kDa E2 E2 * Ubq-FL Ubq-FL Ubq-FL Ubq-CLV Ubq-CLV Ubq-GG 12 12345678 1234 Coomassie Coomassie d 2+ y 2+ y16 9 1113.66 y b8 b b 664.63 13 10 11 b b 100 100 2 7 90 90 y16 y15 y14 y10 y8 y7 y6 y5 y4 y y y 80 80 10 9 8 2+ b10 y 2+ 70 10 738.99 70 587.94 60 60 50 50 40 40 2+ y b11 10 30 789.79 30 b 2+ b 8 2+ 8 y y13 Relative abundance 20 6 2+ y 20 2+ y 14 Relative abundance y 5 y 451.28 10 7 y8 y 2+ b 509.24 15 2 b 2+ 2+ 708.18 10 y 1254.59 10 201.14 7 y8 4 385.60 b 0 1434.91 1580.16 1803.72 1960.76 0 7 901.57 971.75 1182.79 1243.77 400 600 800 1000 1200 1400 1600 1800 2000 200 400 600 800 1000 1200 1400 m/z m/z e b11 100 970.82 y14 y13 y12 y11 y10 y9 y8 90 80 70 979.72 60 2+ b11 50 647.59 2+ 2+ y9 y11 40 2+ y10 y 2+ y 2+ 30 8 12 609.95 828.19 2+ 980.62 y14 Relative abundance 20 2+ y13 10 570.04 486.17 1211.47 0 330.46 1332.37 1671.47 200400 600 800 1000 1200 1400 1600 1800 2000 m/z Fig. 2 Rpn11/JAMM metalloprotease cleavage of the C. subterraneum ubiquitin precursor to generate a canonical C-terminal di-glycine motif that is subsequently activated by the E1-like enzyme. a C. subterraneum Rpn11 specific cleavage of the C-terminus of the pro-ubiquitin generates a mature ubiquitin species. Lane 1: pro-ubiquitin only; lane 2: pro-ubiquitin plus Rpn11. (Asterisk: C-terminal truncation of Rpn11). b Auto-ubiquitylation of the E1-like and E2-like components by ATP-dependent conjugation of the cleaved ubiquitin species via the exposed ubiquitin di-glycine motif. Lane 1 and 2: uncleaved, full-length, pro-ubiquitin (Ubq-FL) plus E1 in the presence and absence of ATP, respectively; lanes 3 and 4 as in lanes 1 and 2 but with the inclusion of the E2-enzyme; lanes 5–8 as in lanes 1–4 but using Rpn11-cleaved ubiquitin (Ubq-CLV) (assay at 60 °C for 15 min). c Ubiquitin truncated by a stop-codon after the di-glycine motif (Ubq-GG) also auto-ubiquitylates the E1-like and E2-like proteins. Reactions using Ubq-GG in the absence or presence of ATP are displayed in lanes 1 and 2, respectively (cf Ubq-FL shown in lanes 3 and 4). Reactions were performed as described in b. Products in a, b and c separated by SDS-PAGE and visualised by Coomassie staining. The C-terminal Rpn11-specific cleavage site in pro-ubiquitin is displayed below panel a (solid arrow). Trypsin digestion (dashed arrow) results in the generation of the ‘TVGG’ moiety detected as an isopeptide linkage in the tandem mass-spectrometry analyses. d MS/MS spectra of the auto-ubiquitylated E1 peptides following trypsin digestion to generate the ‘TVGG’ isopeptide linked moiety. m/z values of the precursor ions are shown in the top left of each panel. (2+) indicates doubly charged precursor ions. Spectra show the annotated peaks that are due to C-terminal y (coloured red) and N-terminal b (coloured blue) fragment ions. In each case, the m/z values of the precursor ions and the m/z values of the fragment ions are consistent with lysine residues modified with the ‘TVGG’ moiety. The amino-acid sequence of the trypsin-generated peptide, including the ‘TVGG’ modified lysine, is shown within in each example. e MS/MS spectra of the auto-ubiquitylated E2 peptide, as described in d ultimately results in the covalent attachment of the small ubi- activated modifier is then transferred to the catalytic cysteine of quitin modifier to a target lysine on a substrate. The first protein, the E1 enzyme, forming a covalent thioester intermediate4. Upon referred to as the E1 enzyme, activates the modifier by adeny- interaction with a second protein, the E2 conjugating enzyme, lating the C-terminal residue of the di-glycine motif that is a this activated ubiquitin moiety is then shuttled from the catalytic feature of almost all ubiquitin-like modifier proteins15, 16. The centre of the E1 protein to the catalytic cysteine of the E2 protein NATURE COMMUNICATIONS | 8: 1120 | DOI: 10.1038/s41467-017-01162-7 | www.nature.com/naturecommunications 3 ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-01162-7 a K100 K225 Zinc-coordinating C-cluster Catalytic C* Zinc-coordinating Catalytic C* C-cluster H.
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