Protein family review The ring between ring fingers (RBR) family Birgit Eisenhaber*, Nina Chumak†, Frank Eisenhaber* and Marie-Theres Hauser†

Addresses: *Research Institute of Molecular Pathology (IMP), Dr Bohr-Gasse, A-1030 Vienna, Austria. †Institute of Applied Genetics and Cell Biology, Department of Plant Science and Plant Biotechnology, University of Natural Resources and Applied Life Sciences, Muthgasse, A-1190 Vienna, Austria.

Correspondence: Birgit Eisenhaber. Email: [email protected]. Marie-Theres Hauser. Email: [email protected]

Published: 15 March 2007 Genome Biology 2007, 8:209 (doi:10.1186/gb-2007-8-3-209) The electronic version of this article is the complete one and can be found online at http://genomebiology.com/2007/8/3/209 © 2007 BioMed Central Ltd

Summary

Proteins of the ring between ring fingers (RBR)-domain family are characterized by three groups of specifically clustered (typically eight) cysteine and histidine residues. Whereas the amino- terminal ring domain (N-RING) binds two zinc ions and folds into a classical cross-brace ring finger, the carboxy-terminal ring domain (C-RING) involves only one zinc ion. The three- dimensional structure of the central ring domain, the IBR domain, is still unsolved. About 400 coding for RBR have been identified in the genomes of uni- and multicellular eukaryotes and some of their viruses, but the family has not been found in archaea or bacteria. The RBR proteins are classified into 15 major subfamilies (besides some orphan cases) by the phylogenetic relationships of the RBR segments and the conservation of their sequence architecture. The RBR domain mediates protein-protein interactions and a subset of RBR proteins has been shown to function as E3 ubiquitin ligases. RBR proteins have attracted interest because of their involvement in diseases such as parkinsonism, dementia with Lewy bodies, and Alzheimer’s disease, and in susceptibility to some intracellular bacterial pathogens. Here, we present an overview of the RBR-domain containing proteins and their subcellular localization, additional domains, function, specificity, and regulation.

Gene organization and evolutionary history (RING1 or N-RING) is thought to bind two zinc ions and to The ring between ring fingers (RBR) proteins are a large and fold into a classical cross-braced ring finger, whereas the diverse group of proteins characterized by a compact sequence carboxy-terminal ring (RING2 or C-RING) appears to bind module that is predicted to form three ring finger-type, or only one metal ion and forms a hydrophobic core different ‘ring’, domains separated by loops [1,2]. The RBR domain from that of classical ring fingers [3]. The central usually occurs as part of a multidomain protein with diverse cysteine/histidine cluster is likely to also form a ring finger- functional modules, and appears to mediate protein-protein type structure. Morett and Bork [4] derived a general interaction. The function of most of the family members has sequence profile-based characterization of this domain and not yet been explored experimentally, but a subset of RBR called it IBR (in between rings). Independently, van der proteins is known to have E3 ubiquitin ligase activity. Reijden et al. [5] identified this domain - with a more

restricted PROSITE-like pattern [6] C6HC - as DRIL (double The sequence of each ring domain in the RBR region contains ring finger linked) domain, and called the family of RING- a cluster of, typically, eight cysteine and histidine residues that DRIL-RING-containing proteins TRIAD. The two definitions potentially bind metal ions. The amino-terminal ring domain are largely overlapping but not identical. The approach of

Genome Biology 2007, 8:209 209.2 Genome Biology 2007, Volume 8, Issue 3, Article 209 Eisenhaber et al. http://genomebiology.com/2007/8/3/209

* * * * * * * XXX * * * * * * * * XX * * . * . * * * AAm|XP_396912.1| CGICFTIQP--SAMMTGLE-CGHR-FCTGCWGEYLTTKIMEEGVGQT--IACAA----HACDIL-XX-RWCPS-PDCNNAIK---VQYVEARP-----VTCK---CGHT-FCFHCGE---NWHDPVK-CHLLR-XX-KECPKCNVTIEKD-GG-CNHMVCKNQ--NCKTDFCWVC 140 AAm|XP_624643.1| CSVCVAIYS--AEKFSTLT-CGHS-FCKDCWCMHFEVQITQ-GISTG--ISCMA----QDCNVL-XX-RFCPG-PNCQMIMRSK-EQRAKR------VMCSS--CKTV-FCFRCGI---DYHAPTD-CNTMK-XX-KDCPKCHICIEKN-GG-CNHMQCYN----CKYDFCWMC 138 AAt|NP_189408.1| CDVCMEDDLP-SNVMTRME-CGHR-FCNDCWIGHFTVKINE-GESKR--ILCMA----HECKAI-XX-KWCPSKPHCGSAIRKI-EDGHDVVEV-----GCS---CGLQ-FCFSCLS---ESHSPCS-CLMWK-XX-KLCPKCSKPIQKR-DG-CNLMTCK-----CGQHFCWLC 140 AAt|NP_567966.1| CDVCMEDL-P-GDHMTRMD-CGHC-FCNNCWTEHFTVQINE-GQSKR--IRCMA----HQCNAI-XX-KWCPSTPHCGNAIRAE-DD--KLCEV-----ECS---CGLQ-FCFSCLC---QAHSPCS-CLMWE-XX-KLCPKCYKPVEKN-GG-CNLVRCI-----CGQCFCWLC 137 AAt|NP_179206.2| CDICVEDV-P-GYQLTRMD-CGHS-FCNNCWTGHFTVKINE-GQSKR--IICMA----HKCNAI-XX-KWCPSTPHCGNAIRVE-DD--ELCEV-----ECS---CGLQ-FCFSCSS---QAHSPCS-CVMWE-XX-KPCPKCHKPVEKN-GG-CNLVTCL-----CRQSFCWLC 137 ACe|AAB93645.3| CDICCSLG----E-LSGLS-CNHR-ACTQCWKAYLTNKIANNAQSE---IECMA----PNCKLL-XX-KWCPG-IDCGKAVR---VSHWEPRL-----VVCS---CGSR-FCFSCGH---DWHEPVN-CRLLK-XX-KECPKCMITIEKD-GG-CNHMTCKNT--ACRFEFCWMC 136 ACe|AAB93643.1| CDICCSMD----E-LSGLS-CNHR-ACAECWQAYLTNKIVSDAQSE---IECMA----PNCKLL-XX-RWCPG-IDCGKAVK---VSHWEPRL-----VVCS---CGTC-FCFSCGQ---NWHEPLN-CRHLK-XX-KDCPKCMIPIEKN-GG-CNRMLCTNS--GCRYEFCWMC 136 ACe|AAB93644.1| CDICCSMD----E-LSGLS-CNHR-ACAECWQAYLTNKIVSDAQSE---IECMA----PNCKLL-XX-KWCPG-VDCGRTVK---VSHGEPRL-----VVCT---CGSR-FCFSCGQ---DWHEPVN-CRLLK-XX-KECPKCMATIEKN-GG-CNQITCKNT--GCKFQFCWMC 136 ACe|AAB03121.2| CSVCAMDG---YTELPHLT-CGHC-FCEHCWKSHVESRLSE-GVASR--IECME----SECEVY-XX-KFCVG-NECPVIIRST-EVKPKR------VTCMQ--CHTS-FCVKCGA---DYHAPTS-CETIK-XX-KDCPQCHSCIEKA-GG-CNHIQCTR----CRHHFCWMC 137 ACe|CAB70234.1| CDVCCSMT----R-LSGLA-CAHR-ACDECWKAYLTEKIVDVGQSE---IECMM----MDCKLL-XX-KWCPG-AGCGKAVKGE-PSDREP------AVCT---CGER-FCFACAQ---DWHDPLS-CRHMK-XX-KPCPKCSVTIEKN-GG-CNHMSCKSS--SCRYEFCWLC 136 ADd|XP_636442.1| CLVCFSDVTK--QKAYSLQ-CNHS-YCIDCWYSYLAISVDS-GKTCL-YTKCIE----PNCKYV-XX-RWCTNPQSCSMAIHYSGVDLPNIIN-----VTCS---CNWR-FCFHCGD---EYHTPST-CVQVS-XX-KKCPKCKIHIEKN-EG-CAHLTCLN----CKHEFCWLC 142 ADd|XP_637482.1| CLICLEDYP--PTQTFALI-CNHR-YCLPCYKNYLEIKVSEGPECIY--TPCPA----PKCKVI-XX-KWCPA-PGCIYSIRCD-RK--ERKEA----VNCK---CGFQ-YCFNCNDYEIGDHMPCP-CSQVD-XX-KKCPECRSPIEKN-GG-CMHMTCRKNAGGCGFEFCWLC 146 ADd|XP_643727.1| CIVCFENQSN-DDSFYSLS-CGHGPYCKGCWKSYLHQEMQTCGGEII-HSKCIY----PLCNGK-XX-EFCPNP-SCGNAIRYSGVGRPNDV------VECH---CGTR-FCFSCGS---EKHNPVS-CAQLV-XX-KPCYHCGMPTERI-MG-CNHIICRKEQGGCGGEWCWMC 147 ADm|AAN11912.1| CGICFCSC---DE-LIGLG-CGHN-FCAACWKQYLANKTCSEGLANT--IKCPA----ANCEIL-XX-RWCPA-PNCSHAVK---AVCAEPRA-----VLCK---CGHE-FCFACGE---NWHEPAS-CSSLK-XX-KECPKCNVTIEKD-GG-CNHMVCKNP--SCRYDFCWVC 138 ADm|AAS65398.1| CEICFSQLP--PDSMAGLE-CGHR-FCMPCWHEYLSTKIVAEGLGQT--ISCAA----HGCDIL-XX-RWCPS-VDCTYAVK---VPYAEPRR-----VHCK---CGHV-FCFACGE---NWHDPVK-CRWLK-XX-KECPRCSVTIEKD-GG-CNHMVCKNQ--NCKNEFCWVC 140 ADm|AAF46823.1| CPVCASSQL--GDKFYSLA-CGHS-FCKDCWTIYFETQIFQ-GISTQ--IGCMA----QMCNVR-XX-RFCPG-PNCQIIVQSS-EISAKR------AICKA--CHTG-FCFRCGM---DYHAPTD-CQVIK-XX-KDCPKCHICIEKN-GG-CNHMQCFN----CKHDFCWMC 138 ADr|AAH53248.1| CGVCLQLVR--RDALLSLP-CQHS-FCKGCWEQHCTVLVKD-GVGVE--ISCMA----QDCSLR-XX-QLCPG-ADCPIVIQVQ-EPRARR------VQCSR--CEEV-FCFKCRQ---MYHAPTD-CATIR-XX-KDCPKCNICIEKN-GG-CNHMQCSK----CKHDFCWMC 138 ADr|AAH67684.1| CQICYLNYP--NSYFTGLE-CGHK-FCMQCWGDYLTTKIIEEGMGQT--ISCPA----HNCDIL-XX-KWCPA-PDCHHVVK---VQYPDAKP-----VRCK---CGRQ-FCFNCGE---NWHDPVK-CKWLR-XX-KECPKCHVTIEKD-GG-CNHMVCRNQ--NCKAEFCWVC 140 ADr|XP_687241.1| CQICYLNYP--NSYFTGLE-CGHK-FCMQCWGDYLTTKIIEEGMGQT--ISCPA----HSCDIL-XX-KWCPA-PDCHHVVK---VQYPDAKP-----VRCK---CGRQ-FCFNCGE---NWHDPVK-CKWLR-XX-KECPKCHVTIEKD-GG-CNHMVCRNQ--NCKAEFCWVC 140 AGz|EAA76603.1| CDICCEDDD--GLESFAMK-CGHR-YCVDCYRHYLTQKIREEGEAAR--IQCPS----DGCGRI-XX-KWCPA-PDCPNALECG-VKKKDLGRIVPT-VECR---CGFR-FCFGCPN---PDHQPAP-CDLVK-XX-KECPKCNSTIEKN-GG-CNHMTCR----KCKYEFCWMC 144 AHs|AAH00422.1| CAVCMQFVR--KENLLSLA-CQHQ-FCRSCWEQHCSVLVKD-GVGVG--VSCMA----QDCPLR-XX-QLCPG-ADCPMVIRVQ-EPRARR------VQCNR--CNEV-FCFKCRQ---MYHAPTD-CATIR-XX-KDCPKCNICIEKN-GG-CNHMQCSK----CKHDFCWMC 138 AHs|NP_055904.1| CPVCVSPLGC-DDDLPSLC-CMHY-CCKSCWNEYLTTRIEQ-NLVLN--CTCPI----ADCPAQ-XX-TWCTNPQGCDRILCRQ-GLGCGT------TCSK--CGWA-SCFNCSFP--EAHYPAS-CGHMS-XX-KRCPSCQAPIEKN-EG-CLHMTCAK----CNHGFCWRC 140 AHs|AAH51877.1| CQICYLNYP--NSYFTGLE-CGHK-FCMQCWSEYLTTKIMEEGMGQT--ISCPA----HGCDIL-XX-KWCPA-PDCHHVVK---VQYPDAKP-----VRCK---CGRQ-FCFNCGE---NWHDPVK-CKWLK-XX-KECPKCHVTIEKD-GG-CNHMVCRNQ--NCKAEFCWVC 140 ANc|XP_327168.1| CDICCEDGD--GLESFAIK-CGHR-YCVDCYRQYLSQKIREEGEAAR--IQCPA----DGCNLI-XX-KWCPS-PDCANAVECG-VKKKDLTKVVPT-VSCL---CGHR-FCFGCIY---TDHQPAP-CELVK-XX-KECPKCNSTIEKN-GG-CNHMTCR----KCKYEFCWMC 144 ASp|CAB95997.1| CEICYDEG---CLPFFSAE-CDHE-FCLACYRQYLDSRISE-GESV---IQCPE----ESCTQI-XX-RWCPA-PDCEFAIECH-VTQASLSSVVPT-VTCN---CGKQ-FCFGCGH---DNHQPTI-CPLVK-XX-KECPKCSTTIEKN-GG-CNHMTCK----KCKYEFCWVC 141 BAm|XP_624683.1| CKICFVDKI--GEHCTQFLPCGHI-FCKDCITGYLEVRIKDGN-VQ--NIYCPEE----KCTS--XX-VYCPR-RSCQYPVSR----EPN--EQMA---NCPI--CQYA-FCVYCKM---VYHG-IEPCKVYS-XX-QKCPKCQAAIEKS-DG-CNKMVCWR----CNTFFCWLC 137 BAt|NP_174512.1| CCICFSES--AGIDFVKL-PCQHF-FCLKCMKTYTDIHVTEGT-VN--KLKCPDSK----CGE--XX--YCPR---CETPCIED---EEQ----L---ALCFK--CYFS-FCTLCKE---KRHVGVA-CMSPE-XX-KQCPSCKIAISRT-GG-CNKMVCNN----CGQYFCYRC 132 BCe|AAB52683.1| CQVCFESQM--GQHCIKFQPCSHV-FCKSCTFNYY-ISIAKGF-VSKP-MSCLAE----GCENE-XX-MECPN-ENCQMVAYLT---DSQ--RNL---VECSY--CNYS-FCNLCKG---TFHG-VSRCKFRK-XX-KQCPKCLVYIEKD-EG-CNKMHCTK----CNASFCWLC 139 BDr|NP_001002087.1| CGICYSEKL--GCDCLLFKECEHV-YCKACIKEYFQIQIKDGK-VQ--CLNCPEP----KCAS--XX-VYCPR-MSCCMAVMV----EPDSTMG-----ICPS--CRYA-FCTLCRR---SYHG-LSHCIATA-XX-KQCPCCGTNIQKA-HG-CNKMTCSS----CQKYFCWIC 137 BDr|XP_687162.1| CGICFTENL--GSSFVLFNECQHV-YCKTCVKDYFEIQIKDGK-VQ--FLSCPEA----ECTS--XX-VYCPR-KSCGMAVML----EPDRTMG-----ICPS--CKFV-FCTLCNR---VYHA-LALCNES--XX-KQCPTCGVKIQKD-MG-CDMMTCSS----CQQFFCWTC 136 BHs|NP_899648.1| CSICFCEKL--GSECMYFLECRHV-YCKACLKDYFEIQIRDGQ-VQ--CLNCPEP----KCPS--XX-VYCPR-PCCQLPVMQ----EPGCTMG-----ICSS--CNFA-FCTLCRL---TYHG-VSPCKVTA-XX-KSCPCCGTPIEKL-DG-CNKMTCTG----CMQYFCWIC 137 BSp|CAB65614.1| CNVCFDEFN--GTDCFQLTRCGHV-SCQSCLRDYYTMCIQEGM-FSQ--IKCIDL----DCGKD-XX--FCPR-SFCQGPSKRD--PGQK--LA-----ICQK--CDFA-FCSFCQAT---WHGDLSPCKLEG-XX-QRCPTCDRVVERI-DG-CCHMNCL-----CGTHFCFLC 137 CAm|XP_393708.2| CKLCLVDTS-LSK-TFKIEGCGCS-YCKDCMKAYIEFEIEEG--AY--EISCPD----AQCEH--XX-AWCPR-AGCETICSINS-TGSNGTPIGP--VHCPN--CSID-FCSICRE---SWHTG--PCSDIS-XX-KCCPMCSVPIEKD-EG-CAQMMCKR----CKHVFCWYC 141 CDr|AAH76084.1| CKLCLGEFP-LEQ-MTTISQCQCI-FCSLCLKQYVELLIKEGL-ET--AISCPD----SACPK--XX-TWCPS-SSCQAVCQLNE--AEVQLPQP---VQCPE--CSLR-FCSACRA---DCHTGQA-CQEML-XX-KRCPKCKVYIERD-EG-CAQMMCKN----CKHAFCWYC 141 CDr|CAI20730.1| CKLCLGEFP-LEQ-MTTITQCQCV-FCTMCLKQYVELLIKEGF-ET--AISCPD----SACPK--XX-TWCPS-STCQAVCQLKE--SDTVLPQL---VRCSV--CTLE-FCSACKA---SWHPDQD-CQENV-XX-KRCPKCKVYIERD-EG-CAQMMCKN----CKHAFCWYC 141 CHs|AAH50373.1| CKLCLGEYP-VEQ-MTTIAQCQCI-FCTLCLKQYVELLIKEGL-ET--AISCPD----AACPK--XX-TWCPA-STCQAVCQLQDVGLQTPQP-----VQCKA--CRME-FCSTCKA---SWHPGQG-CPETM-XX-KRCPKCKVYIERD-EG-CAQMMCKN----CKHAFCWYC 141 CHs|NP_877434.2| CKLCLCEQS-LDK-MTTLQECQCI-FCTACLKQYMQLAIREGC-GS--PITCPD----MVCLN--XX-TWCPV-ADCQTVCPVA-S-SDPGQPVL---VECPS--CHLK-FCSCCKD---AWHAEVS-CRDSQ-XX-KQCPVCRVYIERN-EG-CAQMMCKN----CKHTFCWYC 141 DAm|XP_623728.1| CPLCLAEL-PMEF-FPVVQSCHHR-SCYDCYQQYLKVEISESR--V--NIACP------ECSEP-XX-RWCPA-PDCSFAVIASGCASCP-K------LRCERPGCDSY-FCYHCKA---RWHPNQT-CDAAR-XX-KPCPRCQVLIVKMDDGSCNHMVCAV----CGAEFCWLC 141 DCe|AAK68189.1| CPLCAAKM-PGSA-FPKLKGCQHR-SCRACLRQYVELSITENR--V--EVPCP------ECSSY-XX-RWCPA-PDCGFVFIATKCAACP-Q------LKCQRPDCGTL-FCYHCKR---EWHSNQT-CDEAR-XX-KACPRCKTYIVKMDDGSCNHMVCTM----CNAEFCWLC 141 DDr|CAI21211.1| CPLCLLSQ-PRAH-FPRLSSCQHR-ACTDCLRQYLRIEISESR--V--GIACP------QCPEA-XX-RWCPA-PDCSYAVIAYGCAECP-K------LSCGREGCETE-FCYHCRQ---LWHPDQT-CDQAR-XX-KPCPRCGAYIMKTNDGSCNRMNCTV----CACQFCWLC 141 DDr|XP_690873.1| CPLCLVRQ-PAEQ-LPELQGCSHR-SCLCCLRQYLRIEITESR--V--QLSCP------ECAER-XX-RWCPA-PDCGFAVIASGCASCP-R------LVCRREGCGAE-FCYHCKQ---AWHPNQT-CDSAR-XX-KPCPRCGAYIIKMNDGSCNHMTCAV----CGCEFCWLC 141 DHs|NP_904355.1| CPLCLLRH-SKDR-FPDIMTCHHR-SCVDCLRQYLRIEISESR--V--NISCP------ECTER-XX-RWCPA-PDCGYAVIAFGCASCP-K------LTCGREGCGTE-FCYHCKQ---IWHPNQT-CDAAR-XX-KPCPRCAAYIIKMNDGSCNHMTCAV----CGCEFCWLC 141 EGz|EAA77947.1| CSVCFGEAEES---LETS--CGHI-YCNICFVNMCQSGESS-S-GDFS-IKCVG--ASDACKKI-XX-RYCPT-PDCDQVYRVS--SPEK--VPFM--FTCSR--CFTS-TCTACNAS----HPG-ISCSKNK-XX-KDCPKCTTAIQKS-EG-CNHMTCFA----CRTHICWVC 139 FGz|EAA76086.1| CIACAESA---HG---RA-PCGCN-YCVTCYRQIIRIGLRS---QEEFPPKC------CK---XX-VYCYQ-GNCAAFIPP--DLKG------RCPI--CPYK-TCVDCGEK---AHDGWP-CAEGD-XX-VNCPDCGRMIQLS-EA-CNHMTCP-----CGGEFCFLC 123 FGz|EAA77041.1| CGGCMNDFPE-DETAVMA--CTHE-FCEPCFSLMIERSLDG---SSAFPPRC------CDI--XX-TYCSN-VECQTFIPPW-TIESD--I-----GYCPS--CPQR-TCAKCKNPE---HTGR--C-----XX-KPCPRCGQLINKT-SG-CEHVICP-----CGNEFCFHC 127 FGz|EAA77042.1| CLACGEDFPQ-SS-MIFAP-CSHL-FCKPCADNLVSLAMRD---EAYFPARC------CDT--XX-VYCSS-EICATFIPPT-QIDSG--I-----GHCKR--CLTD-TCIACKAKA---HEGV--CG----XX-KRCSKCGHVIEKS-MG-CDHMVCL-----CGHRFCYAC 128 GAt|NP_180108.1| CSICFEETEG-ERMFFTTEKCVHR-HCFPCVKQYVEVKLLSGT--V--PT-CLDDG----CKF--XX-IYCPY-PNCSMLMSKT-ELSSESDLSN-D-RSCVK--CCGL-FCIDCKVPS---HSDLS-CAE---XX-RQCKMCRHMIELS-HA-CNHMTC-----RCGYQFCYQC 139 GAt|NP_179737.1| CCICRENTDA-DRMFF-TENCFHR-QCFSCVNRHVQRMLLCGI--S--PT-CLHFP----CNS--XX-IYCPY-RRCSMLMSKT-ALSRETDQSN-V-RACIK--CCRL-FCIDCKVPS---HAGLS-CVD---XX-RQCVQCSNLVELF-EG-CNHITC-----RCGFEFCYVC 138 GAt|NP_190133.1| CVICLEETKA-DRMFV-MDKCLHR-HCYPCVNQLVEVKLRNGT--V--PT-CLDYE----CKL--XX-IYCPY-INCSTLMSKT-EIS-RSNKSN-D-RACIK--CSGL-VCIDCKVPW---HSDLS-CAE---XX-RQCVKCRHLIELN-QG-CNHMTC-----RCGYQFCYKC 137 GAt|NP_190140.1| CKICLDDDIN-ENQMFCVGKCRHR-FCSDCMRRHIEVRLLEGS--V--MR-CPHYR----CKT--XX-IYCPN-SRCSALMSET-ELSISTKEDEVR-RCCFK--CGQI-FCIKCKVSW---HSNLS-CND---XX-RQCGKCQHMIELS-KG-CVQVKC-----RCGHKFCYRC 140 GAt|NP_190144.1| CSICSDDNFE-PELMFSVALCGHE-FCVECVKRHIEVRLLAGG--V--PR-CLHYQ----CES--XX-VYCPN-PRCSSLMSVT-KLSNSTREDVTM-RSCVK--CGEP-FCINCKLPW---HSNLS-CND---XX-RQCENCKNVIELS-EG-CMHITC-----RCGHQFCYKC 140 GAt|NP_190937.1| CEICVDSKSIIES-FR-IGGCSHF-YCNDCVSKYIAAKLQDNI--L--SIECPVSG----CSG--XX-FYCPY-KDCSALVFLE-ESEVKMKDSE-----CPH--CHRM-VCVECGTQW---HPEMT-CEE---XX-KRCPSCKFYIEKS-QG-CLYMKC-----RCGLAFCYNC 136 GAt|NP_973536.1| CTICFDDDIN-ADMMFYIDQCGHM-FCSECVKRHIEVSLLQGS--L--IT-CPSYR----CKS--XX-VYCPN-PTCSALMSVT-ELDQLTGS----KRCCVK--C-GESFCIKCKVPW---HDNLS-CKR---XX-RQCSKCKHMIELT-QG-CVRVIC-----RCGHEFCYGC 137 GAt|BAB08302.1| CSICSDKTDA-EHMLL-NDKCLHR-HCFSCVKQQVKVKLRSGI--V--PP-CLEDG----CKS--XX-IYCPY-RSCSMLMSKT-ELSREAEQSN--VRACIK--CSEL-FCIDCKVPW---HSDLS-CAD---XX-RQCSECKHMIELT-EG-CNHITC-----RCGYEFCYRC 138 HAt|NP_567206.1| CPICLSEVDD-G-YSLE--GCSHL-FCKACLLEQFEASMRN-FDAFP--ILCSH----IDCGAP-XX-RFCST-PDCPSIYRVA--GPQE--SGEP--FICGA--CHSE-TCTRCHLE---YHP-LITCERYK-XX-KECPICKSTIEKT-DG-CNHLQCR-----CGKHICWTC 138 HAt|NP_196599.2| CPICLSEVDD-G-YSLE--GCSHL-FCKACLLEQFEASMRN-FDAFP--ILCSH----IDCGAP-XX-RFCST-PDCPSVYRVA--GPQE--SGEP--FICGA--CHSE-ICTRCHLE---YHP-LITCERYK-XX-KECPICKSTIEKT-DG-CNHMKCR-----CGKHICWTC 138 UAm|XP_393719.2| CELCTGRFAM-SQ-IVSMLKCIHR-CCNECAKNYFTIQISDRN-IT--DAVCPF------C----XX-KWCIQ---CSSG-----FYADP--DQKR--LICPD--CRSV-TCAQCRRPWEKQHEGIT-CEQ---XX--DCPKCKFRYSLS-RGGCMHFTCS----QCKYEFCCGC 132 UDr|XP_696033.1| C-ICLCSPPQ----MVTMTHCSCT-FCESCFKKYFSSVIKEKN-IV--HAVCPL------C----XX-RWCAH---CCFG-----LLHEA--DRLR--MDCPS--CGKS-TCFKCKRPWAPQHEGIS-CEK---XX--DCPKCKFRFFLA-RGGCLHFRCT----QCQHEFCGGC 129 UHs|AAH12077.1| CAVCGWALPH-NR-MQALTSCECT-ICPDCFRQHFTIALKEKH-IT--DMVCPA------C----XX-LWCAQ---CSFG-----FIYER--EQLE--ATCPQ--CHQT-FCVRCKRQWEEQHRGRS-CED---XX--DCPKCKFSYALA-RGGCMHFHCT----QCRHQFCSGC 132 XAm|XP_393942.2| CPVCFVTYGP-REGVILR-DCLHM-FCRSCIANTIRYCEEA---EV----KCPYRDSEYTCES--XX-FHCKT-PDCPGWCIYD--DDVN------NFLCPV--CGAN-NCLTCQAI----HTGKN-CKQYQ-XX-LACPTCAVVLMKK-WG-CDWLRCS----MCKTEICWVT 136 XDr|AAH74096.1| CAICFGTIMP-GEGAVLR-ECLHS-FCRDCLKGTVVNCLDA---EV----CCPYGDNAYACNC--XX-YHCKT-PDCAGWCIFE--DDVN------EFKCDI--CNET-NCLLCKAI----HKGMN-CKEYQ-XX-MNCPKCQVIVQKK-DG-CDWICCL----MCKTEICWVT 136 XDr|XP_698411.1| CRICYVELES-GEGVLLR-ECLHC-FCKECLRSVILMSEDP---QV----ACPYRDESYACDC--XX-YHCAT-ADCPGWCVYE--DTVN------TFHCPV--CKKQ-NCLLCKAI----HEGMN-CKQYQ-XX-MHCPQCGIIVQKK-EG-CDWLRCT----VCHTEICWVT 136 XHs|NP_112506.1| CPVCYSVLAP-GEAVVLR-ECLHT-FCRECLQGTIRNSQEA---EV----SCPFIDNTYSCSG--XX-YHCKT-PDCKGWCFFE--DDVN------EFTCPV--CFHV-NCLLCKAI----HEQMN-CKEYQ-XX-MRCPQCQIVVQKK-DG-CDWIRCT----VCHTEICWVT 136 ZDd|XP_641886.1| CSVCADDLDSLNG--SYLP-CKHY-SCNDCWNQYLSLKVLE-GGATS--ITCMG----LKCPSV-XX-RWCPA-PKCGNALKAD-SQTEAT------ALCS---CGFK-ICFKCKQ---ESHFPAD-CEKMK-XX-QDCPKCHSAIEKN-GG-CMHMTCK----KCKHEFCWIC 137 ruler 1...... 10...... 20...... 30...... 40...... 50...... 60...... 70...... 80...... 90...... 100...... 110...... 120...... 130...... 140...... 150...... 160...... 170.....

Figure 1 Multiple alignment of RBR domain segments. The figure includes the sequences of 64 representative RBR segments. This alignment is part of the grand alignment used to create the phylogenetic tree in Figure 2. The sequence identifier gives the subfamily code (first letter) followed by the taxon and the accession number in GenBank. The loop segments between the three ring-like domains are indicated by ‘XX’. The gray bars at the bottom represent the degree of amino acid type conservation of alignment positions. The alignment was primarily generated with CLUSTALX [70] and edited manually. Taxon abbreviations: Am, Apis mellifera; At, Arabidopsis thaliana; Ce, Caenorhabditis elegans; Dd, Dictyostelium discoideum; Dm, Drosophila melanogaster; Dr, Danio rerio; Gz, Gibberella zeae; Hs, Homo sapiens; Nc, Neurospora crassa; Sp, Schizosaccharomyces pombe.

Morett and Bork [4] is consistent with the concept of file 1, available online with this article. Additional information and the criterion of statistically (sequence lists, subgroup alignments, annotations, and significant sequence similarity and is therefore the supplementary text and tables) is also available at our preferred one. website [7].

RBR proteins make up a large, diverse family with, at Genome sequencing projects have added considerably to the present, around 400 representatives in sequence databases. information on RBR genes; they comprise a complex and On the basis of sequence conservation within the RBR ancient family that has been found in all eukaryotes segment, RBR proteins are assigned to 15 subfamilies (A-I, examined and in some of their viruses. There are two RBR P, S, T, U, X, Z; Figure 1). There are also some 10 orphan proteins in the yeast Saccharomyces cerevisiae, six in sequences that could be considered as their own groups. Drosophila melanogaster, 10 in Caenorhabditis elegans, Subfamily A (Ariadne-like proteins) can be further sub- about 40 in Arabidopsis thaliana, around 23 in the zebrafish divided into around 10 clusters (A0-A9). Similarly, several (Danio rerio), and about 15 in humans (Additional data file subgroups can be distinguished for subfamilies S (found 1). Genes of RBR proteins are dispersed throughout the only in viruses), T (the TRIAD3 proteins), and Z (found only genome. They vary in chromosomal location and exon in protozoa). The alignment of RBR segments from sequence number. For example, Mladek et al. [8] have studied the representatives of the various groups is shown in Figure 1 Ariadne (A) subfamily in Arabidopsis: the 16 AtARI genes and details with respect to the conservation of the cysteine/ are distributed between all five at 10 loci. histidine pattern and loop sizes are given in Additional data Despite the conserved sequence, they have distinct gene

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structures. The number of exons varies between one bind only one zinc ion. Some RBR proteins seem to remain (AtARI3) and 15 (AtARI5, AtARI7 and AtARI8). The AtARI functional without C-RING, but parkin and others genes are differentially expressed during plant development apparently require this part of the RBR region for their and in an organ-specific manner. correct function as E3 ubiquitin ligases [2].

Splicing isoforms of RBR proteins are widespread [8-10]. At Unfortunately, little direct structural information is least for some RBR proteins, leads to available for the RBR region. Capili et al. [3] reported the shortened isoforms that control the cellular localization and three-dimensional structure (PDB accession number 1WD2) function of a respective parental larger isoform [9]. For the of the C-RING in the human Ariadne protein HHARI rat transcription factor RBCK1, the RBR domain has a (residues 326-395 of UNIPROT accession number Q9Y4X5, crucial role in its transcriptional activity [11]. RBCK2 has subfamily A1/Ari1), which folds into a novel structure been identified as an alternative splice variant of RBCK1 binding a single zinc ion (Figure 3a). Also, a three- that lacks the carboxy-terminal part of RBCK1, including dimensional structure (PDB 1WIM, structure report unpub- the RBR region [11]. Yoshimoto et al. [9] showed that lished) that essentially represents the N-RING (residues RBCK2 represses the transcriptional activity of RBCK1 by 20-100) in the human UbcM4-interacting protein 4 (subfamily tethering it within the cytoplasm. A similar alternative C/RNF144; UNIPROT accession number P50876) is very splice variant lacking the RBR region has been reported similar to a classical ring-finger domain (Figure 3b). No recently for parkin [12]. structural information is known for any IBR domain.

The genetic module coding for the RBR region has As well as conservation within the RBR segment, the apparently been reused in several gene contexts during sequence architecture conserved among distant taxa is an evolution and has been sequentially modified by point important criterion for the classification of RBR proteins, as mutations and insertions/deletions and shuffled into new shown in Figure 4. The functional significance of the genomic locations. A retrotransposition-based mechanism additional segments and their cooperation with the RBR has been proposed to underlie these changes and evidence part of the sequence is most often not clear, and is certainly hinting at this comes from Arabidopsis [8]. A putative an urgent research task for the near future. For example, it phylogenetic relationship between the RBR segments in would be of interest to determine whether the two hydro- various taxonomic ranges is presented in Figure 2, and it is phobic regions in the dorfins (subfamily D), each the length notable that some RBR protein subfamilies are found only in of a transmembrane helix, do function as membrane-attach- specific taxonomic groups (Additional data file 1); for ment modules or whether they are critical for protein example, subfamilies C (such as RING finger protein 144), I complex formation. The available data are conflicting. The (IBR domain-containing protein 1, IBRDC1), P (parkin), U two long transmembrane helices (each 30-35 residues long (Ariadne-like E3 ubiquitin ligase, PAUL) and X (human with conserved prolines) are predicted by the programs hepatitis B virus X-associated protein, XAP) have been TMHMM [13] and DAS-TMfilter [14,15] at the carboxy- found only in animals. Several other subfamilies are specific terminal side of the dorfin RBR region. A carboxy-terminal for fungi (subfamilies E and F) and plants (subfamilies G deletion that includes the hydrophobic segment but leaves and H). Besides the large number of RBR protein genes in the RBR region untouched results in an enzyme unable to higher eukaryotes, they also notably occur in unicellular bind ubiquitinated substrates [16], suggesting that this eukaryotes and in entomopoxviruses and iridoviruses [7] region is responsible for ubiquitin binding. On the other (Additional data file 1). Interestingly, there are no genes in hand, localization of the protein near the nuclear membrane archaeal or eubacterial genomes that could truly be called and the centrosome, co-localization with vimentin, and inter- RBR family members. action with the calcium-sensing receptor CaR (an integral membrane protein) support membrane-embedding [16,17]. RNF144 (subfamily C) and IBRDC1 (subfamily I) also have a Characteristic structural features predicted transmembrane helix at the carboxy-terminal side The whole RBR segment typically contains some 200 of the RBR region, and localization at the Golgi membrane consecutive amino acids. The ring-like sequence domains has been shown for RNF144 [18]. tend to get smaller going from the amino to the carboxyl terminus - N-RING around 60 residues, IBR around 50 The combination of the RBR region with domains and motifs residues, and C-RING around 40 residues [1,2]. Loops on known from the ubiquitination pathway (such as ubiquitin- the amino-terminal side of the RBR region have a higher associated UBA, ubiquitin UBQ, and ubiquitin-interacting sequence and size variability. In contrast, the motif, UIM) is not a real surprise, whereas the association cysteine/histidine positions are more strongly conserved in with helicase domains (DEXDc and HELICc), nucleic-acid- N-RING and the IBR. Substitutions with non-cysteine or binding domains (KH and RRM) and protein-binding domains non-histidine residues at cysteine/histidine pattern positions (RWD, Armadillo/HEAT repeats and ankyrin segments) are observed only in the C-RING domain, which is known to would make sense in the context of the involvement of RBR

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AAt|CAD52897.1| AAt|CAD52895.1| AAt|NP_180709.3| AAt|NP_172080.2| AAt|NP_176722.2| AAt|NP_180737.2| AAt|NP_180735.1| AAt|NP_180736.1| ADd|XP_643727.1| ADd|XP_636442.1| ADd|XP_637482.1| AAt|NP_179206.2| AAt|NP_567966.1| AAt|NP_189408.1| ADm|AAF46823.1| AAm|XP_624643.1| AHs|AAH00422.1| ADr|AAH53248.1| ACe|AAB03121.2| ACe|AAB93644.1| ACe|AAB93643.1| ACe|AAB93645.3| ACe|CAB70234.1| ADm|AAS65398.1| AAm|XP_396912.1| ADr|XP_687241.1| ADr|AAH67684.1| AHs|AAH51877.1| ADm|AAN11912.1| ANc|XP_327168.1| AGz|EAA76603.1| ASp|CAB95997.1| ZDd|XP_641886.1| ZDd|XP_636573.1| PDm|AAN12155.1| PAm|XP_396426.2| PHs|NP_004553.1| PDr|NP_001017635. PCe|CAB04599.2| AHs|NP_055904.1| AHs|AAH63861.1| ADr|XP_689562.1| DDd|XP_645416.1| DDd|XP_637835.1| DHs|NP_904355.1| DDr|XP_690873.1| DDr|CAI21211.1| DAm|XP_623728.1| DCe|AAK68189.1| DDd|XP_629545.1| CDm|AAF58756.2| CAm|XP_393708.2| CHs|AAH50373.1| CDr|CAI20730.1| CDr|AAH76084.1| CHs|NP_877434.2| BCe|AAB52683.1| BAm|XP_624683.1| BHs|NP_899648.1| BDr|NP_001002087. BDr|XP_687162.1| BAt|NP_174512.1| BNc|CAC28847.2| BGz|EAA76614.1| BSp|CAB65614.1| IHs|CAI22999.1| FGz|EAA77042.1| FGz|EAA77041.1| FGz|EAA76086.1| UDm|AAF52435.2| UAm|XP_393719.2| UDr|XP_696033.1| UHs|AAH12077.1| GAt|NP_973536.1| GAt|NP_190140.1| GAt|NP_190144.1| GAt|NP_200833.1| GAt|NP_190133.1| GAt|NP_180108.1| GAt|BAB08302.1| GAt|NP_179737.1| GAt|NP_193702.1| GAt|NP_190937.1| GAt|NP_973485.1| ZDd|XP_635451.1| ZDd|XP_629844.1| ZDd|XP_639066.1| FNc|XP_324871.1| THs|NP_996994.1| TDr|XP_687871.1| VNc|XP_330958.1| VGz|EAA76165.1| WGz|EAA75506.1| BDd|XP_646567.1| HAt|NP_196599.2| HAt|NP_567206.1| ENc|XP_330102.1| EGz|EAA77947.1| XHs|NP_112506.1| XDr|AAH74096.1| XDr|XP_698411.1| XAm|XP_393942.2|

Figure 2 Phylogenetic tree of RBR domain segments. A grand alignment of RBR segments from 102 proteins representative of the most populated subgroups was used to create the tree. We used the program SEAVIEW and the tool ATV from the Forester package [71-73]. Each entry is labeled as in Figure 1. Typically, subfamily members cluster nicely together and the phylogenetic relationships within subfamilies are determined with significance. Closer to the root of the tree, the branching becomes increasingly uncertain. Some groups of fungal and protozoan sequences are more heterogeneous and appear at several tree positions. The D. discoideum sequence XP_646567.1 does not appear together with other ARA54 sequences but was assigned to the group ‘B (ARA54)’ because of the RWD domain in the sequence architecture.

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(a)

Group Protein family Domain architecture A0 Ariadne/Ank A1 Ariadne/Ari1 A2 Ariadne/Ari2 A3 Ariadne/PARC A4 Ariadne/fungi A5 Ariadne/plantA A6 Ariadne/plantB A7 Ariadne/plantC A8 Ariadne/prot A9 Ariadne/worms B ARA54 C RNF144 D Dorfin E Fungi1 F Fungi2 G Plant2 H Plant1 I IBRDC1 P Parkin S1 Viruses/Irido S2 Viruses/Pox T1 TRIAD3/animals T2 TRIAD3/fun1 T3 TRIAD3/fun2 U PAUL X XAP3 Z1 Protozoa1 (b) Z2 Protozoa2

500 amino acids

APC10 DUF1605 Proline-rich Cullin HA2 UBA DEXDc HELICc UBQ RBR KH UIM RBR containing atypical IBR ANK ZnF_RBZ RWD Negative- Hydrophobic Region with ARM/HEAT-like charge cluster region helical repeats

Figure 4 Sequence architecture of RBR proteins. The detailed sequence architecture of the subfamilies of RBR proteins is shown. The globular domains (such as cullin) and non-globular regions (negative-charge clusters or proline-rich regions) are color-coded as shown in the key. It should be noted that the typical sequence architecture is shown. There are several exceptions: for example, PAUL proteins mostly contain three additional zinc fingers, but a few representatives have only one. Similarly, dorfins have two predicted Figure 3 hydrophobic helices with the exception of the protozoan members, which Structures of ring domains in the RBR segments. The structure graphics have only one or none. The two sequences of the Fungi1 group are very were generated with the program VMD [74]. The backbone trace and the diverse and have only the RBR segment in common. Among the Plant1 secondary structure are shown as ribbons and the zinc ions as red representatives, one protein contains two RRM domains instead of the spheres. (a) Carboxy-terminal part of the RBR region (C-RING) in the usual two KH domains. Domain accession numbers: APC10, PF03256; human Ariadne-1 homolog protein HHARI (PDB accession number cullin, SM00182; DEXDc, SM00487; IBR, SM00647; RWD, SM00591; 1WD2; residues 326-395 of UNIPROT accession number Q9Y4X5; DUF1605, PF07717; HA2, PF04408; HELICc, SM00490; KH, SM00322; subfamily A1/Ari1). (b) The N-RING of the RBR segment of the human RRM, SM 00360; ANK, SM002481; UBA, SM00165; UBQ, SM00213; UIM, UbcM4-interacting protein 4 (PDB accession number 1WIM; residues 20- SM00726; and ZnF_RBZ, SM00547. The first two letters ‘PF’ and ‘SM’ 100 of UNIPROT accession number P50876; subfamily C/RNF144). indicate the PFAM and SMART databases, respectively.

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proteins in the regulation of . A functional such as the parkin-associated endothelin receptor-like link between ubiquitination and RNA metabolism appears to receptor (Pael-R) [45], synaptotagmin XI [46], the AAA- be a general phenomenon [19]. ATPase valosin-containing protein [47], and the dopamine transporter [48], before they accumulate in the endoplasmic reticulum. Moreover, plasma membrane receptors such as Localization and function the Toll-like receptors TLR4 and TLR9 are substrates for the RBR proteins fulfill diverse functions, ranging from the RBR protein TRIAD3A [10]. This led to the hypothesis that control of protein quality to the regulation of translation and TRIAD3A controls the intensity and duration of pro- signaling [10,20-22]. This diversity is highlighted by the inflammatory responses mediated by Toll signaling. A recent manner in which some RBR proteins were originally dis- study proposes a novel role for parkin in regulating signaling covered. Protein-protein interaction studies first attracted from the epidermal growth factor receptor (EGFR) through attention to the RBR proteins. For example, XAP3 [23] and its ability to bind EGFR and the EGFR pathway substrate 15 rat RBCK1 [11] were discovered with the regulatory domain (EPS15) and to ubiquitinate EPS15, consequently regulating of protein kinase C-β-interacting protein as a bait, and the the internalization and degradation of EGFR [22]. Ariadne proteins were found as interaction partners of ubiquitin-conjugating enzymes (E2s) in fruit flies, mice and At least three RBR proteins are involved in the regulation of humans [24-27]. The putative Ariadne-like E3 ubiquitin the cell cycle and apoptosis. PARC acts as negative regulator ligase PAUL appeared in a complex extracted with the of the tumor suppressor protein p53. Overexpression of cytoplasmic domain of the muscle-specific kinase (MuSK) as PARC was shown to sequester p53 in the cytoplasm without bait [28]. Parkin is the best characterized RBR protein ubiquitinating it for degradation [49]. In contrast, the RBR functionally [29,30]. In a clinical context, mutations in the protein p53RFP, a member of the RNF144 subfamily (C), gene encoding parkin are associated with sporadic early- targets an inhibitor of cell cycle progression (p21WAF1) for onset parkinsonism and autosomal recessive juvenile degradation. The zinc finger protein inhibiting NF-κB protein parkinsonism [29-30] and parkin has recently also been (ZIN), a splicing variant of TRIAD3, similarly supports the associated with susceptibility to intracellular pathogens such degradation of an inhibitor of NF-κB activation (RIP). as Salmonella typhi, S. paratyphi and Mycobacterium Overexpression of p53RFP or RIP induces apoptosis [50,51]. leprae and cancer [31-35]. Several other RBR proteins are involved in human neurodegenerative diseases, The protein Vif, encoded by the human immunodeficiency susceptibility to infections, and cancer [2, 21,36,37]. virus (HIV), induces the translocation of ZIN to the nucleus, suggesting that RBR proteins might be attractive candidates E3 ubiquitin ligase activity has been reported for 15 RBR for interfering with virus replication. Vif is important for the proteins, and 25 of their substrates and more than 70 assembly of HIV-1 particles and the stability of the reverse interactors have been identified so far (Figure 5; Additional transcription complex [37]. Another possible regulator of data file 1). As typical E3 enzymes, RBR proteins interact virus infection is the RBR protein heme-oxidized IRP2 with ubiquitin-conjugating enzymes (E2s) and catalyze the ubiquitin ligase (HOIL-1), which interacts with the X protein covalent attachment of ubiquitin to target proteins [38]. For of hepatitis B virus and enhances its ability to activate most of the characterized RBR proteins, the N-RING is X-responsive promoters [23]. essential for recruiting specific E2s and binding substrates. There are exceptions to this rule, however, and substrate RBR proteins of the Ariadne subfamily are probably interactions have been defined for non-RBR regions as well involved in translational regulation, as the Ariadne-sub- (Figure 5; Additional data file 1). RBR proteins are family protein HHARI ubiquitinates the eukaryotic mRNA considered single-molecule E3 ubiquitin ligases (E3s). cap-binding protein 4EHP [20], which apparently alters the However, parkin and the parkin-like cytoplasmic protein binding efficiency of 4EHP to mRNA caps. 4EHP ubiquitina- (PARC) interact with components of the SCF-like E3 ubiquitin tion may also be a signal for the intracellular compart- ligase complex, such as cullin and F-box proteins [39,40]. mentalization of specific mRNA populations.

Parkin and dorfin protect dopaminergic neurons from the RBR proteins can be recruited by their interaction partners consequences of mitochondrial damage [41-43] and from to particular subcellular compartments. For example, harmful levels of aggregation-prone proteins by ubiquitin- RBCK1 is translocated to the nucleus via an interaction with mediated proteasomal degradation and/or subcellular its RBR-domain-deficient splicing variant RBCK2 [9]. Such tethering of such proteins [29,30,44]. They are also nucleo-cytoplasmic shuttling is frequent with RBR proteins involved in the endoplasmic reticulum-associated and is supported by the protein-interaction map of degradation pathway (ERAD). This is supported by their Drosophila [52], which revealed an interaction of the RBR interaction with E2s associated with the endoplasmic protein ARI-2 with a classical nuclear transport receptor, reticulum and by their role in promoting the degradation of karyopherin 3. On the other hand, some RBR members may unfolded or misfolded forms of transmembrane proteins, be able to regulate nucleo-cytoplasmic transport themselves.

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B: UBE2E2, UBCH6, UBE2E3 P: EPS15 C: UBCH7, UBCH8 D: UBCH7, UBCH8 VCP P: Pael-R U: MuSK Septin5 Microtubules P: 14-3-3 T: RIP BAG5

UBL (P) UBA, ZnF_RBZ (U) Cullin, N-RING IBR C-RING H Φ ARM/HEAT, APC10 (A3)

A3: p53 A: 4EHP P: Synphilin-1 D: CaR Cul7 UBCM4, UBCH7, UBCD10 LRRK2 Synphilin-1 P: O-glyco-α-synuclein (UBCH6, UBCH13) UBC6, UBC7, UBCH8 SOD1 IRP2 P: p38 Rpn10 Septin4 PRT-2 Synaptotagmin XI FLRF/Nrdp1 CHIP P: SIM2 HRS Hsp70 UBC-2, -18,-15 SUMO-1 C: UBCH7, UBCM4 CHN-1 X: SOCS6 X: UBCM4 Proteasomal subunit alpha 4 BAG5

Further substrates and interactors A: UBCH7 P: hSel-10 A,P: SIM2 B: androgen receptor AR C: p21WAF1 P: DAT cyclin E alpha/beta tubulin Poly Q protein FBP1 RANBP2 Glup/PACRG T: Vif HIV-1 TLR4, TLR9 X: hepatitis B virus X protein CBP PML

Figure 5 Known interactors with the RBR protein. The interacting proteins for each region are boxed and are preceded by the name of the subfamily of RBR proteins with which they interact: A, Ariadne; A3, Parc; B, ARA54; D, dorfin; P, parkin; C, RNF144; X, XAP; U, Paul; T, TRIAD3. The additional amino- terminal domains present in some subfamilies of RBR proteins are highlighted in light blue and indicated by the domain abbreviation with the subfamily in which they are found in parentheses. UBL, ubiquitin-like domain; UBA, ubiquitin-associated domain; ZnF_RBZ, zinc finger; cullin, cullin-like domain; ARM/HEAT, Armadillo and HEAT repeats; APC10, anaphase-promoting complex subunit 10. A carboxy-terminal hydrophobic segment is present in the dorfins. Additional substrates and interactors are listed in Additional data file 1.

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For example, parkin interacts with and ubiquitinates the course of microbial infection. These glimpses into the Ran-binding protein 2 (RanBP2), which is related to the diverse functions of RBR proteins have mainly been gained small ubiquitin-related modifier (SUMO) E3 ligase family by detailed analyses of the multipurpose neuroprotective and is a component of the nuclear pore complex [53]. agent parkin, some ten other animal RBR proteins and a Binding of parkin to SUMO-1 enhances parkin’s nuclear single member in Arabidopsis. Essentially nothing is known translocation and auto-ubiquitination, indicating that both about the remaining 350 or so RBR proteins, in particular its E3 activity and subcellular localization are modulated the animal-specific IBRDC1, and the plant-, fungus-, through association with SUMO-1 [54]. protozoa- and virus-specific subfamilies (H, G, F, E, Z and S). It is likely that some of these RBR proteins also have E3 Mechanisms of regulation activities. It is not yet clear, however, whether RBR proteins RBR proteins are also subject to posttranslational regulation act as single molecules and/or in SCF-like E3 ligase complexes, that predominantly tends to inhibit their E3 activity. and whether they catalyze mono- or polyubiquitination and Parkin’s stability is controlled by the RING finger domain E3 lysine 48- or 63-types of linkages. Apart from the E3 ligase ligase FLRF/Nrdp1, which interacts with the amino terminus function, RBR segments might serve as activation domains, and reduces parkin’s half-life and enzymatic activity [55]. interact with cytoskeletal components, and act as tethering Auto-ubiquitination has been shown to inhibit E3 activity in modules. Therefore, one future challenge is to analyze their members of the Ariadne, androgen receptor-associated changing subcellular distributions. protein 54 (ARA54), RNF144, dorfin, parkin and TRIAD3 subfamilies [10,16,49,56-62] (Additional data file 1). Several RBR proteins are associated with neurodegenerative Parkin’s E3 activity is also suppressed through the binding and infectious diseases, and the three-dimensional struc- of the chaperone-like protein 14-3-3η to its linker region tures of the IBR and the complete RBR domain, and (Figure 5). 14-3-3η is released from this inhibitory complex knowledge of the residues responsible for interactions and upon its tight binding of α-synuclein. Thus, parkin’s activity structural stabilizations, will be a prerequisite for identifying is mutually regulated by 14-3-3η and α-synuclein [63]. A specific target sites for the possible design of therapeutic comparable antagonistic regulation of parkin by the carboxyl drugs. Future work should reveal the functional significance terminus of the Hsc-70-interacting protein (CHIP) and the of the additional domains and their cooperation with the chaperone Hsp70 is involved in the degradation of unfolded RBR domain. This is particularly important for members of Pael-R [45]. Whereas Hsp70 inhibits parkin’s E3 activity by the neglected organism-specific subfamilies, as these are forming a complex with unfolded Pael-R and parkin, CHIP found in organisms that pose pathogenic, agricultural and induces the dissociation of Hsp70 and enhances parkin’s aquacultural threats (for example, the human pathogenic activity and the degradation of Pael-R. Parkin is also protozoan Entamoeba histolytica and fungus Aspergillus inhibited through its enhanced sequestration to protein fumigatus, the plant pathogenic fungi Gibberella zeae and aggregates on interaction with Bcl-2-associated athanogene Magnaporthe grisea, and the fish and amphibian viruses 5 (BAG5) and Hsp70 [64]. lymphocystis disease virus and grouper iridovirus). More surprises and astonishing and valuable discoveries are Phosphorylation is involved in the negative regulation of expected from the future analysis of RBR proteins. parkin, as stress-induced reduction of phosphorylation results in an increase in its activity [65]. Parkin is controlled by nitric-oxid (NO) modifications in a biphasic manner Additional data file [66,67]. Within the first two hours of S-nitrosylation, Additional data file 1, containing supplementary tables and parkin’s catalytic activity increases. Then it declines additional references, is available online. gradually and is inhibited 24 hours after NO exposure [68]. Whereas phosphorylation and S-nitrosylation are reversible, a novel irreversible covalent adduct, a dopamine-derived Acknowledgements catechol modification of parkin, has been detected that BE and FE are grateful to Boehringer Ingelheim for continuous support. This project benefited from a grant to FE under the Gen-AU BIN scheme decreases E3 activity and solubility. Parkin is particularly of the Austrian Federal Ministry of Education, Science and Culture. MTH sensitive to this modification, as the adduct could not be and NC are supported by the Austrian Science Fund (FWF project transferred to the Ariadne-subfamily member HHARI [69]. P17888) with resources from the Nationalstiftung.

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