ubiquitination is an important posttranslational modification that regulates diverse processes , including protein degradation , intracellular trafficking , transcription , kinase activation , and the dna damage response ( hershko and ciechanover , 1998 ; komander and rape , 2012 ) . this variety of functions is mediated by eight different types of polyubiquitin ( polyub ) linkages , and , although the roles of lys48- and lys63-linked polyub have been studied in great detail , much less is known about the remaining  atypical  ub chains ( behrends and harper , 2011 ; kulathu and komander , 2012 ) . met1-linked polyub ( met1-polyub ) is the source of the cellular ub pool , given that ub is translated as a polyprotein ( ozkaynak et  al . , 1984 ) and posttranslationally processed by dedicated dubs , such as usp5 ( also known as isot ) ( amerik ayu et  al . , 1997 ) . this chain type can also be assembled by the linear ub chain assembly complex ( lubac ) , a multisubunit e3 ligase consisting of hoip , hoil-1l , and sharpin ( gerlach et  al . , 2011 ; ikeda et  al . , 2011 ; kirisako et  al . , 2006 ; tokunaga et  al . , lubac has roles in nf-b activation ( haas et  al . , 2009 ; tokunaga and iwai , 2012 ; tokunaga et  al . , 2009 ; , 2012 ) and is required for full activation of the inhibitor of b ( ib ) kinase ( ikk ) complex . ikk activation leads to the phosphorylation and degradation of ib and the activation of the nf-b transcription factor ( karin and ben - neriah , 2000 ) . it is not fully understood how met1-polyub regulates this process , but it involves the binding and modification of the ikk subunit nemo with met1-linked chains . nemo harbors a met1-specific ub - binding domain ( ubd ) that is important for nf-b signaling ( komander et  al . , 2009 ; rahighi et  al . much less is known about dubs that regulate met1-polyub chains , and a specific dub for met1-linkages has not been identified . of the roughly 80 active dubs in the human genome , a potential reason is the distinct chemistry of a peptide versus an isopeptide linkage ( figure  1a ) . a20 regulates nf-b signaling ( hymowitz and wertz , 2010 ) , otud5 ( also known as duba ) regulates irf3 signaling ( kayagaki et  al . , 2007 ) , and otub1 regulates the dna damage response ( nakada et  al . , 2010 ) structural work has revealed the basis for otub1 lys48 specificity ( juang et  al . , 2012 ; wiener et  al . , 2012 ) and trabid specificity against lys29 and lys33 linkages ( licchesi et  al . , 2012 ) . moreover , viral otu dubs have been reported that are highly divergent in sequence but are structurally similar ( frias - staheli et  al . , 2007 ) . here , we identify a previously unannotated human dub , fam105b / otulin , which is specific for met1-linked ub chains . structural studies reveal that this specificity is due to met1-specific ub - binding sites and a mechanism of substrate - assisted catalysis where a residue in a met1-linked chain directly participates in the organization of the catalytic triad of the enzyme . overexpression and knockdown analysis of otulin suggest that the protein binds lubac and regulates lubac - mediated processes in cells . given the high sequence divergence of otu domains , we set out to identify unstudied otu enzymes using a bioinformatical screen based on generalized profile analysis ( bucher et  al . , 1996 ) . iterative profile refinement , starting from a multiple - sequence alignment of experimentally validated otus , indicated an otu domain with a complete catalytic triad in the uncharacterized human protein fam105b ( figure  1b ) . fam105b comprises 352 amino acids ( aa ) , and the otu domain spans the majority of the protein ( aa 80352 ) and an n - terminal region with predicted helical content ( figure  1b , 1c ) . the catalytic domain is highly conserved between species ( figure  s1a available online ) . bacterially expressed full - length fam105b did not hydrolyze common fluorescent substrates such as ub - amc ( figure  s1b ) . ub - based suicide inhibitors that comprise an electrophilic group at the ub c terminus ( borodovsky et  al . , 2002 ) covalently modify most otu domain dubs but showed no reactivity against fam105b ( figure  s1c ) . however , dub assays against diubiquitin ( diub ) of all eight linkage types revealed that fam105b exclusively hydrolyzed met1-diub ( figure  1d ) . the enzyme was active at 10  nm concentration ( figure  1d ) and remained met1 linkage specific at a 1  m concentration ( figure  s1d ) . specificity is maintained when longer ub chains are used as substrates ( figure  1e ) but depended on an intact ub gly - met linkage sequence between ub moieties , given that mutant tetraub with ser - met linkages was not hydrolyzed ( figures 1e and s1e ) . catalytic mutants of fam105b ( c129a , h339a , and n341a ) did not hydrolyze met1-diub ( figure  1f ) . having established fam105b as a met1-linkage - specific otu dub , we named the enzyme otulin ( otu dub with linear linkage specificity ) . otulin is unique , given that the 14 annotated human otu dubs can not hydrolyze met1-diub ( mevissen et al . , 2013 ) . the catalytic domain of otulin ( otulincat , aa 80352 ) is sufficient for linkage specificity ( figure  1 g ) , and otulincat c129a bound met1-diub with a similarly high affinity as full - length otulin c129a , as revealed by fluorescence anisotropy measurements ( kd 196 versus 178  nm , figure  1h ) . otulincat was crystallized , and its structure was determined to 1.3   resolution with semet phasing ( figure  2a and table s1 ) . otulincat adopts an otu fold most similar to otub1 ( root - mean - square deviation [ rmsd ] 2.1   , dali z score 8.7 ) ( figure  2b ) . interestingly , catalytic triad residues his339 and cys129 display two alternate conformations . in the  active  conformation ( occupancy 30% ) , the catalytic triad is formed by interactions between asn341 , his339 , and cys129 ( figure  2c ) ; e.g. , as observed in otub1 in complex with ub suicide inhibitor ( wiener et  al . , 2012 ) inhibited  conformation ( occupancy 70% ) , asp336 pulls his339 away from its catalytic position ( figure  2c ) , and cys129 flips to an inactive rotamer . next , we determined the structure of otulin d336a to 1.35   resolution ( figures 2d and s2 g and table s1 ) . there were no global structural perturbations ( figures s2e and s2f ) , but his339 was now in the active rotamer , and cys129 showed increased occupancy of the active rotamer ( figure  2d ) . consistently , ub suicide inhibitors that did not modify wild - type ( wt ) otulin modified otulin d336a and also otulin n341d , in which the catalytic asn341 was changed to a negatively charged asp ( figure  2e ) . both mutants stabilize his339 in the active conformation , generating a more reactive enzyme . to understand how otulin acted on met1-polyub specifically , we determined the structure of otulincat c129a bound to met1-diub to 1.9   resolution ( figures 2f and s2 and table s1 ) . the distal and proximal ub moieties occupy extensive s1 and s1 ub - binding sites on otulin , respectively ( figures 2f2h ) . residues mediating ub binding are highly conserved in  otulin orthologs ( figure  s2i ) . the binding interface with the  distal ub covers 1,045   and involves the ile44 patch that  interacts with a helical arm ( aa 254264 ) conserved in all otu domains ( figures 2f2h ) . however , compared to the otub1ub structure ( wiener et  al . , 2012 ) ( figure  s2j ) , the distal ub rotates by 18 in the s1 binding site ( figure  s2k ) . the proximal ub binds with an interface of 880   to an s1 ub - binding site formed by helices 1 and 2 of otulincat via an unusual binding surface on ub involving the ub helix and the phe4 patch ( figure  2h ) . point mutations in the s1 ( l259e and e314r ) or s1 ( w96a and tqk100 - 102aaa ) ub - binding sites reduced otulin activity toward met1-diub ( figure  2i ) by decreasing met1-diub affinity ( figure  2j ) . the extensive s1 ub - binding site is likely to be important for otulin specificity in that it orients the proximal ub such that only met1 points toward the catalytic center ( figure  3a ) . in this orientation of the proximal ub , all lys residues are remote from the catalytic center , except for lys63 , which is spatially close to met1 ( figure  3b ) . importantly , otulin wedges these linkage points apart by two loops ( aa 125127 and 282284 ) that fix lys63 in a dedicated binding pocket ( figure  3b ) . nonetheless , a differently linked ub chain including a lys63-linked diub would rotate the proximal ub moiety by several degrees , and such orientation would likely not fit the otulin s1 binding site . indeed , lys63-linked diub bound with 100-fold reduced affinity ( < 112  nm for met1-linked diub versus 12  m for lys63-linked diub ) , and no binding was detected for lys48-linked diub ( figure  3c ) . this shows that the ub - binding sites of otulin already distinguish between structurally similar met1 and lys63 chain types by two orders of magnitude . the complex structure revealed that the proximal ub directly participates in the organization of the catalytic center . autoinhibition of the catalytic triad in the absence of substrate ( figure  3d ) is released by the binding of the met1-linked proximal ub ( figures 3e and 3f ) . the carbonyl group of met1 sterically interferes with the inhibited conformation of his339 , pushing it into an active conformation ( figure  3f ) . lys - linked ub chains lack a structural equivalent of this carbonyl moiety in the linkage ( figure  1a ) . more significantly , glu16 of the proximal ub is inserted into the catalytic center , displacing the inhibitory otulin residue asp336 , further restricting the mobility of his339 ( figures 3e and 3f ) . in addition , glu16 coordinates the third residue in the catalytic triad asn341 , aligning it toward his339 ( figure  3e ) . importantly , met1-diub with mutations of glu16 in the proximal ub was hydrolyzed with significantly lower activity in qualitative gel - based assays , whereas mutation of nearby gln2 ( which also interacts with otulin , figure  3e ) had no effect ( figures 3 g and s3a ) . all mutants were hydrolyzed by the nonspecific dub usp21 ( ye et  al . , 2011 ) ( figure  s3b ) and bound to otulincat c129a in analytical gel filtration ( figure  s3c ) , and met1-diub e16a affinity toward otulin c129a was only slightly decreased ( 612 versus < 112  nm , figure  s3d ) . we used a quantitative fluorescent kinetic assay for diub cleavage ( virdee et  al . , 2010 ) to examine whether glu16 on a proximal ub had a direct role in catalysis ( figures 3h and 3i ) . met1-diub e16a decreased kcat 240-fold and enzyme efficiency ( kcat / km ) 74-fold ( because of a 3.5-fold lower km for the mutant ) in comparison to met1-diub ( figures 3h and 3k ) . interestingly , the negative charge on ub is important for otulin activity , given that met1-diub e16q still showed a 35-fold lower kcat for wt otulin . next , we tested whether the more reactive otulin n341d or d336a mutants ( figure  2e ) showed improved diub hydrolysis activity . wt met1-diub is a poor substrate for otulin n341d , most likely because of the repulsion of negative charges ( ub glu16 and otulin asp341 ) in the catalytic center ( figures 3i and k ) . indeed , otulin n341d showed improved kinetics when ub glu16 was mutated , and both kcat and km improved , although wt activity was not regained . the otulin n341d mutant worked best with met1-diub e16q , suggesting that the requirement for a negative charge was partially compensated ( figures 3j and 3k ) . the coordination of asn341 is a key event in otulin activation , as was confirmed when the only otulin residue coordinating the asn341 side chain , tyr91 , was mutated to phe , resulting in 20-fold reduction of kcat while not affecting km ( figures 2i , 2j , 3k , and s3f ) . strong effects of glu16 mutation were also observed in the otulin d336a mutant ( figures 3j and 3k ) . altogether , this showed that the coordination of the catalytic triad through ub interaction is important for otulin activation ( figure  3l ) . hence , we reveal a mechanism of substrate - assisted catalysis in which glu16 of the proximal ub activates the catalytic triad of otulin by both restricting the movement of the catalytic his339 and introducing a negative charge , presumably for the proper coordination of asn341 for catalysis . it appears that otulin has evolved met1-linkage - specific ub - binding sites to specifically interact with linear chains . additionally , to further distinguish chain types , otulin invokes a mechanism of substrate - assisted catalysis in order to ensure  that only met1 linkages are hydrolyzed . remarkable specificity suggests that met1-linked polyub have to be tightly regulated independently of other ubiquitination events in cells . the identification of otulin as a met1-linkage - specific dub prompted us to study its role in cells . a polyclonal antiserum detected otulin in human embryonic kidney ( hek ) 293et  and other cell lines ( figure  s4a c - terminal gfp - tagged otulin is cytoplasmic , active , and met1 specific ( figure  s4d otulin is evolutionarily restricted to vertebrates and selected invertebrate lineages but is not detected in d.  melanogaster and c.  elegans . interestingly , all otulin - comprising taxa also contain genes for components of the met1-polyub chain assembly machinery lubac . expression of the lubac components hoip , hoil-1l , and sharpin induced met1-polyub , which was removed when otulin was coexpressed ( figure  4a , lanes 2 and 3 ) . inactive otulin c129a led to the significant enrichment of met1-polyub in cells , and otulin ub chain - binding mutants w96a and l259e ( figures 2i and 2j ) increased met1 linkages in cells , albeit not to the same extent as c129a ( figure  4a , lanes 46 ) . , 2011 ; tokunaga et  al . , 2009 ) , which was suppressed when otulin was transiently coexpressed ( figure  4b ) and also in stable cell lines overexpressing otulin ( figures s4 g and s4h ) . despite the enrichment of met1-polyub ( figure  4a ) , otulin catalytic or ub - binding mutants still inhibited lubac - driven nf-b activity to some extent ( figure  4b ) . apparently , the increase in met1-polyub alone is not sufficient to augment nf-b activity ( figures 4a and 4b ) . nf-b activation by tnf leads to the translocation of the cytosolic p65 nf-b subunit to the nucleus ( hayden and ghosh , 2008 ) ( figures 4c , s5c , and s5d ) . c129a blocked p65 nuclear translocation after tnf stimulation , whereas otulin w96a or otulin l259e had no effect ( figures 4c , s5c , and s5d ) . it appears that otulin overexpression antagonizes nf-b activation by removing met1-polyub , whereas otulin c129a acts as a high - affinity ubd that competes with other met1-specific ubds required for nf-b signaling in a similar manner to the one recently reported for the overexpression of the nemo uban domain ( van wijk et  al . , 2012 ) . one of the few reported targets of lubac is nemo ( tokunaga et  al . , 2009 ) . transfection of gst - tagged nemo resulted in nemo modification which was  prevented in a nemo k285/309r double mutant where ubiquitination sites are mutated ( tokunaga et  al . , co - overexpression of lubac resulted in an additional ub band on nemo , and the met1-specific antibody indicated that this additional band was a short ub chain . this was absent in otulin - overexpressing stable cell lines ( figures 4d and s5f )  suggesting that otulin could remove met1-polyub from nemo . glutathione s - transferase ( gst ) pulldown of nemo precipitated hoip , hoil1 , sharpin , and otulin , suggesting that these proteins might form a complex ( figures 4d and s5f ) . immunoprecipitation ( ip ) of endogenous sharpin precipitated endogenous hoip and , interestingly , also endogenous otulin under unstimulated conditions ( figures 4e and s5 g ) . upon tnf stimulation , the tnf - r1 was enriched in sharpin ips ( figure  4e ) , further supporting the idea that a lubac - otulin complex is formed and that this complex may translocate to the tnf receptor signaling complex ( tnf - rsc ) . met1-linked polyub is known to affect complex assembly at the tnf - rsc ( haas et  al . importantly , overexpression of otulin abrogated nemo - ripk1 complex formation ( figure  4f ) . this supports the idea that the interaction between nemo and ripk1 is stabilized by met1 linkages and reveals a mechanism of how otulin may affect nf-b activation in response to tnf stimulation . next , with a cellular readout for otulin overexpression at hand , we set out to test otulin s mechanism of substrate - assisted catalysis in cells by characterizing the effects of an ub e16a mutant . control experiments assessing whether this mutant is still assembled in met1-polyub by hoip gave the surprising result that a minimal hoip ligase construct ( smit et  al . , 2012 ; stieglitz et  al . , 2012 ) was impaired in assembling met1-linked chains from ub e16a ( figure  5a ) . furthermore , fluorescent met1-diub e16a bound to the nemo uban domain with reduced affinity ( figure  5b ) , which is consistent with the known interaction between the nemo uban domain and ub glu16 ( figure  5c ) ( rahighi et  al . , 2009 ) . despite this , co - overexpression of ub e16a and lubac in hek 293et cells still activated nf-b , and this could not be completely reversed by otulin ( figure  5d ) , consistent with otulin s inability to efficiently hydrolyze ub e16a - containing polymers ( figure  3 ) . transient overexpression of otulin or the well - studied nf-b inhibitor a20 ( hymowitz and wertz , 2010 ) blocks poly(i : c ) induced nf-b activity ( figure  6a ) . in comparison , otulin inhibits tnf-mediated nf-b by 50% , which correlates with effects observed by lubac downregulation ( haas et  al . , 2009 ; tokunaga et  al . , 2009 ) ( figure  6a ) . in otulin - expressing stable cell lines , transcription of nf-b target genes was reduced in response to 10  ng / ml tnf ( figure  6b ) , and , although ib was rapidly phosphorylated and almost completely degraded after 15  min of tnf stimulation in control cell lines , ib phosphorylation , degradation , and nf-b activation was delayed in otulin - overexpressing cells ( figure  6c ) . moreover , stable overexpression of otulin sensitized cells to tnf-induced cell death ( figures 6d and 6e ) , consistent with observations in cpdm mice that lack sharpin ( gerlach et  al . , 2011 ; , 2011 ; tokunaga et  al . , 2011 ; walczak et  al . , 2012 ) or in humans with mutations in hoil1 ( boisson et  al . , 2012 ) . otulin overexpression promoted enhanced and persistent jnk activation and c - jun phosphorylation at later time points , leading to the cleavage of parp and caspase-3 , two key players in the regulation of cell - death induction ( figure  6f ) . as for ib phosphorylation , otulin overexpression also resulted in a delay of jnk activation kinetics ( figures 6c and 6f ) . small interfering rna ( sirna ) against otulin resulted in an  increase in lubac - induced nf-b activation in hek 293et and u2os cells and also in t - rex 293 cells stably expressing a doxycycline - inducible otulin - targeting microrna ( mirna ) ( figures 7a and s6a  s6c ) . lubac - dependent induction of nf-b could be prevented by co - overexpression of active , but not inactive , otulin ( figure  7a ) . interestingly , western blotting against the overexpressed lubac components revealed that hoip was ubiquitinated in otulin mirna cell lines ( figure  7b ) , and coexpression of otulin removed these chains , showing that they are met1 linked ( figure  7b ) . this suggested that one role of otulin in the lubac complex ( figure  4e ) is to prevent the autoubiquitination of hoip . however , lubac recruitment to the tnf - rsc was unchanged in otulin knockdown cell lines , and met1-ub linkages were enriched in the tnf - rsc , implying that otulin also regulates met1-ub on other components ( figure  7c ) . accordingly , we observed a slight increase in ripk1 ubiquitination enriched with a met1-linkage - specific ub - binding matrix in otulin - depleted u2os cells ( figure  7d ) , suggesting that ripk1 met1 ubiquitination in response to tnf is targeted by otulin ( figure  4f ) . stable otulin knockdown affected cytokine responses and led to enhanced nf-b activation after poly(i : c ) and tnf ( figure  7e ) , and , even though the initial induction of nf-b target genes was not severely altered after otulin knockdown , expression of a20 , ib , il-8 , and tnf was sustained in response to tnf ( figure  7f ) . initial phosphorylation and degradation of ib after tnf stimulation was only slightly enhanced in otulin knockdown cells ( figure  s6d ) . however , jnk and c - jun phosphorylation was sustained at late time points , again leading to cell death ( figures s6d  s6f ) . the fact that both overexpression and knockdown of otulin led to cell death was surprising and requires further investigation . otulin knockdown cell lines were deregulated in their nf-b response , but we could not observe a strong effect on canonical nf-b signaling . jurkat cells express higher amounts of otulin ( figure  s4c ) , and otulin knockdown by sirna led to an increase in phospho - ib and subsequent enhancement of nf-b dna binding ( figure  7 g ) . this was due to increased ikk activation ( figure  7h ) , consistent with current models of lubac function ( tokunaga and iwai , 2012 ; walczak et  al . , 2012 ) . altogether , our data are consistent with a role of otulin in lubac - mediated met1-polyub signaling . here , we identify fam105b / otulin as a human otu dub specific for met1-linked ub chains . otulin is a missing piece in met1-ub chain biology , for which no linkage - specific dub has been described to date . our data suggest that otulin is an antagonist of lubac in  vitro and in cells . we reveal the molecular basis for the remarkable met1 linkage specificity by structural and biophysical studies and give insights into potential roles of otulin as a lubac interactor and antagonist in cells . consistent with the biochemical findings , overexpression of otulin removes met1-polyub and dampens lubac - mediated nf-b signaling , whereas knockdown of otulin leads to increased met1-polyub , ikk activation , and nf-b signaling after tnf stimulation . however , both overexpression and knockdown of otulin sensitized cells to tnf-induced cell death , suggesting more complex roles for met1 linkages in balancing signaling pathways downstream of the tnf receptor . linkage specificity in dubs is not well understood , and the structure of apo otulin did not explain its met1 specificity , because of its structural similarity to the lys48-specific otub1 . the key insight into linkage specificity for these two enzymes came from the complex structures of the enzyme with two ub molecules bound across the active site . a high - affinity ub - binding site in otulin allows it to select met1-linked chains with 100-fold preference over lys63 linkages and disallows the binding of lys48-linked and presumably other chains . this mechanism is shared by many dubs ; e.g. , trabid ( licchesi et  al . , 2012 ) and otub1 ( juang et  al . , 2012 ; wiener et  al . , 2012 ) . however , in a physiological situation where multiple ub chain linkages are often mixed , substrate - targeted dubs may cleave additional ub chain types . otulin prevents such promiscuity by requiring a properly positioned ub residue , glu16 , for catalytic efficiency . even in lys63-linked chains , therefore , otulin is the first dub for which substrate - assisted catalysis has been demonstrated to achieve linkage specificity . it is possible that substrate - assisted catalysis explains the linkage ( or substrate ) specificity of other dubs . it will also be interesting to see whether dub activators exist that complement the catalytic triad in an analogous manner as that observed in otulin . ub - mediated substrate - assisted catalysis was recently shown to be crucial for the assembly of lys11-linked polyub , where ub glu34 complements the active site of the e2  ub - conjugating enzyme ube2s ( wickliffe et  al . , 2011 ) . the fact that glu16 also impairs hoip activity also suggests the functionality of this side chain in assembly reactions . altogether , this shows that ub is more than just a binding partner for other proteins and that it can actively participate in enzymatic reactions . in transfection experiments , we demonstrated that otulin could counteract cellular lubac responses , suggesting that otulin helps to balance the amount of met1-polyub in cells . furthermore , the mechanism of substrate - assisted catalysis suggests that otulin may not target individual proteins but , instead , target met1-linked ub chains , regardless of where they are attached . indeed , we identified three proteins that change in their ubiquitination status when otulin levels are modulated . nemo and ripk1 are among the few reported targets of lubac ( gerlach et  al . we provide evidence that otulin directly interacts with lubac . the observation that depletion of otulin leads to the modification of hoip with met1-polyub suggests that hoip , like many other e3 ligases , undergoes autoubiquitination and that otulin has the ability to prevent this . this most likely explains why the overexpression of otulin chain - binding mutants lead to increased met1-polyub in cells ( figure  4a ) . however , the functional consequences of a lubac - otulin interaction are unclear , given that hoip stability , recruitment , and activity appear to be unaffected , and this requires further investigation . regarding the functional requirement for met1-linked chains in nf-b responses , key questions remain to be answered . first , in  contrast to lys63-linked chains , for which roles in nf-b signaling have long been verified , for example , by elegant replacement strategies ( xu et  al . , 2009 ) , met1-linked polymers have a very low abundance , and are hard to detect in cell lysates . this may change with the discovery that catalytically inactive otulin ( figure  4a ) or otulin knockdown ( figure  7c ) leads to the stabilization of met1 linkages . a second , more important issue is the lack of proteins modified with met1-polyub in  vivo after a physiological stimulus . we believe that otulin can also be instrumental here , given that its specificity can be exploited as a met1-specific ub - binding protein when inactivated or in mass spectrometry methods . we anticipate that the identification of lubac and otulin targets may reveal a surprising variety of cellular proteins not restricted to nf-b signaling . our data identify roles of otulin in tnf signaling , which is in agreement with reported roles of met1-polyub in this pathway ( haas et  al . however , in tnf signaling , the putative redundant or nonredundant involvement of many different ub chain types leads to a complicated interplay of ub signaling ( kulathu and komander , 2012 ) . the functional importance of met1-linked chains in the mix is supported by genetic evidence from cpdm mice lacking sharpin ( gerlach et  al . , 2011 ; ikeda et  al . , 2011 ; tokunaga et  al . , 2011 ) and data from human patients with mutations in hoil1 ( boisson et  al . , 2012 ) . so far , there is no unifying molecular mechanism that explains why so many different forms of polyub are seemingly important for the activation of identical kinase cascades upon tnf stimulation . interestingly , the effects of otulin overexpression or knockdown seem more pronounced in poly(i : c ) versus tnf signaling ( figures 6a and 7e ) , and it will be important to study roles of otulin in other pathways that depend on met1-linked chains , such as nod2 signaling ( damgaard et  al . , 2012 ) . our data support a role of met1 linkages in providing an important scaffold for productive complex formation , given that the loss of met1-linked chains induced by otulin overexpression prevents the association of nemo with ubiquitinated ripk1 . ubiquitinated ripk1 is targeted also by the dubs a20 ( hymowitz and wertz , 2010 ) and cyld ( sun , 2010 ) . however , although a20 and cyld are induced by tnf for the establishment of a negative feedback loop , neither otulin ( figure  7f ) nor lubac ( haas et  al . , 2009 ) are induced by tnf , again suggesting that they function as a differentially regulated signaling module . otulin is subject to phosphorylation , acetylation , and ubiquitination in cells , and it will be interesting to study whether these modifications regulate its activity or function . overall , our data suggest that the identified dub otulin is an antagonist of the lubac e3 ligase complex . consistent with reported roles for met1-polyub , changing otulin expression affects lubac - mediated processes , including nf-b signaling . detailed genetic analysis will be necessary in order to understand how lubac and otulin balance met1-polyub chains in  vivo . this might confirm some reported roles and may reveal new cellular roles for this atypical ub chain type . fam105b / outlin was identified bioinformatically with generalized profile analysis , as described in the extended experimental procedures . fam105b was cloned from brain complementary dna ( invitrogen ) , expressed in e.  coli from popin - f vector , and purified by anion exchange and gel filtration . apo , semet - substituted , and mutant otulin were crystalized from 100  mm mes and imidazole , 30  mm mgcl2 , 30  mm cacl2 , 10% ( w / v ) peg 4k , and 20% glycerol ( ph 6.5 ) . the structure of apo otulin was determined by single anomalous dispersion , and subsequent structures were determined by molecular replacement . qualitative deubiquitination assays were performed as previously described ( komander et  al . , 2009 ) . ( 2011 ) , and kinetic studies were performed as in virdee et  al . otulin and a20 were expressed from popin - f or pcdna4/to / mrgs6h , hoip , sharpin and hoil-1l from pcdna3.1 , and gst - tagged nemo were expressed from pebg vectors . stable t - rex 293 cell lines overexpressing pcdna4/to / mrgs6h - otulin or a pdest30-emgfp construct with an mirna targeting the 3 untranslated region of fam105b were generated according to the manufacturer s protocol ( invitrogen ) . knockdown analysis was performed in mirna cell lines or with eight different sirna sequences , as listed in the extended experimental procedures . nf-b activity was assessed by luciferase assays , for which cells were cotransfected with m3p sin rev b firefly and prl - tk renilla ( promega ) luciferase plasmids by immunofluorescence with anti - p65 staining or by quantitative pcr , as described in the extended experimental procedures . tnf signaling was analyzed by western blotting with the antibodies listed in the extended experimental procedures . extended experimental proceduresbioinformaticsmultiple sequence alignments were created by using the l - ins - i algorithm of the mafft package ( katoh et  al . , 2002 ) . sequence profiles were constructed , scaled , and subjected to iterative refinement using programs from the pftools package ( bucher et  al . , 1996 ) . only database hits with p - values the significance of novel otu assignments was further assessed by hmm - hmm comparison using the hhsearch program ( sding , 2005).materials and reagentsdi- and tetraub for lys6 , lys11 , lys48 , lys63 were generated using published procedures ( bremm et  al . , 2010 ; hospenthal et  al . , 2013 ; komander et  al . , proteins and epitope tags were detected with the following antibodies : anti - a20 ( 59a426 ) ( imgenex ) ; anti - caspase-3 ( # 9662 ) , anti - c - jun ( 60a8 ) , anti - phospho - ib ( 14d4 or 5a5 ) , anti - ib ( l35a5 ) , anti - jnk1 ( 2c6 ) , anti - parp ( # 9542 ) , anti - phospho - c - jun ( d47g9 ) , anti - phospho - jnk ( 81e11 ) and anti - nemo ( da10 - 12 ) , ( cell signaling ) ; anti - hoil-1/c20orf18 , anti - hoip / rnf31 , anti - tubulin ( sigma ) ; anti - actin ( sc-1616 ) ( or millipore ) , anti - ib ( c15 ) , ikk / ( sc-7607 ) , anti - nemo ( sc-8330 ) , anti - p65 ( sc372 ) , anti - ub ( santa cruz biotechnology ) ( p4d1 ) , ( millipore ( fk2 ) or imgenex ) ; anti - ripk1 ( bd biosciences ) ; anti - sharpin , ( proteintech ) ; anti - ha , ( roche diagnostics ) ; anti-6his ( clontech ) and anti - rgs4his ( qiagen ) . the met1-linkage specific ub chain antibody was kindly provided by vishva dixit ( genentech ) . a fam105b / otulin antibody was generated by cambridge research biochemicals from rabbits that were immunized with full - length recombinant his6-otulin . recombinant human tnf ( r&d systems ) ; poly(i : c ) and blasticidine ( invivogen ; , zeocin ( invitrogen ) ; g418 ( paa ) and gtm20 gfp - trap ( chromotek).dna oligos were obtained from sigma aldrich and sirna duplexes against otulin and hoip from dharmacon ( smart pools , sp , thermo scientific ) , sigma and eurogentech.the prl - tk renilla ( invitrogen ) and the m3p sin rev b firefly luciferase ( coding for 4 nf-b binding sites in the firefly luciferase promoter ) plasmids were a kind gift of felix randow ( mrc lmb ) , pcdna3-hoip1-/hoil1-/sharpin - v5-his vectors were kindly provided by henning walczak ( imperial college , london ) . pcdna4/to / n - mrgs6h ( a modified pcdna4/to / myc - his b , invitrogen ) was a kind gift of gerrit j.k . pebg - nemo fl was a kind gift from sally swift ( icr london).molecular biologythe coding sequence for full - length fam105b / otulin was obtained following pcr amplification of a human adult normal brain cdna library ( invitrogen ) using kod hotstart dna polymerase ( novagen ) and the following primers : otulin fwd 5- aagttctgtttcagggcccgatgagtcgggggactatgcccc and otulin rev 5-atggtctagaaagctttatagactggtctcctcacaca ctctg . the pcr product was cloned into popinf and popin - gfp ( berrow et  al . , 2007 ) using infusion hd ( clontech ) . the otulin catalytic domain ( otulincat , aa 80 - 352 ) was subcloned into popinf from full - length otulin using otulin80 fwd primer 5-aagttctgtttcagggcccgttaagcgtagctcctgaaatggatatcatgg-3. otulin mutants ( c129a , y91f , w96a , l259e , e314r , and a triple mutant tqk100 - 102 to aaa ) were generated in the full - length otulin sequence in popin - f by site directed mutagenesis using the quikchange method with kod hotstart dna polymerase according to manufacturer s protocol , and used for bacterial protein expression as well as for luciferase analysis . otulin fl variants were subcloned into a modified pcdna4/to / mrgs6h using the following primers 5-gcggatccatgagtcgggggactatgcccc and 3-ggctcgagttatcatagactggtctcctcacacactctg . ha - tagged otulin ( ha - otulin ) was generated by pcr amplification of otulin from pcdna4/to / mrgs6h - otulin and cloning into not1 and xhoi sites in pcdna5/frt / to / n-2xha-2xstreptag ( a kind gift from pascal meier and tencho tenev , institute of cancer research , london , uk ) using the following primers 5'-aaaagcggccgcatgagtcgggggactatgccc and 5'-aaactcgagtcatagactggtctcctcacacactc . ub and met1-diub was expressed from pet17 plasmids ( komander et  al . , 2009 ) . mutated ( g76s ) met1-tetraub was generated by cloning ub fragments in which the c - terminal gly - gly coding sequence was exchanged for a bamhi site encoding gly - ser . flash - tagged diub constructs in which a c - terminal wccpgcc motif replaces aa 72 - 76 were described previously ( ye et  al . , 2011 ) . to generate untagged monoub wild - type and e16a for mammalian expression , the following primers were used : pcdna - ub fwd gcggatccgccgccaccatgcagatcttcgtg aagaccctgac and pcdna - ub rev cgaattcttatcacccacctctgaga cggaggac . note that sequencing revealed that the pebg - nemo k285,309r construct had an additional e287 g mutation.expression and purification of otulinotulin was expressed in e.  coli strain rosetta2 ( de3 ) placi . cells were grown at 30c in 2xty containing 50  g / ml ampicillin , 34  g / ml chloramphenicol to an od600 of 0.7 . the culture was cooled to 20c prior to induction with 150 - 400  m iptg and harvested 20  hr postinduction.cells were resuspended and lysed by sonication in lysis buffer ( 20  mm tris ph 8.0 , 500  mm nacl , 50  mm imidazole , 5  mm -mercaptoethanol , lysozyme dnasei ( sigma ) , protease inhibitor cocktail ( roche ) ) . otulin was purified by immobilized metal - affinity chromatography using ni resin ( ge life sciences ) . otulin was subjected to size exclusion chromatography ( sec ) ( hiload 16/60 superdex 75 , ge life sciences ) in buffer a ( 20  mm tris , 150  mm nacl , 5  mm -mercaptoethanol , ph 8.0 ) . for crystallographic analysis , otulincat was dialyzed into 20  mm tris , 5  mm dtt ( ph 8.5 ) and subject to anion exchange chromatography ( hitrapq ff , ge life sciences ) , prior to sec in buffer a. purified proteins were concentrated and flash frozen.qualitative dub linkage specificity assayqualitative deubiquitination assays were performed as previously described ( komander et  al . , briefly , dubs were diluted in 25  mm tris ( ph 7.5 ) , 150  mm nacl , and 10  mm dtt and activated at 23c for 10  min . subsequently , 1  m di- or tetraub was incubated with 10  nm , 50  nm or 1  m dubs in 50  mm tris ( ph 7.5 ) , 50  mm nacl and 5  mm dtt at 37c . samples were taken at different time points and directly mixed with 4x sds sample buffer to stop the reaction . the reaction was resolved on 4%12% sds - page gradient gels using mes running buffer ( invitrogen ) , and visualized by silver staining using the biorad silverstain plus kit.ub suicide probe generation and analysisub thioester was generated following ub - intein cleavage as described previously ( akutsu et  al . , 2011 ) . haloalkylamine haloacid salts of 2-chloroethylamine ( c2cl ) , 2-bromoethylamine ( c2br ) and 3-bromopropylamine ( c3br ) were dissolved to a final concentration of 0.7  m in 20  mm tris , 200  mm nacl , 5  mm dtt , ph 8.0 and 0.2  mmol of each salt was mixed with 40  m ub - thioester . the reaction was initiated with 100  l of 2  m naoh and incubated at room temperature for 40  min . the reaction was quenched following addition of equimolar hcl and the reacted ub - probes were buffer exchanged into 25  mm tris , 200  mm nacl , 5  mm dtt , ph 8.0.for monitoring ub - probe reactivity , votu was used as a positive control , which displays strong reactivity toward different ub - probes ( akutsu et  al . , 2011 ) . enzymes were diluted to a concentration of 3.6  m and reacted with 15  m ub - probe for 30  min at room temperature . the reaction was stopped by the addition of sds sample buffer ( invitrogen ) and resolved by sds-page.crystallizationinitial hits of all crystals were obtained by sitting - drop vapor diffusion method . native ( 15  mg / ml ) , selenomethionine ( semet)-substituted ( 0.1  mg / ml ) and d336a ( 12  mg / ml ) otulincat crystals were grown from drops containing an equal volume of protein and reservoir ( 100  mm mes / imidazole , 30  mm mgcl2 , 30  mm cacl2 , 10% ( w / v ) peg 4k , 20% ( v / v ) glycerol , ph 6.5 ) . for semet - substituted crystals , native crystals were used to seed hanging drops containing semet - substituted otulincat . for met1-diub complex crystals , otulincat c129a was mixed with met1-diub in a 1:1.2 molar ratio , and set up at a concentration of 13  mg / ml . crystals grew from 100  mm bis - tris , 2  m ( nh4)2so4 , ph 6.5 . prior to data collection wt , d336a and semet - otulincat crystals were harvested and vitrified in liquid nitrogen . otulincat c129a - met1-diub crystals were soaked in 2.5  m li2so4 prior to vitrification.data collection , structure determination , and refinementdiffraction data were collected at the european synchrotron radiation facility ( esrf ) , beam lines id14 - 4 and id23 - 1 , and at diamond light source , beam line i04 - 1 . diffraction images were processed and integrated using imosflm ( battye et  al . , 2011 ) and scaled using scala ( evans , 2006 ) . the structure of otulincat was determined by sad phasing using data collected from a semet - substituted crystal . phasing and density modification were performed using the shelx software pipeline ( sheldrick , 2008 ) . the structure of otulincat d336a was determined using a refined model of the apo otulincat structure with residues from the catalytic site omitted during the early stages of refinement . the structure of otulincat c129a in complex with met1-diub was determined by molecular replacement using phaser ( mccoy et  al . , 2007 ) with otulincat and ub as initial search models . iterative rounds of model building and refinement were performed with coot ( emsley et  al . , 2010 ) and phenix ( afonine et  al . , 2012 ) , respectively . final refinement of wt and d336a otulincat structures employed refmac5 ( murshudov et  al . , 2011 ) where the occupancies of cys129 and his339 were independently refined . simulated - annealing composite omit maps were calculated using phenix ( afonine et  al . , 2012 ) . data collection and refinement statistics can be found in table s1.expression , purification , and labeling of ub constructsub constructs ( mono- and met1-diub ) were expressed and purified as described ( pickart and raasi , 2005 ) . in the case of flash - tagged diub constructs , 5  mm -mercaptoethanol was included in all purification steps to prevent oxidation of the wccpgcc motif . flash - tagged met1-diub constructs were labeled by lumio green ( invitrogen ) as described in ( ye et  al . , 2011 ) , flash - frozen and stored in aliquots at 80c.ub - amc assayub - amc ( boston biochem ) was diluted in reaction buffer ( 20  mm tris , 100  mm nacl , 1  mm -mercaptoethanol , ph 7.4 ) . for each reaction 10  l of diluted substrate in a black 384-well low volume plate ( corning ) was mixed with 10  l of either : 10 pm uch - l3 ( virdee et  al . , 2010 ) or 500  nm otulin at 37c . the rate of amc generation was measured using a pherastar plate reader ( bmg labtech ) . fluorescent intensities were recorded following excitation at 340  nm and emission at 440  nm . initial rates of ub - amc cleavage were recorded at each substrate concentration and fitted to a michaelis - menten equation using graphpad prism 5.binding studies performed with size exclusion chromatographyanalytical sec analysis was performed on an akta micro system ( ge life sciences ) using a superdex 75 pc 3.2/30 column equilibrated in sec buffer ( 20  mm tris , 50  mm nacl , 5  mm dtt ph 8.0 ) . inactive otulin 80 - 352 c129a ( otulincat c129a ) was mixed with met1-diub variants in a 1:1.3 molar ratio ( 33.5  m : 43  m ) and incubated at room temperature for 30  min . 125  g of complex was loaded onto the column . fractions containing protein were mixed with sds loading buffer prior to sds - page analysis.binding studies performed with fluorescence anisotropyto measure binding affinities of otulin c129a variants to met1-diub variants , 10  l of 100  nm flash - tagged met1-diub was aliquoted into a 384 -well low volume plate ( corning ) . serial dilutions in flash - buffer ( 20  mm tris ( ph 7.4 ) , 100  mm nacl , 5  mm -mercaptoethanol , 0.1  mg / ml bovine serum albumin ) were prepared of otulin c129a variants and 10  l of this was added to flash - tagged met1-diub containing wells . fluorescence polarization was recorded on a pherastar plate reader ( bmg labtech ) using an optics module with ex  = 485  nm and em  = 520  nm . fluorescence polarization values were fitted to a one - site binding model using graphpad prism 5 to derive binding constants ( kd).chain cleavage kinetics assessed with fluorescence anisotropychange in fluorescence anisotropy upon cleavage of the peptide bond between the distal and proximal ubiquitin molecules was used to derive michaelis - menten rates ( virdee et  al . , 2010 ) . reactions were performed in a black 384-well low volume plate ( corning ) and measured on a pherastar plate reader ( bmg labtech ) carrying a fluorescence polarization module with ex  = 485  nm and em  = 520  nm . met1-diub variants were serially diluted into flash - buffer and contained a fixed concentration of 300  nm flash - tagged met1-diub , to detect changes in fluorescence anisotropy . to each well , 10  l of substrate was mixed with 10  l otulin variants in flash - buffer . the observed fluorescence polarization values were converted to percentage of substrate cleavage by comparing to baseline values from intact substrate and flash - tagged monoub that were acquired for each experiment . all measurements were corrected by subtracting changes in fluorescent anisotropy for flash - tagged met1-diub alone . initial rates of the enzymatic reaction were calculated and plotted against substrate concentration at a fixed otulin concentration , allowing determination of michaelis - menten parameters ( figure  3).linear chain assembly assaysthe rbr and c - terminal region of hoip ( residues 6991,072 ) ( smit et  al . , 2012 ; stieglitz et  al . , 2012 ) was cloned into popink . cells were grown at 37c until an od of 0.9 , induced with 50  m iptg , 200  m zncl2 and grown at 19c overnight . cells were lysed in gst lysis buffer ( 270  mm sucrose , 10  mm glycerol 2-phosphate , 50  mm naf , 14  mm -mercaptoethanol , 50  mm tris ( ph 8.0 ) ) and the lysate was incubated with glutathione sepharose 4b ( ge life sciences ) . cleaved protein was subjected to sec ( hiload superdex75 16/60 , ge healthcare ) in 200  mm nacl , 10  mm dtt , 25  mm tris ph 8.5.the ligation assay was performed by mixing 100  nm e1 , 2.2  m ube2l3 , 2.5  m hoip 699 - 1072 and 0.25  mg / ml ub or ub e16a in reaction buffer ( 40  mm tris ph 7.5 , 10  mm mgcl2 , 0.6  mm dtt ) . a sample was taken before addition of 10  mm atp ( t  = 0  min ) . the reaction was incubated at 37c for 60  min before another sample was removed . samples were analyzed by sds - page and stained using silverstain plus kit ( biorad).cell culturehek 293et , hela , u2os , hct116 and mcf7 cells were cultured in dmem  + glutamax - i ( gibco ) , rko and jurkat cells in rpmi ( gibco ) both supplemented with 10% ( v / v ) fbs ( hyclone , thermo scientific ) , 100  u / ml penicillin,100  g / ml streptomycin at 37c and 5% co2 . t - rex293 cells ( invitrogen ) were grown at similar conditions in dmem  + glutamax - i ( gibco ) , 10% ( v / v ) tet - approved fbs ( clontech ) , 100  u / ml penicillin , 100  g / ml streptomycin , 5  g / ml blasticidin ( invivogen).immunoprecipitation of endogenous otulin from hek 293et cellsendogenous otulin was immunoprecipitated from hek 293et cells using otulin - specific antibodies coupled to protein a sepharose ( roche ) via dimethylpimelimidate ( sigma ) . cells were lysed in pbs , 0.1% ( v / v ) triton x-100 , complete protease inhibitor cocktail ( roche ) , 2  mm nem and lysates were incubated overnight with protein a - coupled otulin antibodies . beads were washed five times with pbs , 0.01% ( v / v ) triton x-100 , complete protease inhibitor cocktail ( roche ) , 2  mm nem and eluted in pbs/0.5% sds ph 9.0 or by boiling in 2x sds sample buffer.immunoprecipitation of endogenous nemohela cells were transfected and treated as indicated . cells were lysed in ip buffer ( 25  mm hepes ph 7.4 , 150  mm kcl , 2  mm mgcl2 , 1  mm egta , 0.5% ( v / v ) triton x-100 ) supplemented with 5  mm n - ethylmaleimide ( nem ; sigma aldrich ) , complete protease inhibitor cocktail ( roche ) and phosstop ( roche ) for 30  min on ice . lysates were cleared by centrifugation and were incubated at 4c overnight with anti - ikk -coupled beads ( santa cruz biotechnology ) . beads were washed four times in 500  l ice - cold ip buffer and bound material eluted with 0.2  m glycine , ph 2.5.purification of ubiquitinated proteinsubiquitin conjugates from cell lysates were pulled down in u2os flpin - t - rex cells using affinity reagents . for isolation of met1-ub chains , recombinant protein containing one copy of the uban domain from human nemo ( residues 257 - 346 ) fused to glutatione - s - transferase ( gst ) was used ( m1-sub ) . tube1 , consisting of four uba domains in tandem fused to gst , was used to purify all ubiquitin chains . one confluent 10  cm dish per condition was lysed in 600  l lysis buffer ( 20  mm na2hpo4 , 20  mm nah2po4 , 1% ( v / v ) np-40 , 2  mm edta ) supplemented with 1  mm dtt , 5  mm nem , complete protease inhibitor cocktail and phosstop . lysates were divided in two and 50  g tube1 or 100  g m1-sub was added . lysates were cleared , mixed with glutathione sepharose 4b beads ( ge healthcare ) and incubated at 4c for a minimum of 2  hr with rotation . beads were washed four times in 500  l ice - cold pbs tween-20 ( 0.1% ( v / v ) ) . bound material was eluted with 1xsds sample buffer.isolation of otulin - gfp from cells for specificity assays2   10 hek 293et cells were transiently transfected with popin - gfp - otulin wt or c129a for 24  hr using genejuice ( novagen ) . cells were lysed in 200  l gfp lysis buffer ( 10  mm tris ph 7.5 , 150  mm nacl , 0.5  mm edta , 0.5% ( v / v ) np40 , complete protease inhibitor cocktail ( roche ) ) for 30  min on ice . diluted lysates were incubated with 120  l equilibrated magnetic gfp - trap beads ( chromotek ) for 16  hr at 4c and washed following the manufacturer s protocol . beads were then washed twice in dub dilution buffer ( 25  mm tris ph 7.5 , 150  mm nacl , and 10  mm dtt ) and specificity assays were performed as described above using approximately 500  nm gfp - otulin bound to gfp - trap beads.generation of stable inducible cell linest - rex293 cells were stably transfected with pcdna / to / n - mrgs6h - otulin using gene juice ( novagen ) . selection was carried out with 150  g / ml zeocin ( invivogen ) and individual colonies were sub - cloned , expanded and screened for otulin expression . otulin expression was induced with 1  g / ml doxycycline for 24  hr.for stable cell lines inducibly expressing nontargeting or otulin targeting mirna , the respective sequences were cloned into the  mammalian pt - rex - dest30 plasmid following the block - it pol ii mir rnai expression vector kit with emgfp protocol ( invitrogen ) . the following sequences were used : otulin mirna top strand 5-tgctgtgctgttgaatccagacccaagttttggccactgactgacttgggtctattcaacagca-3 , otulin mirna bottom strand 5-cctgtgctgttgaatagacccaagtcagtcagtggccaaaacttgggtctggattcaacagcac-3. a nontargeting control mirna sequence was used according to the manufacturer s protocol . after stable transfection , selection was carried out with 500  g / ml g418 ( paa ) and individual clones were screened for gfp expression and otulin downregulation . otulin knock down was induced by 1  g / ml doxycycline for 60 - 72  hr.transient knockdown studiesfor transient knockdown studies , 20  nm sirna duplexes against otulin or hoip were transfected into hek 293et or u2os cells using interferin sirna transfection reagent ( polyplus transfection ) or lipofectamine rnaimax ( invitrogen ) according to the manufacturer s instructions and analyzed 48  hr posttransfection . for rna interference in jurkat t  cells , 100  nm sirna was transfected with atufect transfection reagent ( 0.51  mg / ml ) ( silence therapeutics ) and analyzed after 72  hr.sirna sequenceshuman fam105b / otulin on - targetplus smartpool ( sp , dharmacon / thermo scientific ) contained the following sirnas : gcuuaacugucucgggaaa , gggcaucagaaccgagauu , uagcaaaggcagggcgcaa , cuuuaguaguaacggguuu . hoip1/rnf31 on - targetplus smartpool contained the following sirnas : gcagaauacucauccaaga , ccuagaaccugaucuugca , ggcguggugucaaguuuaa , gccgagaugugcugcgauu . the on - targetplus nontargeting sirna # 1 ( dharmacon / thermo scientific ) or sequence gggauaccuagacguucua served as a nontargeting control . single sirnas ( sigma , eurogentech ) had the following sequences : # 1 : gacugaaauuugaugggaa , # 2 : caaaugaggcggaggaaua , # 3 : acagauagcuugugaugaa , # 4 : gcaucagaaccgagauuaa . sequence # 4 partly overlaps with one of the sp sequences.nfb activity assayscells were transiently transfected with the m3p sin rev b firefly and prl - tk renilla ( promega ) luciferase plasmids , and with plasmids encoding indicated proteins ( popinf - otulin variants , popinf - a20 , and/or lubac composed of pcdna3.1-hoil-1 , pcdna3.1-hoip1 or pcdna3.1-sharpin using genejuice ( novagen ) . cells were lysed in passive lysis buffer ( promega ) 15 or 24  hr posttransfection and luminescence was measured using a microplate reader ( berthold detection systems ) . for knockdown studies , cells were transfected with sirnas ( see above ) 48  hr before lysis . after 24 hr , luciferase plasmids were transfected either alone or together with lubac ; luciferase activity was assessed as described above . tnf or poly(i : c ) stimulation was carried out using the indicated concentrations for 6  hr or 20  hr , respectively.immunofluorescence staining and confocal microscopyhela cells were grown on glass cultureslides ( bd biosciences ) and transfected with pcdna4/to / n - mrgs6h - otulin wt , c129a , w96a or l259e 24  hr prior to stimulation with 20  ng / ml tnf for 30  min . cells were fixed in pbs , 4% paraformaldehyde ( w / v ) , permeabilized in pbs , 0.3% ( w / v ) saponin and blocked with pbs , 0.3% ( w / v ) saponin , 4% ( w / v ) bsa . slides were embedded in mounting medium with dapi ( vector laboratories ) and data were obtained using a lsm 710 on the inverse axio observer ax10 microscope and the zen2009 software ( zeiss).isolation of gst - nemo variants from stable cell linesgst - nemo wt or k285/309r ( kr ) was transfected with or without lubac into doxycycline induced control cells or otulin wt overexpressing cells . 24  hr later , cells were lysed in pbs , 0.1% ( v / v ) triton x-100 , complete protease inhibitor cocktail ( roche ) , 2  mm nem and 2.5  mg of total protein for each sample was incubated with 50  l gsh - coupled sepharose beads 4b ( ge healthcare ) overnight ( o / n ) . beads were washed five times with pbs , 0.01% triton x-100 ( v / v ) , complete protease inhibitor cocktail ( roche ) , 2  mm nem and proteins were eluted by boiling in 2x sds sample buffer . samples and inputs were subjected to sds - page and western blotting , and detection was carried out using indicated antibodies.nf-b target gene analysisstable cell lines downregulating or overexpressing otulin and their respective control cell lines were induced for expression with 1  g / ml doxycycline for 72  hr ( mirna ) or 24  hr ( overexpression ) , respectively . finally , cells were stimulated with 10  ng / ml tnf for the indicated times . cellular mrna was isolated using the rneasy mini kit , qiashredder and the rnase - free dnase set ( all qiagen ) . 1  g mrna was then reverse transcribed ( quantitect reverse transcription kit , qiagen ) and cdna quantification was measured by real - time pcr using the quantifast sybr green pcr kit ( qiagen ) in a rotorgene 6000 ( corbett research ) . data were analyzed with the rotorgene 6000 software and visualized in excel ( microsoft ) . all samples were normalized to their respective gapdh levels.electrophoretic mobility shift assaysfor electrophoretic mobility shift assay ( emsa ) , cells were lysed in whole cell lysis buffer ( 20  mm hepes ph 7.9 , 350  mm nacl , 20% ( v / v ) glycerol , 1  mm mgcl2 , 0.5  mm edta , 0.1 egta , 1% ( v / v ) nonidet p-40 , 0.5  m naf , 1  m dtt , 1  m -glycerophosphate , 200  mm na vanadate , and 50x complete protease inhibitor ( roche ) ) according to standard protocols . electrophoretic mobility shift assays ( emsa ) were performed by using a p - datp  labeled , double - stranded nf-b oligonucleotide probe ( 5-cagggctggggattccccatctccacagg-3 ) . the samples were separated on native polyacrylamide gels prior to autoradiography.cell viability assaysstable inducible cell lines overexpressing or downregulating otulin and their respective control cell lines were induced for expression with 1  g / ml doxycycline . after 24  hr ( overexpression ) or 72  hr ( mirna downregulation ) , on day 0 , cells were stimulated or not with 50  ng / ml tnf for 24  hr in doxycycline containing media . on day 1 , for samples of day 2 - 4 , media was exchanged to doxycycline containing media without tnf. thereby , detached cells were collected and re - plated into their respective wells . for all samples , attached and detached cells were collected after the indicated days and counted using a vi - cell xr cell viability analyzer ( beckman coulter ) . the staining procedure included fixing in pbs , 4% ( v / v ) paraformaldehyde for 5  min and staining in 0.05% crystal violet ( sigma ) for 30  min.analysis of signaling cascadesto analyze tnf-induced nf-b signaling , stable cell lines downregulating or overexpressing otulin and their respective control cell lines were induced for expression with 1  g / ml doxycycline for 72  hr ( mirna ) and 24  hr ( overexpression ) , respectively . finally , cells were stimulated with 10 ( mirna ) or 100  ng / ml tnf ( overexpression ) for the indicated times . cells were lysed in ice - cold lysis buffer ( pbs , 0.1% triton x-100 ( v / v ) , protease inhibitor cocktail ( roche ) , 2  mm nem , phosphostop ( roche ) ) for 30  min on ice . lysate protein concentration was measured by bradford assay , and equal protein amounts were subjected to sds - page and western blotting . analysis was carried out with the indicated antibodies.immunoprecipitation of the tnf receptor signaling complextnf - rsc was purified from the indicated cell lines after stimulation with 100  ng / ml flag - tnf ( ( human tnf , from alexis ) for the indicated times . following lysis , tnf - rsc was purified by incubation with flag m2 agarose beads ( sigma ) . tnfr was purified from the unstimulated sample by adding 1  g of tnf during lysis . the purified tnf - rsc was analyzed by western blotting for linear ubiquitination , hoip and tnfr1.kinase assayfor kinase assays , jurkat t  cells were lysed in 900  l co - ip buffer ( 25  mm hepes ph 7.5 , 150  mm nacl , 0.2% ( v / v ) np-40 , 10% ( v / v ) glycerol , 1  mm dtt , 10  mm sodium fluoride , 8  mm -glycerophosphate , 300  m sodium vanadate and protease inhibitor cocktail ) . after immunoprecipitation with nemo antibody ( santa cruz , sc-8330 ) , pellets were washed and incubated in kinase assay buffer ( 20  mm hepes ph 7.5 , 10  mm mgcl2 , 20  m atp , 20  mm -glycerophosphate , 50  m na vanadate , 1  mm dtt ) in the presence of gst - ib ( 1 - 53 ) for 25  min at 37c . after boiling in loading buffer , multiple sequence alignments were created by using the l - ins - i algorithm of the mafft package ( katoh et  al . , 2002 ) . sequence profiles were constructed , scaled , and subjected to iterative refinement using programs from the pftools package ( bucher et  al . , 1996 ) . only database hits with p - values the significance of novel otu assignments was further assessed by hmm - hmm comparison using the hhsearch program ( sding , 2005 ) . di- and tetraub for lys6 , lys11 , lys48 , lys63 were generated using published procedures ( bremm et  al . , 2010 ; hospenthal et  al . , 2013 ; komander et  al . , proteins and epitope tags were detected with the following antibodies : anti - a20 ( 59a426 ) ( imgenex ) ; anti - caspase-3 ( # 9662 ) , anti - c - jun ( 60a8 ) , anti - phospho - ib ( 14d4 or 5a5 ) , anti - ib ( l35a5 ) , anti - jnk1 ( 2c6 ) , anti - parp ( # 9542 ) , anti - phospho - c - jun ( d47g9 ) , anti - phospho - jnk ( 81e11 ) and anti - nemo ( da10 - 12 ) , ( cell signaling ) ; anti - hoil-1/c20orf18 , anti - hoip / rnf31 , anti - tubulin ( sigma ) ; anti - actin ( sc-1616 ) ( or millipore ) , anti - ib ( c15 ) , ikk / ( sc-7607 ) , anti - nemo ( sc-8330 ) , anti - p65 ( sc372 ) , anti - ub ( santa cruz biotechnology ) ( p4d1 ) , ( millipore ( fk2 ) or imgenex ) ; anti - ripk1 ( bd biosciences ) ; anti - sharpin , ( proteintech ) ; anti - ha , ( roche diagnostics ) ; anti-6his ( clontech ) and anti - rgs4his ( qiagen ) . the met1-linkage specific ub chain antibody was kindly provided by vishva dixit ( genentech ) . a fam105b / otulin antibody was generated by cambridge research biochemicals from rabbits that were immunized with full - length recombinant his6-otulin . recombinant human tnf ( r&d systems ) ; poly(i : c ) and blasticidine ( invivogen ; , zeocin ( invitrogen ) ; g418 ( paa ) and gtm20 gfp - trap ( chromotek ) . dna oligos were obtained from sigma aldrich and sirna duplexes against otulin and hoip from dharmacon ( smart pools , sp , thermo scientific ) , sigma and eurogentech . the prl - tk renilla ( invitrogen ) and the m3p sin rev b firefly luciferase ( coding for 4 nf-b binding sites in the firefly luciferase promoter ) plasmids were a kind gift of felix randow ( mrc lmb ) , pcdna3-hoip1-/hoil1-/sharpin - v5-his vectors were kindly provided by henning walczak ( imperial college , london ) . pcdna4/to / n - mrgs6h ( a modified pcdna4/to / myc - his b , invitrogen ) was a kind gift of gerrit j.k . pebg - nemo fl was a kind gift from sally swift ( icr london ) . the coding sequence for full - length fam105b / otulin was obtained following pcr amplification of a human adult normal brain cdna library ( invitrogen ) using kod hotstart dna polymerase ( novagen ) and the following primers : otulin fwd 5- aagttctgtttcagggcccgatgagtcgggggactatgcccc and otulin rev 5-atggtctagaaagctttatagactggtctcctcacaca ctctg . the pcr product was cloned into popinf and popin - gfp ( berrow et  al . , 2007 ) using infusion hd ( clontech ) . the otulin catalytic domain ( otulincat , aa 80 - 352 ) was subcloned into popinf from full - length otulin using otulin80 fwd primer 5-aagttctgtttcagggcccgttaagcgtagctcctgaaatggatatcatgg-3. otulin mutants ( c129a , y91f , w96a , l259e , e314r , and a triple mutant tqk100 - 102 to aaa ) were generated in the full - length otulin sequence in popin - f by site directed mutagenesis using the quikchange method with kod hotstart dna polymerase according to manufacturer s protocol , and used for bacterial protein expression as well as for luciferase analysis . otulin fl variants were subcloned into a modified pcdna4/to / mrgs6h using the following primers 5-gcggatccatgagtcgggggactatgcccc and 3-ggctcgagttatcatagactggtctcctcacacactctg . ha - tagged otulin ( ha - otulin ) was generated by pcr amplification of otulin from pcdna4/to / mrgs6h - otulin and cloning into not1 and xhoi sites in pcdna5/frt / to / n-2xha-2xstreptag ( a kind gift from pascal meier and tencho tenev , institute of cancer research , london , uk ) using the following primers 5'-aaaagcggccgcatgagtcgggggactatgccc and 5'-aaactcgagtcatagactggtctcctcacacactc . ub and met1-diub was expressed from pet17 plasmids ( komander et  al . , 2009 ) . mutated ( g76s ) met1-tetraub was generated by cloning ub fragments in which the c - terminal gly - gly coding sequence was exchanged for a bamhi site encoding gly - ser . flash - tagged diub constructs in which a c - terminal wccpgcc motif replaces aa 72 - 76 were described previously ( ye et  al . , 2011 ) . to generate untagged monoub wild - type and e16a for mammalian expression , the following primers were used : pcdna - ub fwd gcggatccgccgccaccatgcagatcttcgtg aagaccctgac and pcdna - ub rev cgaattcttatcacccacctctgaga cggaggac . note that sequencing revealed that the pebg - nemo k285,309r construct had an additional e287 g mutation . cells were grown at 30c in 2xty containing 50  g / ml ampicillin , 34  g / ml chloramphenicol to an od600 of 0.7 . the culture was cooled to 20c prior to induction with 150 - 400  m iptg and harvested 20  hr postinduction . cells were resuspended and lysed by sonication in lysis buffer ( 20  mm tris ph 8.0 , 500  mm nacl , 50  mm imidazole , 5  mm -mercaptoethanol , lysozyme dnasei ( sigma ) , protease inhibitor cocktail ( roche ) ) . otulin was purified by immobilized metal - affinity chromatography using ni resin ( ge life sciences ) . otulin was subjected to size exclusion chromatography ( sec ) ( hiload 16/60 superdex 75 , ge life sciences ) in buffer a ( 20  mm tris , 150  mm nacl , 5  mm -mercaptoethanol , ph 8.0 ) . for crystallographic analysis , otulincat was dialyzed into 20  mm tris , 5  mm dtt ( ph 8.5 ) and subject to anion exchange chromatography ( hitrapq ff , ge life sciences ) , prior to sec in buffer a. purified proteins were concentrated and flash frozen . qualitative deubiquitination assays were performed as previously described ( komander et  al . , 2009 ) . briefly , dubs were diluted in 25  mm tris ( ph 7.5 ) , 150  mm nacl , and 10  mm dtt and activated at 23c for 10  min . subsequently , 1  m di- or tetraub was incubated with 10  nm , 50  nm or 1  m dubs in 50  mm tris ( ph 7.5 ) , 50  mm nacl and 5  mm dtt at 37c . samples were taken at different time points and directly mixed with 4x sds sample buffer to stop the reaction . the reaction was resolved on 4%12% sds - page gradient gels using mes running buffer ( invitrogen ) , and visualized by silver staining using the biorad silverstain plus kit . ub thioester was generated following ub - intein cleavage as described previously ( akutsu et  al . , 2011 ) . haloalkylamine haloacid salts of 2-chloroethylamine ( c2cl ) , 2-bromoethylamine ( c2br ) and 3-bromopropylamine ( c3br ) were dissolved to a final concentration of 0.7  m in 20  mm tris , 200  mm nacl , 5  mm dtt , ph 8.0 and 0.2  mmol of each salt was mixed with 40  m ub - thioester . the reaction was initiated with 100  l of 2  m naoh and incubated at room temperature for 40  min . the reaction was quenched following addition of equimolar hcl and the reacted ub - probes were buffer exchanged into 25  mm tris , 200  mm nacl , 5  mm dtt , ph 8.0 . for monitoring ub - probe reactivity , votu was used as a positive control , which displays strong reactivity toward different ub - probes ( akutsu et  al . enzymes were diluted to a concentration of 3.6  m and reacted with 15  m ub - probe for 30  min at room temperature . the reaction was stopped by the addition of sds sample buffer ( invitrogen ) and resolved by sds - page . native ( 15  mg / ml ) , selenomethionine ( semet)-substituted ( 0.1  mg / ml ) and d336a ( 12  mg / ml ) otulincat crystals were grown from drops containing an equal volume of protein and reservoir ( 100  mm mes / imidazole , 30  mm mgcl2 , 30  mm cacl2 , 10% ( w / v ) peg 4k , 20% ( v / v ) glycerol , ph 6.5 ) . for semet - substituted crystals , native crystals were used to seed hanging drops containing semet - substituted otulincat . for met1-diub complex crystals , otulincat c129a was mixed with met1-diub in a 1:1.2 molar ratio , and set up at a concentration of 13  mg / ml . crystals grew from 100  mm bis - tris , 2  m ( nh4)2so4 , ph 6.5 . prior to data collection wt , d336a and semet - otulincat crystals were harvested and vitrified in liquid nitrogen . otulincat c129a - met1-diub crystals were soaked in 2.5  m li2so4 prior to vitrification . diffraction data were collected at the european synchrotron radiation facility ( esrf ) , beam lines id14 - 4 and id23 - 1 , and at diamond light source , beam line i04 - 1 . diffraction images were processed and integrated using imosflm ( battye et  al . , 2011 ) and scaled using scala ( evans , 2006 ) . the structure of otulincat was determined by sad phasing using data collected from a semet - substituted crystal . phasing and density modification the structure of otulincat d336a was determined using a refined model of the apo otulincat structure with residues from the catalytic site omitted during the early stages of refinement . the structure of otulincat c129a in complex with met1-diub was determined by molecular replacement using phaser ( mccoy et  al . , 2007 ) with otulincat and ub as initial search models . iterative rounds of model building and refinement were performed with coot ( emsley et  al . , 2010 ) and phenix ( afonine et  al . , 2012 ) , respectively . final refinement of wt and d336a otulincat structures employed refmac5 ( murshudov et  al . , 2011 ) where the occupancies of cys129 and his339 were independently refined . simulated - annealing composite omit maps were calculated using phenix ( afonine et  al . , 2012 ) . ub constructs ( mono- and met1-diub ) were expressed and purified as described ( pickart and raasi , 2005 ) . in the case of flash - tagged diub constructs , 5 mm -mercaptoethanol was included in all purification steps to prevent oxidation of the wccpgcc motif . flash - tagged met1-diub constructs were labeled by lumio green ( invitrogen ) as described in ( ye et  al . , 2011 ) , flash - frozen and stored in aliquots at 80c . ub - amc ( boston biochem ) was diluted in reaction buffer ( 20  mm tris , 100  mm nacl , 1  mm -mercaptoethanol , ph 7.4 ) . for each reaction 10  l of diluted substrate in a black 384-well low volume plate ( corning ) was mixed with 10  l of either : 10 pm uch - l3 ( virdee et  al . , 2010 ) or 500  nm otulin at 37c . the rate of amc generation was measured using a pherastar plate reader ( bmg labtech ) . fluorescent intensities were recorded following excitation at 340  nm and emission at 440  nm . initial rates of ub - amc cleavage were recorded at each substrate concentration and fitted to a michaelis - menten equation using graphpad prism 5 . analytical sec analysis was performed on an akta micro system ( ge life sciences ) using a superdex 75 pc 3.2/30 column equilibrated in sec buffer ( 20  mm tris , 50  mm nacl , 5  mm dtt ph 8.0 ) . inactive otulin 80 - 352 c129a ( otulincat c129a ) was mixed with met1-diub variants in a 1:1.3 molar ratio ( 33.5  m : 43  m ) and incubated at room temperature for 30  min . fractions containing protein were mixed with sds loading buffer prior to sds - page analysis . to measure binding affinities of otulin c129a variants to met1-diub variants , 10  l of 100  nm flash - tagged met1-diub was aliquoted into a 384 -well low volume plate ( corning ) . serial dilutions in flash - buffer ( 20  mm tris ( ph 7.4 ) , 100  mm nacl , 5  mm -mercaptoethanol , 0.1  mg / ml bovine serum albumin ) were prepared of otulin c129a variants and 10  l of this was added to flash - tagged met1-diub containing wells . fluorescence polarization was recorded on a pherastar plate reader ( bmg labtech ) using an optics module with ex  = 485  nm and em  = 520  nm . fluorescence polarization values were fitted to a one - site binding model using graphpad prism 5 to derive binding constants ( kd ) . change in fluorescence anisotropy upon cleavage of the peptide bond between the distal and proximal ubiquitin molecules was used to derive michaelis - menten rates ( virdee et  al . , reactions were performed in a black 384-well low volume plate ( corning ) and measured on a pherastar plate reader ( bmg labtech ) carrying a fluorescence polarization module with ex  = 485  nm and em  = 520  nm . met1-diub variants were serially diluted into flash - buffer and contained a fixed concentration of 300  nm flash - tagged met1-diub , to detect changes in fluorescence anisotropy . to each well , 10  l of substrate was mixed with 10  l otulin variants in flash - buffer . the observed fluorescence polarization values were converted to percentage of substrate cleavage by comparing to baseline values from intact substrate and flash - tagged monoub that were acquired for each experiment . all measurements were corrected by subtracting changes in fluorescent anisotropy for flash - tagged met1-diub alone . initial rates of the enzymatic reaction were calculated and plotted against substrate concentration at a fixed otulin concentration , allowing determination of michaelis - menten parameters ( figure  3 ) . the rbr and c - terminal region of hoip ( residues 6991,072 ) ( smit et  al . , 2012 ; stieglitz et  al . , 2012 ) was cloned into popink . protein was expressed in e.  coli rosetta2 placi cells . cells were grown at 37c until an od of 0.9 , induced with 50  m iptg , 200  m zncl2 and grown at 19c overnight . cells were lysed in gst lysis buffer ( 270  mm sucrose , 10  mm glycerol 2-phosphate , 50  mm naf , 14  mm -mercaptoethanol , 50  mm tris ( ph 8.0 ) ) and the lysate was incubated with glutathione sepharose 4b ( ge life sciences ) . cleaved protein was subjected to sec ( hiload superdex75 16/60 , ge healthcare ) in 200  mm nacl , 10  mm dtt , 25  mm tris ph 8.5 . the ligation assay was performed by mixing 100  nm e1 , 2.2  m ube2l3 , 2.5  m hoip 699 - 1072 and 0.25  mg / ml ub or ub e16a in reaction buffer ( 40  mm tris ph 7.5 , 10  mm mgcl2 , 0.6  mm dtt ) . a sample was taken before addition of 10  mm atp ( t  = 0  min ) . the reaction was incubated at 37c for 60  min before another sample was removed . samples were analyzed by sds - page and stained using silverstain plus kit ( biorad ) . hek 293et , hela , u2os , hct116 and mcf7 cells were cultured in dmem  + glutamax - i ( gibco ) , rko and jurkat cells in rpmi ( gibco ) both supplemented with 10% ( v / v ) fbs ( hyclone , thermo scientific ) , 100  u / ml penicillin,100  g / ml streptomycin at 37c and 5% co2 . t - rex293 cells ( invitrogen ) were grown at similar conditions in dmem  + glutamax - i ( gibco ) , 10% ( v / v ) tet - approved fbs ( clontech ) , 100  u / ml penicillin , 100  g / ml streptomycin , 5  g / ml blasticidin ( invivogen ) . endogenous otulin was immunoprecipitated from hek 293et cells using otulin - specific antibodies coupled to protein a sepharose ( roche ) via dimethylpimelimidate ( sigma ) . cells were lysed in pbs , 0.1% ( v / v ) triton x-100 , complete protease inhibitor cocktail ( roche ) , 2  mm nem and lysates were incubated overnight with protein a - coupled otulin antibodies . beads were washed five times with pbs , 0.01% ( v / v ) triton x-100 , complete protease inhibitor cocktail ( roche ) , 2  mm nem and eluted in pbs/0.5% sds ph 9.0 or by boiling in 2x sds sample buffer . cells were lysed in ip buffer ( 25  mm hepes ph 7.4 , 150  mm kcl , 2  mm mgcl2 , 1  mm egta , 0.5% ( v / v ) triton x-100 ) supplemented with 5  mm n - ethylmaleimide ( nem ; sigma aldrich ) , complete protease inhibitor cocktail ( roche ) and phosstop ( roche ) for 30  min on ice . lysates were cleared by centrifugation and were incubated at 4c overnight with anti - ikk -coupled beads ( santa cruz biotechnology ) . beads were washed four times in 500  l ice - cold ip buffer and bound material eluted with 0.2  m glycine , ph 2.5 . ubiquitin conjugates from cell lysates were pulled down in u2os flpin - t - rex cells using affinity reagents . for isolation of met1-ub chains , recombinant protein containing one copy of the uban domain from human nemo ( residues 257 - 346 ) fused to glutatione - s - transferase ( gst ) was used ( m1-sub ) . tube1 , consisting of four uba domains in tandem fused to gst , was used to purify all ubiquitin chains . one confluent 10  cm dish per condition was lysed in 600  l lysis buffer ( 20  mm na2hpo4 , 20  mm nah2po4 , 1% ( v / v ) np-40 , 2  mm edta ) supplemented with 1  mm dtt , 5  mm nem , complete protease inhibitor cocktail and phosstop . lysates were divided in two and 50  g tube1 or 100  g m1-sub was added . lysates were cleared , mixed with glutathione sepharose 4b beads ( ge healthcare ) and incubated at 4c for a minimum of 2  hr with rotation . beads were washed four times in 500  l ice - cold pbs tween-20 ( 0.1% ( v / v ) ) . 2   10 hek 293et cells were transiently transfected with popin - gfp - otulin wt or c129a for 24  hr using genejuice ( novagen ) . cells were lysed in 200  l gfp lysis buffer ( 10  mm tris ph 7.5 , 150  mm nacl , 0.5  mm edta , 0.5% ( v / v ) np40 , complete protease inhibitor cocktail ( roche ) ) for 30  min on ice . diluted lysates were incubated with 120  l equilibrated magnetic gfp - trap beads ( chromotek ) for 16  hr at 4c and washed following the manufacturer s protocol . beads were then washed twice in dub dilution buffer ( 25  mm tris ph 7.5 , 150  mm nacl , and 10  mm dtt ) and specificity assays were performed as described above using approximately 500  nm gfp - otulin bound to gfp - trap beads . t - rex293 cells were stably transfected with pcdna / to / n - mrgs6h - otulin using gene juice ( novagen ) . selection was carried out with 150  g / ml zeocin ( invivogen ) and individual colonies were sub - cloned , expanded and screened for otulin expression . otulin expression was induced with 1  g / ml doxycycline for 24  hr . for stable cell lines inducibly expressing nontargeting or otulin targeting mirna , the respective sequences were cloned into the  mammalian pt - rex - dest30 plasmid following the block - it pol ii mir rnai expression vector kit with emgfp protocol ( invitrogen ) . the following sequences were used : otulin mirna top strand 5-tgctgtgctgttgaatccagacccaagttttggccactgactgacttgggtctattcaacagca-3 , otulin mirna bottom strand 5-cctgtgctgttgaatagacccaagtcagtcagtggccaaaacttgggtctggattcaacagcac-3. a nontargeting control mirna sequence was used according to the manufacturer s protocol . after stable transfection , selection was carried out with 500  g / ml g418 ( paa ) and individual clones were screened for gfp expression and otulin downregulation . otulin knock down was induced by 1  g / ml doxycycline for 60 - 72  hr . for transient knockdown studies , 20  nm sirna duplexes against otulin or hoip were transfected into hek 293et or u2os cells using interferin sirna transfection reagent ( polyplus transfection ) or lipofectamine rnaimax ( invitrogen ) according to the manufacturer s instructions and analyzed 48  hr posttransfection . for rna interference in jurkat t  cells , 100  nm sirna was transfected with atufect transfection reagent ( 0.51  mg / ml ) ( silence therapeutics ) and analyzed after 72  hr . human fam105b / otulin on - targetplus smartpool ( sp , dharmacon / thermo scientific ) contained the following sirnas : gcuuaacugucucgggaaa , gggcaucagaaccgagauu , uagcaaaggcagggcgcaa , cuuuaguaguaacggguuu . hoip1/rnf31 on - targetplus smartpool contained the following sirnas : gcagaauacucauccaaga , ccuagaaccugaucuugca , ggcguggugucaaguuuaa , gccgagaugugcugcgauu . the on - targetplus nontargeting sirna # 1 ( dharmacon / thermo scientific ) or sequence gggauaccuagacguucua served as a nontargeting control . single sirnas ( sigma , eurogentech ) had the following sequences : # 1 : gacugaaauuugaugggaa , # 2 : caaaugaggcggaggaaua , # 3 : acagauagcuugugaugaa , # 4 : gcaucagaaccgagauuaa cells were transiently transfected with the m3p sin rev b firefly and prl - tk renilla ( promega ) luciferase plasmids , and with plasmids encoding indicated proteins ( popinf - otulin variants , popinf - a20 , and/or lubac composed of pcdna3.1-hoil-1 , pcdna3.1-hoip1 or pcdna3.1-sharpin using genejuice ( novagen ) . cells were lysed in passive lysis buffer ( promega ) 15 or 24  hr posttransfection and luminescence was measured using a microplate reader ( berthold detection systems ) . for knockdown studies , cells were transfected with sirnas ( see above ) 48  hr before lysis . after 24 hr , luciferase plasmids were transfected either alone or together with lubac ; luciferase activity was assessed as described above . tnf or poly(i : c ) stimulation was carried out using the indicated concentrations for 6  hr or 20  hr , respectively . hela cells were grown on glass cultureslides ( bd biosciences ) and transfected with pcdna4/to / n - mrgs6h - otulin wt , c129a , w96a or l259e 24  hr prior to stimulation with 20  ng / ml tnf for 30  min . cells were fixed in pbs , 4% paraformaldehyde ( w / v ) , permeabilized in pbs , 0.3% ( w / v ) saponin and blocked with pbs , 0.3% ( w / v ) saponin , 4% ( w / v ) bsa . slides were embedded in mounting medium with dapi ( vector laboratories ) and data were obtained using a lsm 710 on the inverse axio observer ax10 microscope and the zen2009 software ( zeiss ) . gst - nemo wt or k285/309r ( kr ) was transfected with or without lubac into doxycycline induced control cells or otulin wt overexpressing cells . 24  hr later , cells were lysed in pbs , 0.1% ( v / v ) triton x-100 , complete protease inhibitor cocktail ( roche ) , 2  mm nem and 2.5  mg of total protein for each sample was incubated with 50  l gsh - coupled sepharose beads 4b ( ge healthcare ) overnight ( o / n ) . beads were washed five times with pbs , 0.01% triton x-100 ( v / v ) , complete protease inhibitor cocktail ( roche ) , 2  mm nem and proteins were eluted by boiling in 2x sds sample buffer . samples and inputs were subjected to sds - page and western blotting , and detection was carried out using indicated antibodies . stable cell lines downregulating or overexpressing otulin and their respective control cell lines were induced for expression with 1  g / ml doxycycline for 72  hr ( mirna ) or 24  hr ( overexpression ) , respectively . finally , cells were stimulated with 10  ng / ml tnf for the indicated times . cellular mrna was isolated using the rneasy mini kit , qiashredder and the rnase - free dnase set ( all qiagen ) . 1  g mrna was then reverse transcribed ( quantitect reverse transcription kit , qiagen ) and cdna quantification was measured by real - time pcr using the quantifast sybr green pcr kit ( qiagen ) in a rotorgene 6000 ( corbett research ) . data were analyzed with the rotorgene 6000 software and visualized in excel ( microsoft ) . for electrophoretic mobility shift assay ( emsa ) , cells were lysed in whole cell lysis buffer ( 20  mm hepes ph 7.9 , 350  mm nacl , 20% ( v / v ) glycerol , 1  mm mgcl2 , 0.5  mm edta , 0.1 egta , 1% ( v / v ) nonidet p-40 , 0.5  m naf , 1  m dtt , 1  m -glycerophosphate , 200  mm na vanadate , and 50x complete protease inhibitor ( roche ) ) according to standard protocols . electrophoretic mobility shift assays ( emsa ) were performed by using a p - datp  labeled , double - stranded nf-b oligonucleotide probe ( 5-cagggctggggattccccatctccacagg-3 ) . stable inducible cell lines overexpressing or downregulating otulin and their respective control cell lines were induced for expression with 1  g / ml doxycycline . after 24  hr ( overexpression ) or 72  hr ( mirna downregulation ) , on day 0 , cells were stimulated or not with 50  ng / ml tnf for 24  hr in doxycycline containing media . on day 1 , for samples of day 2 - 4 , media was exchanged to doxycycline containing media without tnf. thereby , detached cells were collected and re - plated into their respective wells . for all samples , attached and detached cells were collected after the indicated days and counted using a vi - cell xr cell viability analyzer ( beckman coulter ) . the staining procedure included fixing in pbs , 4% ( v / v ) paraformaldehyde for 5  min and staining in 0.05% crystal violet ( sigma ) for 30  min . to analyze tnf-induced nf-b signaling , stable cell lines downregulating or overexpressing otulin and their respective control cell lines were induced for expression with 1  g / ml doxycycline for 72  hr ( mirna ) and 24  hr ( overexpression ) , respectively . finally , cells were stimulated with 10 ( mirna ) or 100  ng / ml tnf ( overexpression ) for the indicated times . cells were lysed in ice - cold lysis buffer ( pbs , 0.1% triton x-100 ( v / v ) , protease inhibitor cocktail ( roche ) , 2  mm nem , phosphostop ( roche ) ) for 30  min on ice . lysate protein concentration was measured by bradford assay , and equal protein amounts were subjected to sds - page and western blotting . tnf - rsc was purified from the indicated cell lines after stimulation with 100  ng / ml flag - tnf ( ( human tnf , from alexis ) for the indicated times . following lysis , tnf - rsc was purified by incubation with flag m2 agarose beads ( sigma ) . tnfr was purified from the unstimulated sample by adding 1  g of tnf during lysis . the purified tnf - rsc was analyzed by western blotting for linear ubiquitination , hoip and tnfr1 . for kinase assays , jurkat t  cells were lysed in 900  l co - ip buffer ( 25  mm hepes ph 7.5 , 150  mm nacl , 0.2% ( v / v ) np-40 , 10% ( v / v ) glycerol , 1  mm dtt , 10  mm sodium fluoride , 8  mm -glycerophosphate , 300  m sodium vanadate and protease inhibitor cocktail ) . after immunoprecipitation with nemo antibody ( santa cruz , sc-8330 ) , pellets were washed and incubated in kinase assay buffer ( 20  mm hepes ph 7.5 , 10  mm mgcl2 , 20  m atp , 20  mm -glycerophosphate , 50  m na vanadate , 1  mm dtt ) in the presence of gst - ib ( 1 - 53 ) for 25  min at 37c . after boiling in loading buffer ,
summarythe linear ubiquitin ( ub ) chain assembly complex ( lubac ) is an e3 ligase that specifically assembles met1-linked ( also known as linear ) ub chains that regulate nuclear factor b ( nf-b ) signaling . 
 deubiquitinases ( dubs ) are key regulators of ub signaling , but a dedicated dub for met1 linkages has not been identified . here , 
 we reveal a previously unannotated human dub , otulin ( also known as fam105b ) , which is exquisitely specific for met1 linkages . 
 crystal structures of the otulin catalytic domain in complex with diubiquitin reveal met1-specific ub - binding sites and a mechanism of substrate - assisted catalysis in which the proximal ub activates the catalytic triad of the protease . 
 mutation of ub glu16 inhibits otulin activity by reducing kcat 240-fold . 
 otulin overexpression or knockdown affects nf-b responses to lubac , tnf , and poly(i : c ) and sensitizes cells to tnf-induced cell death . 
 we show that otulin binds lubac and that overexpression of otulin prevents tnf-induced nemo association with ubiquitinated ripk1 . 
 our data suggest that otulin regulates met1-polyub signaling .