ubiquitin is an evolutionarily highly conserved small protein of 76 amino acids ( 8.6  kda ) .
ubiquitination is a post - translational protein modification , carried out by three classes of enzymes , namely the ubiquitin - activating- ( e1 ) , ubiquitin - conjugating- ( e2 ) , and ubiquitin - ligating - enzymes ( e3 ) .
the consecutive activity of these enzymes leads to the attachment of ubiquitin via its c terminus to a target protein ( hershko and ciechanover , 1998 ) .
ubiquitin itself can be ubiquitinated by attachment of the incoming ubiquitin to either of seven different lysine ( k ) residues ( k6 , k11 , k27 , k29 , k33 , k48 , k63 ) or the n - terminal methionine ( m1 ) .
thus , depending on the linkage type(s ) target proteins can be decorated with ubiquitin chains that are diverse in their compositions and exhibit different three - dimensional conformations ( kulathu and komander , 2012 ) . whereas k48-ubiquitin linkages serve to signal for protein degradation by the proteasome ( hershko and ciechanover , 1998 ) , non
- degradative ubiquitin chains have emerged as important regulators of signals emanating from diverse immune receptors including tnfr1 , nod2 , cd40 , tlr2 , tlr4 , and il-1r . upon stimulation by their respective ligands , components within the primary receptor - associated signaling complexes ( scs )
are modified by addition of k63- and m1-linked and , in certain cases , also other types of ubiquitin chains ( fiil and gyrd - hansen , 2014 , iwai et  al . , 2014 , shimizu et  al . , 2015 ,
formation of k63 chains is mediated by various e3 ubiquitin ligases specific for individual scs .
the linear ubiquitin chain assembly complex ( lubac ) , consisting of hoil-1 , sharpin , and the catalytically active subunit hoip , is the only currently known e3 capable of forming m1 chains de novo ( gerlach et  al . , 2011 , haas et  al . , 2009 , ikeda et  al . , 2011 ,
, 2006 , tokunaga et  al . , 2011 ) . in all of the above signaling pathways
, lubac has been determined to be responsible for m1 chain formation ( damgaard et  al .
, 2012 , emmerich et  al . , 2013 , gerlach et  al . , 2011 , rodgers et  al . , 2014 ) .
k63 chains are recognized by the ubiquitin binding domains of tab2 or tab3 ( kanayama et  al . , 2004 , wang et  al . , 2001 ) , resulting in recruitment of the tak / tab complex as well as lubac ( haas et  al . , 2009 ,
lubac then enables efficient recruitment of nemo and , consequently , of the nemo / ikk/ikk ( nemo / ikk ) complex ( haas et  al . ,
these two functional units then cooperatively trigger activation of the nf-b and mapk signaling pathways ( walczak et  al . ,
absence of lubac therefore attenuates gene induction by the above receptors and causes early embryonic lethality in mice due to aberrant tnfr1-induced endothelial cell death .
importantly , this cell death is due to increased formation of complex ii of tnfr1 and not caused by attenuated gene activation from the tnf - rsc ( peltzer et  al . , 2014 ) .
to signal at the physiological level in response to a given stimulus , it is not only required that the corresponding sc forms , but it also has to disassemble with the appropriate kinetics . regulated assembly and disassembly of ubiquitin chains within scs are essential to achieve this .
the enzymes responsible for removing ubiquitin moieties from target proteins and cleaving polyubiquitin chains are deubiquitinases ( dubs ) .
dubs implicated in the regulation of signaling by tnfr1 and other immune receptors are cyld , a20 ( harhaj and dixit , 2012 ) , and the m1-specific dub otulin , which was recently proposed to specifically antagonize lubac at scs , including in the context of the tnf - rsc and the nod2-sc ( fiil et  al . , 2013 , keusekotten et  al . , 2013 , rivkin et  al . , 2013 ) .
, 2003 , wright et  al . , 2007 ) , it cleaves various linkages in  vitro , albeit with preference for k63 and m1 linkages ( komander et  al . , 2008 , ritorto et  al . , 2014 ) .
a20 is induced by nf-b upon stimulation of various immune receptors and hydrolyzes k11 , k63 , and k48 but not m1 linkages ( mevissen et  al .
, 2013 , ritorto et  al . , 2014 , wertz et  al . , 2004 ) .
a20 binds to both k63 and m1 linkages via its zinc finger ( znf ) domains 4 and 7 , respectively ( bosanac et  al . , 2010 , tokunaga et  al . , 2012 ,
otulin deficiency is embryonically lethal due to vascular defects ( rivkin et  al . , 2013 ) .
cyld deficiency causes cylindromatosis in humans , a disease characterized by formation of benign tumors in the skin of affected individuals ( bignell et  al . , 2000 ,
deficiency in a20 , but interestingly not inactivation of its dub activity , causes early death in mice due to severe inflammation , implying that a20 likely exerts major functions independently from its dub activity ( lee et  al . , 2000 ,
recently , hoip was found to directly associate with both cyld and otulin in non - stimulated cells ( elliott et  al . , 2014 ,
, 2013 , schaeffer et  al . , 2014 , takiuchi et  al . , 2014 ) . here
, we analyzed the interplay between lubac , m1-ubiquitin , and the various before - mentioned dubs in assembly and disassembly of immune scs and the functional impact this interplay has on the regulation of their signaling output .
as linear ubiquitination is crucial for various immune signaling pathways ( gerlach et  al . , 2011 , haas et  al . , 2009 ) , we aimed to understand how it is regulated in scs . to do so , we created cell lines that are genetically deficient in hoip , the m1-chain - forming component of lubac , and re - expressed tandem affinity purification ( tap)-tagged ( 2   strep - tag ii followed by a prescission cleavage site and 1   flag ) hoip in these cells ( figure  s1a ) .
we first analyzed the unstimulated lubac obtained from these cells by mass spectrometry following tap .
in line with recent reports ( elliott et  al . , 2014 , fu et  al . , 2014 , rivkin et  al . , 2013 , schaeffer et  al
, our data showed that prior to stimulation , both cyld and otulin interacted with hoip ( figures s1b  s1d ) .
we therefore deemed it likely that lubac would mediate recruitment of both of these dubs to scs .
unexpectedly , however , a kinetic analysis following tumor necrosis factor ( tnf ) stimulation revealed that otulin was not recruited to the tnf - rsc following tnf stimulation , despite being present in lysates , whereas recruitment of cyld was evident , interestingly with kinetics reminiscent of lubac recruitment ( figure  1a ) .
it is currently unclear how cyld is recruited to scs , including the tnf - rsc , although optineurin was previously suggested to be involved ( nagabhushana et  al . , 2011 ) . as cyld interacts with lubac prior to stimulation , and
because it is recruited to the tnf - rsc with similar kinetics as lubac , we wondered whether recruitment of cyld may require hoip
. analyzing the native tnf - rsc in hoip - proficient versus -deficient cells revealed that while cyld was present in the tnf - rsc of hoip - proficient a549 ( figure  1b ) and hacat ( figure  s1e ) cells , it was absent from it in their hoip - deficient counterparts ( figures 1b and s1e ) .
thus , hoip is required for recruitment of cyld to the tnf - rsc . to determine whether absence of m1 chains from the tnf - rsc affected cyld presence and otulin absence , we reconstituted hoip - deficient cells with either tap - tagged wild - type ( wt ) or enzymatically inactive hoip - c885s ( smit et  al . , 2012 , stieglitz et  al . , 2012 ) .
when examining the effects of absence of lubac activity on the constitutive interaction of hoip with cyld and otulin , we found that neither of these interactions required hoip activity ( figure  1c ) . regarding the tnf - rsc , in line with our previous results ,
this complex is less stable in the absence of hoip or its activity ( haas et  al . , 2009 ) .
consequently , all proteins were retained less efficiently in the complex ( figure  1d ) .
importantly , however , cells expressing hoip - c885s maintained the capacity to recruit cyld to the tnf - rsc , yet otulin remained absent from this complex ( figure  1d ) .
we therefore conclude that the enzymatic activity of hoip is not required for recruitment of cyld to the tnf - rsc . as mentioned above , the mechanism of cyld recruitment to scs
was responsible for tnf - rsc recruitment , we wondered whether lubac might also be responsible for recruitment of cyld to other scs . to test this hypothesis
, we analyzed the receptor - associated complex that forms upon stimulation of nod2 because , despite of being composed quite differently from the tnf - rsc , signaling via nod2 also involves lubac ( damgaard et  al .
nod2 is an intracellular pattern - recognition receptor ( prr ) recognizing components of the bacterial cell wall , and it plays a critical role in gastrointestinal host defense ( chen et  al . , 2009 ) . as previous studies employed a system triggered by overexpression of nod2 that does not require ligand - induced stimulation and to avoid possible non - physiological events resulting from ligand - independent signaling by nod2 overexpression , we generated stable clones expressing tap - tagged nod2 at intermediate levels . in these cells ,
nod2-sc formation requires ligand - induced stimulation , which more closely resembles the physiological situation .
stimulation of these cells with synthetic muramyl dipeptide ( mdp ) l18-mdp , a known ligand for nod2 ( grimes et  al . , 2012 ) , induced strong nf-b activation in hoip - proficient , but not in hoip - deficient cells ( figures 2a and s2a ) . in line with reports on hoip
s requirement for erk activation upon cd40 and tnfr1 stimulation ( peltzer et  al .
, 2014 , sasaki et  al . , 2013 ) , we found that activation of erk was also substantially inhibited in hoip - deficient nod2-stimulated cells ( figure  s2a ) .
we next analyzed the nod2-sc and found that cyld also forms part of this complex ( figures 2b and s2b ) .
as with the tnf - rsc , hoip is essential for recruitment of cyld to the nod2-sc ( figures 2b and s2b ) , while otulin also does not form part of this complex ( figure  2b ) .
cyld was previously described as an inhibitor of tnf signaling ( brummelkamp et  al . , 2003 ,
, we isolated bone - marrow derived macrophages ( bmdms ) from wt and cyld - deficient mice and stimulated them with l18-mdp , as these cells constitutively express nod2 .
this revealed that in the absence of cyld , nod2-induced gene activation via nf-b and mapks ( erk , p38 , and jnk ) is enhanced ( figure  2c ) .
as it was unclear why otulin could be absent from lubac - containing scs despite interacting with lubac in the cytosol prior to stimulation , we next aimed to find a biochemical explanation for this unexpected phenomenon .
the crystal structure of the hoip pub domain bound to the otulin pim peptide ( aa 4967 ) shows that tyr56 in otulin and asn102 in hoip are crucial for their interaction ( elliott et  al . , 2014 , schaeffer et  al . , 2014 ) .
accordingly , phosphorylation of an otulin - derived peptide on the residue corresponding to tyr56 prevented its association with hoip s pub domain ( elliott et  al .
therefore , one possible explanation of otulin s absence from scs could be its release from lubac as a result of tyr56 phosphorylation .
this event would have to be postulated to occur at , or shortly following , recruitment .
tnf stimulation , however , did not abolish the interaction between hoip and otulin ( figures 1c and s3a ) .
furthermore , induction of global tyrosine phosphorylation by pervanadate treatment , which induces irreversible inhibition of phosphatases , did not prevent this association either ( figure  3a ) .
conversely , reduction of overall tyrosine phosphorylation by phosphatase treatment did not increase it ( figure  3a ) .
thus , stimulation - associated tyrosine phosphorylation of otulin , including at tyr56 , can not be responsible for otulin
intriguingly , hoip s relatively small pub domain mediates the interaction with both cyld and otulin and even short deletions in this domain abolished interaction with both factors ( figures s3b  s3d ) .
this suggested that steric hindrance may prevent simultaneous interaction of hoip with cyld and otulin .
if that was the case , they would compete for binding to hoip so that cyld - interacting lubac would be devoid of otulin and vice versa . to test this possibility , we precipitated tap - tagged otulin and checked for binding of lubac components and cyld .
while lubac components were co - precipitated , cyld was not ( figure  3b ) .
thus , cyld and tap - tagged otulin do not form part of the same individual lubac complexes , implying that they indeed can not simultaneously interact with an individual hoip protein . to address whether this also holds true for endogenous otulin , we next quantitatively immunoprecipitated otulin from wt cells , which would result  in removal of the fraction of lubac bound to endogenous otulin , yet without co - precipitating cyld . in accord with this hypothesis , cyld did not form part of endogenous otulin - associated lubac complexes .
subsequent cyld immunoprecipitation from otulin - depleted samples revealed that a second fraction of lubac is bound to cyld ( figure  3c ) .
hence , there are two distinct fractions of lubac in the cell , one associated with cyld , and another one bound to otulin
. the hoip - otulin interaction can be disrupted by mutation of a critical residue ( n102 ) in hoip s pub domain ( elliott et  al .
, 2014 ) . to test whether the cyld - hoip interaction would also be affected by this mutation , we reconstituted hoip - deficient cells with hoip - n102a and checked for association with cyld .
this revealed that hoip - n102a was unable to bind to cyld ( figure  3d ) .
together , these results show that cyld and otulin interact with hoip via the same or an overlapping site and that hoip s interactions with cyld and otulin are mutually exclusive .
based on these results , in combination with our findings regarding the composition of the tnf - rsc ( figure  1 ) and the nod2-sc ( figure  2 ) , it can be concluded that cyld - associated lubac is recruited to scs , whereas lubac associated with otulin is not .
tnf stimulation induces expression of various response genes , which help counteract invading pathogens but also promote autoinflammation ( walczak , 2011 ) .
we therefore analyzed the tnf - rsc in cells expressing hoip n102a to assess functional consequences of the loss of dub interaction with hoip .
the recruitment of cyld was abrogated in cells expressing this hoip variant ( figure  3e ) , confirming that the interaction with lubac is required for cyld recruitment . following tnf stimulation
, these cells showed increased m1 ubiquitination at the tnf - rsc ( figure  3e ) and enhanced activation of nf-b ( figure  3f ) .
similar results were obtained with cells expressing hoip devoid of the pub domain ( figure  s3e ) .
furthermore , the lack of hoip s pub domain resulted in increased tnf - induced expression of irf1 , icam1 , tnf , and ib ( figure  s3f ) .
nemo was previously shown to be linearly ubiquitinated ( gerlach et  al . , 2011 , tokunaga et  al . , 2009 ) , and
this polyubiquitination event , albeit weak , could be observed in cells expressing hoip - wt but not in cells lacking hoip or expressing inactive hoip . in cells expressing hoip - n102a , however , the polyubiquitination of nemo was enhanced ( figure  s3
, abolishing hoip s capacity to interact with both cyld and otulin enhances m1 ubiquitination in the tnf - rsc and , consequently , tnf - induced gene activation . as hoip - associated otulin
was not recruited to scs , we next studied whether otulin deficiency may increase levels of linear ubiquitin found in the cytosol . using crispr - cas9 , we generated otulin - deficient cells . already prior to stimulation ,
these cells contained significantly more m1-linked ubiquitin chains in their cytosol than control cells ( figures 4a and s4a ) .
these aberrant m1 chains were not free chains but conjugated to substrates ( figure  s4b ) .
the accumulation of linear ubiquitin linkages was found to be due to absence of otulin s enzymatic activity ( figure  s4c ) .
however , in accordance with our observation that otulin is not present at the tnf - rsc , linear ubiquitination within the tnf - rsc was not increased in otulin - ko as compared to wt cells ( figures 4a and s4a ) .
these circumstances led us to suspect that , instead of antagonizing m1-ubiquitin at scs , otulin might regulate lubac components themselves . to identify which proteins show
increased linear ubiquitination in lysates of otulin - deficient cells , we devised a new strategy to enrich for m1-ubiquitinated proteins .
this method , which we will refer to as m1-affinity  purification ( m1-ap ) , employs a bead - resin - immobilized , enzymatically inactive portion of otulin ( aa 58352 , c129a ) that binds to , but does not cleave , m1 linkages with high affinity and specificity ( keusekotten et  al . , 2013 )
prior to m1-ap , proteins were completely denatured in 1% sds and subsequently renatured so that only proteins directly modified by ubiquitin chains containing m1 linkages , and not proteins that are only non - covalently associated with such chains , could be detected in these assays .
notably , m1-ap showed that m1 ubiquitination of hoil-1 and sharpin was substantially increased in otulin - deficient cells ( figure  4c ) .
in accord with the results of the tnf immunoprecipitation , analysis of the components of the tnf - rsc by m1-ap showed that m1 ubiquitination of rip1 , a known lubac target in the tnf - rsc ( gerlach et  al .
, 2011 ) , was not increased in otulin - deficient cells ( figure  4d ) .
hence , rather than serving as a negative regulator of lubac activity at scs , otulin keeps components of lubac free of aberrant m1 linkages prior to stimulation .
since its association with lubac was the recruiting principle for cyld and because expression of hoip - n102a , unable to recruit cyld to the tnf - rsc , increased the amount of m1 linkages in this sc , we next sought to determine whether cyld could be responsible for antagonizing lubac activity in scs .
we employed crispr - cas9 to create cyld - deficient a549 cells and stimulated them with tnf before subjecting them to m1-ap or k63-ap , the latter being based on isolation of k63-ubiquitin linkages with tuim ( sims et  al . , 2012 )
when determining which proteins were modified by these linkages , we found that both m1 and k63 ubiquitination of rip1 , tnfr1 , and tradd were increased in the absence of cyld ( figures 5a and s5b ) .
this suggested that each one of these proteins carries m1 and k63 chains when present in the tnf - rsc , and that cyld antagonizes both of them on all of these proteins . to determine whether tnfr1 and tradd are bona fide lubac targets , subsequent to tnf stimulation and isolation by m1-ap we treated m1-ubiquitinated proteins with two different recombinant dubs ( figure  s5c ) .
treatment with otulin resulted in removal of all m1-linked chains from these proteins ( figure  5b ) .
crucially , this treatment significantly reduced the high - molecular - weight species of tnfr1 , tradd , and rip1 ( figure  5b ) .
thus , prior to otulin treatment m1-linked ubiquitin chains had been present on these proteins .
it should be noted that complete removal of ubiquitin chains by otulin would be expected only for exclusively linearly ubiquitinated targets .
however , in line with our previous results regarding rip1 ( gerlach et  al . , 2011 ) ,
tradd and tnfr1 also carry other chain types , and the degree of reduction in their overall ubiquitination by otulin treatment is indicative of the ratio between m1 linkages and other linkages present on these proteins . in contrast , the dub votu is capable of cleaving all ubiquitin linkages except for the m1 linkage ( akutsu et  al . , 2011 ) .
treatment with votu resulted in removal of all linkages from m1-affinity purified tnfr1 , tradd , and rip1 ( figures 5b and s5d ) .
as this includes the ubiquitin moieties through which m1 chains are linked to these proteins , a ladder of linear ubiquitin chains that are shed from these proteins by votu treatment becomes apparent .
as they are completely hydrolyzed when otulin is added together with votu , it can be concluded that these chains are pure m1-linked chains ( figure  5b ) .
importantly , no linear ubiquitination of rip1 , tnfr1 , or tradd was observed in cells lacking hoip ( figure  s5d ) .
these results identify tnfr1 and tradd as previously unrecognized , additional bona fide targets of lubac in the tnf - rsc .
in addition , they show that the linear chains present on these targets are of considerable length . as absence of cyld enhanced m1 ubiquitination of tnf - rsc components , we assessed whether the recombinant usp domain ( aa 583956 ) of cyld ( cyld - usp ) , encompassing its catalytic dub activity , could be capable of removing ubiquitin chains from them .
isolation of ubiquitinated proteins following tnf stimulation and subsequent treatment with recombinant cyld - usp or votu showed that cyld s dub domain is capable of removing the majority of overall ubiquitination from tnfr1 , tradd , and rip1 ( figure  5c ) .
the fact that treatment with cyld , in contrast to treatment with votu , did not release m1 chains demonstrates that cyld indeed hydrolyzes m1 linkages present on these target proteins ( figure  5c ) .
thus , cyld acts as a dub that antagonizes both linear and k63 ubiquitin linkages in the tnf - rsc . as cyld
was previously described to be a positive regulator of tnf - induced necroptosis ( odonnell et  al . , 2011 ) and because we previously showed that linear ubiquitination in the tnf - rsc protects from tnf - induced cell death ( peltzer et  al . , 2014 ) , we next wanted to assess the impact of cyld s activity on m1 chains in the tnf - rsc in relation to tnf - induced cell death . in line with previous studies
, we found that cyld - reconstituted but not -deficient mefs were prone to tnf- and tnf / zvad - induced cell death ( figure  5d ) .
importantly , this coincided with a decrease in both linear ubiquitination of tnf - rsc components and tnf - induced gene activation in cyld - reconstituted mefs ( figures 5e and 5f ) .
thus , cyld - mediated removal of ubiquitin chains , including of linear chains , from components of the tnf - rsc results in diminished tnf - induced gene activation and , at the same time , enhanced cell death . another major dub involved in tnf
the znf4 domain of a20 selectively recognizes k63-linked ubiquitin , and mutations in this domain were reported to impair a20 recruitment to the tnf - rsc ( bosanac et  al . , 2010 , lu et  al . , 2013 ) .
interestingly , the znf7 domain of a20 is involved in a20 s ability to suppress nf-b and cell death ( tokunaga et  al . , 2012 , verhelst et  al . ,
in addition , this domain binds with high affinity to linear ubiquitin chains , and tnf - rsc recruitment of a20 devoid of znf7 is reduced ( tokunaga et  al . ,
this prompted us to study the biochemical and functional interplay between lubac , linear ubiquitination , and a20 in tnfr1 and nod2 signaling .
a20 is induced by various stimuli in an nf-b - dependent manner ( catrysse et  al . , 2014 ) .
in all cell lines tested , we observed , however , that a20 was already present before stimulation .
consequently , it formed part of the tnf - rsc already 5  min after tnf stimulation , yet interestingly only in wt but not hoip - deficient cells ( figures 6a , s6a , and s6b ) . prolonged stimulation of up to 3  hr resulted in increased a20 expression and recruitment to the tnf - rsc , again only in control and not in hoip - deficient cells ( figure  s6c ) .
a20 functions as a negative regulator of nod2 signaling ( hitotsumatsu et  al . , 2008 ) .
we therefore next analyzed the nod2-sc for a20 presence and , if there , what the role of lubac would be in its recruitment .
this analysis revealed that a20 forms part of the nod2-sc and that hoip is required for this ( figure  6b ) .
as lubac deficiency reduces gene activation , we next addressed the role of hoip s enzymatic activity in induction of a20 .
as expected , hoip - deficient cells showed substantially decreased activation of nf-b and , consequently , reduced production of ccl2 and il-8 as well as a comparably weak upregulation of a20 upon tnf stimulation ( figures 6c6e ) . in cells expressing catalytically inactive hoip - c885s the tnf - induced stimulation of gene - activatory signaling pathways and cytokine production
interestingly , these cells had increased basal expression of a20 before stimulation , but 3  hr after stimulation the a20 levels were almost identical to the ones observed in hoip - wt cells ( figure  6e ) . to evaluate whether the enzymatic activity of hoip is required for a20 recruitment to the tnf - rsc
, we therefore compared the tnf - rsc that forms after 3  hr in cells expressing hoip - wt and hoip - c885s .
this showed that hoip - c885s expressing cells are almost completely defective in a20 recruitment to the tnf - rsc ( figure  6f ) .
together , this identifies lubac - generated m1 chains as required for recruitment of a20 to the tnf - rsc . even though a20 is unable to cleave m1 linkages , we hypothesized that it could affect m1 chains in scs indirectly through cleavage of other linkages .
we therefore next assessed the impact of a20 absence on the presence of linear ubiquitin chains in the tnf - rsc .
to do so , we again employed crispr - cas9 , this time to generate a20-deficient a549 cells .
unexpectedly , rather than being increased , m1 ubiquitination was markedly decreased in the tnf - rsc of a20-deficient as compared to control cells , a finding that also applied to tnfr1 ( figure  7a ) .
a20 deficiency also reduced the amount of m1-linked ubiquitin present in the nod2-sc ( figure  7b ) and on rip2 in this complex ( figure  s7b ) .
finally , also mefs deficient in a20 showed a marked reduction in linear ubiquitination at the tnf - rsc ( figure  s7c ) .
even though this result made it unlikely that a20 s dub activity was responsible for this effect , we could not formally exclude it based on the experiments performed so far .
independently thereof , we reasoned that because a20 is recruited to scs via linear ubiquitin chains , this interaction could be responsible for their a20-endowed stabilization . since a20
s znf7 was known to bind m1 chains and to be required for tnf - rsc recruitment ( tokunaga et  al . , 2012 ) , we tested whether znf7 could be responsible for a20-mediated stabilization of linear ubiquitin chains at the tnf - rsc
. to do so , we used crispr - cas9 to prepare cells lacking only the znf7 domain of a20 ( figure  s7d ) . in line with previous results ( tokunaga et  al . , 2012 ) , we found in both a549 and hacat cells that absence of znf7 severely compromised recruitment of a20 to the tnf - rsc and accumulation of ubiquitin chains therein ( figures 7c and s7e ) . to assess
whether the dub activity of a20 or its m1-binding function was responsible for its capacity to stabilize m1 linkages in the tnf - rsc , we reconstituted a20-deficient mefs with a20-wt , dub - inactive a20 ( a20-c103s ) , or an a20 znf7 point mutant c779a / c782a ( a20-znf7mut ) , which is unable to interact with m1 chains ( tokunaga et  al . , 2012 ) .
both a20-wt and a20-c103s , but not a20-znf7mut , were recruited to the tnf - rsc and stabilized linear ubiquitin linkages within this complex ( figures 7d and s7f ) .
thus , the linear - ubiquitin - binding activity of a20 , but not its dub activity , is required for stabilization of linear chains . in summary , these results show that direct binding of a20 to linear ubiquitin chains enables recruitment of a20 to the tnf - rsc and that this , in turn , results in stabilization of m1-ubiquitin chains in this complex .
the observation that znf7 is necessary for recruitment of a20  to the tnf - rsc fits with the finding that a20-wt and a20-c103s , but not a20-znf7mut , inhibit tnf - induced gene activation ( figures 7e and s7 g ) and protect cells from tnf / zvad - induced necroptosis ( figure  7f ) ( yamaguchi and yamaguchi , 2015 ) .
hence , the activity of a20 as a binder and stabilizer of linear ubiquitin linkages appears to be functionally more significant than its activity as a dub .
our analysis of lubac obtained from non - stimulated cells confirmed previous reports that cyld and otulin bind to the pub domain of hoip ( takiuchi et  al . , 2014 ) .
to our surprise , we found that , unlike cyld , otulin formed part of neither the native tnf - rsc nor the nod2-sc .
( 2013 ) reported that transient overexpression of flag - tagged nod2 resulted in its interaction with otulin .
however , this system does not require stimulation by a nod2 ligand , and it is therefore possible that the detected interaction could be a non - physiological event related to nod2 overexpression .
even though we were able to detect cyld in two different scs , including the tnf - rsc , in all cell lines studied , we never found otulin to be recruited .
these results implied that sc recruited hoip was not associated with otulin , despite the fact that otulin and hoip interacted prior to stimulation .
indeed , we found that otulin antagonizes lubac - mediated linear ubiquitination in the cytoplasm but not at scs .
the function of otulin is therefore likely to prevent accumulation of m1-ubiquitin linkages outside of scs ( figure  7 g ) . aiming to find a mechanistic explanation for the unexpected absence of otulin from scs
, we first turned our attention to phosphorylation , as phosphorylation of otulin tyr56 was previously reported to be capable of disrupting the interaction between hoip and otulin ( elliott et  al . , 2014 ) .
our data , however , show that phosphorylation is not responsible for otulin absence from scs .
the explanation was provided by our discovery that hoip can not simultaneously bind otulin and cyld as both require hoip - asn102 for binding .
we next demonstrated the existence of two separate pools of cytoplasmic lubac : one associated with otulin , the other one with cyld .
importantly , whereas cyld - bound lubac is recruited to the tnf - rsc and the nod2-sc , otulin - associated lubac is not
. it will be interesting to determine how otulin s interaction with hoip prevents its recruitment to scs , e.g. , whether otulin binding could interfere with hoip s ability to bind ubiquitin chains , a requirement for lubac recruitment to scs ( gerlach et  al .
we show here that cyld is recruited to scs due to its interaction with hoip , independently of lubac s enzymatic activity .
furthermore , in cells expressing hoip - n102a , which can interact with neither cyld nor otulin , cyld is not recruited to the tnf - rsc , demonstrating that cyld s interaction with hoip is essential for cyld recruitment .
cyld is a dub with broad specificity , yet the enzymatically active usp domain of cyld , when produced recombinantly , was previously shown to most efficiently hydrolyze m1- and k63-linked tetra - ubiquitin in  vitro ( komander et  al . , 2009 ) .
it has been unclear to date , however , whether endogenous cyld would be able to regulate m1-ubiquitin , in addition to its previously demonstrated role as a dub that cleaves k63 linkages on targets within the tnf - rsc ( brummelkamp et  al . , 2003 , kovalenko et  al . , 2003 , trompouki et  al .
employing newly devised protocols for k63-ap and m1-ap , we demonstrate here that , as a consequence of cyld deficiency , both k63- and m1-ubiquitin are increased on several components of the tnf - rsc , including tnfr1 , tradd , and rip1 .
furthermore , the recombinant usp domain of cyld completely removes m1 linkages , and indeed the majority of other linkage types , from components of the tnf - rsc .
importantly , m1 linkages are fully hydrolyzed and not merely released from complex components , as no free m1 chains appear following treatment with cyld .
collectively , these results identify cyld as an antagonist of linear ubiquitination in scs , in addition to its previously described role as an antagonist of k63-linked ubiquitination ( figure  7h ) .
we show that reconstitution of cyld - deficient mefs with cyld , but not with vector control , decreases m1-ubiquitin in the tnf - rsc .
this is accompanied by decreased gene activation , yet interestingly , also with enhanced tnf - induced cell death .
this is in line with the fact that cyld has previously been described as being both an inhibitor of gene activation and promoter of cell death , the latter by enhancing complex ii formation upon tnf stimulation ( hitomi et  al . , 2008 ,
we previously showed that lack of linear ubiquitination in the tnf - rsc enhances formation of complex ii of tnf signaling ( peltzer et  al . , 2014 ) .
based on the results presented here , cyld requires hoip for tnf - rsc recruitment .
hence , in cells lacking hoip , cyld does not form part of the tnf - rsc . consequently in these cells
, cyld can not mediate the transition from complex i of tnfr1 signaling ( i.e. , the tnf - rsc ) to complex ii .
decisively , however , in hoip - deficient cells this event does not require cyld activity ; it readily occurs without it ( peltzer et  al . ,
this identifies the lack of m1 chains in complex i as decisive to render this complex unstable so that complex ii can readily form .
we thus conclude that the m1-chain - antagonizing activity of cyld in complex i of tnfr1 signaling is responsible for its pro - cell death role .
whether this is due to direct cleavage of m1 chains and/or their indirect removal through the previously demonstrated cleavage of k63 linkages that are extended by linear chains remains to be determined .
equally , whether removal of a specific m1 chain from a particular target enables complex ii formation , or multiple such events together account for it , remains to be resolved .
with respect to a20 , we show that in absence of hoip or m1-ubiquitin a20 recruitment to scs is almost completely prevented and that genomic deletion of a20 s znf7 , in turn , drastically impairs recruitment .
thus , lubac , by placing m1 chains on sc components , recruits a20 to these scs via its znf7 ( figure  7h ) .
these results are in line with the previous finding that a20 can bind to m1-ubiquitin via its znf7 domain ( tokunaga et  al .
additionally , they provide the biochemical explanation for the observation that tnf - rsc recruitment of a20 lacking znf7 was significantly reduced ( tokunaga et  al . , 2012 ) . even though a20 was shown to be incapable of cleaving m1 chains ( mevissen et  al . , 2013 ) , we were surprised to find that a20 significantly stabilized m1-ubiquitin in scs .
given that m1 chains are required for full gene - activatory signaling ( fiil and gyrd - hansen , 2014 , iwai et  al .
, 2014 , walczak et  al . , 2012 ) and that a20 is a dub previously described to inhibit nf-b signaling ( bosanac et  al . , 2010 , tokunaga et  al . , 2012 , wertz et  al . , 2004 ) , this finding seemed counterintuitive , at first .
indeed , results obtained in reconstitution experiments showed that it is znf7 and not a20 s enzymatic dub activity or znf4 that is required for a20-mediated restriction of gene activation ( skaug et  al . , 2011 ) . combined with our observation that znf7 is required for a20 recruitment to scs due to its interaction with lubac - generated m1 chains , which are , in turn , stabilized by it ,
these findings now offer a possible alternative explanation for a20 s nf-b - inhibitory activity .
a20 binding to m1 chains in scs could compete with the binding of other gene - activatory factors , e.g. , the nemo / ikk complex , to them .
rising levels of a20 protein , induced as a consequence of nf-b activation , would render m1 chains less available for nemo / ikk retention over time so that the negative feedback loop would be completed .
a20 has also been implicated as a negative regulator of tnf - induced cell death ( lee et  al . , 2000 ,
yamaguchi and yamaguchi , 2015 ) , whereas cyld was shown to promote it ( hitomi et  al .
thus , while they cooperatively restrict gene activation from various scs , they act in opposing ways on tnf - induced cell death .
linear ubiquitination in the tnf - rsc prevents tnf - induced cell death by restricting complex ii formation ( gerlach et  al . , 2011 , ikeda et  al . , 2011 ,
we show here that a20 binding to m1 chains in the tnf - rsc stabilizes them , whereas cyld antagonizes m1 chains in this complex .
together this implies that the ability of a20 to bind m1 chains protects them from cleavage by dubs that are capable of cleaving linear ubiquitin chains in signaling complexes .
having identified herein cyld as a dub with precisely this activity at the tnf - rsc , we therefore propose a model according to which a20 protects m1 chains in the tnf - rsc from cyld - mediated cleavage , thereby providing the sought after explanation for the opposing roles played by a20 and cyld with regards to tnf - induced cell death .
intriguingly , both roles depend on lubac - generated linear ubiquitin in the tnf - rsc .
hence , an intricate interplay between lubac , linear ubiquitination , a20 , and cyld is crucial for assembly and disassembly of scs to enable efficient , yet properly controlled , immune signaling .
for description of cell lines , antibodies , and plasmids , see supplemental experimental procedures .
tap - tnf , untagged tnf , recombinant deubiquitinases , and proteins used for m1-ap , k63-ap , and ubi - ap were produced in e.  coli .
hoip- and hoil-1-deficient k562 and hoip - deficient a549 and hela cell lines were prepared by transfecting mrna encoding gene - specific zinc finger nucleases ( sigma ) .
cyld- , otulin- , a20-deficient , and a20znf7-expressing cells were prepared by lentiviral transduction with lenticrispr v2 vectors ( sanjana et  al . , 2014 ) provided by feng zhang ( addgene plasmid # 52961 ) .
coding sequences of hoip - wt , hoip - c885s , hoip - n102a , deletion mutants of human hoip fused or not at the c terminus to the tap - tag and tap - tagged nod2 ( aa 281040 ) were inserted into the retroviral mscv vector containing gfp as selection marker . upon infection , cells were sorted using moflo facs ( beckman coulter ) .
samples were subsequently diluted to 0.1% sds before m1- , k63- , or total ubiquitin - specific recombinant affinity protein coupled to halo beads was added for overnight incubation at 4c .
beads were washed , and samples were subjected to treatment with 1  m recombinant deubiquitinase for 1  hr at 37c or eluted with reducing sample buffer . to analyze the native tnf - rsc ,
after cell lysis , samples were subjected to anti - flag immunoprecipitation using m2 beads ( sigma ) or incubated with protein a / g - agarose beads ( santa cruz biotechnology ) coupled to indicated antibodies . for pervanadate treatment
, cells were incubated with 1  mm pervanadate for 20  min before lysis .
phosphatase treatment was performed on lysates using 50u of fastap ( thermo scientific ) per mg of protein in absence of phosphatase inhibitors .
performed experiments ; s.s . performed mass spectrometric analysis of tap - purified lubac ; l.t . and e.r .
 summaryubiquitination and deubiquitination are crucial for assembly and disassembly of signaling complexes . 
 lubac - generated linear ( m1 ) ubiquitin is important for signaling via various immune receptors . 
 we show here that the deubiquitinases cyld and a20 , but not otulin , are recruited to the tnfr1- and nod2-associated signaling complexes ( tnf - rsc and nod2-sc ) , at which they cooperate to limit gene activation . 
 whereas cyld recruitment depends on its interaction with lubac , but not on lubac s m1-chain - forming capacity , a20 recruitment requires this activity . 
 intriguingly , cyld and a20 exert opposing effects on m1 chain stability in the tnf - rsc and nod2-sc . while cyld cleaves m1 chains , and thereby sensitizes cells to tnf - induced death , a20 binding to them prevents their removal and , consequently , inhibits cell death . 
 thus , cyld and a20 cooperatively restrict gene activation and regulate cell death via their respective activities on m1 chains . 
 hence , the interplay between lubac , m1-ubiquitin , cyld , and a20 is central for physiological signaling through innate immune receptors . 
Introduction
Results
Discussion
Experimental Procedures
Author Contributions