Inflammasome regulation by adaptor isoforms, ASC and ASCb, via differential self-assembly

Abstract

ASC is an essential adaptor of the inflammasome, a micrometer-size multiprotein complex that processes proinflammatory cytokines. Inflammasome formation depends on ASC self-association into large assemblies via homotypic interactions of its two death domains, PYD and CARD. ASCb, an alternative splicing isoform, activates the inflammasome to a lesser extent compared with ASC. Thus, it has been postulated that adaptor isoforms differentially regulate inflammasome function. At the amino acid level, ASC and ASCb differ only in the length of the linker connecting the two death domains. To understand inflammasome regulation at the molecular level, we investigated the self-association properties of ASC and ASCb using real-time NMR, dynamic light scattering (DLS), size-exclusion chromatography, and transmission electron microscopy (TEM). The NMR data indicate that ASC self-association is faster than that of ASCb; a kinetic model for this oligomerization results in differing values for both the reaction order and the rate constants. Furthermore, DLS analysis indicates that ASC self-associates into more compact macrostructures compared with ASCb. Finally, TEM data show that ASCb has a reduced tendency to form densely packed filaments relative to ASC. Overall, these differences can only be explained by an effect of the linker length, as the NMR results show structural equivalence of the PYD and CARD in both proteins. The effect of linker length was corroborated by molecular with the CARD results indicate that faster and is more inflammasome differences by ASC isoforms at the molecular ASC is an essential adaptor of the inflammasome, a micrometer-size multiprotein complex that processes proinflammatory cytokines. Inflammasome formation depends on ASC self-association into large assemblies via homotypic interactions of its two death domains, PYD and CARD. ASCb, an alternative splicing isoform, activates the inflammasome to a lesser extent compared with ASC. Thus, it has been postulated that adaptor isoforms differentially regulate inflammasome function. At the amino acid level, ASC and ASCb differ only in the length of the linker connecting the two death domains. To understand inflammasome regulation at the molecular level, we investigated the self-association properties of ASC and ASCb using real-time NMR, dynamic light scattering (DLS), size-exclusion chromatography, and transmission electron microscopy (TEM). The NMR data indicate that ASC self-association is faster than that of ASCb; a kinetic model for this oligomerization results in differing values for both the reaction order and the rate constants. Furthermore, DLS analysis indicates that ASC self-associates into more compact macrostructures compared with ASCb. Finally, TEM data show that ASCb has a reduced tendency to form densely packed filaments relative to ASC. Overall, these differences can only be explained by an effect of the linker length, as the NMR results show structural equivalence of the PYD and CARD in both proteins. The effect of linker length was corroborated by molecular with the CARD results indicate that faster and is more inflammasome differences by ASC isoforms at the molecular is a that and The is by a of that been as and molecular and The of and in of inflammasome The of these the formation of a complex as the The of the inflammasome is by self-association and oligomerization of of and of inflammasome that show for and by the and in and and regulation of and The of of inflammasome and Inflammasome and at a The Inflammasome and death PYD and CARD and The death in of and and of the Inflammasome by and the oligomerization of the inflammasome adaptor ASC a a with an PYD and a CARD and regulation of and The of of inflammasome and a of and of a CARD The inflammasome adaptor as a molecular by and of the and the and regulation of and interactions of the inflammasome ASC by The inflammasome ASC into filaments with of its two death domains, PYD and Inflammasome to the of it of and into to the to the in the of by the oligomerization and self-association of and Inflammasome of and to the The is of the inflammasome is in and and in the of the inflammasome in an form in the and into a micrometer-size complex with the adaptor ASC. in a and The of of and the oligomerization and as the of the the of the inflammasome and The of of and and of inflammasome and and of the in a that into an regulation of inflammasome into the a of ASC and of and The of the inflammasome is to as the of an of and ASC its in The of the inflammasome in and and in into the in inflammasome regulation and is to for isoforms amino acid and and to in by and function. Thus, the of the molecular by isoforms is to understand regulation at the molecular The inflammasome adaptor ASC has ASCb, and differ in amino acid splicing of the a ASC and ASCb the as both the two domains, PYD and CARD and only differ in the length of the linker and amino for ASC and ASCb, the CARD and a of the only a PYD and CARD. has been that only ASC and ASCb as both the of of ASC and and been to with and splicing of the a ASCb activates the inflammasome to a lesser extent as by the of and form the of splicing of the a and with and to the that these isoforms inflammasome the of by ASC is in the of that the as an inflammasome splicing of the a effect on inflammasome has been for and the of this is splicing of the a To understand the of inflammasome by the ASC and ASCb, we the self-association properties of both using and NMR, we the and of to to data for ASC and and of ASC a CARD NMR that the PYD and CARD of both isoforms as to the amino acid NMR oligomerization was by real-time NMR at the for both a of on these we that ASC and ASCb for NMR data indicate that the PYD and CARD of ASC differences for the two in ASCb. was by dynamic light scattering in ASC and ASCb as a of the self-association in that with the The effect of and on the oligomerization processes was by size-exclusion that ASC has a self-association compared with ASCb. the of the macrostructures by ASCb investigated by transmission electron microscopy and compared with TEM data for ASC. The analysis of that ASCb filaments of to by ASC to form of a of Finally, the of linker length on the ASC ASCb and the CARD of was in by structural via molecular The NMR of ASC that the PYD and CARD the to death The two by a amino linker and of a CARD that the of the PYD and CARD of ASC and ASCb be the two isoforms only differ in the linker length, and the PYD and CARD of both amino acid To structural of the two isoforms the NMR we the and of ASCb and compared this with that for ASC and and of ASC a CARD NMR NMR, the the and of the is as the it is for interactions of the inflammasome ASC by an of the that the of the PYD and CARD of ASC and ASCb as The more of the ASC the the the The in for and of ASC and ASCb the is that both PYD and CARD in ASCb into the that to the linker in the amino acid in the to show in for both is the for amino and and show these amino to the linker can that the of these be to the properties of ASC and ASCb. a of the and for both isoforms indicates that the of both is as ASC has a tendency to and filaments and at The inflammasome ASC into filaments with of its two death domains, PYD and is a of ASC as the inflammasome self-association and of the inflammasome via has been that ASC self-association is reduced at and values for the of ASC by NMR and of a CARD interactions of the inflammasome ASC by ASC self-associates at ASC self-association can be by a of NMR with and of a CARD results the formation of large that in NMR to to the the oligomerization of the by real-time of by NMR this to in ASC and ASCb self-association by real-time in form and to as in the NMR of as a of NMR for The kinetic on at values to the of on the of The of the the of the with for ASC and ASCb to the formation of The rate of these large in NMR, in analysis of the with that ASC faster than ASCb is an for both ASC and ASCb, as self-association at ASC faster than ASCb at the the the two isoforms is compared with the at is to that we the of ASC ASCb self-association for these the the kinetic large to be by NMR, that to to be The to and and to oligomerization rate and is to that and a to the self-association for the of both proteins. of the NMR to a for ASC for ASCb. that ASC oligomerization at two with rate constants. The to a results in and rate for and Thus, kinetic in ASC oligomerization is faster than the the formation of an that at a rate a The for ASC at of the at the of at the of the kinetic the at this and values and the to the data at and for for rate in values and the to the data at and for for rate in of values and the to the data at and for for rate in in a the of the ASCb NMR to a in compared with a Thus, ASCb to with a kinetic with an rate at the of the kinetic the of ASCb is The of for ASC and ASCb to the a in and that results in and of for ASC and ASCb, in with the of the of the in To of the two death in we the of the two isoforms at the amino acid as the of amino in the PYD and CARD of ASC and ASCb to the and in the PYD and CARD of ASC show this is for ASCb. The and values of amino the show that the two death of ASC in the faster The values of the PYD than of the and the values for both and for that interactions the in the oligomerization and that both in the PYD and CARD of ASCb show values of the oligomerization rate and to of both in the of the rate is than the rate for ASC self-association that ASCb at a rate compared with ASC. The in ASCb be explained by an and with rate constants. The oligomerization of ASC is a complex as by NMR and that ASC the of ASC filaments and that the to form The ASC homotypic interactions the and of the two PYD and CARD that and The and for the self-association of ASC and ASCb on the of kinetic and the of and differences in the self-association of the these to the oligomerization kinetic to a kinetic model for ASC and ASCb self-association on for The oligomerization of been in using kinetic of and of as by the kinetic of of and of The model by has been and to the oligomerization of and of to on of a of of the of to and to this kinetic of in the formation of an of a the of the the is the of to this is to and of to To for the of this and on been with and and of the the formation of the on the of of of of for an via a of of in and the of by a to that of the kinetic to the data and of the a model has been to the kinetic of and in form is to the in the oligomerization of to this the that results in formation two the formation of an the of a of a that is and the of a of to this in a that is to be of The can be by to both of the as and formation by of into of this model kinetic data on and a of this the the formation of that by and processes as the model is in form in The kinetic to the in as and the of and and the and reaction rate for the formation of the and is the rate for the reaction of formation The model that the kinetic of these it has been that the of the can on and to kinetic analysis of these by that the of is at of the self-association this the of the results in an of the of at as and with the data the of as self-association the of the data to the values of and for the formation of the rate for ASC than for ASCb. is to two of for is order of that ASC has a tendency to form compared with ASCb, and that the has tendency to to The values at and as the values show these to the in that the and the rate for the self-association of ASC and ASCb kinetic model in a an of the reaction rate order for formation can be by the rate these the of is to be and is The of the reaction was the of the by the of the NMR at the values for ASC and ASCb the data at the values to the reaction order for ASC and for results indicate that the formation of ASCb has a on the compared with ASC. the results the kinetic model to the oligomerization of ASC and ASCb with more the reaction rate and the order rate values as The NMR that of oligomerization indicate that formation is a the in the kinetic model is the model to with the NMR a of to an and of the NMR the oligomerization of ASC and ASCb is the of the in the of the in values of the rate of the reaction in the NMR is to in ASC and ASCb the of the self-association this the to to The for ASC indicates the of and with molecular of and the for ASCb only a with molecular of The data the molecular of the and of ASC and ASCb, The ASC and ASCb with acid and by the to into the of an ASC was The data indicate that ASC is of to the with data on the oligomerization of ASC using NMR and indicate that the ASC is the of the ASC The inflammasome ASC into filaments with of its two death domains, PYD and the of a in in ASCb, that a ASCb is as in the self-association of the two we to the in ASC and ASCb we in death oligomerization ASC self-associates and using and and and and this we an with a molecular of The and in these to interactions the and the NMR and we that the of death in the of the PYD of ASC and of the inflammasome ASC self-associates and using Thus, self-association these compared with in the kinetic for analysis ASC and ASCb that to to in the that the in the in and at for to and in of in the of the is in in the values for the of the results at in the in a of to ASC and ASCb at an of to the and a and of ASC and ASCb at and The the and is to the of the two The at to the of both ASC and ASCb as the NMR data the of a and of a CARD The and both ASC and ASCb at the with indicates that the by ASC and ASCb is the for the molecular of the the of the of in the ASC and ASCb is on the molecular of the proteins. ASCb, an at an in the that of ASCb is these data indicate that both ASC and ASCb at in the of results on the effect of in death The of on was At ASC in the with a at ASCb in the a at the and a at a tendency of ASC to compared with ASCb. to the effect of on ASC and ASCb on ASC and ASCb at values than to of an effect for ASC and of a CARD ASC self-associates and using At ASC an at the at the is ASCb two the at and the at the At a only is for both isoforms at with results that by ASC and ASCb more at large of in the in at The data to to the of the oligomerization and in the of and at we can of for ASC and ASCb. Overall, the results that ASC has a tendency to compared with ASCb on the effect of and to The results indicate that of that a light scattering was to the self-association of ASC and ASCb as a of The of the light was for ASC and ASCb at to The of the depends on the of the and is to the of the in with of the light compared with at is to that a is for the of the the of in ASC and this is The inflammasome ASC into filaments with of its two death domains, PYD and the on the of the the data to of and in the kinetic DLS data for ASC show and with an of and The of ASC in the is in the DLS data by the the large in The to at the of the kinetic indicate that the more the of the at with the compared with the we can that the of the is At the of the kinetic ASC at The of the is to the in of the Thus, we the has more compact by and to the of the DLS data for ASCb show at and the more the kinetic the into two with of and ASC and ASCb form of and the to to a at the of the kinetic the of the and a tendency to form of more the values for ASC and ASCb the DLS kinetic and is as it that ASC self-associates into of ASCb with it has been that ASC and ASCb into macrostructures the ASC a of and ASCb and filaments splicing of the a The for oligomerization in the in in of and and in the with DLS results that ASC is of ASCb is analysis of the macrostructures by ASC using has been The inflammasome ASC into filaments with of its two death domains, PYD and to this ASC to form filaments with an of and length and The inflammasome ASC into filaments with of its two death domains, PYD and The filaments form of with and more is a of the of the the of filaments in the that the by of the TEM show that ASC filaments by with the of an ASC to of the these to filaments a for and analysis was for ASCb to differences in the of the macrostructures ASCb into filaments that to form differences ASCb of and The analysis of filaments indicates the of of and of Thus, for ASCb, more The of and is and values indicate that ASCb filaments of compared with for ASC and that by of length was to be in the than ASC to filaments for ASCb. is to that the for ASC and ASCb formation the for oligomerization using formation is at and the for NMR TEM results indicate that ASC can form filaments of to the filaments at and The inflammasome ASC into filaments with of its two death domains, PYD and to ASC and ASCb and as of in inflammasome Thus, both isoforms in oligomerization processes proteins. investigated differences ASC and ASCb in this at the an to the of inflammasome we and The of to the of ASC and ASCb to CARD of of ASC and ASCb with using the interactions death and interactions in the death that and of and and of the Thus, CARD in ASC ASCb is of with two via the by and The and to by The of the and compared with the of a with interactions the of the CARD of and its by a The of the CARD and PYD of ASC and ASCb to the linker is to of the PYD and CARD that the of with we to the and linker of ASC and ASCb, in the to The of the and and in at the of and the of ASC with the ASC and ASC for and at the of and interactions ASCb and ASC and ASCb of via interactions in a in ASC and ASCb in in light The of this is to the by the of the of and its by a in light a is only the by ASC the of the CARD on the and The in the ASCb is in a that the CARD and it is the in that the with the PYD of ASCb in the of the CARD. the ASCb with ASCb in results in the of the in a is with the PYD of ASCb, the on the ASC and ASCb in an the formation of the The the PYD and CARD of ASCb interactions of with linker results in of the two domains, interactions with Overall, the with the to a of oligomerization of ASC and ASCb with ASC and ASCb with inflammasome to a of for in and this the effect by PYD and CARD in ASCb more Finally, on the model for a structural of ASC ASCb in differences in the of the and in its has been that ASC and its ASCb to with and splicing of the a of ASC and both isoforms into the inflammasome ASCb to an in inflammasome compared with ASC on the of of splicing of the a The in inflammasome ASC and ASCb can on the regulation of the it is to understand this at the molecular The of ASC as an inflammasome adaptor is to and with the and Thus, we that the of inflammasome depends on the properties in ASC and ASCb. this we this by the self-association of ASC and ASCb using for a more The of ASC and ASCb self-association by to kinetic by the data to The results of the indicate that ASC self-associates with two kinetic ASCb a kinetic two with rate data indicate that ASC oligomerization a kinetic and a kinetic the kinetic by ASCb is than both kinetic of ASC. Furthermore, NMR of the of the self-association this of NMR to differences in oligomerization at the we that the self-association of the PYD and CARD of ASC and ASCb the PYD and CARD of ASC show two kinetic rate The values of the PYD relative to the CARD that the kinetic the interactions in the to by by The kinetic is by the of both the PYD and CARD as by the PYD and CARD of ASCb show kinetic rate that both in the formation of the data on ASC by show that ASC the of ASC into filaments The inflammasome ASC into filaments with of its two death domains, PYD and is that and into of a that into The kinetic data for ASC that interactions formation by to the results of the kinetic data for ASCb that the PYD and CARD the oligomerization oligomerization depends on the of the in the PYD and CARD and on the of these to for be The PYD and CARD of ASC and ASCb on structural data and the amino acid Thus, the of the two in ASC is to the linker connecting the two domains. ASC linker to a extent compared with ASCb. it is to that the oligomerization tendency in ASC for the PYD relative to the CARD is in ASCb to of as a of the To the effect of the linker length, we via molecular and structural of of ASC ASCb and two The of The indicate that ASCb linker in an of the that the of the of and its by a The DLS kinetic that ASC and ASCb into DLS data the of a at the of ASC The of the the formation of of ASCb is more as by the of two of the of the self-association results in with on ASC and ASCb by that ASC a of in ASCb filaments Inflammasome of and to the splicing of the a ASC and ASCb show differences at the The analysis filaments by ASCb compared with ASC and a tendency to into by a large of be to the macrostructures in the for ASC and ASCb. Furthermore, we show that oligomerization and ASC and ASCb indicate that ASC has a tendency to compared with ASCb. in the of at a to both ASC and ASCb. with that ASC into of at a in a of ASC at the oligomerization and the of indicate that the oligomerization and the of kinetic data and analysis that and a the of an ASC in the the of large the of the to its compared with ASCb. data indicate that ASC faster and is of into of more compared with ASCb. this we show that these differences only to the of ASC and ASCb with of that an in the macrostructures in the splicing of the a The only at the amino acid is the linker length, to be in ASC to self-association and the formation of more compact to ASC and ASCb can in the regulation of the as a of the of of the of the and ASC is The a the inflammasome, of and and The of the inflammasome as for the of to an of the in its The ASC formation of ASCb into filaments be in results indicate that ASC self-association is by the interactions by the of that to the of the and tendency of ASCb to the of the Furthermore, the of ASCb in a of an in inflammasome with for ASC ASCb. and in using as and using and at for by at for at was in and was at to the The was at for at The was a ASC and ASCb using The was using and and to of and ASC and ASCb by with a to and at of ASC and ASCb was in and the was to the for the proteins. on the amino acid as molecular amino acid and was the The of ASC and of a CARD was as to a model of the of ASCb using the for and this amino the linker of ASC only amino to the linker of ASCb. ASCb NMR at in a of and at NMR at in a with a at of ASCb was the and NMR and in with and with for NMR on of ASC to differences and and of ASC a CARD NMR The differences ASC and ASCb using the and to to ASC and ASCb in and at of ASC and ASCb was using at a of and values of both ASC and ASCb by at to the at and at on a with a NMR The of the of the for was to to and the of was by to using values for both ASC and ASCb with to the an of and as a of The with of NMR to and and for an of kinetic data to the the kinetic model to values of the kinetic for ASC and ASCb at and for the PYD and CARD by the at using for NMR values and to with the of values in was using a at a rate of to the was with a molecular The was using ASC and ASCb at and in of ASC and ASCb at and into the at two and The effect of on ASC and ASCb oligomerization was at for at and into the values at a of The with DLS using a at using a of The of ASC and ASCb to be using an The of ASC and ASCb at was ASC and ASCb at a of by in at was to by at for both ASC and ASCb. in a for and for at to at and of the light to by the the and values of the light at and the data and with ASCb filaments by ASCb in a and The was with to ASCb. The of the was by at The was and and the was of of the was on a the was in of and in of The was for and to of the of ASCb filaments using a with a at of was for of ASCb filaments and was with NMR of ASC and ASCb at for the the of the oligomerization in an to an The with a acid and in a at a rate of The with with was with the The of using ASC by NMR and of a CARD the ASCb model with for and and a CARD the of the CARD by of and its by a of of ASC ASCb and of to the molecular the interactions and of CARD with and of the CARD and The of ASC ASC ASCb ASCb The of of the on the was to of by the of as ASC ASC ASCb ASCb The of of the on the is in the and acid linker of ASC and ASCb, was to for The and The of was of for was with as for the The for the and with for and The data that the of this the and the data for this the The that of with the of this to for and at the of for to to for the dynamic light scattering in The of has the NMR to NMR data in this and and and and and and and and was by the of and of the of to and by the for and at the of to The of this is the of the and the of the of the

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