Design principles for inflammasome inhibition by pyrin-only-proteins
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In this useful and potentially important manuscript, Mazanek and colleagues combine computational analysis and in vitro experiments to develop a comprehensive analysis of the ability of pyrin-only proteins (POPs) to inhibit inflammasome assembly. The results lead the authors to propose that a mixture of favorable and unfavorable interaction surfaces is required for a POP to inhibit a given inflammasome component. The results presented are solid, but additional experimentation is required to fully justify the authors' model.
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Abstract
Inflammasomes are filamentous signaling platforms essential for host defense against various intracellular calamities such as pathogen invasion and genotoxic stresses. However, dysregulated inflammasomes cause an array of human diseases including autoinflammatory disorders and cancer. It was recently identified that endogenous pyrin-only-proteins (POPs) regulate inflammasomes by directly inhibiting their filament assembly. Here, by combining Rosetta in silico, in vitro, and in cellulo methods, we investigate the target specificity and inhibition mechanisms of POPs. We find here that POP1 is ineffective in directly inhibiting the central inflammasome adaptor ASC. Instead, POP1 acts as a decoy and targets the assembly of upstream receptor pyrin-domain (PYD) filaments such as those of AIM2, IFI16, NLRP3, and NLRP6. Moreover, not only does POP2 directly suppress the nucleation of ASC, but it can also inhibit the elongation of receptor filaments. In addition to inhibiting the elongation of AIM2 and NLRP6 filaments, POP3 potently suppresses the nucleation of ASC. Our Rosetta analyses and biochemical experiments consistently suggest that a combination of favorable and unfavorable interactions between POPs and PYDs is necessary for effective recognition and inhibition. Together, we reveal the intrinsic target redundancy of POPs and their inhibitory mechanisms.
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eLife assessment
In this useful and potentially important manuscript, Mazanek and colleagues combine computational analysis and in vitro experiments to develop a comprehensive analysis of the ability of pyrin-only proteins (POPs) to inhibit inflammasome assembly. The results lead the authors to propose that a mixture of favorable and unfavorable interaction surfaces is required for a POP to inhibit a given inflammasome component. The results presented are solid, but additional experimentation is required to fully justify the authors' model.
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Reviewer #1 (Public Review):
Pyrin domains (PYD) in inflammasome proteins oligomerize into filamentous assemblies and mediate inflammasome formation. Mammalian pyrin-only-proteins (POPs) exert inhibitory effects on inflammasome as they mimic the pyrin domains while lacking the effector domain. In this manuscript, Mazanek and colleagues combined computational prediction with cellular and in vitro experiments to investigate the mechanism and target specificity for three POPs, POP1, POP2, and POP3, in inflammasome activation.
The authors first modeled the structures of complex formed by POPs with inflammasomal PYDs, including ASCPYD, AIM2PYD, IFI16PYD, NLRP6PYD, and NLRP3PYD, then calculated their Rosetta interface energies(∆Gs). By comparing the ∆Gs of inflammasomal PYD(∆GPYD•PYD) with inflammasomal PYD/POPs complex (∆GPOP•PYD), they …
Reviewer #1 (Public Review):
Pyrin domains (PYD) in inflammasome proteins oligomerize into filamentous assemblies and mediate inflammasome formation. Mammalian pyrin-only-proteins (POPs) exert inhibitory effects on inflammasome as they mimic the pyrin domains while lacking the effector domain. In this manuscript, Mazanek and colleagues combined computational prediction with cellular and in vitro experiments to investigate the mechanism and target specificity for three POPs, POP1, POP2, and POP3, in inflammasome activation.
The authors first modeled the structures of complex formed by POPs with inflammasomal PYDs, including ASCPYD, AIM2PYD, IFI16PYD, NLRP6PYD, and NLRP3PYD, then calculated their Rosetta interface energies(∆Gs). By comparing the ∆Gs of inflammasomal PYD(∆GPYD•PYD) with inflammasomal PYD/POPs complex (∆GPOP•PYD), they defined favorable and unfavorable interaction surfaces (∆∆G = ∆GPYD•PYD- ∆GPOP•PYD ). Their initial computational model indicates POP1 may have the strongest inhibitory effect on ASC, as it exhibits the most favorable interfaces. But the experiment results showed otherwise, with POP2 and POP3, which contain both favorable and unfavorable interfaces, exhibiting stronger inhibitory effects. They then revised the model and proposed the combination of favorable (recognition) and unfavorable interfaces (repulsion) is necessary for POPs to interfere with the assembly of inflammasome PYDs, which was further tested by other inflammasomal PYDs.
This is a timely study that enhanced our current understanding of inflammasome regulation by POPs, it is also interesting as it combined the newest computational prediction method with biological experimental validation. The explanations on 1.) sequence homology may not dictate the target specificity of POPs, and 2.) excess POPs are required to inhibit the polymerization of inflammasome assembly, are well supported; however, some questions about the target specificity need to be addressed/clarified:
1. The authors showed MBP tag affected the oligomerization of POPs, while the POPs used in Figures 2A, 3A, and 4A contain a GFP tag. It should be considered GFP may affect the property of POPs, such may change the inhibitory effect of POPs on ASC filament formation.
2. The authors take the reduction of PYD filamentation as an indication of inhibition, but it was not clear how they ruled out the possibility that POP1 co-assembles into ASCPYD filaments and inhibits inflammasome formation by repressing the recruitment of Caspase-1, as it lacks CARD the effector domain. Especially the model predicted comparable energy between POP1 and ASC, which could indicate POP1 co-assembled into ASC filament.
3. Further computational analysis should be performed to evaluate the interpretation of Rosetta interface energies. Could the "combination of favorable and unfavorable interfaces" theory apply to other PYD/PYD interactions and CARD/CARD interactions?
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Reviewer #2 (Public Review):
In this manuscript, Mazanek et al use Rosetta to calculate the relative binding energies of the six distinct PYD/PYD interactions between the pyrin-only proteins (POPs) and the pyrin domains (PYD) of various inflammasome components. Following these calculations, the authors measure the ability of the POPs to disrupt PYD spec formation or disrupt PYD oligomerization. From these experiments the authors propose that the POPs do not simply disrupt ASC oligomerization, but instead that each POP has unique specificity for the various PYDs and can thusly act upstream of ASC filamentation through their direct interactions with the inflammasome PYDs. Furthermore, the authors propose the ability of the POPs to inhibit PYD filament formation is not solely dictated by sequence similarity between the POP and the PYDs, …
Reviewer #2 (Public Review):
In this manuscript, Mazanek et al use Rosetta to calculate the relative binding energies of the six distinct PYD/PYD interactions between the pyrin-only proteins (POPs) and the pyrin domains (PYD) of various inflammasome components. Following these calculations, the authors measure the ability of the POPs to disrupt PYD spec formation or disrupt PYD oligomerization. From these experiments the authors propose that the POPs do not simply disrupt ASC oligomerization, but instead that each POP has unique specificity for the various PYDs and can thusly act upstream of ASC filamentation through their direct interactions with the inflammasome PYDs. Furthermore, the authors propose the ability of the POPs to inhibit PYD filament formation is not solely dictated by sequence similarity between the POP and the PYDs, but instead that a combination of both strong and weak interactions between the POP and PYD is required to disrupt PYD filament formation. These observations help to elucidate the individual roles of the different POPs.
In total this manuscript presents a rigorous and careful biochemical analysis of how the POPs act to modulate PYD oligomerization. However, there are several weaknesses that need to be addressed. First, while the authors propose that the combination of strong and weak interactions dictates the ability of the POPs to disrupt PYD oligomerization this hypothesis is not directly tested. Second, while the author's careful examination demonstrates the ability of the POPs to disrupt PYD spec formation in a reconstituted system, they do not confirm that their in vitro measurements correlate with the ability to restrict inflammasome activity in an endogenous system and as such the physiological consequences of their measurements remain unclear.
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Reviewer #3 (Public Review):
The authors use a combination of computational and experimental analyses to study how Pyrin-only proteins (POPs) could regulate either the abundant ASC effector protein or the PYDs of ALRs AIM2 and IFI16 or NLRs NLRP3 and NLRP6. This systematic approach shows differences in the free energy of binding interfaces within the potential filament assemblies. Fluorescence anisotropy experiments are performed on PYD filament formation, using FRET-donor and -acceptor labeled recombinant PYDs (e.g., ASC) and increasing concentrations of unlabeled POPs. These experiments indicate how the lag phase of PYD nucleation and the kinetics of the filament elongation phase is perturbed. Fluorescence microscopy images of HEK cells co-transfected with, e.g., mCherry-tagged ASC-PYD and eGFP-labelled POPs indicate co-localization …
Reviewer #3 (Public Review):
The authors use a combination of computational and experimental analyses to study how Pyrin-only proteins (POPs) could regulate either the abundant ASC effector protein or the PYDs of ALRs AIM2 and IFI16 or NLRs NLRP3 and NLRP6. This systematic approach shows differences in the free energy of binding interfaces within the potential filament assemblies. Fluorescence anisotropy experiments are performed on PYD filament formation, using FRET-donor and -acceptor labeled recombinant PYDs (e.g., ASC) and increasing concentrations of unlabeled POPs. These experiments indicate how the lag phase of PYD nucleation and the kinetics of the filament elongation phase is perturbed. Fluorescence microscopy images of HEK cells co-transfected with, e.g., mCherry-tagged ASC-PYD and eGFP-labelled POPs indicate co-localization and overall filament content (as % puncta). Finally, negative stain EM imaging shows assemblies into ordered filaments or aggregates for the recombinant PYD proteins in the presence or absence of POPs. In conclusion, the authors propose a decoy receptor mechanism for the POPs and NLRs/ALRs with different specificities for each individual PYD.
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