Catalytic activity and autoprocessing of murine caspase-11 mediate noncanonical inflammasome assembly in response to cytosolic LPS
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eLife assessment
Brodsky and colleagues report here an unexpected cis-activation mechanism of caspase-11. The authors use cellular imaging methods and cleavage site mutants to show that the LPS-induced speck formation by caspase-11 depends on the autoprocessing between two subdomains. This new finding opens multiple doors for further investigating how this non-canonical inflammasome is regulated and activated at the molecular level.
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Abstract
Inflammatory caspases are cysteine protease zymogens whose activation following infection or cellular damage occurs within supramolecular organizing centers (SMOCs) known as inflammasomes. Inflammasomes recruit caspases to undergo proximity-induced autoprocessing into an enzymatically active form that cleaves downstream targets. Binding of bacterial LPS to its cytosolic sensor, caspase-11 (Casp11), promotes Casp11 aggregation within a high-molecular-weight complex known as the noncanonical inflammasome, where it is activated to cleave gasdermin D and induce pyroptosis. However, the cellular correlates of Casp11 oligomerization and whether Casp11 forms an LPS-induced SMOC within cells remain unknown. Expression of fluorescently labeled Casp11 in macrophages revealed that cytosolic LPS induced Casp11 speck formation. Unexpectedly, catalytic activity and autoprocessing were required for Casp11 to form LPS-induced specks in macrophages. Furthermore, both catalytic activity and autoprocessing were required for Casp11 speck formation in an ectopic expression system, and processing of Casp11 via ectopically expressed TEV protease was sufficient to induce Casp11 speck formation. These data reveal a previously undescribed role for Casp11 catalytic activity and autoprocessing in noncanonical inflammasome assembly, and shed new light on the molecular requirements for noncanonical inflammasome assembly in response to cytosolic LPS.
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Author Response
Reviewer #3 (Public Review):
Comment 1: I'm having some difficulty understanding the logic of Figure 5 in determining cis processing. It is an inverse of figure 4, and in my view, provides further evidence of trans processing. A better experiment would be to use WT-citrine tagged protein with catalytic dead mcherry and image them together. This would show WT cis processing occurs faster than trans processing as citrine specks should appear earlier than the mCherry ones. Can also do colocalization and FRET-based assays with the pair.
We thank the reviewer for pointing this out. While our data demonstrate that the same molecule must be catalytically active and competent for processing at the IDL (Figure 5), we agree that the data do not rule out trans-processing as a mechanism for speck formation. We have therefore …
Author Response
Reviewer #3 (Public Review):
Comment 1: I'm having some difficulty understanding the logic of Figure 5 in determining cis processing. It is an inverse of figure 4, and in my view, provides further evidence of trans processing. A better experiment would be to use WT-citrine tagged protein with catalytic dead mcherry and image them together. This would show WT cis processing occurs faster than trans processing as citrine specks should appear earlier than the mCherry ones. Can also do colocalization and FRET-based assays with the pair.
We thank the reviewer for pointing this out. While our data demonstrate that the same molecule must be catalytically active and competent for processing at the IDL (Figure 5), we agree that the data do not rule out trans-processing as a mechanism for speck formation. We have therefore modified the interpretation of these findings accordingly (pp. 7-8). We agree that some of the quantitative assays the reviewer has suggested would strengthen this logic, and we are making efforts to carry out a kinetic FRET-based assay for our upcoming biochemistry-focused manuscript to better characterize the enzymatic affinity of Casp11 for cis- vs. trans- based autoprocessing, and how either impacts Casp11 speck assembly.
Comment 2: Do those casp11 specks still contain CARDs?- i.e. is the second cleavage necessary for speck formation? Is CARD necessary at all? Would adding the TEV site at CDL and b/w p20 and p10 rescue? i.e. trans-activate?
We are grateful to the reviewer for these insightful questions, which we also had considered. We addressed this question in two ways – first by replacing the CARD with a DmrB dimerizable domain that undergoes inducible dimerization of Casp11 in the presence of the dimerizing drug AP20187. Critically, inducible dimerization of DmrB-ΔCARD-Casp11-mCherry significantly enhances Casp11-mCherry speck formation, and this speck formation requires catalytic activity, even in the presence of dimerizer (Figure 6A-C). Moreover, we generated CARD-less Casp11-mCherry constructs containing wild-type p20-p10 and catalytically inactive p20-p10. Intriguingly, the CARD was dispensable for spontaneous Casp11-mCherry speck formation, which again was dependent on catalytic activity (Figure 6-figure supplement 2A-B). While we do not currently have data with a TEV-cleavable CDL construct, our data here demonstrate that the CARD is dispensable for speck formation in an overexpression system, implying that the p20/p10 contains all the information that is necessary and sufficient to mediate spontaneous assembly of Casp11 specks in HEK293T cells. Nonetheless, as forced dimerization enhances speck formation (Figure, we hypothesize that CARD-LPS interactions act to facilitate catalytic activity and push cooperative assembly of the Casp11 speck.
To address whether both the N-terminal CARD and C-terminal p10 domains are present in Casp11 specks, we performed a dual-fluorophore co-localization assay in which we transiently expressed C-terminal mCherry-tagged Casp11 constructs (Casp11-mCherry) in HEK293T cells that stably express N-terminal Flag-tagged Casp11 (2xFLAG-Casp11). As expected, Casp11-mCherry formed specks spontaneously in this setting (Figure 3-figure supplement 1). Critically, both the N-terminal FLAG and C-terminal mCherry were found together in these specks, indicating the presence of both Casp11 N- and C- termini within the specks. Moreover, the wild-type Casp11-mCherry also recruited catalytically inactive 2xFLAG-Casp11C254A, again supporting the finding that wild-type Casp11 can recruit a catalytic mutant to noncanonical inflammasome complexes.
Comment 3: What are the equations that fit experimental data points and R2 for? E.g. Figure 1E. What are the parameters being fitted/compared and how are those interpreted? A table of fitted values and proper interpretation should be provided.
We thank the reviewer for this request to clarify how the curves were fit to the experimental data points. We have modified our ‘Statistical Analysis’ section and all figure legends that contain dose-response curves to reflect the equations used to fit each curve. Additionally, please find a table of raw values in the corresponding source data provided for each dose-response curve (Figure 2 Source Data 5; Figure 4 Source Data 3, 6; Figure 5 Source Data 3, 4; Figure 7 Source Data 2; and Figure 4-figure supplement 1 Source Data 1).
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eLife assessment
Brodsky and colleagues report here an unexpected cis-activation mechanism of caspase-11. The authors use cellular imaging methods and cleavage site mutants to show that the LPS-induced speck formation by caspase-11 depends on the autoprocessing between two subdomains. This new finding opens multiple doors for further investigating how this non-canonical inflammasome is regulated and activated at the molecular level.
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Reviewer #1 (Public Review):
In their work, Akuma and colleagues identify the autoprocessing in cis of casp11 as a key step that allows the aggregation of casp11, and its capacity to cleave GSDMD and induce pyroptosis. The authors utilize, for the first time, a fluorescent casp11 that allowed us to visualize its aggregation (formation of specks). This is a key event that was largely overlooked for casp11. Indeed, casp11 directly binds LPS and initiates pyroptosis in the absence of other NLR members and adaptors such as ASC. While NLRs and adaptors form the structure that allows the recruitment and cleavage of casp1, how casp11 specks are formed remained unknown so far. Using casp11 mutants that lack the catalytic activity or the autoprocessing site, as well as casp11 that can be cleaved by other proteases, the authors demonstrate that …
Reviewer #1 (Public Review):
In their work, Akuma and colleagues identify the autoprocessing in cis of casp11 as a key step that allows the aggregation of casp11, and its capacity to cleave GSDMD and induce pyroptosis. The authors utilize, for the first time, a fluorescent casp11 that allowed us to visualize its aggregation (formation of specks). This is a key event that was largely overlooked for casp11. Indeed, casp11 directly binds LPS and initiates pyroptosis in the absence of other NLR members and adaptors such as ASC. While NLRs and adaptors form the structure that allows the recruitment and cleavage of casp1, how casp11 specks are formed remained unknown so far. Using casp11 mutants that lack the catalytic activity or the autoprocessing site, as well as casp11 that can be cleaved by other proteases, the authors demonstrate that self-cleavage of casp11 is a pre-requisite for aggregation and speck formation. Also, by using their mutants the authors demonstrated that casp11 acts in cis, rather than in trans, to exert this function. So far, mostly based on casp1 biology, the main view was that aggregation is a prerequisite for cleaving. Here the authors changed this view for casp11, and found that casp11 autocleavage is upstream of its aggregation induced upon LPS sensing. They found that initial dimerization and subsequent oligomerization are two distinct events and that LPS binding of casp11 is insufficient to assemble the non-canonical inflammasome.
The paper makes use of elegant mutant caspases and is based on solid bases. Some experiments lack analyses of the functional consequences of non-canonical inflammasome formation, and the paper would benefit from this type of analysis.
Another key finding is that Cys-254 plays more roles than "simply" cleaving casp11 at D285. This finding needs to be better highlighted also in the abstract because it opens more future investigations.
Also, the separation between dimerization and oligomerization may open to future studies and may be briefly mentioned also in the abstract.
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Reviewer #2 (Public Review):
Casp11 is a cytosolic sensor for LPS in mice (orthologue of Casp4/5 in human). It is an important innate sensor of intracellular infection. Casp11 activity results in cleavage and activation of the pore-forming protein Gasdemin D (GSDMD) leading to lytic death (pyroptosis), of an infected cell. How exactly Casp11 signals upon LPS detection is beginning to be understood, but the picture is incomplete. Previous reports suggested that upon LPS detection, Casp11 dimerizes and undergoes auto-processing to form a pyroptosis-competent enzyme. The prediction from these studies was that the formation of a fully functional Casp11 signalling complex involves two steps: inducible dimerization and auto-processing.
In this study, authors used fluorescently tagged Casp11 reporter fusions, to report that detection of …
Reviewer #2 (Public Review):
Casp11 is a cytosolic sensor for LPS in mice (orthologue of Casp4/5 in human). It is an important innate sensor of intracellular infection. Casp11 activity results in cleavage and activation of the pore-forming protein Gasdemin D (GSDMD) leading to lytic death (pyroptosis), of an infected cell. How exactly Casp11 signals upon LPS detection is beginning to be understood, but the picture is incomplete. Previous reports suggested that upon LPS detection, Casp11 dimerizes and undergoes auto-processing to form a pyroptosis-competent enzyme. The prediction from these studies was that the formation of a fully functional Casp11 signalling complex involves two steps: inducible dimerization and auto-processing.
In this study, authors used fluorescently tagged Casp11 reporter fusions, to report that detection of cytosolic LPS induces Casp11 assembly into a large perinuclear speck to form a signalling complex, where GSDMD can be processed. Such signalling complex resembles signalling specks formed upon the activation of other canonical inflammasomes.
Strengths:
Results are clean, experiments well controlled, and support the conclusions. Overall conclusions fit nicely in the general principle of innate signalling, whereby activation of many innate sensors results in their inducible assembly into higher-order oligomeric signalling complexes, called supra-molecular organizing centers (SMOCs).
A surprising finding from this work was that catalytically inactive Casp11 (C254A mutant) did not form signalling specks, despite being able to bind LPS and dimerise. This model is proposed where LPS binding to the CARD domain of Casp11 and Casp11 dimerization is necessary but not sufficient to mediate Casp11 speck formation within cells. The Casp11 catalytic activity is needed to facilitate the assembly of the higher-order, pyroptosis-competent Casp11 signalling platform. The model is further supported by experimental evidence that auto-processing of Casp11, by an exogenous protease TEV, (i.e. in the absence of LPS), is sufficient to mediate speck assembly in cells expressing wild type, but not catalytically inactive Casp11 mutant.
Possible technical improvements:
In general, the authors achieved their aims, and the results support the conclusions.
For technical robustness, it would be nice to consider a few controls:
(a) Visualise Casp11 specks using constructs with smaller tags, and test whether tag placement on N or C terminus matters for speck formation; or
(b) Biochemically crosslink and isolate endogenous, untagged Casp11 specks upon LPS transfection of primed macrophages (e.g. after priming through IFNs or TLRs). This would mimic the natural upregulation and activation of endogenous Casp11.
(c) Test what happens after actual intracellular pathogen detection when the pathogen itself serves as a signalling platform? Are specks stills formed (or even needed)?The broad impact of the work, implication, and questions for future work:
Results of this study would suggest that the enzymatic activity of Casp11 in macrophages may be highly restricted to the speck location, similar to what was described for Casp1. This may explain the very restricted substrate repertoire of Casp11 in cells, likely controlled by the substrate recruitment to the speck. This also opens avenues for follow-up work to answer several emerging questions:
1. After LPS binding and dimerization, why Casp11 must undergo intra-molecular processing to induce the formation of a pyroptosis-competent speck? Is there any substrate for LPS-bound, uncleaved Casp11 (beyond Casp11 itself), before Casp11 forms a full speck for GSDMD processing? The only currently known targets of Casp11 activity are itself, and GSDMD. Also, after intradomain linker cleavage of Casp11, what additional substrate must the cleaved Casp11 process to allow full speck formation?
2. Can activity probes be designed to detect the location of the active Casp11, and if so, would the activity of Casp11 be restricted to the speck? Is there a second cleavage event that would eventually dissociate Casp11 from the speck, to terminate its signalling? If not, how is speck activity terminated? If specks are released by lysis, are they capable of seeding new speck formation in neighbouring phagocytes, in prion-like behaviour previously described for canonical ASC speck?
3. What is the role of macrophage priming in speck formation, and what roles, if any GBPs play in speck formation?
4. Does this model apply to human orthologues, Casp4/5?
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Reviewer #3 (Public Review):
Casp11 plays an important role in host defense against a wide range of pathogens; however, it also promotes autoinflammatory disorders when dysregulated. Unlike other ASC-dependent receptors, Casp11 forms a non-canonical inflammasome via LPS-indued self-assembly. Here, Brodsky and colleagues report that the catalytic activity of casp11 is required to form LPS-induced "SMOCs."
Here are my concerns/questions:
• I'm having some difficulty understanding the logic of Figure 5 in determining cis processing. It is an inverse of figure 4, and in my view, provides further evidence of trans processing. A better experiment would to be use WT-citrine tagged protein with catalytic dead mcherry and image them together. This would show WT cis processing occurs faster than trans processing as citrine specks should appear …
Reviewer #3 (Public Review):
Casp11 plays an important role in host defense against a wide range of pathogens; however, it also promotes autoinflammatory disorders when dysregulated. Unlike other ASC-dependent receptors, Casp11 forms a non-canonical inflammasome via LPS-indued self-assembly. Here, Brodsky and colleagues report that the catalytic activity of casp11 is required to form LPS-induced "SMOCs."
Here are my concerns/questions:
• I'm having some difficulty understanding the logic of Figure 5 in determining cis processing. It is an inverse of figure 4, and in my view, provides further evidence of trans processing. A better experiment would to be use WT-citrine tagged protein with catalytic dead mcherry and image them together. This would show WT cis processing occurs faster than trans processing as citrine specks should appear earlier than the mCherry ones. Can also do colocalization and FRET-based assays with the pair.
• Do those casp11 specks still contain CARDs?- i.e. is the second cleavage necessary for speck formation? Is CARD necessary at all? Would adding the TEV site at CDL and b/w p20 and p10 rescue? i.e. trans-activate?
• What are the equations that fit experimental data points and R2 for? E.g. Figure 1E. What are the parameters being fitted/compared and how are those interpreted? A table of fitted values and proper interpretation should be provided.
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