A Chemical Strategy for Protease Substrate Profiling

Griswold, Andrew R.; Cifani, Paolo; Rao, Sahana D.; Axelrod, Abram; Miele, Matthew M.; Hendrickson, Ronald C.; Kentsis, Alex; Bachovchin, Daniel A.

doi:10.1016/j.chembiol.2019.03.007

Cited by 59 publications

(76 citation statements)

References 47 publications

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“…2c ). Consistent with previous studies 8 , 9 , 25 , N-terminal sequencing indicated that the seven non-structured, N-terminal residues of the UPA N-loop were not cleaved by DPP9 (Extended Data Fig. 5a ).…”

Section: Specific Interaction Between Rnlrp1 and Rdpp9supporting

confidence: 90%

Structural and biochemical mechanisms of NLRP1 inhibition by DPP9

Huang

Zhang

Toh

et al. 2021

Nature

112

108

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Nucleotide-binding domain, leucine-rich repeat receptors (NLRs) mediate innate immunity by forming inflammasomes. Activation of the NLR protein NLRP1 requires autocleavage within its function-to-find domain (FIIND)1–7. In resting cells, the dipeptidyl peptidases DPP8 and DPP9 interact with the FIIND of NLRP1 and suppress spontaneous NLRP1 activation8,9; however, the mechanisms through which this occurs remain unknown. Here we present structural and biochemical evidence that full-length rat NLRP1 (rNLRP1) and rat DPP9 (rDPP9) form a 2:1 complex that contains an autoinhibited rNLRP1 molecule and an active UPA–CARD fragment of rNLRP1. The ZU5 domain is required not only for autoinhibition of rNLRP1 but also for assembly of the 2:1 complex. Formation of the complex prevents UPA-mediated higher-order oligomerization of UPA–CARD fragments and strengthens ZU5-mediated NLRP1 autoinhibition. Structure-guided biochemical and functional assays show that both NLRP1 binding and enzymatic activity are required for DPP9 to suppress NLRP1 in human cells. Together, our data reveal the mechanism of DPP9-mediated inhibition of NLRP1 and shed light on the activation of the NLRP1 inflammasome.

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Section: Specific Interaction Between Rnlrp1 and Rdpp9supporting

confidence: 90%

Structural and biochemical mechanisms of NLRP1 inhibition by DPP9

Huang

Zhang

Toh

et al. 2021

Nature

112

108

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“…18−22 DPP8/9 are highly related intracellular serine dipeptidyl peptidases that cleave N-terminal dipeptides from polypeptide substrates. 23,24 DPP8/9 inhibitors, like LF, induce the proteasome-mediated degradation of the inflammasome N-terminal fragments, but the mechanistic basis of this response remains unknown. 13,20 Zhong and co-workers recently reported that DPP9 directly binds to both human NLRP1 and CARD8 and suggested that the DPP9 protein itself, in addition to its catalytic activity, may play a role in inhibiting NLRP1.…”

mentioning

confidence: 99%

DPP9’s Enzymatic Activity and Not Its Binding to CARD8 Inhibits Inflammasome Activation

et al. 2019

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Inflammasomes are multiprotein complexes formed in response to pathogens. NLRP1 and CARD8 are related proteins that form inflammasomes, but the pathogen-associated signal(s) and the molecular mechanisms controlling their activation have not been established. Inhibitors of the serine dipeptidyl peptidases DPP8 and DPP9 (DPP8/9) activate both NLRP1 and CARD8. Interestingly, DPP9 binds directly to NLRP1 and CARD8, and this interaction may contribute to the inhibition of NLRP1. Here, we use activity-based probes, reconstituted inflammasome assays, and mass spectrometry-based proteomics to further investigate the DPP9–CARD8 interaction. We show that the DPP9–CARD8 interaction, unlike the DPP9–NLRP1 interaction, is not disrupted by DPP9 inhibitors or CARD8 mutations that block autoproteolysis. Moreover, wild-type, but not catalytically inactive mutant, DPP9 rescues CARD8-mediated cell death in DPP9 knockout cells. Together, this work reveals that DPP9’s catalytic activity and not its binding to CARD8 restrains the CARD8 inflammasome and thus suggests the binding interaction likely serves some other biological purpose.

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“…63,64 Interestingly, the neo-N-terminus of the NLRP1 C-terminal fragment is NH 2 -Ser-Pro, raising the possibility that DPP8/9 directly cleaves NLRP1 itself to restrain inflammasome assembly. However, a study using Chemical Enrichment of Protease Substrates (CHOPS) found that the mNLRP1B allele 1 C-terminal fragment is not a direct DPP8/9 substrate, 65 and immunoprecipitation-mass spectrometry (IP-MS) experiments failed to identify an N-terminal peptide consistent with DPP8/9 cleavage of hNLRP1. 62 Moreover, the N-terminal sequence of CARD8's neo-C-terminal fragment is NH 2 -Ser-Leu, which is not a preferred DPP8/9 substrate.…”

Section: The D Ipep Tidyl Pep Tida S E S 8 and 9 (D Pp8/9)mentioning

confidence: 99%

“…It is possible that some, as yet unknown, DPP8/9 substrate(s) regulate NLRP1 and CARD8 activation. 56,62 Intriguingly, DPP9 substrate profiling studies using terminal amine isotopic labelling of substrates (TAILS) in SKOV3 cells 66 and CHOPS in THP-1 cells 65 identified very few potential protein substrates. Instead, CHOPS analysis indicated that DPP8/9 preferentially cleaved after proline residues in unstructured peptides, rather than globular proteins.…”

Section: The D Ipep Tidyl Pep Tida S E S 8 and 9 (D Pp8/9)mentioning

confidence: 99%

The NLRP1 and CARD8 inflammasomes

Taabazuing

Griswold

Bachovchin

2020

Immunological Reviews

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127

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Inflammasomes are multiprotein complexes that activate inflammatory cytokines and induce pyroptosis in response to intracellular danger‐associated signals. NLRP1 and CARD8 are related germline‐encoded pattern recognition receptors that form inflammasomes, but their activation mechanisms and biological purposes have not yet been fully established. Both NLRP1 and CARD8 undergo post‐translational autoproteolysis to generate two non‐covalently associated polypeptide chains. NLRP1 and CARD8 activators induce the proteasome‐mediated destruction of the N‐terminal fragment, liberating the C‐terminal fragment to form an inflammasome. Here, we review the danger‐associated stimuli that have been reported to activate NLRP1 and/or CARD8, including anthrax lethal toxin, Toxoplasma gondii, Shigella flexneri and the small molecule DPP8/9 inhibitor Val‐boroPro, focusing on recent mechanistic insights and highlighting unresolved questions. In addition, we discuss the recently identified disease‐associated mutations in NLRP1 and CARD8, the potential role that DPP9’s protein structure plays in inflammasome regulation, and the emerging link between NLRP1 and metabolism. Finally, we summarize all of this latest research and consider the possible biological purposes of these enigmatic inflammasomes.

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A Chemical Strategy for Protease Substrate Profiling

Cited by 59 publications

References 47 publications

Structural and biochemical mechanisms of NLRP1 inhibition by DPP9

Structural and biochemical mechanisms of NLRP1 inhibition by DPP9

DPP9’s Enzymatic Activity and Not Its Binding to CARD8 Inhibits Inflammasome Activation

The NLRP1 and CARD8 inflammasomes

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