Date Published: December 28, 2016
Publisher: Public Library of Science
Author(s): Chong-Shan Shi, John H. Kehrl, Taishin Akiyama.
The release of cytochrome c from the inner mitochondrial membrane, where it is anchored by caridolipin, triggers the formation of the Apaf-1 apoptosome. Cardiolipin also interacts with NLRP3 recruiting NLRP3 to mitochondria and facilitating inflammasome assembly. In this study we investigated whether cytosolic cytochrome c impacts NLRP3 inflammasome activation in macrophages. We report that cytochrome c binds to the LRR domain of NLRP3 and that cytochrome c reduces the interactions between NLRP3 and cardiolipin and between NLRP3 and NEK7, a recently recognized component of the NLRP3 inflammasome needed for NLRP3 oligomerization. Protein transduction of cytochrome c impairs NLRP3 inflammasome activation, while partially silencing cytochrome c expression enhances it. The addition of cytochrome c to an in vitro inflammasome assay severely limited caspase-1 activation. We propose that there is a crosstalk between the NLRP3 inflammasome and apoptosome pathways mediated by cytochrome c, whose release during apoptosis acts to limit NLRP3 inflammasome activation.
Apoptosis is an active, programmed process of autonomous cellular death. Signals that trigger apoptosis lead to the assembly of the apoptosome, a cytosolic protein complex that uses Apaf-1 as a sensor to detect cytosolic cytochrome c released from stressed or damaged mitochondria . Upon oligomeration Apaf-1 recruits and activates caspase-9 and in-turn caspase-3, leading to apoptosis . Apoptosis is considered a non-inflammatory form of cell death. Similar to the apoptosome, an inflammasome is a cytosolic protein complex that activates a caspase. NLRP3 (NLR family, pyrin domain containing 3) inflammasomes use NLRP3 as a sensor protein, which in the course of inflammasome activation recruits the adaptor Asc and caspase-1 resulting in caspase activation . The activated caspase cleaves pro-IL-1β and pro-IL-18 releasing the mature cytokines and in some instances causing pyroptosis, an inflammatory form of cell death .
The conceptual similarity between the NLRP3 inflammasome (NLR sensor, ASC, and caspase-1) and the apoptosome (Apaf-1, cytochrome c, and caspase-9) has been increasingly recognized [18, 19]. In response to specific activating signals both assemble multiprotein complexes that control inflammation and cell death. Mitochondrial damage and lysosomal membrane destabilization have been implicated in the activation of both of them [14, 20, 21]. There is also increasing evidence of cross-regulation between the signaling pathways that control their assembly. The inhibitors of apoptosis protein (IAPs), which are critical inhibitors of apoptotic signaling, have both positive and negative effects on inflammasome activation [22, 23]. In this study we confirmed the release of cytochrome c into the cytosol by signals that assemble NLRP3 inflammasomes. We found that cytochrome c interferes with the binding of NLRP3 to cardiolipin and NEK7. By introducing small amounts of cytochrome c into the cytosol by protein transduction we reduced NLRP3, but not AIM2 inflammasome activity. Conversely by reducing cytochrome c levels by gene silencing we enhanced IL-1β production following NLRP3 inflammasome activation. Using an in vitro caspase-1 activation assay that depended upon mitochondria purified from cells treated with ATP we found that the addition of exogenous cytochrome c markedly reduced the activation of caspase-1. Our findings contrast with experiments that showed a lack of involvement of cytochrome c in NALP-1 (NLRP1) inflammasome activation . Human NLRP1 features an amino-terminal PYD, a NOD, LRRs, a function-to-find domain and a carboxy-terminal CARD domain. Although NLRP1 and NLRP3 both have LRR repeats, NLRP3 has nine repeats, while NLRP1 has only six. Alignments of their LRR repeats show approximately 50% identity. This suggests that the LRR repeats of NLRP3 and NALP1 differ such that cytochrome can interact with NLRP3, but not with NLRP1. Following apoptotic signals the release of large amounts of cytochrome c likely limits the concurrent activation of NLRP3 inflammasomes. Further study of the interactions between NEK7, cardiolipin and cytochrome c with NLRP3 may provide a means to modulate NLRP3 inflammasome activity.