Enzymatically Inactive Procaspase 1 stabilizes the ASC Pyroptosome and Supports Pyroptosome Spreading during Cell Division

Abstract

Caspase-1 is a key player during the initiation of pro-inflammatory innate immune responses, activating pro-IL-1␤ in socalled inflammasomes. A subset of patients with recurrent febrile episodes and systemic inflammation of unknown origin harbor mutations in CASP1 encoding caspase-1. CASP1 variants result in reduced enzymatic activity of caspase-1 and impaired IL-1␤ secretion. The apparent paradox of reduced IL-1␤ secretion but systemic inflammation led to the hypothesis that CASP1 mutations may result in variable protein interaction clusters, thus activating alternative signaling pathways. To test this hypothesis, we established and characterized an in vitro system of transduced immortalized murine macrophages expressing either WT or enzymatically inactive (p.C284A) procaspase-1 fusion reporter proteins. Macrophages with variant p.C284A caspase-1 did not secrete IL-1␤ and exhibited reduced inflammatory cell death, referred to as pyroptosis. Caspase-1 and apoptosis-associated speck-like protein containing a CARD (ASC) formed cytosolic macromolecular complexes (so-called pyroptosomes) that were significantly increased in number and size in cells carrying the p.C284A caspase-1 variant compared with WT caspase-1. Furthermore, enzymatically inactive caspase-1 interacted with ASC longer and with increased intensity compared with WT caspase-1. Applying live cell imaging, we documented for the first time that pyroptosomes containing enzymatically inactive variant p.C284A caspase-1 spread during cell division. In conclusion, variant p.C284A caspase-1 stabilizes pyroptosome formation, potentially enhancing inflammation by two IL-1␤-independent mechanisms: pyroptosomes convey an enhanced inflammatory stimulus through the recruitment of additional proteins (such as RIP2, receptor interacting protein kinase 2), which is further amplified through pyroptosome and cell division.Caspase-1 is a key player during innate immune responses. It aids to control pathogens and danger signals by triggering pyroptotic cell death. Together with caspase-11 and -12 in mice and caspase-4, -5, and -12 in humans, caspase-1 belongs to the family of inflammatory caspases, which are produced as inactive forms. After the detection of pathogens or cytoplasmic danger signals, inactive caspase-1 monomers recruit to multiprotein complexes referred to as inflammasomes (1-3). In such inflammasomes, inactive procaspases are autoprocessed into caspase activation and recruitment domains (CARDs), 2 the p10 and the p20 subunits (4). Two p10 and two p20 proteins subsequently form the active caspase-1 tetramer, which then cleaves and thereby activates IL-1␤ in the so-called canonical inflammasome pathway (5, 6). Furthermore, inflammatory caspases induce pro-inflammatory cell death, referred to as pyroptosis, which involves cell swelling, cell lysis, and the release of cytoplasmic contents (7,8). Pyroptosis depends on the activation of caspase-4, -5, or -11 and caspase-1 (9 -11). Caspase-1 or -11 cleaves gasdermin D, a gasdermin domain-containing protein, at amin...

Inflammasome and Caspase-1 Activity Characterization and Evaluation: An Imaging Flow Cytometer–Based Detection and Assessment of Inflammasome Specks and Caspase-1 Activation

“…When only ASC is expressed, the diameter of ASC specks is larger, at ∼5 mm. Thus, ICCE can be used to evaluate speck area and diameter in a high-throughput system, yielding quantitative results similar to those reported by others (41,42). These changes in speck area suggest a possible higher order organization of the speck upon NLRP3 activation and implicate NLRP3 activation in dictating features of such organization (Fig.…”

“…ICCE can also be used for qualitative assessment of ASC speck features such as area, which is not possible using TOFIE. Variations in ASC speck size have recently been associated with the presence of active caspase-1 and the duration of caspase-1 activity (41,42). Further, in the absence of NLRP3, hypotonic conditions lead to ASC speck formation without caspase-1 activation (45), reminiscent of the specks formed when ASC is overexpressed (17).…”

“…Enzymatically inactive caspase-1 activates receptor-interacting protein kinase-2 (RIP-2) to drive inflammation independently of the inflammasome (40). Moreover, the area of ASC specks is larger when caspase-1 is enzymatically inactive (41). Thus, speck area provides qualitative information regarding potential speck function and the type of the pathway involved in the inflammatory response.…”

Procaspase-1 patrolled to the nucleus of proatherogenic lipid LPC-activated human aortic endothelial cells induces ROS promoter CYP1B1 and strong inflammation

“…Previous reports showed that 1) protease-dead mutant of pro-caspase-1 can still be translocated to the nucleus [ 31 ], suggesting that pro-caspase-1 may travel to nucleus for its enzymatic activities-independent functions such as promotion of gene transcription as we reported [ 28 ]; and 2) enzymatically inactive pro-caspase-1 stabilizes the apoptosis-associated speck-like protein containing a CARD (ASC, PYCARD) pyroptosome (a supramolecular assembly of ASC dimers mediating inflammatory cell death via caspase-1 activation [ 88 ]) and supports pyroptosome spreading during cell division [ 89 ]. We then hypothesized that LPC modulate RNA transcriptomes in HAECs in the presence and absence of caspase-1 inhibitor ( Fig.…”

The ASC Speck and NLRP3 Inflammasome Function Are Spatially and Temporally Distinct

“…Whether the ASC speck is a SMOC that facilitates the formation and release of small inflammasome complexes is unclear and demonstrating this presents numerous challenges. ASC specks are larger in cells with enzymatically inactive caspase-1 (Caspase-1 DEAD) compared to cells with wildtype caspase-1 (Stein et al, 2016). Moreover, ASC specks formed by ASC alone are larger than NLRP3:ASC specks and the size of NLRP3:ASC specks is further reduced after nigericin stimulation (Nagar et al, 2019).…”