Interleukin (IL)-1beta maturation is accomplished by caspase-1-mediated proteolysis, an essential element of innate immunity. NLRs constitute a recently recognized family of caspase-1-activating proteins, which contain a nucleotide-binding oligomerization domain and leucine-rich repeat (LRR) domains and which assemble into multiprotein complexes to create caspase-1-activating platforms called "inflammasomes." Using purified recombinant proteins, we have reconstituted the NALP1 inflammasome and have characterized the requirements for inflammasome assembly and caspase-1 activation. Oligomerization of NALP1 and activation of caspase-1 occur via a two-step mechanism, requiring microbial product, muramyl-dipeptide, a component of peptidoglycan, followed by ribonucleoside triphosphates. Caspase-1 activation by NALP1 does not require but is enhanced by adaptor protein ASC. The findings provide the biochemical basis for understanding how inflammasome assembly and function are regulated, and shed light on NALP1 as a direct sensor of bacterial components in host defense against pathogens.

Low-grade, chronic inflammation has been associated with many diseases of aging, but the mechanisms responsible for producing this inflammation remain unclear. Inflammasomes can drive chronic inflammation in the context of an infectious disease or cellular stress, and they trigger the maturation of interleukin-1β (IL-1β). Here we find that the expression of specific inflammasome gene modules stratifies older individuals into two extremes: those with constitutive expression of IL-1β, nucleotide metabolism dysfunction, elevated oxidative stress, high rates of hypertension and arterial stiffness; and those without constitutive expression of IL-1β, who lack these characteristics. Adenine and N4-acetylcytidine, nucleotide-derived metabolites that are detectable in the blood of the former group, prime and activate the NLRC4 inflammasome, induce the production of IL-1β, activate platelets and neutrophils and elevate blood pressure in mice. In individuals over 85 years of age, the elevated expression of inflammasome gene modules was associated with all-cause mortality. Thus, targeting inflammasome components may ameliorate chronic inflammation and various other age-associated conditions.

Summary Controversy surrounds the role and mechanism of mitochondrial cristae remodeling in apoptosis. Here we show that pro-apoptotic BH3-only proteins Bid and Bim induced full cytochrome c release but only a subtle alteration of crista junctions, which involved the disassembly of Opa1 complexes. Both mitochondrial outer membrane permeabilization (MOMP) and crista junction opening (CJO) were caspase-independent and required a functional BH3 domain and Bax/Bak. However, MOMP and CJO were experimentally separable. Pharmacological blockade of MOMP did not prevent Opa1 disassembly and CJO; moreover, expression of a disassembly-resistant mutant Opa1 (Q297V) blocked cytochrome c release and apoptosis but not Bax activation. Thus, apoptosis requires a subtle form of Opa1-dependent crista remodeling induced by BH3-only proteins and Bax/Bak, but independent of MOMP.

Mitochondria are key players of apoptosis and can irreversibly commit the cell to death by releasing cytochrome c (Cyt.c) to the cytosol, where caspases 9 and 3 subsequently get activated. Under conditions of oxidative stress, opening of the mitochondrial permeability transition pore (PTP) represents an early trigger and is crucial in causing Cyt.c release. To account for the latter, current models propose that PTP gating would result, as is the case in vitro, in the rupture of the outer mitochondrial membrane caused by mitochondrial matrix swelling. Using live cell imaging and recombinant fluorescent probes based on the green fluorescent protein (GFP) and its mutants, we report that directed repetitive gating of the PTP triggers a delayed Cyt.c efflux, which is not associated with mitochondrial swelling. Instead, subcellular imaging shows that PTP opening signals the redistribution of the cytosolic protein Bax to the mitochondria, where it secondarily forms clusters that appear to be a prerequisite for Cyt.c release. Fluorescence resonance energy transfer imaging further reveals that Bax clustering coincides with the formation of Bax multimers. We conclude that the PTP is not itself a component of the Cyt.c release machinery, but that it acts indirectly by signaling Bax translocation and multimerization.