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.

The study of mitochondrial diseases has revealed dramatic variability in the phenotypic presentation of mitochondrial genetic defects. To attempt to understand this variability, different authors have studied energy metabolism in transmitochondrial cell lines carrying different proportions of various pathogenic mutations in their mitochondrial DNA. The same kinds of experiments have been performed on isolated mitochondria and on tissue biopsies taken from patients with mitochondrial diseases. The results have shown that, in most cases, phenotypic manifestation of the genetic defect occurs only when a threshold level is exceeded, and this phenomenon has been named the 'phenotypic threshold effect'. Subsequently, several authors showed that it was possible to inhibit considerably the activity of a respiratory chain complex, up to a critical value, without affecting the rate of mitochondrial respiration or ATP synthesis. This phenomenon was called the 'biochemical threshold effect'. More recently, quantitative analysis of the effects of various mutations in mitochondrial DNA on the rate of mitochondrial protein synthesis has revealed the existence of a 'translational threshold effect'. In this review these different mitochondrial threshold effects are discussed, along with their molecular bases and the roles that they play in the presentation of mitochondrial diseases.

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.

Caspases are intracellular proteases that cleave substrates involved in apoptosis or inflammation. In C. elegans, a paradigm for caspase regulation exists in which caspase CED-3 is activated by nucleotide-binding protein CED-4, which is suppressed by Bcl-2-family protein CED-9. We have identified a mammalian analog of this caspase-regulatory system in the NLR-family protein NALP1, a nucleotide-dependent activator of cytokine-processing protease caspase-1, which responds to bacterial ligand muramyl-dipeptide (MDP). Antiapoptotic proteins Bcl-2 and Bcl-X(L) bind and suppress NALP1, reducing caspase-1 activation and interleukin-1beta (IL-1beta) production. When exposed to MDP, Bcl-2-deficient macrophages exhibit more caspase-1 processing and IL-1beta production, whereas Bcl-2-overexpressing macrophages demonstrate less caspase-1 processing and IL-1beta production. The findings reveal an interaction of host defense and apoptosis machinery.

To investigate the physiological diversity in the regulation and control of mitochondrial oxidative phosphorylation, we determined the composition and functional features of the respiratory chain in muscle, heart, liver, kidney, and brain. First, we observed important variations in mitochondrial content and infrastructure via electron micrographs of the different tissue sections. Analyses of respiratory chain enzyme content by Western blot also showed large differences between tissues, in good correlation with the expression level of mitochondrial transcription factor A and the activity of citrate synthase. On the isolated mitochondria, we observed a conserved molar ratio between the respiratory chain complexes and a variable stoichiometry for coenzyme Q and cytochrome c, with typical values of [1–1.5]:[30–135]:[3]:[9–35]:[6.5–7.5] for complex II:coenzyme Q:complex III:cytochrome c:complex IV in the different tissues. The functional analysis revealed important differences in maximal velocities of respiratory chain complexes, with higher values in heart. However, calculation of the catalytic constants showed that brain contained the more active enzyme complexes. Hence, our study demonstrates that, in tissues, oxidative phosphorylation capacity is highly variable and diverse, as determined by different combinations of 1) the mitochondrial content, 2) the amount of respiratory chain complexes, and 3) their intrinsic activity. In all tissues, there was a large excess of enzyme capacity and intermediate substrate concentration, compared with what is required for state 3 respiration. To conclude, we submitted our data to a principal component analysis that revealed three groups of tissues: muscle and heart, brain, and liver and kidney.

Salmonella enterica translocates virulent factors into host cells using type III secretion systems to promote host colonization, intracellular bacterial replication and survival, and disease pathogenesis. Among many effectors, the type III secretion system encoded in Salmonella pathogenicity island 2 translocates a Salmonella-specific protein, designated Salmonella secreted factor L (SseL), a putative virulence factor possessing deubiquitinase activity. In this study, we attempt to elucidate the mechanism and the function of SseL in vitro, in primary host macrophages and in vivo in infected mice. Expression of SseL in mammalian cells suppresses NF-κB activation downstream of IκBα kinases and impairs IκBα ubiquitination and degradation, but not IκBα phosphorylation. Disruption of the gene encoding SseL in S. enterica serovar typhimurium increases IκBα degradation and ubiquitination, as well as NF-κB activation in infected macrophages, compared with wild-type bacteria. Mice infected with SseL-deficient bacteria mount stronger inflammatory responses, associated with increased production of NF-κB-dependent cytokines. Thus, SseL represents one of the first bacterial deubiquitinases demonstrated to modulate the host inflammatory response in vivo.

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SummaryChlamydia trachomatis is an obligate intracellular bacterial pathogen that causes various human diseases, including blindness caused by ocular infection and sexually transmitted diseases resulting from urogenital infection. After infecting host cells, Chlamydiae avoid alarming the host's immune system. Among the immune evasion mechanisms, Chlamydiae can inhibit NF-kB activation, a crucial pathway for host inflammatory responses. In this study, we show that ChlaDub1, a deubiquitinating and deNeddylating protease from C. trachomatis, is expressed in infected cells. In transfection experiments, ChlaDub1 suppresses NF-kB activation induced by several pro-inflammatory stimuli and binds the NF-kB inhibitory subunit IkBa, impairing its ubiquitination and degradation. Thus, we provide further insight into the mechanism by which C. trachomatis may evade the host inflammatory response by demonstrating that ChlaDub1, a protease produced by this microorganism, is capable of inhibiting IkBa degradation and blocking NF-kB activation.