The NLRP3 inflammasome fires up heme-induced inflammation in hemolytic conditions.

Overactive inflammatory responses are central to the pathophysiology of many hemolytic conditions including sickle cell disease. Excessive hemolysis leads to elevated serum levels of heme due to saturation of heme scavenging mechanisms. Extracellular heme has been shown to activate the NLRP3 inflammasome, leading to activation of caspase-1 and release of pro-inflammatory cytokines IL-1ß and IL-18. Heme also activates the non-canonical inflammasome pathway, which may contribute to NLRP3 inflammasome formation and leads to pyroptosis, a type of inflammatory cell death. Some clinical studies indicate there is a benefit to blocking the NLRP3 inflammasome pathway in patients with sickle cell disease and other hemolytic conditions. However, a thorough understanding of the mechanisms of heme-induced inflammasome activation is needed to fully leverage this pathway for clinical benefit. This review will explore the mechanisms of heme-induced NLRP3 inflammasome activation and the role of this pathway in hemolytic conditions including sickle cell disease.

Visualization of Inflammatory Caspases Induced Proximity in Human Monocyte-Derived Macrophages.

Inflammatory caspases include caspase-1, -4, -5, -11, and -12 and belong to the subgroup of initiator caspases. Caspase-1 is required to ensure correct regulation of inflammatory signaling and is activated by proximity-induced dimerization following recruitment to inflammasomes. Caspase-1 is abundant in the monocytic cell lineage and induces maturation of the pro-inflammatory cytokines interleukin (IL)-1ß and IL-18 to active secreted molecules. The other inflammatory caspases, caspase-4 and -5 (and their murine homolog caspase-11) promote IL-1ß release by inducing pyroptosis. Caspase Bimolecular Fluorescence Complementation (BiFC) is a tool used to measure inflammatory caspase induced proximity as a readout of caspase activation. The caspase-1, -4, or -5 prodomain, which contains the region that binds to the inflammasome, is fused to non-fluorescent fragments of the yellow fluorescent protein Venus (Venus-N [VN] or Venus-C [VC]) that associate to reform the fluorescent Venus complex when the caspases undergo induced proximity. This protocol describes how to introduce these reporters into primary human monocyte-derived macrophages (MDM) using nucleofection, treat the cells to induce inflammatory caspase activation, and measure caspase activation using fluorescence and confocal microscopy. The advantage of this approach is that it can be used to identify the components, requirements, and localization of the inflammatory caspase activation complex in living cells. However, careful controls need to be considered to avoid compromising cell viability and behavior. This technique is a powerful tool for the analysis of dynamic caspase interactions at the inflammasome level as well as for the interrogation of the inflammatory signaling cascades in living MDM and monocytes derived from human blood samples.

Cellular autophagy, an unbidden effect of caspase inhibition by zVAD-fmk.

zVAD-fmk is a widely used pan-caspase inhibitor that blocks apoptosis but has undesirable side effects, including autophagy. In this issue, Needs et al. propose that zVAD-fmk induces autophagy by inhibiting the N-glycanase NGLY1 rather than caspases. NGLY1 is essential for the ERAD response and patients with inactivating mutations in NGLY1 present with neurodevelopmental defects and organ dysfunction. The ability of NGLY1 to inhibit basal levels of autophagy may contribute to this pathology. This study demonstrates possible crosstalk between protein turnover and autophagy while also underscoring the importance of specificity when using chemical tools to interrogate these pathways. Comment on https://doi.org/10.1111/febs.16345.

Noncanonical Roles of Caspase-4 and Caspase-5 in Heme-Driven IL-1ß Release and Cell Death.

Excessive release of heme from RBCs is a key pathophysiological feature of several disease states, including bacterial sepsis, malaria, and sickle cell disease. This hemolysis results in an increased level of free heme that has been implicated in the inflammatory activation of monocytes, macrophages, and the endothelium. In this study, we show that extracellular heme engages the human inflammatory caspases, caspase-1, caspase-4, and caspase-5, resulting in the release of IL-1ß. Heme-induced IL-1ß release was further increased in macrophages from patients with sickle cell disease. In human primary macrophages, heme activated caspase-1 in an inflammasome-dependent manner, but heme-induced activation of caspase-4 and caspase-5 was independent of canonical inflammasomes. Furthermore, we show that both caspase-4 and caspase-5 are essential for heme-induced IL-1ß release, whereas caspase-4 is the primary contributor to heme-induced cell death. Together, we have identified that extracellular heme is a damage-associated molecular pattern that can engage canonical and noncanonical inflammasome activation as a key mediator of inflammation in macrophages.

Inflammatory caspase regulation: maintaining balance between inflammation and cell death in health and disease.

Members of the mammalian inflammatory caspase family, including caspase-1, caspase-4, caspase-5, caspase-11, and caspase-12, are key regulators of the innate immune response. Most studies to date have focused on the role of caspase-1 in the maturation of the proinflammatory cytokine interleukin-1ß and its upstream regulation by the inflammasome signaling complexes. However, an emerging body of research has supported a role for caspase-4, caspase-5, and caspase-11 in both regulating caspase-1 activation and inducing the inflammatory form of cell death called pyroptosis. This inflammatory caspase pathway appears essential for the regulation of cytokine processing. Consequently, insight into this noncanonical pathway may reveal important and, to date, understudied targets for the treatment of autoinflammatory disorders where the inflammasome pathway is dysregulated. Here, we will discuss the mechanisms of inflammasome and inflammatory caspase activation and how these pathways intersect to promote pathogen clearance.

Studies of the Binding of Modest Modulators of the Human Enzyme, Sirtuin†6, by STD NMR.

Pyrazinamide (PZA), an essential constituent of short-course tuberculosis chemotherapy, binds weakly but selectively to Sirtuin 6 (SIRT6). Despite the structural similarities between nicotinamide (NAM), PZA, and pyrazinoic acid (POA), these inhibitors modulate SIRT6 by different mechanisms and through different binding sites, as suggested by saturation transfer difference (STD) NMR. Available experimental evidence, such as that derived from crystal structures and kinetic experiments, has been of only limited utility in elucidation of the mechanistic details of sirtuin inhibition by NAM or other inhibitors. For instance, crystallographic structural analysis of sirtuin binding sites does not help us understand important differences in binding affinities among sirtuins or capture details of such dynamic process. Hence, STD NMR was utilized throughout this study. Our results not only agreed with the binding kinetics experiments but also gave a qualitative insight into the binding process. The data presented herein suggested some details about the geometry of the binding epitopes of the ligands in solution with the apo- and holoenzyme. Recognition that SIRT6 is affected selectively by PZA, an established clinical agent, suggests that the rational development of more potent and selective NAM surrogates might be possible. These derivatives might be accessible by employing the malleability of this scaffold to assist in the identification by STD NMR of the motifs that interact with the apo- and holoenzymes in solution.

Sirtuin 6: a review of biological effects and potential therapeutic properties.

Sirtuins, possessing either histone deacetylase or mono-ribosyltransferase activity, regulate important pathways in bacteria, archaea and eukaryotes. SIRT6, an enzyme highly expressed in skeletal muscles, brain, heart, liver, and thymus, affects transcriptional regulation in a tissue-specific manner. This enzyme has a two-domain structure that consists of a large Rossmann fold and a smaller and structurally more varied sequence containing a Zn(2+)-binding motif. The C-terminus is required for proper nuclear localization, while the N-terminus is important for chromatin association and for intrinsic catalytic activity. SIRT6 promotes resistance to DNA damage and oxidative stress, the principal defects associated with age-related diseases. The modulation of aging and other metabolic functions by SIRT6 may be indicative of previously unrecognized regulatory systems in the cell. The propensity of individual SIRT6 molecules to undergo intramolecular mono-ADP-ribosylation, suggests this auto-ribosylation may contribute to the self-regulation of SIRT6 function. Until recently, SIRT6 was an orphan enzyme whose catalytic activity and substrates were unclear. It was known that, similar to the yeast Sir2 protein, human SIRT6 deacetylates histones and regulates DNA stability and repair; however, new mechanistic insights can be derived from the discovery of the highly substrate-specific histone deacetylase activity of SIRT6. This deacetylase activity promotes proper chromatin function in several physiologic contexts, to include telomere and genome stabilization, gene expression and DNA repair. By maintaining both the integrity and the expression of the mammalian genome, SIRT6 may help prevent cellular senescence. Moreover, successful molecular modulation of SIRT6 activity may lead to the development of new chemotherapeutic modalities. The action of SIRT6 is described in this review, with an emphasis on the cellular roles of the enzyme and the relation of those enzymatic functions to human biology and disease.