Wolf et al then proceed to interrogate the
Wolf et al. (2016) then proceed to interrogate the mechanism by which NAG activates the NLRP3 inflammasome. When examining potential triggers previously implicated in NLRP3 activation, they found that both PGN and NAG trigger appearance of mitochondrial DNA (mtDNA) in the cytosol, which led them to investigate the role of mitochondria in NAG induced NLRP3 activation. Early investigations into the glycolytic pathway showed that NAG could inhibit glycolysis (Spiro, 1958). Further studies demonstrated that by binding to its active site, NAG competitively inhibits HK, which converts glucose to glucose 6-phosphate in the first step of glycolysis (Bertoni and Weintraub, 1984). Wolf et al. (2016) confirm the ability of NAG to inhibit HK by showing that glucose but not NAG is phosphorylated by HK. Because unacetylated NAG can be phosphorylated by HK, it lacks the inhibitory properties of acetylated NAG. By employing thermal shift technology that identifies sugar binding to a protein by modifying its thermal stability, they further show that NAG directly binds HK. Of the different isoforms of HK, hexokinase 2 (HK2) is normally tethered to the mitochondrial membrane through an interaction with voltage-dependent anion tsa inhibitor (VDACs), which are integral components of the mitochondrial permeability transition pore. HK has previously been identified as an inhibitor of VDAC channel opening, and dissociation of HK from VDAC leads to mitochondrial swelling, cytochrome c release, DNA laddering, and apoptotic cell death (Azoulay-Zohar et al., 2004). Previous work has also shown that ablating VDAC expression by shRNA inhibits NLRP3-dependent IL-1β production (Zhou et al., 2011). Thus, Wolf et al. (2016) hypothesized that HK-VDAC association may prevent release of mitochondrial danger signals into the cytosol through VDAC, and NAG may induce NLRP3 activation by disrupting HK association with mitochondria (Figure 1). To test this hypothesis, they examined the ability of NAG to induce HK2 dissociation from mitochondria and convincingly demonstrate that both PGN and NAG induce specific accumulation of HK protein as well as increased HK enzyme activity in the cytosol, which was not a result of a general loss of mitochondrial integrity. HK release into the cytosol was observed upon phagocytosis of a variety of gram-positive bacteria, and importantly, also in NLRP3-deficient macrophages indicating that it is a proximal event in inflammasome activation. Remarkably, by using a specific, cell-permeable peptide (HKVBD) previously known to induce dissociation of HK2 from mitochondrial VDAC, Wolf et al. (2016) demonstrate that forced dissociation of HK2 from mitochondria is sufficient to induce NLRP3-dependent production of active IL-1β, IL-18, and caspase-1 dependent neutrophil recruitment in vivo. As the first enzyme in the glycolytic pathway, HK can be inhibited by intermediates of both the glycolysis and TCA cycle. Wolf et al. (2016) hypothesized that metabolic disruptions converging on HK inhibition may induce NLRP3 activation and offer insight into how metabolic perturbation impinges on inflammasome activation. Notably, transfection of LPS-primed BMDM with increasing amounts of glucose 6-phosphate (that inhibits HK by feedback inhibition), 2-deoxyglucose (a glycolysis inhibitor), or citrate (an intermediate in the TCA cycle that inhibits the rate-limiting glycolytic enzyme phosphofructokinase, resulting in elevated glucose 6-phosphate levels) mimics the effect of NAG and PGN in inducing NLRP3 inflammasome activation (Figure 1), suggesting an intriguing relationship between metabolism and inflammasome activation. How HK release from mitochondria activates NLRP3 remains unknown. Wolf et al. (2016) speculate that release of mtDNA may constitute one such trigger. It is tempting to speculate that impairment of glycolysis creates a metabolic state permissible to NLRP3 activation, perhaps a pro-oxidant condition that promotes oxidative damage, another trigger previously linked to NLRP3 activation. Mitochondrial regulation of the NLRP3 inflammasome remains a complex puzzle. Nonetheless, the findings of Wolf et al. (2016) have major ramifications for metabolic diseases like type 2 diabetes, atherosclerosis, and obesity in which NLRP3 is implicated. Subject to future investigations into the role of HK and other glycolytic enzymes in the inflammasome response to different NLRP3 activators, their findings may raise the possibility of exploring the development of small molecules that restore glycolytic flux for therapeutic intervention in diverse autoinflammatory, autoimmune, infectious, and metabolic diseases linked to the NLRP3 inflammasome.