Shikonin is a highly lipophilic naphtoquinone found in the roots of Lithospermum erythrorhizon used for its pleiotropic effects in traditional Chinese medicine. Based on its reported antipyretic and anti-inflammatory properties, we investigated whether shikonin suppresses the activation of NLRP3 inflammasome. Inflammasomes are cytosolic protein complexes that serve as scaffolds for recruitment and activation of caspase-1, which, in turn, results in cleavage and secretion of proinflammatory cytokines IL-1β and IL-18. NLRP3 inflammasome activation involves two steps: priming, i.e. the activation of NF-κB pathway, and inflammasome assembly. While shikonin has previously been reported to suppress the priming step, we demonstrated that shikonin also inhibits the second step of inflammasome activation induced by soluble and particulate NLRP3 instigators in primed immortalized murine bone marrow-derived macrophages. Shikonin decreased NLRP3 inflammasome activation in response to nigericin more potently than acetylshikonin. Our results showed that shikonin also inhibits AIM2 inflammasome activation by double stranded DNA. Shikonin inhibited ASC speck formation and caspase-1 activation in murine macrophages and suppressed the activity of isolated caspase-1, demonstrating that it directly targets caspase-1. Complexing shikonin with β-lactoglobulin reduced its toxicity while preserving the inhibitory effect on NLRP3 inflammasome activation, suggesting that shikonin with improved bioavailability might be interesting for therapeutic applications in inflammasome-mediated conditions.
COBISS.SI-ID: 5952026
NLRP3 inflammasome is a multiprotein platform for the activation of caspase-1. Despite the increasing number of reports linking NLRP3 inflammasome to a variety of diseases, the mechanism behind the NLRP3 activation remains elusive, especially in terms of the early stages which are critical to the NLRP3 inflammasome assembly. In the present study we aimed to determine the minimal oligomerization state required for the NLRP3 inflammasome activation. For this purpose, NLRP3 pyrin domain (NLRP3PYD) was fused to various dimerization and trimerization domains. The constructs were expressed under the inducible promoter in mouse macrophages lacking endogenous NLRP3. Dimerization of the NLRP3PYD either in parallel or in antiparallel orientation was insufficient for the inflammasome activation. Trimerization of the NLRP3PYD with the foldon domain, however, induced pyroptosis and robust IL-1β maturation, which was caspase-1 dependent. Interestingly, foldon-induced constitutive activation is resistant to inhibition with NLRP3-specific inhibitor MCC950 and does not lead to ASC speck formation. Although we cannot exclude that wild-type NLRP3 forms higher oligomer species similar to NLRP1 or NLRC4, our results clearly demonstrate that efficient IL-1β response can be achieved by the induced trimerization of the NLRP3PYD domain.
COBISS.SI-ID: 6095642
The project leader contributed two peer-reviewed review papers on the topics of inflammasomes (The NLRC4 inflammasome: The pieces of the puzzle are falling into place) and protein particles in health and disease, with section focusing on signaling multiprotein complexes and their intercellular spread (Prions, prionoid complexes and amyloid: the bad, the good and something in between).
COBISS.SI-ID: 6108698
Oxidative stress is an almost universal hallmark of the response to infection or sterile injury that activates the innate immune response. Here, extracellular vesicles (EVs) from the plasma of patients with rheumatoid arthritis or cells submitted to oxidative stress induced MD2-dependent TLR4 activation via oxidation of phospholipids (oxPLs). EVs from healthy subjects or reconstituted synthetic EVs were converted to TLR4 agonists by a limited oxidation, while prolonged oxidation abrogated TLR4 activation. Furthermore, 15-lipoxygenase was identified as the new enzymatic generator of TLR4 agonists, thus identifying the hydro(pero)xylated PLs as the endogenous agonists of TLR4. Activation of TLR4 by oxPL-containing EVs mimics the molecular mechanism of TLR4 activation by lipopolysaccharide (LPS), which is supported by the role of MD-2, effect of TLR4 binding site mutations and receptor complex dimerization. However, in BMDMs the transcriptional response profile to microbial pathogen (LPS)- and endogenous danger signal (EVs)-induced TLR4 signaling differed, with a strong inflammation-resolving component induced only by the EVs. This identifies EVs as the oxidative stress-induced endogenous danger signal that underlies the pervasive role of TLR4 in inflammatory diseases.
COBISS.SI-ID: 5706266
The mechanism of prion protein (PrP) conversion, the key event in prion diseases, is still not understood. We investigated how perturbations of interactions between the subdomains β1-α1-β2 and α2-α3 affect PrP conversion. In vitro fibrillization and biophysical methods were used to relate mouse PrP conversion kinetics to thermodynamic stability. We show that pathologic mutations H187R and E196K destabilize PrP and accelerate fibrillization. At acidic pH, the major contribution to the destabilization of PrP comes from the protonation of histidine 187 because its replacement by tyrosine led to more stable protein with slower fibrillization. Furthermore, we show that the introduction of a novel histidine residue into the subdomain interface (F198H) acts as a pH-inducible switch that promotes conversion upon histidine protonation, whereas this effect is not observed when His residue is introduced at the protein surface (Y155H). We observed a strong correlation between the stability of native structure and kinetics of fibrillization of PrP variants. Our results show that pathologic mutations promote subdomain separation and suggest that stabilization of the native structure might be a viable strategy for the development of novel therapeutics for prion diseases.
COBISS.SI-ID: 5610266