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
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
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
Inflammasome is intracellular multiprotein complex composed of NLR receptor, adaptor ASC and a caspase-1. NLRP3 or cryopyrin is the most investigated receptor from the NOD-like receptors family. A variety of different triggers are able to activate NLRP3: ATP and other endogenous signals e.g. urate crystals, which accumulate in gout. Aβ fibrils, the main component of senile plaques in Alzheimer’s disease also activate Nlrp3 inflammasome. Currently, we are investigating the mechanism of NLRP3 inflammasome assembly and developing new inhibitors for this detrimental pathway, which is involved in a variety of different diseases. In order to define the roles of selected domains of NLRP3 in NLRP3 inflammasome assembly 21 truncated NLRP3 mutants were expressed in NLRP3-deficient macrophages. We showed that mutants lacking LRR domain are fully responsive to various soluble and particulate instigators. We were able to define minimal fully responsive NLRP3 variant, which is similar in length to some alternative splice variants. For shorter variants, which do not support activation, we were nevertheless able to show that they still respond to triggers, but fail in further steps of inflammasome assembly. We challenged several possible models of NLRP3 activation, including self-propagation model as proposed for homologous protein NLRC4, which seems not to be the case for NLRP3. The revision of the manuscript Hafner-Bratkovič* et al. for Nature Communications is in preparation. Using designed oligomerization domains we showed that trimerization of PYD domains is sufficient for robust activation of inflammasome (Sušjan, Roškar and Hafner-Bratkovič*, BBRC, 2017). We are also exploring the ways to inhibit NLRP3 inflammasome activation. We also participated in development and characterization of novel acrylate NLRP3 inhibitors for treatment of inflammatory bowel disease (Cocco et al., J Med Chem, 2017). Based on available structures of the proteins that comprise the inflammasome and on the NLRP3 regions, carrying pathological mutations, we designed two groups of putative inhibitory peptides, which are most likely to disrupt the formation of the inflammasome. Furthermore, we found that one peptide specifically inhibited the NLRP3 inflammasome and not other inflammasomes and inhibited inflammasome activation in a mouse model of silica-induced peritonitis. Manuscript Sušjan, Lainšček & Hafner-Bratkovič* is to be submitted. Our results were presented in forms of posters and selected talks at international conferences and meetings. We also published two reviews on the topic of inflammasomes. The PI recently wrote a review together with Pablo Pelegrin for Current Opinion in Immunology (accepted for publication). Three MSc theses were defended on this topic and one PhD student and one Msc student are currently involved.
COBISS.SI-ID: 6095642
Protein structures evolved through a complex interplay of cooperative interactions and it is still very challenging to design new protein folds de novo. In Gradišar et al., Nat Chem Biol, 2013 we presented a strategy to design self-assembling polypeptide nanostructured polyhedra, based on modularization using orthogonal dimerizing segments. This design platform provides the basis for construction of new topological polypeptide folds based on the set of orthogonal interacting polypeptide segments. In Ljubetič et al, Nature Biotechnology, 2017 we improved the design of protein nanocages and prepared tetrahedron, four-sided pyramid, and triangular prism. We demonstrated that such structures self-assemble in vivo, in live bacteria, mammalian cells and in mice and do not activate NLRP3 inflammasome or induce ER stress.
COBISS.SI-ID: 6266906