Control of gasdermin D oligomerization and pyroptosis by the Ragulator-Rag-mTORC1 pathway.

The process of pyroptosis is mediated by inflammasomes and a downstream effector known as gasdermin D (GSDMD). Upon cleavage by inflammasome-associated caspases, the N-terminal domain of GSDMD forms membrane pores that promote cytolysis. Numerous proteins promote GSDMD cleavage, but none are known to be required for pore formation after GSDMD cleavage. Herein, we report a forward genetic screen that identified the Ragulator-Rag complex as being necessary for GSDMD pore formation and pyroptosis in macrophages. Mechanistic analysis revealed that Ragulator-Rag is not required for GSDMD cleavage upon inflammasome activation but rather promotes GSDMD oligomerization in the plasma membrane. Defects in GSDMD oligomerization and pore formation can be rescued by mitochondrial poisons that stimulate reactive oxygen species (ROS) production, and ROS modulation impacts the ability of inflammasome pathways to promote pore formation downstream of GSDMD cleavage. These findings reveal an unexpected link between key regulators of immunity (inflammasome-GSDMD) and metabolism (Ragulator-Rag).

Gasdermin D permeabilization of mitochondrial inner and outer membranes accelerates and enhances pyroptosis.

Gasdermin D (GSDMD)-activated inflammatory cell death (pyroptosis) causes mitochondrial damage, but its underlying mechanism and functional consequences are largely unknown. Here, we show that the N-terminal pore-forming GSDMD fragment (GSDMD-NT) rapidly damaged both inner and outer mitochondrial membranes (OMMs) leading to reduced mitochondrial numbers, mitophagy, ROS, loss of transmembrane potential, attenuated oxidative phosphorylation (OXPHOS), and release of mitochondrial proteins and DNA from the matrix and intermembrane space. Mitochondrial damage occurred as soon as GSDMD was cleaved prior to plasma membrane damage. Mitochondrial damage was independent of the B-cell lymphoma 2 family and depended on GSDMD-NT binding to cardiolipin. Canonical and noncanonical inflammasome activation of mitochondrial damage, pyroptosis, and inflammatory cytokine release were suppressed by genetic ablation of cardiolipin synthase (Crls1) or the scramblase (Plscr3) that transfers cardiolipin to the OMM. Phospholipid scramblase-3 (PLSCR3) deficiency in a tumor compromised pyroptosis-triggered anti-tumor immunity. Thus, mitochondrial damage plays a critical role in pyroptosis.

NLRP3 is its own gatekeeper: a group hug of NLRP3 monomers controls inflammation.

A recent study by Hochheiser et al. describes the cryo-electron microscopy (cryoEM) structure of an autoinhibited nucleotide-binding domain-, leucine-rich repeat (LRR)- and pyrin domain-containing protein 3 (NLRP3) decamer that assembles via LRR interactions and is further stabilized by the small-molecule NLRP3-specific inhibitor CRID3 binding into a cleft within the NACHT domain. The study provides a springboard for the development of novel NLRP3-based therapies.

Sequestration of membrane cholesterol by cholesterol-binding proteins inhibits SARS-CoV-2 entry into Vero E6 cells.

Membrane lipids and proteins form dynamic domains crucial for physiological and pathophysiological processes, including viral infection. Many plasma membrane proteins, residing within membrane domains enriched with cholesterol (CHOL) and sphingomyelin (SM), serve as receptors for attachment and entry of viruses into the host cell. Among these, human coronaviruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), use proteins associated with membrane domains for initial binding and internalization. We hypothesized that the interaction of lipid-binding proteins with CHOL in plasma membrane could sequestrate lipids and thus affect the efficiency of virus entry into host cells, preventing the initial steps of viral infection. We have prepared CHOL-binding proteins with high affinities for lipids in the plasma membrane of mammalian cells. Binding of the perfringolysin O domain four (D4) and its variant D4E458L to membrane CHOL impaired the internalization of the receptor-binding domain of the SARS-CoV-2 spike protein and the pseudovirus complemented with the SARS-CoV-2 spike protein. SARS-CoV-2 replication in Vero E6 cells was also decreased. Overall, our results demonstrate that the integrity of CHOL-rich membrane domains and the accessibility of CHOL in the membrane play an essential role in SARS-CoV-2 cell entry.

Disruption of disulfides within RBD of SARS-CoV-2 spike protein prevents fusion and represents a target for viral entry inhibition by registered drugs.

The SARS-CoV-2 pandemic imposed a large burden on health and society. Therapeutics targeting different components and processes of the viral infection replication cycle are being investigated, particularly to repurpose already approved drugs. Spike protein is an important target for both vaccines and therapeutics. Insights into the mechanisms of spike-ACE2 binding and cell fusion could support the identification of compounds with inhibitory effects. Here, we demonstrate that the integrity of disulfide bonds within the receptor-binding domain (RBD) plays an important role in the membrane fusion process although their disruption does not prevent binding of spike protein to ACE2. Several reducing agents and thiol-reactive compounds are able to inhibit viral entry. N-acetyl cysteine amide, L-ascorbic acid, JTT-705, and auranofin prevented syncytia formation, viral entry into cells, and infection in a mouse model, supporting disulfides of the RBD as a therapeutically relevant target.

The Relevance of Physico-Chemical Properties and Protein Corona for Evaluation of Nanoparticles Immunotoxicity-In Vitro Correlation Analysis on THP-1 Macrophages.

Alongside physiochemical properties (PCP), it has been suggested that the protein corona of nanoparticles (NPs) plays a crucial role in the response of immune cells to NPs. However, due to the great variety of NPs, target cells, and exposure protocols, there is still no clear relationship between PCP, protein corona composition, and the immunotoxicity of NPs. In this study, we correlated PCP and the protein corona composition of NPs to the THP-1 macrophage response, focusing on selected toxicological endpoints: cell viability, reactive oxygen species (ROS), and cytokine secretion. We analyzed seven commonly used engineered NPs (SiO2, silver, and TiO2) and magnetic NPs. We show that with the exception of silver NPs, all of the tested TiO2 types and SiO2 exhibited moderate toxicities and a transient inflammatory response that was observed as an increase in ROS, IL-8, and/or IL-1ß cytokine secretion. We observed a strong correlation between the size of the NPs in media and IL-1ß secretion. The induction of IL-1ß secretion was completely blunted in NLR family pyrin domain containing 3 (NLRP3) knockout THP-1 cells, indicating activation of the inflammasome. The correlations analysis also implicated the association of specific NP corona proteins with the induction of cytokine secretion. This study provides new insights toward a better understanding of the relationships between PCP, protein corona, and the inflammatory response of macrophages for different engineered NPs, to which we are exposed on a daily basis.

Selective inhibition of NLRP3 inflammasome by designed peptide originating from ASC.

NACHT, LRR, and PYD domains-containing protein 3 (NLRP3) inflammasome is a multiprotein complex which forms within cells in response to various microbial and self-derived triggers. Mutations in the gene encoding NLRP3 cause rare cryopyrin-associated periodic syndromes (CAPS) and growing evidence links NLRP3 inflammasome to common diseases such as Alzheimer´s disease. In order to modulate different stages of NLRP3 inflammasome assembly nine peptides whose sequences correspond to segments of inflammasome components NLRP3 and apoptosis-associated speck-like protein containing a CARD (ASC) were selected. Five peptides inhibited IL-1ß release, caspase-1 activation and ASC oligomerization in response to soluble and particulate NLRP3 triggers. Modulatory peptides also attenuated IL-1ß maturation induced by constitutive CAPS-associated NLRP3 mutants. Peptide corresponding to H2-H3 segment of ASC pyrin domain selectively inhibited NLRP3 inflammasome by binding to NLRP3 pyrin domain in the micromolar range. The peptide had no effect on AIM2 and NLRC4 inflammasomes as well as NF-κB pathway. The peptide effectively dampened neutrophil infiltration in the silica-induced peritonitis and when equipped with Antennapedia or Angiopep-2 motifs crossed the blood-brain barrier in a mouse model. Our study demonstrates that peptides represent an important tool for targeting multiprotein inflammatory complexes and can serve as the basis for the development of novel anti-inflammatory strategies for neurodegeneration.

Differential Effect of Extracellular Acidic Environment on IL-1ß Released from Human and Mouse Phagocytes.

Areas of locally decreased pH are characteristic for many chronic inflammatory diseases such as atherosclerosis and rheumatoid arthritis, acute pathologies such as ischemia reperfusion, and tumor microenvironment. The data on the effects of extracellular acidic pH on inflammation are conflicting with respect to interleukin 1 beta (IL-1ß) as one of the most potent proinflammatory cytokines. In this study, we used various mouse- and human-derived cells in order to identify potential species-specific differences in IL-1ß secretion pattern in response to extracellular acidification. We found that a short incubation in mild acidic medium caused significant IL-1ß release from human macrophages, however, the same effect was not observed in mouse macrophages. Rather, a marked IL-1ß suppression was observed when mouse cells were stimulated with a combination of various inflammasome instigators and low pH. Upon activation of cells under acidic conditions, the cytosolic pH was reduced while metabolic activity and the expression of the main inflammasome proteins were not affected by low pH. We show that IL-1ß secretion in mouse macrophages is reversible upon restoration of physiological pH. pH sensitivity of NLRP3, NLRC4 and AIM2 inflammasomes appeared to be conferred by the processes upstream of the apoptosis-associated speck-like protein containing a CARD (ASC) oligomerization and most likely contributed by the cell background rather than species-specific amino acid sequences of the sensor proteins.

Analysis of the Direct and Indirect Effects of Nanoparticle Exposure on Microglial and Neuronal Cells In Vitro.

Environmental or biomedical exposure to nanoparticles (NPs) can results in translocation and accumulation of NPs in the brain, which can lead to health-related problems. NPs have been shown to induce toxicity to neuronal cells through several direct mechanisms, but only a few studies have also explored the indirect effects of NPs, through consequences due to the exposure of neighboring cells to NPs. In this study, we analysed possible direct and indirect effects of NPs (polyacrylic acid (PAA) coated cobalt ferrite NP, TiO2 P25 and maghemite NPs) on immortalized mouse microglial cells and differentiated CAD mouse neuronal cells in monoculture (direct toxicity) or in transwell co-culture system (indirect toxicity). We showed that although the low NP concentrations (2-25 µg/mL) did not induce changes in cell viability, cytokine secretion or NF-κB activation of microglial cells, even low NP concentrations of 10 µg/mL can affect the cells and change their secretion of protein stress mediators. These can in turn influence neuronal cells in indirect exposure model. Indirect toxicity of NPs is an important and not adequately assessed mechanism of NP toxicity, since it not only affects cells on the exposure sites, but through secretion of signaling mediators, can also affect cells that do not come in direct contact with NPs.

The mechanism of NLRP3 inflammasome initiation: Trimerization but not dimerization of the NLRP3 pyrin domain induces robust activation of IL-1ß.

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-1ß 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-1ß response can be achieved by the induced trimerization of the NLRP3PYD domain.

Anchorless forms of prion protein - Impact of truncation on structure destabilization and prion protein conversion.

Prion diseases are a group of fatal neurodegenerative diseases caused by scrapie form of prion protein, PrPSc. Prion protein (PrP) is bound to the cell via glycophosphatidylinositol (GPI) anchor. The role of GPI anchor in PrPSc replication and propagation remains unclear. It has been shown that anchorless and truncated PrP accelerate the formation and propagation of prions in vivo and further increases the risk for transmission of prion diseases among species. To explain the role of anchorless forms of PrP in the development of prion diseases, we have prepared five C-terminal PrP truncated variants, determined their thermodynamic properties and analyzed the kinetics of conversion into amyloid fibrils. According to our results thermodynamic and kinetic properties are affected both by pH and truncation. We have shown that the shortest variant was the most destabilized and converted faster than other variants in acidic pH. Other variants converted with longer lag time of fibrillization than WT despite comparable or even decreased stability in acidic pH. Our results indicate that even the change in length for 1 amino acid residue can have a profound effect on in vitro conversion.

Pathological mutations H187R and E196K facilitate subdomain separation and prion protein conversion by destabilization of the native structure.

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 ß1-α1-ß2 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 (by 3.2 and 1.1 kJ/mol, respectively, at pH 7) 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 (by 4.2 kJ/mol at pH 4) 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.

Design of a single-chain polypeptide tetrahedron assembled from coiled-coil segments.

Protein structures evolved through a complex interplay of cooperative interactions, and it is still very challenging to design new protein folds de novo. Here we present a strategy to design self-assembling polypeptide nanostructured polyhedra based on modularization using orthogonal dimerizing segments. We designed and experimentally demonstrated the formation of the tetrahedron that self-assembles from a single polypeptide chain comprising 12 concatenated coiled coil-forming segments separated by flexible peptide hinges. The path of the polypeptide chain is guided by a defined order of segments that traverse each of the six edges of the tetrahedron exactly twice, forming coiled-coil dimers with their corresponding partners. The coincidence of the polypeptide termini in the same vertex is demonstrated by reconstituting a split fluorescent protein in the polypeptide with the correct tetrahedral topology. Polypeptides with a deleted or scrambled segment order fail to self-assemble correctly. This design platform provides a foundation for constructing new topological polypeptide folds based on the set of orthogonal interacting polypeptide segments.

NLRP3 inflammasome activation in macrophage cell lines by prion protein fibrils as the source of IL-1ß and neuronal toxicity.

Prion diseases are fatal transmissible neurodegenerative diseases, characterized by aggregation of the pathological form of prion protein, spongiform degeneration, and neuronal loss, and activation of astrocytes and microglia. Microglia can clear prion plaques, but on the other hand cause neuronal death via release of neurotoxic species. Elevated expression of the proinflammatory cytokine IL-1ß has been observed in brains affected by several prion diseases, and IL-1R-deficiency significantly prolonged the onset of the neurodegeneration in mice. We show that microglial cells stimulated by prion protein (PrP) fibrils induced neuronal toxicity. Microglia and macrophages release IL-1ß upon stimulation by PrP fibrils, which depends on the NLRP3 inflammasome. Activation of NLRP3 inflammasome by PrP fibrils requires depletion of intracellular K(+), and requires phagocytosis of PrP fibrils and consecutive lysosome destabilization. Among the well-defined molecular forms of PrP, the strongest NLRP3 activation was observed by fibrils, followed by aggregates, while neither native monomeric nor oligomeric PrP were able to activate the NLRP3 inflammasome. Our results together with previous studies on IL-1R-deficient mice suggest the IL-1 signaling pathway as the perspective target for the therapy of prion disease.

Assessing the ATP Binding Ability of NLRP3 from Cell Lysates by a Pull-down Assay.

NACHT-, LRR-, and PYD-containing protein 3 (NLRP3) is a member of AAA+ ATPase family that upon activation forms inflammasomes. Several studies demonstrated that ATP binding and hydrolysis are important for NLRP3 function as an inflammasome sensor. Furthermore, compounds targeting ATP binding motifs and interfering with ATPase activity of NLRP3 inhibit NLRP3 inflammasome formation. Measuring ATPase activity of proteins and binding of radiolabeled ATP to specified proteins are well-established methods that require purified protein. Here, we describe a method for assessing NLRP3 binding to ATP using ATP-conjugated beads and lysates of cells that either express endogenous NLRP3 or are transfected with plasmids encoding NLRP3. Efficiency of binding is followed after elution from the beads and detection with Western blot and immunolabelling. The method can be used to evaluate the functionality of NLRP3 variants or to check whether compounds or NLRP3 binding partners interfere with binding of ATP.

Gasdermin D pore-forming activity is redox-sensitive.

Reactive oxygen species (ROS) regulate the activities of inflammasomes, which are innate immune signaling organelles that induce pyroptosis. The mechanisms by which ROS control inflammasome activities are unclear and may be multifaceted. Herein, we report that the protein gasdermin D (GSDMD), which forms membrane pores upon cleavage by inflammasome-associated caspases, is a direct target of ROS. Exogenous and endogenous sources of ROS, and ROS-inducing stimuli that prime cells for pyroptosis induction, promote oligomerization of cleaved GSDMD, leading to membrane rupture and cell death. We find that ROS enhance GSDMD activities through oxidative modification of cysteine 192 (C192). Within macrophages, GSDMD mutants lacking C192 show impaired ability to form membrane pores and induce pyroptosis. Reciprocal mutagenesis studies reveal that C192 is the only cysteine within GSDMD that mediates ROS responsiveness. Cellular redox state is therefore a key determinant of GSDMD activities.

Engineered combinatorial cell device for wound healing and bone regeneration.

Growth factors are the key regulators that promote tissue regeneration and healing processes. While the effects of individual growth factors are well documented, a combination of multiple secreted growth factors underlies stem cell-mediated regeneration. To avoid the potential dangers and labor-intensive individual approach of stem cell therapy while maintaining their regeneration-promoting effects based on multiple secreted growth factors, we engineered a "mix-and-match" combinatorial platform based on a library of cell lines producing growth factors. Treatment with a combination of growth factors secreted by engineered mammalian cells was more efficient than with individual growth factors or even stem cell-conditioned medium in a gap closure assay. Furthermore, we implemented in a mouse model a device for allogenic cell therapy for an in situ production of growth factors, where it improved cutaneous wound healing. Augmented bone regeneration was achieved on calvarial bone defects in rats treated with a cell device secreting IGF, FGF, PDGF, TGF-ß, and VEGF. In both in vivo models, the systemic concentration of secreted factors was negligible, demonstrating the local effect of the regeneration device. Finally, we introduced a genetic switch that enables temporal control over combinations of trophic factors released at different stages of regeneration mimicking the maturation of natural wound healing to improve therapy and prevent scar formation.

Globular domain of the prion protein needs to be unlocked by domain swapping to support prion protein conversion.

Prion diseases are fatal transmissible neurodegenerative diseases affecting many mammalian species. The normal prion protein (PrP) converts into a pathological aggregated form, PrPSc, which is enriched in the ß-sheet structure. Although the high resolution structure of the normal PrP was determined, the structure of the converted form of PrP remains inaccessible to high resolution techniques. To map the PrP conversion process we introduced disulfide bridges into different positions within the globular domain of PrP, tethering selected secondary structure elements. The majority of tethered PrP mutants exhibited increased thermodynamic stability, nevertheless, they converted efficiently. Only the disulfides that tether subdomain B1-H1-B2 to subdomain H2-H3 prevented PrP conversion in vitro and in prion-infected cell cultures. Reduction of disulfides recovered the ability of these mutants to convert, demonstrating that the separation of subdomains is an essential step in conversion. Formation of disulfide-linked proteinase K-resistant dimers in fibrils composed of a pair of single cysteine mutants supports the model based on domain-swapped dimers as the building blocks of prion fibrils. In contrast to previously proposed structural models of PrPSc suggesting conversion of large secondary structural segments, we provide evidence for the conservation of secondary structural elements of the globular domain upon PrP conversion. Previous studies already showed that dimerization is the rate-limiting step in PrP conversion. We show that separation and swapping of subdomains of the globular domain is necessary for conversion. Therefore, we propose that the domain-swapped dimer of PrP precedes amyloid formation and represents a potential target for therapeutic intervention.

The Role of Inflammasomes in Osteoarthritis and Secondary Joint Degeneration Diseases.

Osteoarthritis is age-related and the most common form of arthritis. The main characteristics of the disease are progressive loss of cartilage and secondary synovial inflammation, which finally result in pain, joint stiffness, and functional disability. Similarly, joint degeneration is characteristic of systemic inflammatory diseases such as rheumatoid arthritis and gout, with the associated secondary type of osteoarthritis. Studies suggest that inflammation importantly contributes to the progression of the disease. Particularly, cytokines TNFα and IL-1ß drive catabolic signaling in affected joints. IL-1ß is a product of inflammasome activation. Inflammasomes are inflammatory multiprotein complexes that propagate inflammation in various autoimmune and autoinflammatory conditions through cell death and the release of inflammatory cytokines and damage-associated molecule patterns. In this article, we review genetic, marker, and animal studies that establish inflammasomes as important drivers of secondary arthritis and discuss the current evidence for inflammasome involvement in primary osteoarthritis. The NLRP3 inflammasome has a significant role in the development of secondary osteoarthritis, and several studies have provided evidence of its role in the development of primary osteoarthritis, while other inflammasomes cannot be excluded. Inflammasome-targeted therapeutic options might thus provide a promising strategy to tackle these debilitating diseases.

Supramolecular organizing centers at the interface of inflammation and neurodegeneration.

The pathogenesis of neurodegenerative diseases involves the accumulation of misfolded protein aggregates. These deposits are both directly toxic to neurons, invoking loss of cell connectivity and cell death, and recognized by innate sensors that upon activation release neurotoxic cytokines, chemokines, and various reactive species. This neuroinflammation is propagated through signaling cascades where activated sensors/receptors, adaptors, and effectors associate into multiprotein complexes known as supramolecular organizing centers (SMOCs). This review provides a comprehensive overview of the SMOCs, involved in neuroinflammation and neurotoxicity, such as myddosomes, inflammasomes, and necrosomes, their assembly, and evidence for their involvement in common neurodegenerative diseases. We discuss the multifaceted role of neuroinflammation in the progression of neurodegeneration. Recent progress in the understanding of particular SMOC participation in common neurodegenerative diseases such as Alzheimer's disease offers novel therapeutic strategies for currently absent disease-modifying treatments.