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1.
Biochem Biophys Res Commun ; 716: 149954, 2024 Jul 05.
Article En | MEDLINE | ID: mdl-38704887

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.


Cell Membrane , Cholesterol , SARS-CoV-2 , Spike Glycoprotein, Coronavirus , Virus Internalization , Vero Cells , Chlorocebus aethiops , Cholesterol/metabolism , Animals , SARS-CoV-2/metabolism , SARS-CoV-2/physiology , Cell Membrane/metabolism , Cell Membrane/virology , Spike Glycoprotein, Coronavirus/metabolism , Spike Glycoprotein, Coronavirus/chemistry , Humans , Carrier Proteins/metabolism , COVID-19/virology , COVID-19/metabolism , Protein Binding
2.
Immunity ; 56(11): 2523-2541.e8, 2023 Nov 14.
Article En | MEDLINE | ID: mdl-37924812

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.


Gasdermins , Pyroptosis , Neoplasm Proteins/metabolism , Cardiolipins/metabolism , Intracellular Signaling Peptides and Proteins/genetics , Intracellular Signaling Peptides and Proteins/metabolism , Inflammasomes/metabolism
3.
Methods Mol Biol ; 2696: 257-267, 2023.
Article En | MEDLINE | ID: mdl-37578728

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.


Inflammasomes , NLR Family, Pyrin Domain-Containing 3 Protein , Inflammasomes/metabolism , NLR Family, Pyrin Domain-Containing 3 Protein/metabolism , Adenosine Triphosphate/pharmacology , Adenosine Triphosphatases , Interleukin-1beta/metabolism
4.
Front Bioeng Biotechnol ; 11: 1168330, 2023.
Article En | MEDLINE | ID: mdl-37234478

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.

5.
Cell Rep ; 42(1): 112008, 2023 01 31.
Article En | MEDLINE | ID: mdl-36662620

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.


Inflammasomes , Intracellular Signaling Peptides and Proteins , Intracellular Signaling Peptides and Proteins/genetics , Intracellular Signaling Peptides and Proteins/metabolism , Inflammasomes/metabolism , Gasdermins , Reactive Oxygen Species/metabolism , Cysteine/metabolism , Neoplasm Proteins/metabolism , Oxidation-Reduction
6.
Front Immunol ; 13: 940969, 2022.
Article En | MEDLINE | ID: mdl-35979366

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.


Alzheimer Disease , Neurodegenerative Diseases , Neurotoxicity Syndromes , Alzheimer Disease/pathology , Humans , Inflammasomes , Inflammation/pathology , Neurodegenerative Diseases/pathology , Signal Transduction/physiology
7.
Int J Mol Sci ; 23(11)2022 May 31.
Article En | MEDLINE | ID: mdl-35682872

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.


Nanoparticles , Protein Corona , Inflammasomes/metabolism , Interleukin-1beta/metabolism , Macrophages/metabolism , NLR Family, Pyrin Domain-Containing 3 Protein/metabolism , Nanoparticles/chemistry , Nanoparticles/toxicity , Protein Corona/metabolism , Reactive Oxygen Species/metabolism , Silicon Dioxide/metabolism , Silicon Dioxide/toxicity , Silver/metabolism , Silver/toxicity
8.
Life (Basel) ; 12(5)2022 May 13.
Article En | MEDLINE | ID: mdl-35629398

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.

9.
Trends Biochem Sci ; 47(8): 635-637, 2022 08.
Article En | MEDLINE | ID: mdl-35382945

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.


Inflammasomes , NLR Family, Pyrin Domain-Containing 3 Protein , Cryoelectron Microscopy , Humans , Inflammasomes/metabolism , Inflammation
10.
Sci Adv ; 7(38): eabf4468, 2021 Sep 17.
Article En | MEDLINE | ID: mdl-34524838

The NLRP3 inflammasome is activated by a wide range of stimuli and drives diverse inflammatory diseases. The decrease of intracellular K+ concentration is a minimal upstream signal to most of the NLRP3 activation models. Here, we found that cellular K+ efflux induces a stable structural change in the inactive NLRP3, promoting an open conformation as a step preceding activation. This conformational change is facilitated by the specific NLRP3 FISNA domain and a unique flexible linker sequence between the PYD and FISNA domains. This linker also facilitates the ensemble of NLRP3PYD into a seed structure for ASC oligomerization. The introduction of the NLRP3 PYD-linker-FISNA sequence into NLRP6 resulted in a chimeric receptor able to be activated by K+ efflux­specific NLRP3 activators and promoted an in vivo inflammatory response to uric acid crystals. Our results establish that the amino-terminal sequence between PYD and NACHT domain of NLRP3 is key for inflammasome activation.

11.
Cell ; 184(17): 4495-4511.e19, 2021 08 19.
Article En | MEDLINE | ID: mdl-34289345

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).


Adaptor Proteins, Signal Transducing/metabolism , Intracellular Signaling Peptides and Proteins/metabolism , Mechanistic Target of Rapamycin Complex 1/metabolism , Monomeric GTP-Binding Proteins/metabolism , Phosphate-Binding Proteins/metabolism , Protein Multimerization , Pyroptosis , Signal Transduction , Amino Acids/metabolism , Animals , Cell Adhesion Molecules, Neuronal/metabolism , Cell Line , Genetic Testing , Humans , Inflammasomes/metabolism , Intracellular Signaling Peptides and Proteins/chemistry , Macrophages/metabolism , Mechanistic Target of Rapamycin Complex 2/metabolism , Mice, Inbred C57BL , Mitochondria/metabolism , NLR Family, Pyrin Domain-Containing 3 Protein/metabolism , Nerve Growth Factors/metabolism , Phosphate-Binding Proteins/chemistry , Protein Domains , RNA, Guide, Kinetoplastida/metabolism , Reactive Oxygen Species/metabolism , TOR Serine-Threonine Kinases/metabolism
12.
FASEB J ; 35(6): e21651, 2021 06.
Article En | MEDLINE | ID: mdl-34004056

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.


Acetylcysteine/analogs & derivatives , Amides/pharmacology , Ascorbic Acid/pharmacology , Auranofin/pharmacology , COVID-19 Drug Treatment , COVID-19 , Disulfides/metabolism , Esters/pharmacology , SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/metabolism , Sulfhydryl Compounds/pharmacology , Virus Internalization/drug effects , Acetylcysteine/pharmacology , COVID-19/metabolism , COVID-19/pathology , HEK293 Cells , Humans
13.
Vaccines (Basel) ; 9(5)2021 Apr 27.
Article En | MEDLINE | ID: mdl-33925446

The response of the adaptive immune system is augmented by multimeric presentation of a specific antigen, resembling viral particles. Several vaccines have been designed based on natural or designed protein scaffolds, which exhibited a potent adaptive immune response to antigens; however, antibodies are also generated against the scaffold, which may impair subsequent vaccination. In order to compare polypeptide scaffolds of different size and oligomerization state with respect to their efficiency, including anti-scaffold immunity, we compared several strategies of presentation of the RBD domain of the SARS-CoV-2 spike protein, an antigen aiming to generate neutralizing antibodies. A comparison of several genetic fusions of RBD to different nanoscaffolding domains (foldon, ferritin, lumazine synthase, and ß-annulus peptide) delivered as DNA plasmids demonstrated a strongly augmented immune response, with high titers of neutralizing antibodies and a robust T-cell response in mice. Antibody titers and virus neutralization were most potently enhanced by fusion to the small ß-annulus peptide scaffold, which itself triggered a minimal response in contrast to larger scaffolds. The ß-annulus fused RBD protein increased residence in lymph nodes and triggered the most potent viral neutralization in immunization by a recombinant protein. Results of the study support the use of a nanoscaffolding platform using the ß-annulus peptide for vaccine design.

14.
Front Immunol ; 12: 790258, 2021.
Article En | MEDLINE | ID: mdl-35069570

Coordination among multiple signaling pathways ensures an appropriate immune response, where a signaling pathway may impair or augment another signaling pathway. Here, we report a negative feedback regulation of signaling through the key innate immune mediator MyD88 by inflammasome-activated caspase-1. NLRP3 inflammasome activation impaired agonist- or infection-induced TLR signaling and cytokine production through the proteolytic cleavage of MyD88 by caspase-1. Site-specific mutagenesis was used to identify caspase-1 cleavage site within MyD88 intermediary segment. Different cleavage site location within MyD88 defined the functional consequences of MyD88 cleavage between mouse and human cells. LPS/monosodium urate-induced mouse inflammation model corroborated the physiological role of this mechanism of regulation, that could be reversed by chemical inhibition of NLRP3. While Toll/interleukin-1 receptor (TIR) domain released by MyD88 cleavage additionally contributed to the inhibition of signaling, Waldenström's macroglobulinemia associated MyD88L265P mutation is able to evade the caspase-1-mediated inhibition of MyD88 signaling through the ability of its TIRL265P domain to recruit full length MyD88 and facilitate signaling. The characterization of this mechanism reveals an additional layer of innate immunity regulation.


Caspase 1/immunology , Immunity, Innate , Inflammasomes/immunology , Myeloid Differentiation Factor 88/immunology , Signal Transduction/immunology , Animals , Caspase 1/genetics , Enzyme Activation/immunology , HEK293 Cells , Humans , Inflammasomes/genetics , Mice , Mice, Knockout , Myeloid Differentiation Factor 88/genetics , Signal Transduction/genetics , THP-1 Cells
15.
Int J Mol Sci ; 21(19)2020 Sep 30.
Article En | MEDLINE | ID: mdl-33007998

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.


Acids/pharmacology , Inflammation/genetics , Interleukin-1beta/genetics , Phagocytes/metabolism , Animals , CARD Signaling Adaptor Proteins/genetics , Calcium-Binding Proteins/genetics , Cellular Microenvironment/genetics , DNA-Binding Proteins/genetics , Humans , Hydrogen-Ion Concentration , Inflammation/metabolism , Inflammation/pathology , Macrophages/drug effects , Macrophages/metabolism , Mice , NLR Family, Pyrin Domain-Containing 3 Protein/genetics , Phagocytes/drug effects
16.
Int J Mol Sci ; 21(19)2020 Sep 24.
Article En | MEDLINE | ID: mdl-32987760

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.


Cell Survival/drug effects , Microglia/drug effects , Nanoparticles/toxicity , Neurons/drug effects , Animals , Cell Line , Cytokines/metabolism , Mice , Microglia/cytology , Neurons/cytology
17.
FASEB J ; 34(8): 11068-11086, 2020 08.
Article En | MEDLINE | ID: mdl-32648626

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.


CARD Signaling Adaptor Proteins/metabolism , Inflammasomes/metabolism , NLR Family, Pyrin Domain-Containing 3 Protein/metabolism , Peptides/metabolism , Animals , Apoptosis Regulatory Proteins/metabolism , Blood-Brain Barrier/metabolism , Caspase 1/metabolism , Cells, Cultured , Cryopyrin-Associated Periodic Syndromes/metabolism , DNA-Binding Proteins/metabolism , Female , Inflammation/metabolism , Interleukin-1beta/metabolism , Male , Mice, Inbred C57BL , NF-kappa B/metabolism , Neutrophil Infiltration/physiology , Peritonitis/metabolism
18.
FEBS Lett ; 594(15): 2452-2461, 2020 08.
Article En | MEDLINE | ID: mdl-32401336

The considerable potential of engineered cells compels the development of strategies for the stringent control of gene expression. A promising approach is the introduction of a premature stop codon (PTC) into a selected gene that is expressed only in the presence of noncanonical amino acids through nonsense suppression. Here, different strategies of amber PTC readthrough in mammalian cells were tested. The use of a tRNA synthetase together with a TAG codon-specific tRNA achieved PTC readthrough depending on the addition of a noncanonical amino acid (4-benzoyl-L-phenylalanine; Bpa). While single TAG codon incorporation exhibited detectable expression of the reporter protein even in the absence of Bpa, the use of a double PTC enabled virtually leakage-free functional gene translation. The introduction of an additional 5'-PTC, therefore, represents a generally applicable strategy to increase stringency in gene translation.


Amino Acyl-tRNA Synthetases , Benzophenones , Codon, Nonsense , Phenylalanine/analogs & derivatives , Protein Biosynthesis , RNA, Transfer , Amino Acyl-tRNA Synthetases/genetics , Amino Acyl-tRNA Synthetases/metabolism , Benzophenones/metabolism , HEK293 Cells , Humans , Phenylalanine/genetics , Phenylalanine/metabolism , RNA, Transfer/genetics , RNA, Transfer/metabolism
19.
Nat Commun ; 9(1): 5182, 2018 12 05.
Article En | MEDLINE | ID: mdl-30518920

NLRP3 is a cytosolic sensor triggered by different pathogen- and self-derived signals that plays a central role in a variety of pathological conditions, including sterile inflammation. The leucine-rich repeat domain is present in several innate immune receptors, where it is frequently responsible for sensing danger signals and regulation of activation. Here we show by reconstitution of truncated and chimeric variants into Nlrp3-/- macrophages that the leucine-rich repeat domain is dispensable for activation and self-regulation of NLRP3 by several different triggers. The pyrin domain on the other hand is required to maintain NLRP3 in the inactive conformation. A fully responsive minimal NLRP3 truncation variant reconstitutes peritonitis in Nlrp3-/- mice. We demonstrate that in contrast to pathogen-activated NLRC4, the constitutively active NLRP3 molecule cannot engage wild-type NLRP3 molecules in a self-catalytic oligomerization. This lack of signal amplification is likely a protective mechanism to decrease sensitivity to endogenous triggers to impede autoinflammation.


Inflammasomes/immunology , Inflammation/immunology , Leucine/immunology , NLR Family, Pyrin Domain-Containing 3 Protein/chemistry , NLR Family, Pyrin Domain-Containing 3 Protein/immunology , Animals , Apoptosis Regulatory Proteins/genetics , Apoptosis Regulatory Proteins/immunology , Calcium-Binding Proteins/genetics , Calcium-Binding Proteins/immunology , Female , Humans , Inflammasomes/chemistry , Inflammasomes/genetics , Inflammation/genetics , Leucine/genetics , Macrophages/immunology , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , NLR Family, Pyrin Domain-Containing 3 Protein/genetics , Protein Domains
20.
Curr Opin Immunol ; 52: 8-17, 2018 06.
Article En | MEDLINE | ID: mdl-29555598

The NLRP3 inflammasome is a multiprotein platform for the activation of caspase-1, which in turn drives inflammation through the activation of proinflammatory cytokines, such as IL-1ß. In contrast to the majority of pattern recognition receptors, NLRP3 inflammasome can be triggered by a plethora of pathogen-derived or endogenous activators, which perturb intracellular ion homeostasis. Here, we discuss how the complex interplay of ion fluxes contributes to canonical, non-canonical, and alternative NLRP3 activation pathways that induce IL-1ß secretion from immune cells. Particular attention is given to the concrete evidence for the involvement of ion channels, which may present viable therapeutic targets for the modulation of NLRP3 inflammasome activation.


Homeostasis , Inflammasomes/metabolism , Ion Channels/metabolism , Ions/metabolism , NLR Family, Pyrin Domain-Containing 3 Protein/metabolism , Animals , Biological Transport , Humans , Intracellular Space/metabolism , Ion Channel Gating , Ion Channels/genetics , NLR Family, Pyrin Domain-Containing 3 Protein/genetics , Receptors, Purinergic/genetics , Receptors, Purinergic/metabolism
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