39 results on '"Hafner-Bratkovič I"'
Search Results
2. A DNA origami of Slovenia in nano dimensions
- Author
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Jerala, M., Jerala, R., and Hafner-Bratkovič, I.
- Abstract
The principle of the rapidly evolving DNA nanotechnology is the design of nanostructures based only on the Watson- Crick base pairing and the oligonucleotide sequence. DNA origami technique is able to produce a variety of different shapes by constraining a long single stranded DNA molecule with alarge number of short oligonucleotides. We designed 227 short oligonucleotides in order to scaffold the long strand of M13 bacteriophage single-stranded DNA into the shape of Slovenia. After annealing DNA origamis of Slovenia were observed by atomic force microscopy showing that most of the structures followed the design. Our results demonstrate that DNA origami technique can be used for construction of irregular asymmetric shapes with curvy edges and prove the feasibility of this technique.
- Published
- 2011
3. The complete genome sequence of a Crimean-Congo Hemorrhagic Fever virus isolated from an endemic region in Kosovo
- Author
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Dedushaj Iusuf, Petrovec Miroslav, Hafner-Bratkovič Iva, Saksida Ana, Khristova Marina L, Nichol Stuart T, Duh Darja, Ahmeti Salih, and Avšič-Županc Tatjana
- Subjects
Infectious and parasitic diseases ,RC109-216 - Abstract
Abstract The Balkan region and Kosovo in particular, is a well-known Crimean-Congo hemorrhagic fever (CCHF) endemic region, with frequent epidemic outbreaks and sporadic cases occurring with a hospitalized case fatality of approximately 30%. Recent analysis of complete genome sequences of diverse CCHF virus strains showed that the genome plasticity of the virus is surprisingly high for an arthropod-borne virus. High levels of nucleotide and amino acid differences, frequent RNA segment reassortment and even RNA recombination have been recently described. This diversity illustrates the need to determine the complete genome sequence of CCHF virus representatives of all geographically distinct endemic areas, particularly in light of the high pathogenicity of the virus and its listing as a potential bioterrorism threat. Here we describe the first complete CCHF virus genome sequence of a virus (strain Kosova Hoti) isolated from a hemorrhagic fever case in the Balkans. This virus strain was isolated from a fatal CCHF case, and passaged only twice on Vero E6 cells prior to sequence analysis. The virus total genome was found to be 19.2 kb in length, consisting of a 1672 nucleotide (nt) S segment, a 5364 nt M segment and a 12150 nt L segment. Phylogenetic analysis of CCHF virus complete genomes placed the Kosova Hoti strain in the Europe/Turkey group, with highest similarity seen with Russian isolates. The virus M segments are the most diverse with up to 31 and 27% differences seen at the nt and amino acid levels, and even 1.9% amino acid difference found between the Kosova Hoti and another strain from Kosovo (9553-01). This suggests that distinct virus strains can coexist in highly endemic areas.
- Published
- 2008
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4. Sequestration of membrane cholesterol by cholesterol-binding proteins inhibits SARS-CoV-2 entry into Vero E6 cells.
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Kulma M, Šakanović A, Bedina-Zavec A, Caserman S, Omersa N, Šolinc G, Orehek S, Hafner-Bratkovič I, Kuhar U, Slavec B, Krapež U, Ocepek M, Kobayashi T, Kwiatkowska K, Jerala R, Podobnik M, and Anderluh G
- Subjects
- Vero Cells, Chlorocebus aethiops, Animals, Humans, Carrier Proteins metabolism, COVID-19 virology, COVID-19 metabolism, Protein Binding, Cholesterol metabolism, SARS-CoV-2 metabolism, SARS-CoV-2 physiology, Virus Internalization, Cell Membrane metabolism, Cell Membrane virology, Spike Glycoprotein, Coronavirus metabolism, Spike Glycoprotein, Coronavirus chemistry
- Abstract
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 D4
E458L 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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)- Published
- 2024
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5. Gasdermin D permeabilization of mitochondrial inner and outer membranes accelerates and enhances pyroptosis.
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Miao R, Jiang C, Chang WY, Zhang H, An J, Ho F, Chen P, Zhang H, Junqueira C, Amgalan D, Liang FG, Zhang J, Evavold CL, Hafner-Bratkovič I, Zhang Z, Fontana P, Xia S, Waldeck-Weiermair M, Pan Y, Michel T, Bar-Peled L, Wu H, Kagan JC, Kitsis RN, Zhang P, Liu X, and Lieberman J
- Subjects
- Neoplasm Proteins metabolism, Cardiolipins metabolism, Intracellular Signaling Peptides and Proteins genetics, Intracellular Signaling Peptides and Proteins metabolism, Inflammasomes metabolism, Pyroptosis, Gasdermins
- Abstract
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., Competing Interests: Declaration of interests J.L. and H.W. are cofounders and advisors of Ventus Therapeutics. J.C.K. consults and holds equity in Corner Therapeutics, Larkspur Biosciences, and Neumora Therapeutics., (Copyright © 2023 Elsevier Inc. All rights reserved.)
- Published
- 2023
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6. Engineered combinatorial cell device for wound healing and bone regeneration.
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Kadunc Polajnar L, Lainšček D, Gašperšič R, Sušjan-Leite P, Kovačič U, Butinar M, Turk B, Jerala R, and Hafner-Bratkovič I
- Abstract
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-β, 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., Competing Interests: LKP, DL, RJ and IH-B are co-inventors on the patent application related to the content of this study. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Kadunc Polajnar, Lainšček, Gašperšič, Sušjan-Leite, Kovačič, Butinar, Turk, Jerala and Hafner-Bratkovič.)
- Published
- 2023
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7. Gasdermin D pore-forming activity is redox-sensitive.
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Devant P, Boršić E, Ngwa EM, Xiao H, Chouchani ET, Thiagarajah JR, Hafner-Bratkovič I, Evavold CL, and Kagan JC
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- Gasdermins, Reactive Oxygen Species metabolism, Cysteine metabolism, Neoplasm Proteins metabolism, Oxidation-Reduction, Intracellular Signaling Peptides and Proteins genetics, Intracellular Signaling Peptides and Proteins metabolism, Inflammasomes metabolism
- Abstract
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., Competing Interests: Declaration of interests J.C.K. consults and holds equity in Corner Therapeutics, Larkspur Biosciences, and Neumora Therapeutics. E.T.C. is co-founder, equity holder, and board member of Matchpoint Therapeutics and co-founder and equity holder in Aevum Therapeutics. None of these relationships influenced this study., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)
- Published
- 2023
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8. Assessing the ATP Binding Ability of NLRP3 from Cell Lysates by a Pull-down Assay.
- Author
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Sušjan-Leite P and Hafner-Bratkovič I
- Subjects
- Adenosine Triphosphate pharmacology, Adenosine Triphosphatases, Interleukin-1beta metabolism, Inflammasomes metabolism, NLR Family, Pyrin Domain-Containing 3 Protein metabolism
- Abstract
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., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)
- Published
- 2023
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9. NLRP3 is its own gatekeeper: a group hug of NLRP3 monomers controls inflammation.
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Hafner-Bratkovič I
- Subjects
- Cryoelectron Microscopy, Humans, Inflammation, Inflammasomes metabolism, NLR Family, Pyrin Domain-Containing 3 Protein
- Abstract
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., Competing Interests: Declaration of interests I.H.-B. has no competing interests to declare., (Copyright © 2022 Elsevier Ltd. All rights reserved.)
- Published
- 2022
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10. Supramolecular organizing centers at the interface of inflammation and neurodegeneration.
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Sušjan-Leite P, Ramuta TŽ, Boršić E, Orehek S, and Hafner-Bratkovič I
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- Humans, Inflammasomes, Inflammation pathology, Signal Transduction physiology, Alzheimer Disease pathology, Neurodegenerative Diseases pathology, Neurotoxicity Syndromes
- Abstract
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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Sušjan-Leite, Ramuta, Boršić, Orehek and Hafner-Bratkovič.)
- Published
- 2022
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11. The Relevance of Physico-Chemical Properties and Protein Corona for Evaluation of Nanoparticles Immunotoxicity-In Vitro Correlation Analysis on THP-1 Macrophages.
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Pavlin M, Lojk J, Strojan K, Hafner-Bratkovič I, Jerala R, Leonardi A, Križaj I, Drnovšek N, Novak S, Veranič P, and Bregar VB
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- Inflammasomes metabolism, Interleukin-1beta metabolism, Macrophages metabolism, NLR Family, Pyrin Domain-Containing 3 Protein metabolism, Reactive Oxygen Species metabolism, Silicon Dioxide metabolism, Silicon Dioxide toxicity, Silver metabolism, Silver toxicity, Nanoparticles chemistry, Nanoparticles toxicity, Protein Corona metabolism
- Abstract
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 (SiO
2 , 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-1β cytokine secretion. We observed a strong correlation between the size of the NPs in media and IL-1β secretion. The induction of IL-1β 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.- Published
- 2022
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12. The Role of Inflammasomes in Osteoarthritis and Secondary Joint Degeneration Diseases.
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Roškar S and Hafner-Bratkovič I
- Abstract
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-1β drive catabolic signaling in affected joints. IL-1β 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.
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- 2022
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13. Cleavage-Mediated Regulation of Myd88 Signaling by Inflammasome-Activated Caspase-1.
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Avbelj M, Hafner-Bratkovič I, Lainšček D, Manček-Keber M, Peternelj TT, Panter G, Treon SP, Gole B, Potočnik U, and Jerala R
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- 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, Caspase 1 immunology, Immunity, Innate, Inflammasomes immunology, Myeloid Differentiation Factor 88 immunology, Signal Transduction immunology
- Abstract
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 MyD88
L265P 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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Avbelj, Hafner-Bratkovič, Lainšček, Manček-Keber, Peternelj, Panter, Treon, Gole, Potočnik and Jerala.)- Published
- 2022
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14. Sensing low intracellular potassium by NLRP3 results in a stable open structure that promotes inflammasome activation.
- Author
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Tapia-Abellán A, Angosto-Bazarra D, Alarcón-Vila C, Baños MC, Hafner-Bratkovič I, Oliva B, and Pelegrín P
- Abstract
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+ efflux–specific 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.- Published
- 2021
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15. Control of gasdermin D oligomerization and pyroptosis by the Ragulator-Rag-mTORC1 pathway.
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Evavold CL, Hafner-Bratkovič I, Devant P, D'Andrea JM, Ngwa EM, Boršić E, Doench JG, LaFleur MW, Sharpe AH, Thiagarajah JR, and Kagan JC
- Subjects
- 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, Reactive Oxygen Species metabolism, TOR Serine-Threonine Kinases metabolism, RNA, Guide, CRISPR-Cas Systems, Mice, 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
- Abstract
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)., Competing Interests: Declaration of interests J.C.K. holds equity and consults for IFM Therapeutics and Corner Therapeutics. None of these relationships influenced the work performed in this study. J.G.D. consults for Microsoft Research, Agios, Phenomic AI, Maze Therapeutics, BioNTech, and Pfizer; J.G.D. consults for and has equity in Tango Therapeutics. J.G.D.’s interests were reviewed and are managed by the Broad Institute in accordance with its conflict-of-interest policies., (Copyright © 2021 Elsevier Inc. All rights reserved.)
- Published
- 2021
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16. Disruption of disulfides within RBD of SARS-CoV-2 spike protein prevents fusion and represents a target for viral entry inhibition by registered drugs.
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Manček-Keber M, Hafner-Bratkovič I, Lainšček D, Benčina M, Govednik T, Orehek S, Plaper T, Jazbec V, Bergant V, Grass V, Pichlmair A, and Jerala R
- Subjects
- Acetylcysteine pharmacology, HEK293 Cells, Humans, Acetylcysteine analogs & derivatives, Amides pharmacology, Ascorbic Acid pharmacology, Auranofin pharmacology, COVID-19 metabolism, COVID-19 pathology, Disulfides metabolism, Esters pharmacology, SARS-CoV-2 metabolism, Spike Glycoprotein, Coronavirus metabolism, Sulfhydryl Compounds pharmacology, Virus Internalization drug effects, COVID-19 Drug Treatment
- Abstract
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., (© 2021 The Authors. The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.)
- Published
- 2021
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17. A Nanoscaffolded Spike-RBD Vaccine Provides Protection against SARS-CoV-2 with Minimal Anti-Scaffold Response.
- Author
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Lainšček D, Fink T, Forstnerič V, Hafner-Bratkovič I, Orehek S, Strmšek Ž, Manček-Keber M, Pečan P, Esih H, Malenšek Š, Aupič J, Dekleva P, Plaper T, Vidmar S, Kadunc L, Benčina M, Omersa N, Anderluh G, Pojer F, Lau K, Hacker D, Correia BE, Peterhoff D, Wagner R, Bergant V, Herrmann A, Pichlmair A, and Jerala R
- Abstract
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 β-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 β-annulus peptide scaffold, which itself triggered a minimal response in contrast to larger scaffolds. The β-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 β-annulus peptide for vaccine design.
- Published
- 2021
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18. Differential Effect of Extracellular Acidic Environment on IL-1β Released from Human and Mouse Phagocytes.
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Sušjan P, Benčina M, and Hafner-Bratkovič I
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- 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, Acids pharmacology, Inflammation genetics, Interleukin-1beta genetics, Phagocytes metabolism
- Abstract
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-1β) 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-1β secretion pattern in response to extracellular acidification. We found that a short incubation in mild acidic medium caused significant IL-1β release from human macrophages, however, the same effect was not observed in mouse macrophages. Rather, a marked IL-1β 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-1β 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.
- Published
- 2020
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19. Analysis of the Direct and Indirect Effects of Nanoparticle Exposure on Microglial and Neuronal Cells In Vitro.
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Lojk J, Babič L, Sušjan P, Bregar VB, Pavlin M, Hafner-Bratkovič I, and Veranič P
- Subjects
- Animals, Cell Line, Cytokines metabolism, Mice, Microglia cytology, Neurons cytology, Cell Survival drug effects, Microglia drug effects, Nanoparticles toxicity, Neurons drug effects
- Abstract
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, TiO
2 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.- Published
- 2020
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20. Increased gene translation stringency in mammalian cells by nonsense suppression at multiple permissive sites with a single noncanonical amino acid.
- Author
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Kadunc L, Svetličič M, Forstnerič V, Hafner Bratkovič I, and Jerala R
- Subjects
- HEK293 Cells, Humans, Phenylalanine genetics, Phenylalanine metabolism, Amino Acyl-tRNA Synthetases genetics, Amino Acyl-tRNA Synthetases metabolism, Benzophenones metabolism, Codon, Nonsense, Phenylalanine analogs & derivatives, Protein Biosynthesis, RNA, Transfer genetics, RNA, Transfer metabolism
- Abstract
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., (© 2020 Federation of European Biochemical Societies.)
- Published
- 2020
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21. Selective inhibition of NLRP3 inflammasome by designed peptide originating from ASC.
- Author
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Sušjan P, Lainšček D, Strmšek Ž, Hodnik V, Anderluh G, and Hafner-Bratkovič I
- Subjects
- 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, CARD Signaling Adaptor Proteins metabolism, Inflammasomes metabolism, NLR Family, Pyrin Domain-Containing 3 Protein metabolism, Peptides metabolism
- Abstract
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-1β release, caspase-1 activation and ASC oligomerization in response to soluble and particulate NLRP3 triggers. Modulatory peptides also attenuated IL-1β 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., (© 2020 Federation of American Societies for Experimental Biology.)
- Published
- 2020
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22. NLRP3 lacking the leucine-rich repeat domain can be fully activated via the canonical inflammasome pathway.
- Author
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Hafner-Bratkovič I, Sušjan P, Lainšček D, Tapia-Abellán A, Cerović K, Kadunc L, Angosto-Bazarra D, Pelegrin P, and Jerala R
- Subjects
- 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, Inflammasomes immunology, Inflammation immunology, Leucine immunology, NLR Family, Pyrin Domain-Containing 3 Protein chemistry, NLR Family, Pyrin Domain-Containing 3 Protein immunology
- Abstract
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.- Published
- 2018
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23. Ion homeostasis and ion channels in NLRP3 inflammasome activation and regulation.
- Author
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Hafner-Bratkovič I and Pelegrín P
- Subjects
- 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, Homeostasis, Inflammasomes metabolism, Ion Channels metabolism, Ions metabolism, NLR Family, Pyrin Domain-Containing 3 Protein metabolism
- Abstract
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-1β. 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-1β 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., (Copyright © 2018 Elsevier Ltd. All rights reserved.)
- Published
- 2018
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24. Design of coiled-coil protein-origami cages that self-assemble in vitro and in vivo.
- Author
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Ljubetič A, Lapenta F, Gradišar H, Drobnak I, Aupič J, Strmšek Ž, Lainšček D, Hafner-Bratkovič I, Majerle A, Krivec N, Benčina M, Pisanski T, Veličković TĆ, Round A, Carazo JM, Melero R, and Jerala R
- Subjects
- Models, Molecular, Nanostructures, Protein Folding, Protein Multimerization, Protein Structure, Secondary, Protein Engineering, Proteins chemistry
- Abstract
Polypeptides and polynucleotides are natural programmable biopolymers that can self-assemble into complex tertiary structures. We describe a system analogous to designed DNA nanostructures in which protein coiled-coil (CC) dimers serve as building blocks for modular de novo design of polyhedral protein cages that efficiently self-assemble in vitro and in vivo. We produced and characterized >20 single-chain protein cages in three shapes-tetrahedron, four-sided pyramid, and triangular prism-with the largest containing >700 amino-acid residues and measuring 11 nm in diameter. Their stability and folding kinetics were similar to those of natural proteins. Solution small-angle X-ray scattering (SAXS), electron microscopy (EM), and biophysical analysis confirmed agreement of the expressed structures with the designs. We also demonstrated self-assembly of a tetrahedral structure in bacteria, mammalian cells, and mice without evidence of inflammation. A semi-automated computational design platform and a toolbox of CC building modules are provided to enable the design of protein cages in any polyhedral shape.
- Published
- 2017
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25. Development of an Acrylate Derivative Targeting the NLRP3 Inflammasome for the Treatment of Inflammatory Bowel Disease.
- Author
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Cocco M, Pellegrini C, Martínez-Banaclocha H, Giorgis M, Marini E, Costale A, Miglio G, Fornai M, Antonioli L, López-Castejón G, Tapia-Abellán A, Angosto D, Hafner-Bratkovič I, Regazzoni L, Blandizzi C, Pelegrín P, and Bertinaria M
- Subjects
- Acrylates pharmacokinetics, Acrylates pharmacology, Animals, Energy Transfer, Male, Mice, Mice, Inbred C57BL, Rats, Rats, Sprague-Dawley, Acrylates therapeutic use, Inflammasomes drug effects, Inflammatory Bowel Diseases drug therapy, NLR Family, Pyrin Domain-Containing 3 Protein antagonists & inhibitors
- Abstract
Pharmacological inhibition of NLRP3 inflammasome activation may offer a new option in the treatment of inflammatory bowel disease. In this work, we report the design, synthesis, and biological screening of a series of acrylate derivatives as NLRP3 inhibitors. The in vitro determination of reactivity, cytotoxicity, NLRP3 ATPase inhibition, and antipyroptotic properties allowed the selection of 11 (INF39), a nontoxic, irreversible NLRP3 inhibitor able to decrease interleukin-1β release from macrophages. Bioluminescence resonance energy transfer experiments proved that this compound was able to directly interfere with NLRP3 activation in cells. In vivo studies confirmed the ability of the selected lead to alleviate the effects of colitis induced by 2,4-dinitrobenzenesulfonic acid in rats after oral administration.
- Published
- 2017
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- View/download PDF
26. Prions, prionoid complexes and amyloids: the bad, the good and something in between.
- Author
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Hafner Bratkovič I
- Subjects
- Animals, Humans, Memory physiology, Prion Diseases metabolism, Prion Diseases pathology, Prions physiology, Amyloid metabolism, Prion Proteins, Prions metabolism, Protein Folding
- Abstract
Prions are infectious agents causing transmissible spongiform encephalopathies in humans and animals. These protein-based particles template conformational changes in a host-encoded prion protein to an insoluble self-like conformation. Prions are also present in yeast, where they support protein-based epigenetic inheritance. There is emerging evidence that prion-like (prionoid) particles can support a variety of pathological and beneficial functions. The recent data on the prionoid spread of other pathological amyloids are discussed in light of differences between prions and prion-like aggregates. On the other hand, prion-like action has also been found to support important functions such as memory, and amyloids were shown to have a variety of physiological roles from storage to scaffolding in simple organisms and in humans. Higher-order protein complexes play important roles in signalling. Many death-fold domains can polymerise upon nucleation to enhance sensitivity and induce a robust response. Although these polymers are structurally different from amyloids, some of them are characterised by prionoid activities, such as intercellular spread. The initial activation of these complexes is vital for organismal health, whereas prolonged activation leading to unresolved inflammation underlies autoinflammatory and other diseases. Prionoid complexes play important roles far beyond prion diseases and neurodegeneration.
- Published
- 2017
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27. Cell stress response to two different types of polymer coated cobalt ferrite nanoparticles.
- Author
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Lojk J, Strojan K, Miš K, Bregar BV, Hafner Bratkovič I, Bizjak M, Pirkmajer S, and Pavlin M
- Subjects
- Acrylic Resins chemistry, Animals, Apoptosis drug effects, Cell Line, Tumor, Cell Survival drug effects, Dose-Response Relationship, Drug, Humans, Lymphocyte Antigen 96 genetics, Lymphocyte Antigen 96 metabolism, Melanoma, Experimental metabolism, Melanoma, Experimental pathology, Mice, Myoblasts cytology, Myoblasts metabolism, NF-kappa B genetics, NF-kappa B metabolism, Polyethyleneimine chemistry, Reactive Oxygen Species metabolism, Toll-Like Receptor 4 genetics, Toll-Like Receptor 4 metabolism, Cobalt chemistry, Cobalt toxicity, Ferric Compounds chemistry, Ferric Compounds toxicity, Myoblasts drug effects, Nanoparticles chemistry, Nanoparticles toxicity
- Abstract
Potential nanoparticle (NP) toxicity is one of crucial problems that limit the applicability of NPs. When designing NPs for biomedical and biotechnological applications it is thus important to understand the mechanisms of their toxicity. In this study, we analysed the stress responses of previously designed polyacrylic acid (PAA) and polyethylenimine (PEI) coated NPs on primary human myoblasts (MYO) and B16 mouse melanoma cell line. Negatively charged PAA did not induce cell toxicity, reactive oxygen species (ROS) or activate the transcription factor NF-κB in either cell line even at high concentrations (100μg/ml). On the other hand, positively charged PEI NPs induced a concentration dependent necrotic cell death and an increase in ROS following 24h incubation already at low concentrations (>4μg/ml). Moreover, PEI NPs induced NF-κB activation 15-30min after incubation in MYO cells, most probably through activation of TLR4 receptor. Interestingly, there was no NF-κB response to PEI NPs in B16 cells., (Copyright © 2017 Elsevier B.V. All rights reserved.)
- Published
- 2017
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28. The mechanism of NLRP3 inflammasome initiation: Trimerization but not dimerization of the NLRP3 pyrin domain induces robust activation of IL-1β.
- Author
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Sušjan P, Roškar S, and Hafner-Bratkovič I
- Subjects
- Animals, Caspase 1 metabolism, Cell Line, Enzyme Activation, Inflammasomes immunology, Mice, NLR Family, Pyrin Domain-Containing 3 Protein genetics, Protein Domains, Protein Multimerization, Pyrin chemistry, Recombinant Fusion Proteins chemistry, Recombinant Fusion Proteins genetics, Recombinant Fusion Proteins metabolism, Inflammasomes metabolism, Interleukin-1beta metabolism, NLR Family, Pyrin Domain-Containing 3 Protein chemistry, NLR Family, Pyrin Domain-Containing 3 Protein metabolism
- Abstract
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 (NLRP3
PYD ) 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., (Copyright © 2017 Elsevier Inc. All rights reserved.)- Published
- 2017
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29. Anchorless forms of prion protein - Impact of truncation on structure destabilization and prion protein conversion.
- Author
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Kovač V, Hafner-Bratkovič I, and Čurin Šerbec V
- Subjects
- Binding Sites, Hydrogen-Ion Concentration, Protein Binding, Protein Conformation, Protein Folding, Structure-Activity Relationship, Amyloid chemistry, Amyloid ultrastructure, Prion Proteins chemistry, Prion Proteins ultrastructure
- Abstract
Prion diseases are a group of fatal neurodegenerative diseases caused by scrapie form of prion protein, PrP
Sc . 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., (Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.)- Published
- 2016
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- View/download PDF
30. Correction: Shikonin Suppresses NLRP3 and AIM2 Inflammasomes by Direct Inhibition of Caspase-1.
- Author
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Zorman J, Sušjan P, and Hafner-Bratkovič I
- Abstract
[This corrects the article DOI: 10.1371/journal.pone.0159826.].
- Published
- 2016
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- View/download PDF
31. Shikonin Suppresses NLRP3 and AIM2 Inflammasomes by Direct Inhibition of Caspase-1.
- Author
-
Zorman J, Sušjan P, and Hafner-Bratkovič I
- Subjects
- Animals, DNA-Binding Proteins, Inflammasomes metabolism, Interleukin-1beta metabolism, L-Lactate Dehydrogenase metabolism, Mice, Mice, Inbred C57BL, Nigericin pharmacology, Caspase 1 drug effects, Caspase Inhibitors pharmacology, Cell Line, Transformed metabolism, Inflammasomes drug effects, NLR Family, Pyrin Domain-Containing 3 Protein metabolism, Naphthoquinones pharmacology
- Abstract
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.
- Published
- 2016
- Full Text
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32. Toll-like receptor 4 senses oxidative stress mediated by the oxidation of phospholipids in extracellular vesicles.
- Author
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Manček-Keber M, Frank-Bertoncelj M, Hafner-Bratkovič I, Smole A, Zorko M, Pirher N, Hayer S, Kralj-Iglič V, Rozman B, Ilc N, Horvat S, and Jerala R
- Subjects
- Arthritis, Rheumatoid genetics, Arthritis, Rheumatoid pathology, Cell-Derived Microparticles genetics, Female, HEK293 Cells, Human Umbilical Vein Endothelial Cells, Humans, Lipopolysaccharides pharmacology, Lymphocyte Antigen 96 genetics, Lymphocyte Antigen 96 immunology, Male, Oxidative Stress drug effects, Toll-Like Receptor 4 agonists, Toll-Like Receptor 4 genetics, Arthritis, Rheumatoid immunology, Cell-Derived Microparticles immunology, Oxidative Stress immunology, Toll-Like Receptor 4 immunology
- Abstract
Oxidative stress produced in response to infection or sterile injury activates the innate immune response. We found that extracellular vesicles (EVs) isolated from the plasma of patients with rheumatoid arthritis or secreted from cells subjected to oxidative stress contained oxidized phospholipids that stimulated cells expressing Toll-like receptor 4 (TLR4) in a manner dependent on its co-receptor MD-2. EVs from healthy subjects or reconstituted synthetic EVs subjected to limited oxidation gained the ability to stimulate TLR4-expressing cells, whereas prolonged oxidation abrogated this property. Furthermore, we found that 15-lipoxygenase generated hydro(pero)xylated phospholipids that stimulated TLR4-expressing cells. Molecular modeling suggested that the mechanism of activation of TLR4 by oxidized phospholipids in EVs was structurally similar to that of the TLR4 ligand lipopolysaccharide (LPS). This was supported by experiments showing that EV-mediated stimulation of cells required MD-2, that mutations that block LPS binding to TLR4 abrogated the stimulatory effect of EVs, and that EVs induced TLR4 dimerization. On the other hand, analysis of gene expression profiles showed that genes encoding factors that resolve inflammation were more abundantly expressed in responses to EVs than in response to LPS. Together, these data suggest that EVs act as an oxidative stress-induced endogenous danger signal that underlies the pervasive role of TLR4 in inflammatory diseases., (Copyright © 2015, American Association for the Advancement of Science.)
- Published
- 2015
- Full Text
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33. Pathological mutations H187R and E196K facilitate subdomain separation and prion protein conversion by destabilization of the native structure.
- Author
-
Hadži S, Ondračka A, Jerala R, and Hafner-Bratkovič I
- Subjects
- Amyloid chemistry, Amyloid metabolism, Animals, Calorimetry, Differential Scanning, Circular Dichroism, Hydrogen-Ion Concentration, Kinetics, Mice, Models, Molecular, Prion Diseases genetics, Prion Proteins, Prions metabolism, Protein Folding, Protein Structure, Secondary, Protein Structure, Tertiary, Thermodynamics, Mutation genetics, Prion Diseases pathology, Prions chemistry, Prions genetics
- Abstract
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 (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., (© FASEB.)
- Published
- 2015
- Full Text
- View/download PDF
34. Design of a single-chain polypeptide tetrahedron assembled from coiled-coil segments.
- Author
-
Gradišar H, Božič S, Doles T, Vengust D, Hafner-Bratkovič I, Mertelj A, Webb B, Šali A, Klavžar S, and Jerala R
- Subjects
- Amino Acid Sequence, Circular Dichroism, DNA chemistry, Dimerization, Microscopy, Atomic Force, Microscopy, Electron, Transmission, Models, Molecular, Molecular Conformation, Molecular Sequence Data, Plasmids metabolism, Protein Binding, Peptides chemistry, Protein Engineering methods
- Abstract
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.
- Published
- 2013
- Full Text
- View/download PDF
35. NLRP3 inflammasome activation in macrophage cell lines by prion protein fibrils as the source of IL-1β and neuronal toxicity.
- Author
-
Hafner-Bratkovič I, Benčina M, Fitzgerald KA, Golenbock D, and Jerala R
- Subjects
- Animals, Caspase 1 metabolism, Cell Line, Interleukin-1beta physiology, Lysosomes physiology, Macrophages immunology, Mice, Mice, Inbred C57BL, Microglia metabolism, NF-kappa B metabolism, NLR Family, Pyrin Domain-Containing 3 Protein, Neurons pathology, Phagocytosis, Potassium metabolism, Prion Proteins, Prions metabolism, Carrier Proteins metabolism, Inflammasomes metabolism, Interleukin-1beta metabolism, Macrophages metabolism, Prions pathogenicity
- Abstract
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-1β 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-1β 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.
- Published
- 2012
- Full Text
- View/download PDF
36. Introduction of glutamines into the B2-H2 loop promotes prion protein conversion.
- Author
-
Avbelj M, Hafner-Bratkovič I, and Jerala R
- Subjects
- Amino Acid Sequence, Animals, Asparagine chemistry, Asparagine genetics, Glutamine genetics, Mice, Molecular Sequence Data, Mutation, PrPC Proteins genetics, PrPSc Proteins genetics, Protein Folding, Protein Structure, Secondary, Amyloid chemistry, Glutamine chemistry, PrPC Proteins chemistry, PrPSc Proteins chemistry
- Abstract
In prion diseases cellular prion protein (PrP(C)) undergoes conformational transition into the β-sheet-rich form (PrP(Sc)). PrP(C) consists of the disordered N-terminal part and a C-terminal globular domain containing three α-helices (H1, H2, H3) and an antiparallel beta sheet (B1, B2). B2-H2 loop, which has a focal role in the species barrier, contains the highest density of asparagine (N) and glutamine (Q) residues in the whole sequence. Q/N-rich domains are essential for the conversion of yeast prions. We investigated the role of Q/N residues in the B2-H2 loop in PrP conversion. We prepared mouse PrP mutants with increasing number of consecutive Q/N residues in the B2-H2 loop. Stability of the mutants decreased with the increasing number of inserted glutamines. In vitro conversion of mutants yielded fibrils of similar morphology as the wild-type PrP. Q/N mutants accelerated fibrillization in comparison to the wild-type PrP, with mutant containing the most glutamines having the shortest lag phase. The effect of Q/N residues was specific for the B2-H2 loop and was not due to simple increase in flexibility as the introduction of Gly-Ser or Ala residues slowed the conversion despite their decreased stability. Our results thus suggest that Q/N residues in the B2-H2 loop of PrP promote protein conversion and may represent a link to conversion of Q/N-rich prions., (Copyright © 2011 Elsevier Inc. All rights reserved.)
- Published
- 2011
- Full Text
- View/download PDF
37. Disulfide mapping reveals the domain swapping as the crucial process of the structural conversion of prion protein.
- Author
-
Hafner-Bratkovič I and Jerala R
- Subjects
- Animals, Humans, PrPSc Proteins chemistry, PrPSc Proteins metabolism, Protein Structure, Secondary, Disulfides chemistry, Disulfides metabolism, Prions chemistry, Prions metabolism
- Abstract
Prion diseases are infectious conformational diseases. Despite the determination of many native prion protein (PrP) structures and in vitro production of infectious prions from recombinant PrP the structural background of PrP conversion remains the largest unsolved problem. The aggregated state of PrP (Sc) makes it inaccessible to high resolution techniques, therefore indirect methods have to be used to investigate the conversion process. We engineered disulfide bridges into the structured domain of PrP in order to determine the secondary structure elements that remain conserved upon conversion. Rather surprisingly, introduction of disulfides into each or both of the subdomains B1-H1-B2 and H2-H3 of the C-terminal globular domain retained the robust ability to convert into fibrils with increased content of β-structure, indistinguishable from the wild-type PrP. On the other hand disulfide bridges tethering the two subdomains completely prevented conversion, while their reduction reversed their conversion ability. The same conversion propensity was replicated also in prion infected cell lines. Experiments with combinations of engineered cysteine residues further support that domain swapping, centered on the B2-H2 loop, previously associated to species barrier, leads to PrP swapped dimers as the building block of prion fibrils.
- Published
- 2011
- Full Text
- View/download PDF
38. A DNA origami of slovenia in nano dimensions.
- Author
-
Jerala M, Jerala R, and Hafner-Bratkovič I
- Abstract
The principle of the rapidly evolving DNA nanotechnology is the design of nanostructures based only on the Watson-Crick base pairing and the oligonucleotide sequence. DNA origami technique is able to produce a variety of different shapes by constraining a long single stranded DNA molecule with a large number of short oligonucleotides. We designed 227 short oligonucleotides in order to scaffold the long strand of M13 bacteriophage single-stranded DNA into the shape of Slovenia. After annealing DNA origamis of Slovenia were observed by atomic force microscopy showing that most of the structures followed the design. Our results demonstrate that DNA origami technique can be used for construction of irregular asymmetric shapes with curvy edges and prove the feasibility of this technique.
- Published
- 2011
39. Effect of hydrophobic mutations in the H2-H3 subdomain of prion protein on stability and conversion in vitro and in vivo.
- Author
-
Hafner-Bratkovič I, Gaedtke L, Ondracka A, Veranič P, Vorberg I, and Jerala R
- Subjects
- Amino Acid Substitution genetics, Animals, Cells, Cultured, Endopeptidase K metabolism, Gene Knockout Techniques, Humans, Mice, Microscopy, Atomic Force, Mutant Proteins chemistry, Mutant Proteins metabolism, Prions metabolism, Protein Stability, Protein Structure, Secondary, Protein Structure, Tertiary, Temperature, Hydrophobic and Hydrophilic Interactions, Mutation genetics, Prions chemistry, Prions genetics
- Abstract
Prion diseases are fatal neurodegenerative diseases, which can be acquired, sporadic or genetic, the latter being linked to mutations in the gene encoding prion protein. We have recently described the importance of subdomain separation in the conversion of prion protein (PrP). The goal of the present study was to investigate the effect of increasing the hydrophobic interactions within the H2-H3 subdomain on PrP conversion. Three hydrophobic mutations were introduced into PrP. The mutation V209I associated with human prion disease did not alter protein stability or in vitro fibrillization propensity of PrP. The designed mutations V175I and T187I on the other hand increased protein thermal stability. V175I mutant fibrillized faster than wild-type PrP. Conversion delay of T187I was slightly longer, but fluorescence intensity of amyloid specific dye thioflavin T was significantly higher. Surprisingly, cells expressing V209I variant exhibited inefficient proteinase K resistant PrP formation upon infection with 22L strain, which is in contrast to cell lines expressing wild-type, V175I and T187I mPrPs. In agreement with increased ThT fluorescence at the plateau T187I expressing cell lines accumulated an increased amount of the proteinase K-resistant prion protein. We showed that T187I induces formation of thin fibrils, which are absent from other samples. We propose that larger solvent accessibility of I187 in comparison to wild-type and other mutants may interfere with lateral annealing of filaments and may be the underlying reason for increased conversion efficiency.
- Published
- 2011
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