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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.
Duško Lainšček; Tina Fink; Vida Forstnerič; Iva Hafner-Bratkovič; Sara Orehek; Žiga Strmšek; Mateja Manček-Keber; Peter Pečan; Hana Esih; Špela Malenšek; Jana Aupič; Petra Dekleva; Tjaša Plaper; Sara Vidmar; Lucija Kadunc; Mojca Benčina; Neža Omersa; Gregor Anderluh; Florence Pojer; Kelvin Lau; David Hacker; Bruno Correia; David Peterhoff; Ralf Wagner; Valter Bergant; Alexander Herrmann; Andreas Pichlmair; Roman Jerala. A Nanoscaffolded Spike-RBD Vaccine Provides Protection against SARS-CoV-2 with Minimal Anti-Scaffold Response. Vaccines 2021, 9, 431 .
AMA StyleDuško Lainšček, Tina Fink, Vida Forstnerič, Iva Hafner-Bratkovič, Sara Orehek, Žiga Strmšek, Mateja Manček-Keber, Peter Pečan, Hana Esih, Špela Malenšek, Jana Aupič, Petra Dekleva, Tjaša Plaper, Sara Vidmar, Lucija Kadunc, Mojca Benčina, Neža Omersa, Gregor Anderluh, Florence Pojer, Kelvin Lau, David Hacker, Bruno Correia, David Peterhoff, Ralf Wagner, Valter Bergant, Alexander Herrmann, Andreas Pichlmair, Roman Jerala. A Nanoscaffolded Spike-RBD Vaccine Provides Protection against SARS-CoV-2 with Minimal Anti-Scaffold Response. Vaccines. 2021; 9 (5):431.
Chicago/Turabian StyleDuško Lainšček; Tina Fink; Vida Forstnerič; Iva Hafner-Bratkovič; Sara Orehek; Žiga Strmšek; Mateja Manček-Keber; Peter Pečan; Hana Esih; Špela Malenšek; Jana Aupič; Petra Dekleva; Tjaša Plaper; Sara Vidmar; Lucija Kadunc; Mojca Benčina; Neža Omersa; Gregor Anderluh; Florence Pojer; Kelvin Lau; David Hacker; Bruno Correia; David Peterhoff; Ralf Wagner; Valter Bergant; Alexander Herrmann; Andreas Pichlmair; Roman Jerala. 2021. "A Nanoscaffolded Spike-RBD Vaccine Provides Protection against SARS-CoV-2 with Minimal Anti-Scaffold Response." Vaccines 9, no. 5: 431.
The lack of efficient methods to control the major diseases of crops most important to agriculture leads to huge economic losses and seriously threatens global food security. Many of the most important microbial plant pathogens, including bacteria, fungi, and oomycetes, secrete necrosis- and ethylene-inducing peptide 1 (Nep1)-like proteins (NLPs), which critically contribute to the virulence and spread of the disease. NLPs are cytotoxic to eudicot plants, as they disturb the plant plasma membrane by binding to specific plant membrane sphingolipid receptors. Their pivotal role in plant infection and broad taxonomic distribution makes NLPs a promising target for the development of novel phytopharmaceutical compounds. To identify compounds that bind to NLPs from the oomycetes Pythium aphanidermatum and Phytophthora parasitica, a library of 587 small molecules, most of which are commercially unavailable, was screened by surface plasmon resonance. Importantly, compounds that exhibited the highest affinity to NLPs were also found to inhibit NLP-mediated necrosis in tobacco leaves and Phytophthora infestans growth on potato leaves. Saturation transfer difference-nuclear magnetic resonance and molecular modelling of the most promising compound, anthranilic acid derivative, confirmed stable binding to the NLP protein, which resulted in decreased necrotic activity and reduced ion leakage from tobacco leaves. We, therefore, confirmed that NLPs are an appealing target for the development of novel phytopharmaceutical agents and strategies, which aim to directly interfere with the function of these major microbial virulence factors. The compounds identified in this study represent lead structures for further optimization and antimicrobial product development.
Katja Pirc; Vesna Hodnik; Tina Snoj; Tea Lenarčič; Simon Caserman; Marjetka Podobnik; Hannah Böhm; Isabell Albert; Anita Kotar; Janez Plavec; Jure Borišek; Martina Damuzzo; Alessandra Magistrato; Boris Brus; Izidor Sosič; Stanislav Gobec; Thorsten Nürnberger; Gregor Anderluh. Nep1-like proteins as a target for plant pathogen control. PLOS Pathogens 2021, 17, e1009477 .
AMA StyleKatja Pirc, Vesna Hodnik, Tina Snoj, Tea Lenarčič, Simon Caserman, Marjetka Podobnik, Hannah Böhm, Isabell Albert, Anita Kotar, Janez Plavec, Jure Borišek, Martina Damuzzo, Alessandra Magistrato, Boris Brus, Izidor Sosič, Stanislav Gobec, Thorsten Nürnberger, Gregor Anderluh. Nep1-like proteins as a target for plant pathogen control. PLOS Pathogens. 2021; 17 (4):e1009477.
Chicago/Turabian StyleKatja Pirc; Vesna Hodnik; Tina Snoj; Tea Lenarčič; Simon Caserman; Marjetka Podobnik; Hannah Böhm; Isabell Albert; Anita Kotar; Janez Plavec; Jure Borišek; Martina Damuzzo; Alessandra Magistrato; Boris Brus; Izidor Sosič; Stanislav Gobec; Thorsten Nürnberger; Gregor Anderluh. 2021. "Nep1-like proteins as a target for plant pathogen control." PLOS Pathogens 17, no. 4: e1009477.
Identification of novel agents for bladder cancer treatment is highly desirable due to the high incidence of tumor recurrence and the risk of progression to muscle-invasive disease. The key feature of the cholesterol-dependent toxin listeriolysin O mutant (LLO Y406A) is its preferential activity at pH 5.7, which could be exploited either directly for selective targeting of cancer cells or the release of accumulated therapeutics from acidic endosomes. Therefore, our goal was to compare the cytotoxic effect of LLO Y406A on cancer cells (RT4) and normal urothelial cells (NPU), and to identify which cell membranes are the primary target of LLO Y406A by viability assays, life-cell imaging, fluorescence, and electron microscopy. LLO Y406A decreased viability, altered cell morphology, provoked membrane blebbing, and induced apoptosis in RT4 cells, while it did not affect NPU cells. LLO Y406A did not cause endosomal escape in RT4 cells, while the plasma membrane of RT4 cells was revealed as the primary target of LLO Y406A. It has been concluded that LLO Y406A has the ability to selectively eliminate cancer urothelial cells through pore-forming activity at the plasma membrane, without cytotoxic effects on normal urothelial cells. This promising selective activity merits further testing as an anti-cancer agent.
Nataša Resnik; Larisa Tratnjek; Mateja Kreft; Matic Kisovec; Saša Aden; Apolonija Bedina Zavec; Gregor Anderluh; Marjetka Podobnik; Peter Veranič. Cytotoxic Activity of LLO Y406A Is Targeted to the Plasma Membrane of Cancer Urothelial Cells. International Journal of Molecular Sciences 2021, 22, 3305 .
AMA StyleNataša Resnik, Larisa Tratnjek, Mateja Kreft, Matic Kisovec, Saša Aden, Apolonija Bedina Zavec, Gregor Anderluh, Marjetka Podobnik, Peter Veranič. Cytotoxic Activity of LLO Y406A Is Targeted to the Plasma Membrane of Cancer Urothelial Cells. International Journal of Molecular Sciences. 2021; 22 (7):3305.
Chicago/Turabian StyleNataša Resnik; Larisa Tratnjek; Mateja Kreft; Matic Kisovec; Saša Aden; Apolonija Bedina Zavec; Gregor Anderluh; Marjetka Podobnik; Peter Veranič. 2021. "Cytotoxic Activity of LLO Y406A Is Targeted to the Plasma Membrane of Cancer Urothelial Cells." International Journal of Molecular Sciences 22, no. 7: 3305.
Listeria monocytogenes is an intracellular food-borne pathogen that causes listeriosis, a severe and potentially life-threatening disease. Listeria uses a number of virulence factors to proliferate and spread to various cells and tissues. In this process, three bacterial virulence factors, the pore-forming protein listeriolysin O and phospholipases PlcA and PlcB, play a crucial role. Listeriolysin O belongs to a family of cholesterol-dependent cytolysins that are mostly expressed by gram-positive bacteria. Its unique structural features in an otherwise conserved three-dimensional fold, such as the acidic triad and proline-glutamate-serine-threonine-like sequence, enable the regulation of its intracellular activity as well as distinct extracellular functions. The stability of listeriolysin O is pH- and temperature-dependent, and this provides another layer of control of its activity in cells. Moreover, many recent studies have demonstrated a unique mechanism of pore formation by listeriolysin O, i.e., the formation of arc-shaped oligomers that can subsequently fuse to form membrane defects of various shapes and sizes. During listerial invasion of host cells, these membrane defects can disrupt phagosome membranes, allowing bacteria to escape into the cytosol and rapidly multiply. The activity of listeriolysin O is profoundly dependent on the amount and accessibility of cholesterol in the lipid membrane, which can be modulated by the phospholipase PlcB. All these prominent features of listeriolysin O play a role during different stages of the L. monocytogenes life cycle by promoting the proliferation of the pathogen while mitigating excessive damage to its replicative niche in the cytosol of the host cell.
Nejc Petrišič; Mirijam Kozorog; Saša Aden; Marjetka Podobnik; Gregor Anderluh. The molecular mechanisms of listeriolysin O-induced lipid membrane damage. Biochimica et Biophysica Acta (BBA) - Biomembranes 2021, 1863, 183604 .
AMA StyleNejc Petrišič, Mirijam Kozorog, Saša Aden, Marjetka Podobnik, Gregor Anderluh. The molecular mechanisms of listeriolysin O-induced lipid membrane damage. Biochimica et Biophysica Acta (BBA) - Biomembranes. 2021; 1863 (7):183604.
Chicago/Turabian StyleNejc Petrišič; Mirijam Kozorog; Saša Aden; Marjetka Podobnik; Gregor Anderluh. 2021. "The molecular mechanisms of listeriolysin O-induced lipid membrane damage." Biochimica et Biophysica Acta (BBA) - Biomembranes 1863, no. 7: 183604.
Pore-forming toxins (PFTs) act upon lipid membranes and appropriate model systems are of great importance in researching these proteins. Giant unilamellar vesicles (GUVs) are an excellent model membrane system to study interactions between lipids and proteins. Their main advantage is the size comparable to cells, which means that GUVs can be observed directly under the light microscope. Many PFTs properties can be studied by using GUVs, such as binding specificity, membrane reorganization upon protein binding and oligomerization, pore properties and mechanism of pore formation. GUVs also represent a good model for biotechnological approaches, e.g., in applications in synthetic biology and medicine. Each research area has its own demands for GUVs properties, so several different approaches for GUVs preparations have been developed and will be discussed in this chapter.
Saša Aden; Tina Snoj; Gregor Anderluh. The use of giant unilamellar vesicles to study functional properties of pore-forming toxins. Methods in Enzymology 2021, 649, 219 -251.
AMA StyleSaša Aden, Tina Snoj, Gregor Anderluh. The use of giant unilamellar vesicles to study functional properties of pore-forming toxins. Methods in Enzymology. 2021; 649 ():219-251.
Chicago/Turabian StyleSaša Aden; Tina Snoj; Gregor Anderluh. 2021. "The use of giant unilamellar vesicles to study functional properties of pore-forming toxins." Methods in Enzymology 649, no. : 219-251.
Nanopore-based sensing is a powerful technique for the detection of diverse organic and inorganic molecules, long-read sequencing of nucleic acids, and single-molecule analyses of enzymatic reactions. Selected from natural sources, protein-based nanopores enable rapid, label-free detection of analytes. Furthermore, these proteins are easy to produce, form pores with defined sizes, and can be easily manipulated with standard molecular biology techniques. The range of possible analytes can be extended by using externally added adapter molecules. Here, we provide an overview of current nanopore applications with a focus on engineering strategies and solutions.
Ana Crnković; Marija Srnko; Gregor Anderluh. Biological Nanopores: Engineering on Demand. Life 2021, 11, 27 .
AMA StyleAna Crnković, Marija Srnko, Gregor Anderluh. Biological Nanopores: Engineering on Demand. Life. 2021; 11 (1):27.
Chicago/Turabian StyleAna Crnković; Marija Srnko; Gregor Anderluh. 2021. "Biological Nanopores: Engineering on Demand." Life 11, no. 1: 27.
Aegerolysins are small lipid-binding proteins particularly abundant in fungi. Aegerolysins from oyster mushrooms interact with an insect-specific membrane lipid and, together with MACPF proteins produced by the same organism, form pesticidal pore-forming complexes. The specific interaction with the same membrane lipid was recently demonstrated for nigerolysin A2 (NigA2), an aegerolysin from Aspergillus niger. In Aspergillus species, the aegerolysins were frequently found as secreted proteins, indicating their function in fungal defense. Using immunocytochemistry and live-cell imaging we investigated the subcellular localization of the nigerolysins A in A. niger, while their secretion was addressed by secretion prediction and Western blotting. We show that both nigerolysins A are leaderless proteins that reach the cell exterior by an unconventional protein secretion. NigA proteins are evenly distributed in the cytoplasm of fungal hyphae. A detailed bioinformatics analysis of Aspergillus aegerolysins suggests that the same function occurs only in a limited number of aegerolysins. From alignment, analysis of chromosomal loci, orthology, synteny, and phylogeny it follows that the same or a similar function described for pairs of pesticidal proteins of Pleurotus sp. can be expected in species of the subgenus Circumdati, section Nigri, series Nigri, and some other species with adjacent pairs of putative pesticidal proteins.
Nada Kraševec; Maruša Novak; Simona Barat; Matej Skočaj; Kristina Sepčić; Gregor Anderluh. Unconventional Secretion of Nigerolysins A from Aspergillus Species. Microorganisms 2020, 8, 1973 .
AMA StyleNada Kraševec, Maruša Novak, Simona Barat, Matej Skočaj, Kristina Sepčić, Gregor Anderluh. Unconventional Secretion of Nigerolysins A from Aspergillus Species. Microorganisms. 2020; 8 (12):1973.
Chicago/Turabian StyleNada Kraševec; Maruša Novak; Simona Barat; Matej Skočaj; Kristina Sepčić; Gregor Anderluh. 2020. "Unconventional Secretion of Nigerolysins A from Aspergillus Species." Microorganisms 8, no. 12: 1973.
By using developed ribosomal display, we discovered variants of perfringolysin O, a pore forming toxin from bacteria Clostridium perfringens, with non-conserved amino acid substitutions at regions crucial for cholesterol recognition.
Aleksandra Šakanović; Nace Kranjc; Neža Omersa; Marjetka Podobnik; Gregor Anderluh. More than one way to bind to cholesterol: atypical variants of membrane-binding domain of perfringolysin O selected by ribosome display. RSC Advances 2020, 10, 38678 -38682.
AMA StyleAleksandra Šakanović, Nace Kranjc, Neža Omersa, Marjetka Podobnik, Gregor Anderluh. More than one way to bind to cholesterol: atypical variants of membrane-binding domain of perfringolysin O selected by ribosome display. RSC Advances. 2020; 10 (63):38678-38682.
Chicago/Turabian StyleAleksandra Šakanović; Nace Kranjc; Neža Omersa; Marjetka Podobnik; Gregor Anderluh. 2020. "More than one way to bind to cholesterol: atypical variants of membrane-binding domain of perfringolysin O selected by ribosome display." RSC Advances 10, no. 63: 38678-38682.
Tumor-associated glycolipids such as NeuGc GM3 are auspicious molecular targets in antineoplastic therapies and vaccine strategies. 14F7 is an anti-tumor antibody with high clinical potential, which has extraordinary specificity for NeuGc GM3, but does not recognize the very similar, ubiquitous NeuAc GM3. Here we present the 2.3 Å crystal structure of the 14F7 binding domain (14F7 scFv) in complex with the NeuGc GM3 trisaccharide. Intriguingly, a water molecule appears to shape the specificity of 14F7. Using model membrane systems, we show that 14F7 recognizes NeuGc GM3 only above lipid concentrations that are likely to form glycolipid-rich domains. This “all-or-nothing” effect was exacerbated in giant unilamellar vesicles and multilamellar vesicles, whereas no binding was observed to 100 nm liposomes, emphasizing that the 14F7–NeuGc GM3 interaction is additionally modulated by membrane curvature. Unexpectedly, adding NeuAc GM3 strongly increased binding affinity to NeuGc GM3-containing liposomes. This effect may be important for tumor recognition, where the ubiquitous NeuAc GM3 may enhance 14F7 binding to NeuGc GM3-expressing cancer cells.
Kaare Bjerregaard-Andersen; Hedda Johannesen; Fana Abraha; Aleksandra Šakanović; Daniel Groβer; Ünal Coskun; Gregor Anderluh; Stefan Oscarson; Ernesto Moreno; Michal Grzybek; Ute Krengel. Insight into glycosphingolipid crypticity: Crystal structure of the anti-tumor antibody 14F7 and recognition of NeuGc GM3 ganglioside. 2020, 1 .
AMA StyleKaare Bjerregaard-Andersen, Hedda Johannesen, Fana Abraha, Aleksandra Šakanović, Daniel Groβer, Ünal Coskun, Gregor Anderluh, Stefan Oscarson, Ernesto Moreno, Michal Grzybek, Ute Krengel. Insight into glycosphingolipid crypticity: Crystal structure of the anti-tumor antibody 14F7 and recognition of NeuGc GM3 ganglioside. . 2020; ():1.
Chicago/Turabian StyleKaare Bjerregaard-Andersen; Hedda Johannesen; Fana Abraha; Aleksandra Šakanović; Daniel Groβer; Ünal Coskun; Gregor Anderluh; Stefan Oscarson; Ernesto Moreno; Michal Grzybek; Ute Krengel. 2020. "Insight into glycosphingolipid crypticity: Crystal structure of the anti-tumor antibody 14F7 and recognition of NeuGc GM3 ganglioside." , no. : 1.
Eukaryotic vesicles fuse with the plasmalemma to form the fusion pore, previously considered to be unstable with widening of the pore diameter. Recent studies established that the pore diameter is stable, reflecting balanced forces of widening and closure. Proteins are considered key regulators of the fusion pore, whereas the role of membrane lipids remains unclear. Super-resolution microscopy revealed that lactotroph secretory vesicles discharge cholesterol after stimulation of exocytosis; subsequently, vesicle cholesterol redistributes to the outer leaflet of the plasmalemma. Cholesterol depletion in lactotrophs and astrocytes evokes release of vesicle hormone, indicating that cholesterol constricts the fusion pore. A new model of cholesterol-dependent fusion pore diameter regulation is proposed. High-resolution measurements of fusion pore conductance confirmed that the fusion pore widens with cholesterol depletion and constricts with cholesterol enrichment. In fibroblasts lacking the Npc1 protein, in which cholesterol accumulates in vesicles, the fusion pore is narrower than in controls, showing that cholesterol regulates fusion pore geometry.Graphical AbstractTop: stages through which a vesicle interacts with the plasmalemma. Stage A denotes hemifusion, which proceeds to stage B, with a narrow fusion pore, which can then reversibly open (stage C), before widening fully (stage D). Bottom: redistribution of cholesterol from the vesicle to the outer leaflet of the plasmalemma controls fusion pore constriction.In BriefA membrane pore is formed when the vesicle membrane fuses with the plasmalemma. Proteins were considered key regulators of the opening and closing of this fusion pore. Here, evidence is provided to show that cholesterol, a membrane constituent, determines a radial force constricting the fusion pore, revealing that the fusion pore functions as a proteolipidic structure.HighlightsIntravesicular cholesterol redistributes to the outer leaflet of the plasmalemma.Cholesterol depletion widens the fusion pore, whereas cholesterol enrichment constricts the fusion pore.A model of cholesterol-dependent force preventing fusion pore widening is developed.Disease-related increase in vesicle cholesterol constricts the fusion pore.
Bostjan Rituper; Alenka Gucek; Marjeta Lisjak; Urszula Gorska; Aleksandra Sakanovic; Sasa Trkov Bobnar; Eva Lasic; Mico Bozic; Prabhodh S. Abbineni; Jernej Jorgacevski; Marko Kreft; Alexei Verkhratsky; Frances M. Platt; Gregor Anderluh; Matjaz Stenovec; Bojan Bozic; Jens R. Coorssen; Robert Zorec. Redistribution of cholesterol from vesicle to plasmalemma controls fusion pore geometry. 2020, 1 .
AMA StyleBostjan Rituper, Alenka Gucek, Marjeta Lisjak, Urszula Gorska, Aleksandra Sakanovic, Sasa Trkov Bobnar, Eva Lasic, Mico Bozic, Prabhodh S. Abbineni, Jernej Jorgacevski, Marko Kreft, Alexei Verkhratsky, Frances M. Platt, Gregor Anderluh, Matjaz Stenovec, Bojan Bozic, Jens R. Coorssen, Robert Zorec. Redistribution of cholesterol from vesicle to plasmalemma controls fusion pore geometry. . 2020; ():1.
Chicago/Turabian StyleBostjan Rituper; Alenka Gucek; Marjeta Lisjak; Urszula Gorska; Aleksandra Sakanovic; Sasa Trkov Bobnar; Eva Lasic; Mico Bozic; Prabhodh S. Abbineni; Jernej Jorgacevski; Marko Kreft; Alexei Verkhratsky; Frances M. Platt; Gregor Anderluh; Matjaz Stenovec; Bojan Bozic; Jens R. Coorssen; Robert Zorec. 2020. "Redistribution of cholesterol from vesicle to plasmalemma controls fusion pore geometry." , no. : 1.
Perforin-2 (MPEG1) is thought to enable the killing of invading microbes engulfed by macrophages and other phagocytes, forming pores in their membranes. Loss of perforin-2 renders individual phagocytes and whole organisms significantly more susceptible to bacterial pathogens. Here, we reveal the mechanism of perforin-2 activation and activity using atomic structures of pre-pore and pore assemblies, high-speed atomic force microscopy, and functional assays. Perforin-2 forms a pre-pore assembly in which its pore-forming domain points in the opposite direction to its membrane-targeting domain. Acidification then triggers pore formation, via a 180° conformational change. This novel and unexpected mechanism prevents premature bactericidal attack and may have played a key role in the evolution of all perforin family proteins.
Tao Ni; Fang Jiao; Xiulian Yu; Saša Aden; Lucy Ginger; Sophie I. Williams; Fangfang Bai; Vojtěch Pražák; Dimple Karia; Phillip Stansfeld; Peijun Zhang; George Munson; Gregor Anderluh; Simon Scheuring; Robert J. C. Gilbert. Structure and mechanism of bactericidal mammalian perforin-2, an ancient agent of innate immunity. Science Advances 2020, 6, eaax8286 .
AMA StyleTao Ni, Fang Jiao, Xiulian Yu, Saša Aden, Lucy Ginger, Sophie I. Williams, Fangfang Bai, Vojtěch Pražák, Dimple Karia, Phillip Stansfeld, Peijun Zhang, George Munson, Gregor Anderluh, Simon Scheuring, Robert J. C. Gilbert. Structure and mechanism of bactericidal mammalian perforin-2, an ancient agent of innate immunity. Science Advances. 2020; 6 (5):eaax8286.
Chicago/Turabian StyleTao Ni; Fang Jiao; Xiulian Yu; Saša Aden; Lucy Ginger; Sophie I. Williams; Fangfang Bai; Vojtěch Pražák; Dimple Karia; Phillip Stansfeld; Peijun Zhang; George Munson; Gregor Anderluh; Simon Scheuring; Robert J. C. Gilbert. 2020. "Structure and mechanism of bactericidal mammalian perforin-2, an ancient agent of innate immunity." Science Advances 6, no. 5: eaax8286.
Lipid membranes are becoming increasingly popular in synthetic biology due to their biophysical properties and crucial role in communication between different compartments. Several alluring protein-membrane sensors have already been developed, whereas protein logic gates designs on membrane-embedded proteins are very limited. Here we demonstrate the construction of a two-level protein-membrane logic gate with an OR-AND logic. The system consists of an engineered pH-dependent pore-forming protein listeriolysin O and its DARPin-based inhibitor, conjugated to a lipid vesicle membrane. The gate responds to low pH and removal of the inhibitor from the membrane either by switching to a reducing environment, protease cleavage, or any other signal depending on conjugation chemistry used for inhibitor attachment to the membrane. This unique protein logic gate vesicle system advances generic sensing and actuator platforms used in synthetic biology and could be utilized in drug delivery.
Neža Omersa; Saša Aden; Matic Kisovec; Marjetka Podobnik; Gregor Anderluh. Design of Protein Logic Gate System Operating on Lipid Membranes. ACS Synthetic Biology 2020, 9, 316 -328.
AMA StyleNeža Omersa, Saša Aden, Matic Kisovec, Marjetka Podobnik, Gregor Anderluh. Design of Protein Logic Gate System Operating on Lipid Membranes. ACS Synthetic Biology. 2020; 9 (2):316-328.
Chicago/Turabian StyleNeža Omersa; Saša Aden; Matic Kisovec; Marjetka Podobnik; Gregor Anderluh. 2020. "Design of Protein Logic Gate System Operating on Lipid Membranes." ACS Synthetic Biology 9, no. 2: 316-328.
Perforation of cellular membranes by pore-forming proteins can affect cell physiology, tissue integrity, or immune response. Since many pore-forming proteins are toxins or highly potent virulence factors, they represent an attractive target for the development of molecules that neutralize their actions with high efficacy. There has been an assortment of inhibitors developed to specifically obstruct the activity of pore-forming proteins, in addition to vaccination and antibiotics that serve as a plausible treatment for the majority of diseases caused by bacterial infections. Here we review a wide range of potential inhibitors that can specifically and effectively block the activity of pore-forming proteins, from small molecules to more specific macromolecular systems, such as synthetic nanoparticles, antibodies, antibody mimetics, polyvalent inhibitors, and dominant negative mutants. We discuss their mechanism of inhibition, as well as advantages and disadvantages.
Neža Omersa; Marjetka Podobnik; Gregor Anderluh. Inhibition of Pore-Forming Proteins. Toxins 2019, 11, 545 .
AMA StyleNeža Omersa, Marjetka Podobnik, Gregor Anderluh. Inhibition of Pore-Forming Proteins. Toxins. 2019; 11 (9):545.
Chicago/Turabian StyleNeža Omersa; Marjetka Podobnik; Gregor Anderluh. 2019. "Inhibition of Pore-Forming Proteins." Toxins 11, no. 9: 545.
Necrosis and ethylene-inducing peptide 1 (Nep1)-like proteins (NLPs) are secreted by several phytopathogenic microorganisms. They trigger necrosis in various eudicot plants upon binding to plant sphingolipid glycosylinositol phosphorylceramides (GIPC). Interestingly, HaNLP3 from the obligate biotroph oomycete Hyaloperonospora arabidopsidis does not induce necrosis. We determined the crystal structure of HaNLP3 and showed that it adopts the NLP fold. However, the conformations of the loops surrounding the GIPC headgroup-binding cavity differ from those of cytotoxic Pythium aphanidermatum NLPPya. Essential dynamics extracted from μs-long molecular dynamics (MD) simulations reveals a limited conformational plasticity of the GIPC-binding cavity in HaNLP3 relative to toxic NLPs. This likely precludes HaNLP3 binding to GIPCs, which is the underlying reason for the lack of toxicity. This study reveals that mutations at key protein regions cause a switch between non-toxic and toxic phenotypes within the same protein scaffold. Altogether, these data provide evidence that protein flexibility is a distinguishing trait of toxic NLPs and highlight structural determinants for a potential functional diversification of non-toxic NLPs utilized by biotrophic plant pathogens. The rhizosphere and phyllosphere of terrestrial plants are home to a number of microorganisms, many of which are potentially pathogenic at certain stages during their associations with plants. The pathogens use diverse routes to penetrate physical barriers and colonize host plants with different lifestyles. Necrosis and ethylene inducing peptide 1 (Nep1)-like proteins (NLPs) are widely distributed among prokaryotic and eukaryotic organisms, like fungi, bacteria and oomycetes, and may infect a range of different crops, such as potato, tomato, soybean and tobacco, thus posing major threat to agriculture worldwide. It was shown that NLPs function as cytolytic toxins that induce plasma membrane leakage by binding to plant membrane sphingolipid receptor. Interestingly, non-toxic NLP proteins can also be secreted by several hemibiotrophic and obligate biotrophic pathogens. This study provides a structural and functional characterization of a non-cytotoxic HaNLP3 protein from Hyaloperonospora arabidopsidis that could explain how these proteins evolved a range of new functions in variety of pathogens with different lifestyles.
Tea Lenarčič; Katja Pirc; Vesna Hodnik; Isabell Albert; Jure Borišek; Alessandra Magistrato; Thorsten Nürnberger; Marjetka Podobnik; Gregor Anderluh. Molecular basis for functional diversity among microbial Nep1-like proteins. PLOS Pathogens 2019, 15, e1007951 .
AMA StyleTea Lenarčič, Katja Pirc, Vesna Hodnik, Isabell Albert, Jure Borišek, Alessandra Magistrato, Thorsten Nürnberger, Marjetka Podobnik, Gregor Anderluh. Molecular basis for functional diversity among microbial Nep1-like proteins. PLOS Pathogens. 2019; 15 (9):e1007951.
Chicago/Turabian StyleTea Lenarčič; Katja Pirc; Vesna Hodnik; Isabell Albert; Jure Borišek; Alessandra Magistrato; Thorsten Nürnberger; Marjetka Podobnik; Gregor Anderluh. 2019. "Molecular basis for functional diversity among microbial Nep1-like proteins." PLOS Pathogens 15, no. 9: e1007951.
Cholera is a life-threatening diarrhoeal disease caused by the human pathogen Vibrio cholerae. Infection occurs after ingestion of the bacteria, which colonize the human small intestine and secrete their major virulence factor – the cholera toxin (CT). The GM1 ganglioside is considered the primary receptor of the CT, but recent studies suggest that also fucosylated receptors such as histo-blood group antigens are important for cellular uptake and toxicity. Recently, a special focus has been on the histo-blood group antigen Lewisx (Lex), however, where and how the CT binds to Lex remains unclear. Here we report the high-resolution crystal structure (1.5 Å) of the receptor-binding B-subunits of the CT bound to the Lex trisaccharide, and complementary quantitative binding data for CT holotoxins. Lex, and also l-fucose alone, bind to the secondary binding site of the toxin, distinct from the GM1 binding site. In contrast, fucosyl-GM1 mainly binds to the primary binding site due to high-affinity interactions of its GM1 core. Lex is the first histo-blood group antigen of non-secretor phenotype structurally investigated in complex with CT. Together with the quantitative binding data, this allows unique insight into why individuals with non-secretor phenotype are more prone to severe cholera than so-called ‘secretors’.
Joel B. Heim; Vesna Hodnik; Julie E. Heggelund; Gregor Anderluh; Ute Krengel. Crystal structures of cholera toxin in complex with fucosylated receptors point to importance of secondary binding site. Scientific Reports 2019, 9, 1 -14.
AMA StyleJoel B. Heim, Vesna Hodnik, Julie E. Heggelund, Gregor Anderluh, Ute Krengel. Crystal structures of cholera toxin in complex with fucosylated receptors point to importance of secondary binding site. Scientific Reports. 2019; 9 (1):1-14.
Chicago/Turabian StyleJoel B. Heim; Vesna Hodnik; Julie E. Heggelund; Gregor Anderluh; Ute Krengel. 2019. "Crystal structures of cholera toxin in complex with fucosylated receptors point to importance of secondary binding site." Scientific Reports 9, no. 1: 1-14.
Ketamine is an antidepressant with rapid therapeutic onset and long-lasting effect, although the underlying mechanism(s) remain unknown. Using FRET-based nanosensors we found that ketamine increases [cAMP]i in astrocytes. Membrane capacitance recordings, however, reveal fundamentally distinct mechanisms of effects of ketamine and [cAMP]i on vesicular secretion: a rise in [cAMP]i facilitated, whereas ketamine inhibited exocytosis. By directly monitoring cholesterol-rich membrane domains with a fluorescently tagged cholesterol-specific membrane binding domain (D4) of toxin perfringolysin O, we demonstrated that ketamine induced cholesterol redistribution in the plasmalemma in astrocytes, but neither in fibroblasts nor in PC 12 cells. This novel mechanism posits that ketamine affects density and distribution of cholesterol in the astrocytic plasmalemma, consequently modulating a host of processes that may contribute to ketamine’s rapid antidepressant action.
Eva Lasič; Marjeta Lisjak; Anemari Horvat; Mićo Božić; Aleksandra Šakanović; Gregor Anderluh; Alexei Verkhratsky; Nina Vardjan; Jernej Jorgačevski; Matjaž Stenovec; Robert Zorec. Astrocyte Specific Remodeling of Plasmalemmal Cholesterol Composition by Ketamine Indicates a New Mechanism of Antidepressant Action. Scientific Reports 2019, 9, 1 -12.
AMA StyleEva Lasič, Marjeta Lisjak, Anemari Horvat, Mićo Božić, Aleksandra Šakanović, Gregor Anderluh, Alexei Verkhratsky, Nina Vardjan, Jernej Jorgačevski, Matjaž Stenovec, Robert Zorec. Astrocyte Specific Remodeling of Plasmalemmal Cholesterol Composition by Ketamine Indicates a New Mechanism of Antidepressant Action. Scientific Reports. 2019; 9 (1):1-12.
Chicago/Turabian StyleEva Lasič; Marjeta Lisjak; Anemari Horvat; Mićo Božić; Aleksandra Šakanović; Gregor Anderluh; Alexei Verkhratsky; Nina Vardjan; Jernej Jorgačevski; Matjaž Stenovec; Robert Zorec. 2019. "Astrocyte Specific Remodeling of Plasmalemmal Cholesterol Composition by Ketamine Indicates a New Mechanism of Antidepressant Action." Scientific Reports 9, no. 1: 1-12.
Potato virus Y (PVY) is among the most economically important plant pathogens. Using cryoelectron microscopy, we determined the near-atomic structure of PVY’s flexuous virions, revealing a previously unknown lumenal interplay between extended carboxyl-terminal regions of the coat protein units and viral RNA. RNA–coat protein interactions are crucial for the helical configuration and stability of the virion, as revealed by the unique near-atomic structure of RNA-free virus-like particles. The structures offer the first evidence for plasticity of the coat protein’s amino- and carboxyl-terminal regions. Together with mutational analysis and in planta experiments, we show their crucial role in PVY infectivity and explain the ability of the coat protein to perform multiple biological tasks. Moreover, the high modularity of PVY virus-like particles suggests their potential as a new molecular scaffold for nanobiotechnological applications.
Andreja Kežar; Luka Kavčič; Martin Polák; Jiří Nováček; Ion Gutiérrez-Aguirre; Magda Tušek Žnidarič; Anna Coll; Katja Stare; Kristina Gruden; Maja Ravnikar; David Pahovnik; Ema Žagar; Franci Merzel; Gregor Anderluh; Marjetka Podobnik. Structural basis for the multitasking nature of the potato virus Y coat protein. Science Advances 2019, 5, eaaw3808 .
AMA StyleAndreja Kežar, Luka Kavčič, Martin Polák, Jiří Nováček, Ion Gutiérrez-Aguirre, Magda Tušek Žnidarič, Anna Coll, Katja Stare, Kristina Gruden, Maja Ravnikar, David Pahovnik, Ema Žagar, Franci Merzel, Gregor Anderluh, Marjetka Podobnik. Structural basis for the multitasking nature of the potato virus Y coat protein. Science Advances. 2019; 5 (7):eaaw3808.
Chicago/Turabian StyleAndreja Kežar; Luka Kavčič; Martin Polák; Jiří Nováček; Ion Gutiérrez-Aguirre; Magda Tušek Žnidarič; Anna Coll; Katja Stare; Kristina Gruden; Maja Ravnikar; David Pahovnik; Ema Žagar; Franci Merzel; Gregor Anderluh; Marjetka Podobnik. 2019. "Structural basis for the multitasking nature of the potato virus Y coat protein." Science Advances 5, no. 7: eaaw3808.
Surface plasmon resonance (SPR) is an established method for studying molecular interactions in real time. It allows obtaining qualitative and quantitative data on interactions of proteins with lipids or lipid membranes. In most of the approaches a lipid membrane or a membrane-mimetic surface is prepared on the surface of Biacore (GE Healthcare) sensor chips HPA or L1, and the studied protein is then injected across the surface. Here we provide an overview of SPR in protein-lipid and protein-membrane interactions, different approaches described in the literature and a general protocol for conducting an SPR experiment including lipid membranes, together with some experimental considerations.
Aleksandra Šakanovič; Vesna Hodnik; Gregor Anderluh. Surface Plasmon Resonance for Measuring Interactions of Proteins with Lipids and Lipid Membranes. Cardiovascular Development 2019, 2003, 53 -70.
AMA StyleAleksandra Šakanovič, Vesna Hodnik, Gregor Anderluh. Surface Plasmon Resonance for Measuring Interactions of Proteins with Lipids and Lipid Membranes. Cardiovascular Development. 2019; 2003 ():53-70.
Chicago/Turabian StyleAleksandra Šakanovič; Vesna Hodnik; Gregor Anderluh. 2019. "Surface Plasmon Resonance for Measuring Interactions of Proteins with Lipids and Lipid Membranes." Cardiovascular Development 2003, no. : 53-70.
Diarrhea caused by enterotoxigenic Escherichia coli (ETEC) is one of the leading causes of mortality in children under five years of age and is a great burden on developing countries. The major virulence factor of the bacterium is the heat-labile enterotoxin (LT), a close homologue of the cholera toxin. The toxins bind to carbohydrate receptors in the gastrointestinal tract, leading to toxin uptake and, ultimately, to severe diarrhea. Previously, LT from human- and porcine-infecting ETEC (hLT and pLT, respectively) were shown to have different carbohydrate-binding specificities, in particular with respect to N-acetyllactosamine-terminating glycosphingolipids. Here, we probed 11 single-residue variants of the heat-labile enterotoxin with surface plasmon resonance spectroscopy and compared the data to the parent toxins. In addition we present a 1.45 Å crystal structure of pLTB in complex with branched lacto-N-neohexaose (Galβ4GlcNAcβ6[Galβ4GlcNAcβ3]Galβ4Glc). The largest difference in binding specificity is caused by mutation of residue 94, which links the primary and secondary binding sites of the toxins. Residue 95 (and to a smaller extent also residues 7 and 18) also contribute, whereas residue 4 shows no effect on monovalent binding of the ligand and may rather be important for multivalent binding and avidity.
Julie Elisabeth Heggelund; Joel Benjamin Heim; Gregor Bajc; Vesna Hodnik; Gregor Anderluh; Ute Krengel. Specificity of Escherichia coli Heat-Labile Enterotoxin Investigated by Single-Site Mutagenesis and Crystallography. International Journal of Molecular Sciences 2019, 20, 703 .
AMA StyleJulie Elisabeth Heggelund, Joel Benjamin Heim, Gregor Bajc, Vesna Hodnik, Gregor Anderluh, Ute Krengel. Specificity of Escherichia coli Heat-Labile Enterotoxin Investigated by Single-Site Mutagenesis and Crystallography. International Journal of Molecular Sciences. 2019; 20 (3):703.
Chicago/Turabian StyleJulie Elisabeth Heggelund; Joel Benjamin Heim; Gregor Bajc; Vesna Hodnik; Gregor Anderluh; Ute Krengel. 2019. "Specificity of Escherichia coli Heat-Labile Enterotoxin Investigated by Single-Site Mutagenesis and Crystallography." International Journal of Molecular Sciences 20, no. 3: 703.
Diarrhoea caused by enterotoxigenic Escherichia coli is one of the leading causes of mortality in children under five years of age and is a great burden on developing countries. The major virulence factor of the bacterium is the heat-labile enterotoxin (LT), a close homologue of the cholera toxin. The toxins bind to carbohydrate receptors in the gastrointestinal tract, leading to toxin uptake and, ultimately, to severe diarrhoea. Previously, LT from human- and porcine-infecting ETEC (hLT and pLT, respectively) were shown to have different carbohydrate-binding specificities, in particular with respect to N-acetyllactosamine-terminating glycosphingolipids. Here, we probed eleven single-residue variants of the heat-labile enterotoxin with surface plasmon resonance spectroscopy and compared the data to the parent toxins. In addition we present a 1.45 Å crystal structure of pLTB in complex with branched Lacto-N-neohexaose (Galbeta4GlcNAcbeta6[Galbeta4GlcNAcbeta3]Galbeta4Glc). The largest difference in binding specificity is caused by mutation of residue 94, which links the primary and secondary binding sites of the toxins. Residue 95 (and to a smaller extent also residues 7 and 18) also contribute, whereas residue 4 shows no effect on monovalent binding of the ligand and may rather be important for multivalent binding and avidity.
Julie E. Heggelund; Joel B. Heim; Gregor Bajc; Vesna Hodnik; Gregor Anderluh; Ute Krengel. Specificity of Escherichia coli Heat-Labile Enterotoxin Investigated by Single-Site Mutagenesis and Crystallography. 2019, 1 .
AMA StyleJulie E. Heggelund, Joel B. Heim, Gregor Bajc, Vesna Hodnik, Gregor Anderluh, Ute Krengel. Specificity of Escherichia coli Heat-Labile Enterotoxin Investigated by Single-Site Mutagenesis and Crystallography. . 2019; ():1.
Chicago/Turabian StyleJulie E. Heggelund; Joel B. Heim; Gregor Bajc; Vesna Hodnik; Gregor Anderluh; Ute Krengel. 2019. "Specificity of Escherichia coli Heat-Labile Enterotoxin Investigated by Single-Site Mutagenesis and Crystallography." , no. : 1.