[{"oa":1,"tmp":{"name":"Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International (CC BY-NC-SA 4.0)","legal_code_url":"https://creativecommons.org/licenses/by-nc-sa/4.0/legalcode","image":"/images/cc_by_nc_sa.png","short":"CC BY-NC-SA (4.0)"},"doi":"10.15479/AT:ISTA:8341","language":[{"iso":"eng"}],"acknowledged_ssus":[{"_id":"Bio"},{"_id":"LifeSc"},{"_id":"NanoFab"}],"supervisor":[{"full_name":"Loose, Martin","last_name":"Loose","first_name":"Martin","orcid":"0000-0001-7309-9724","id":"462D4284-F248-11E8-B48F-1D18A9856A87"}],"degree_awarded":"PhD","publication_identifier":{"issn":["2663-337X"]},"month":"09","year":"2020","acknowledgement":"My thanks goes to the Loose lab members, BioImaging, Life Science and Nanofabrication Facilities and the wonderful international community at IST for sharing this experience with me.","publisher":"Institute of Science and Technology Austria","department":[{"_id":"MaLo"}],"publication_status":"published","related_material":{"record":[{"status":"public","relation":"part_of_dissertation","id":"7580"}]},"author":[{"full_name":"Bezeljak, Urban","last_name":"Bezeljak","first_name":"Urban","orcid":"0000-0003-1365-5631","id":"2A58201A-F248-11E8-B48F-1D18A9856A87"}],"date_created":"2020-09-08T08:53:53Z","date_updated":"2023-09-07T13:17:06Z","file_date_updated":"2021-09-16T12:49:12Z","license":"https://creativecommons.org/licenses/by-nc-sa/4.0/","citation":{"ama":"Bezeljak U. In vitro reconstitution of a Rab activation switch. 2020. doi:10.15479/AT:ISTA:8341","ista":"Bezeljak U. 2020. In vitro reconstitution of a Rab activation switch. Institute of Science and Technology Austria.","apa":"Bezeljak, U. (2020). In vitro reconstitution of a Rab activation switch. Institute of Science and Technology Austria. https://doi.org/10.15479/AT:ISTA:8341","ieee":"U. Bezeljak, “In vitro reconstitution of a Rab activation switch,” Institute of Science and Technology Austria, 2020.","mla":"Bezeljak, Urban. In Vitro Reconstitution of a Rab Activation Switch. Institute of Science and Technology Austria, 2020, doi:10.15479/AT:ISTA:8341.","short":"U. Bezeljak, In Vitro Reconstitution of a Rab Activation Switch, Institute of Science and Technology Austria, 2020.","chicago":"Bezeljak, Urban. “In Vitro Reconstitution of a Rab Activation Switch.” Institute of Science and Technology Austria, 2020. https://doi.org/10.15479/AT:ISTA:8341."},"page":"215","date_published":"2020-09-08T00:00:00Z","article_processing_charge":"No","has_accepted_license":"1","day":"08","user_id":"c635000d-4b10-11ee-a964-aac5a93f6ac1","_id":"8341","status":"public","title":"In vitro reconstitution of a Rab activation switch","ddc":["570"],"file":[{"file_id":"8342","relation":"source_file","date_updated":"2021-09-16T12:49:12Z","date_created":"2020-09-08T09:00:29Z","checksum":"70871b335a595252a66c6bbf0824fb02","file_name":"2020_Urban_Bezeljak_Thesis_TeX.zip","access_level":"closed","creator":"dernst","file_size":65246782,"content_type":"application/x-zip-compressed"},{"file_id":"8343","relation":"main_file","date_created":"2020-09-08T09:00:27Z","date_updated":"2021-09-16T12:49:12Z","checksum":"59a62275088b00b7241e6ff4136434c7","file_name":"2020_Urban_Bezeljak_Thesis.pdf","access_level":"open_access","creator":"dernst","content_type":"application/pdf","file_size":31259058}],"oa_version":"Published Version","type":"dissertation","alternative_title":["ISTA Thesis"],"abstract":[{"lang":"eng","text":"One of the most striking hallmarks of the eukaryotic cell is the presence of intracellular vesicles and organelles. Each of these membrane-enclosed compartments has a distinct composition of lipids and proteins, which is essential for accurate membrane traffic and homeostasis. Interestingly, their biochemical identities are achieved with the help\r\nof small GTPases of the Rab family, which cycle between GDP- and GTP-bound forms on the selected membrane surface. While this activity switch is well understood for an individual protein, how Rab GTPases collectively transition between states to generate decisive signal propagation in space and time is unclear. In my PhD thesis, I present\r\nin vitro reconstitution experiments with theoretical modeling to systematically study a minimal Rab5 activation network from bottom-up. We find that positive feedback based on known molecular interactions gives rise to bistable GTPase activity switching on system’s scale. Furthermore, we determine that collective transition near the critical\r\npoint is intrinsically stochastic and provide evidence that the inactive Rab5 abundance on the membrane can shape the network response. Finally, we demonstrate that collective switching can spread on the lipid bilayer as a traveling activation wave, representing a possible emergent activity pattern in endosomal maturation. Together, our\r\nfindings reveal new insights into the self-organization properties of signaling networks away from chemical equilibrium. Our work highlights the importance of systematic characterization of biochemical systems in well-defined physiological conditions. This way, we were able to answer long-standing open questions in the field and close the gap between regulatory processes on a molecular scale and emergent responses on system’s level."}]},{"volume":117,"date_updated":"2023-09-07T13:17:06Z","date_created":"2020-03-12T05:32:26Z","related_material":{"record":[{"status":"public","relation":"dissertation_contains","id":"8341"}],"link":[{"relation":"press_release","description":"News on IST Homepage","url":"https://ist.ac.at/en/news/proteins-as-molecular-switches/"}]},"author":[{"full_name":"Bezeljak, Urban","id":"2A58201A-F248-11E8-B48F-1D18A9856A87","orcid":"0000-0003-1365-5631","first_name":"Urban","last_name":"Bezeljak"},{"first_name":"Hrushikesh","last_name":"Loya","full_name":"Loya, Hrushikesh"},{"last_name":"Kaczmarek","first_name":"Beata M","id":"36FA4AFA-F248-11E8-B48F-1D18A9856A87","full_name":"Kaczmarek, Beata M"},{"full_name":"Saunders, Timothy E.","first_name":"Timothy E.","last_name":"Saunders"},{"first_name":"Martin","last_name":"Loose","id":"462D4284-F248-11E8-B48F-1D18A9856A87","orcid":"0000-0001-7309-9724","full_name":"Loose, Martin"}],"department":[{"_id":"MaLo"},{"_id":"CaBe"}],"publisher":"Proceedings of the National Academy of Sciences","publication_status":"published","year":"2020","language":[{"iso":"eng"}],"acknowledged_ssus":[{"_id":"Bio"},{"_id":"LifeSc"}],"doi":"10.1073/pnas.1921027117","project":[{"name":"Reconstitution of cell polarity and axis determination in a cell-free system","grant_number":"RGY0083/2016","_id":"2599F062-B435-11E9-9278-68D0E5697425"}],"isi":1,"quality_controlled":"1","oa":1,"external_id":{"isi":["000521821800040"]},"main_file_link":[{"url":"https://doi.org/10.1101/776567","open_access":"1"}],"publication_identifier":{"issn":["0027-8424"],"eissn":["1091-6490"]},"month":"03","oa_version":"Preprint","intvolume":" 117","title":"Stochastic activation and bistability in a Rab GTPase regulatory network","status":"public","_id":"7580","user_id":"c635000d-4b10-11ee-a964-aac5a93f6ac1","issue":"12","abstract":[{"text":"The eukaryotic endomembrane system is controlled by small GTPases of the Rab family, which are activated at defined times and locations in a switch-like manner. While this switch is well understood for an individual protein, how regulatory networks produce intracellular activity patterns is currently not known. Here, we combine in vitro reconstitution experiments with computational modeling to study a minimal Rab5 activation network. We find that the molecular interactions in this system give rise to a positive feedback and bistable collective switching of Rab5. Furthermore, we find that switching near the critical point is intrinsically stochastic and provide evidence that controlling the inactive population of Rab5 on the membrane can shape the network response. Notably, we demonstrate that collective switching can spread on the membrane surface as a traveling wave of Rab5 activation. Together, our findings reveal how biochemical signaling networks control vesicle trafficking pathways and how their nonequilibrium properties define the spatiotemporal organization of the cell.","lang":"eng"}],"type":"journal_article","date_published":"2020-03-24T00:00:00Z","page":"6504-6549","article_type":"original","citation":{"ista":"Bezeljak U, Loya H, Kaczmarek BM, Saunders TE, Loose M. 2020. Stochastic activation and bistability in a Rab GTPase regulatory network. Proceedings of the National Academy of Sciences. 117(12), 6504–6549.","apa":"Bezeljak, U., Loya, H., Kaczmarek, B. M., Saunders, T. E., & Loose, M. (2020). Stochastic activation and bistability in a Rab GTPase regulatory network. Proceedings of the National Academy of Sciences. Proceedings of the National Academy of Sciences. https://doi.org/10.1073/pnas.1921027117","ieee":"U. Bezeljak, H. Loya, B. M. Kaczmarek, T. E. Saunders, and M. Loose, “Stochastic activation and bistability in a Rab GTPase regulatory network,” Proceedings of the National Academy of Sciences, vol. 117, no. 12. Proceedings of the National Academy of Sciences, pp. 6504–6549, 2020.","ama":"Bezeljak U, Loya H, Kaczmarek BM, Saunders TE, Loose M. Stochastic activation and bistability in a Rab GTPase regulatory network. Proceedings of the National Academy of Sciences. 2020;117(12):6504-6549. doi:10.1073/pnas.1921027117","chicago":"Bezeljak, Urban, Hrushikesh Loya, Beata M Kaczmarek, Timothy E. Saunders, and Martin Loose. “Stochastic Activation and Bistability in a Rab GTPase Regulatory Network.” Proceedings of the National Academy of Sciences. Proceedings of the National Academy of Sciences, 2020. https://doi.org/10.1073/pnas.1921027117.","mla":"Bezeljak, Urban, et al. “Stochastic Activation and Bistability in a Rab GTPase Regulatory Network.” Proceedings of the National Academy of Sciences, vol. 117, no. 12, Proceedings of the National Academy of Sciences, 2020, pp. 6504–49, doi:10.1073/pnas.1921027117.","short":"U. Bezeljak, H. Loya, B.M. Kaczmarek, T.E. Saunders, M. Loose, Proceedings of the National Academy of Sciences 117 (2020) 6504–6549."},"publication":"Proceedings of the National Academy of Sciences","article_processing_charge":"No","day":"24","scopus_import":"1"},{"abstract":[{"lang":"eng","text":"Inflammation, which is a highly regulated host response against danger signals, may be harmful if it is excessive and deregulated. Ideally, anti-inflammatory therapy should autonomously commence as soon as possible after the onset of inflammation, should be controllable by a physician, and should not systemically block beneficial immune response in the long term. We describe a genetically encoded anti-inflammatory mammalian cell device based on a modular engineered genetic circuit comprising a sensor, an amplifier, a “thresholder” to restrict activation of a positive-feedback loop, a combination of advanced clinically used biopharmaceutical proteins, and orthogonal regulatory elements that linked modules into the functional device. This genetic circuit was autonomously activated by inflammatory signals, including endogenous cecal ligation and puncture (CLP)-induced inflammation in mice and serum from a systemic juvenile idiopathic arthritis (sIJA) patient, and could be reset externally by a chemical signal. The microencapsulated anti-inflammatory device significantly reduced the pathology in dextran sodium sulfate (DSS)-induced acute murine colitis, demonstrating a synthetic immunological approach for autonomous anti-inflammatory therapy."}],"issue":"1","type":"journal_article","file":[{"checksum":"ea8b1b28606dd1edab7379ba4fa3641f","date_created":"2020-03-03T10:55:13Z","date_updated":"2020-07-14T12:47:56Z","file_id":"7561","relation":"main_file","creator":"dernst","content_type":"application/pdf","file_size":3404806,"access_level":"open_access","file_name":"2017_MolecularTherapy_Smole.pdf"}],"oa_version":"Published Version","ddc":["570"],"status":"public","title":"A synthetic mammalian therapeutic gene circuit for sensing and suppressing inflammation","intvolume":" 25","user_id":"2DF688A6-F248-11E8-B48F-1D18A9856A87","_id":"7360","day":"01","article_processing_charge":"No","has_accepted_license":"1","date_published":"2017-01-01T00:00:00Z","article_type":"original","page":"102-119","publication":"Molecular Therapy","citation":{"chicago":"Smole, Anže, Duško Lainšček, Urban Bezeljak, Simon Horvat, and Roman Jerala. “A Synthetic Mammalian Therapeutic Gene Circuit for Sensing and Suppressing Inflammation.” Molecular Therapy. Elsevier, 2017. https://doi.org/10.1016/j.ymthe.2016.10.005.","mla":"Smole, Anže, et al. “A Synthetic Mammalian Therapeutic Gene Circuit for Sensing and Suppressing Inflammation.” Molecular Therapy, vol. 25, no. 1, Elsevier, 2017, pp. 102–19, doi:10.1016/j.ymthe.2016.10.005.","short":"A. Smole, D. Lainšček, U. Bezeljak, S. Horvat, R. Jerala, Molecular Therapy 25 (2017) 102–119.","ista":"Smole A, Lainšček D, Bezeljak U, Horvat S, Jerala R. 2017. A synthetic mammalian therapeutic gene circuit for sensing and suppressing inflammation. Molecular Therapy. 25(1), 102–119.","apa":"Smole, A., Lainšček, D., Bezeljak, U., Horvat, S., & Jerala, R. (2017). A synthetic mammalian therapeutic gene circuit for sensing and suppressing inflammation. Molecular Therapy. Elsevier. https://doi.org/10.1016/j.ymthe.2016.10.005","ieee":"A. Smole, D. Lainšček, U. Bezeljak, S. Horvat, and R. Jerala, “A synthetic mammalian therapeutic gene circuit for sensing and suppressing inflammation,” Molecular Therapy, vol. 25, no. 1. Elsevier, pp. 102–119, 2017.","ama":"Smole A, Lainšček D, Bezeljak U, Horvat S, Jerala R. A synthetic mammalian therapeutic gene circuit for sensing and suppressing inflammation. Molecular Therapy. 2017;25(1):102-119. doi:10.1016/j.ymthe.2016.10.005"},"license":"https://creativecommons.org/licenses/by-nc-nd/4.0/","file_date_updated":"2020-07-14T12:47:56Z","date_created":"2020-01-25T15:55:39Z","date_updated":"2021-01-12T08:13:14Z","volume":25,"author":[{"last_name":"Smole","first_name":"Anže","full_name":"Smole, Anže"},{"full_name":"Lainšček, Duško","first_name":"Duško","last_name":"Lainšček"},{"id":"2A58201A-F248-11E8-B48F-1D18A9856A87","orcid":"0000-0003-1365-5631","first_name":"Urban","last_name":"Bezeljak","full_name":"Bezeljak, Urban"},{"last_name":"Horvat","first_name":"Simon","full_name":"Horvat, Simon"},{"full_name":"Jerala, Roman","last_name":"Jerala","first_name":"Roman"}],"publication_status":"published","publisher":"Elsevier","department":[{"_id":"MaLo"}],"year":"2017","pmid":1,"month":"01","publication_identifier":{"issn":["1525-0016"]},"language":[{"iso":"eng"}],"doi":"10.1016/j.ymthe.2016.10.005","quality_controlled":"1","external_id":{"pmid":["28129106"]},"tmp":{"name":"Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0)","legal_code_url":"https://creativecommons.org/licenses/by-nc-nd/4.0/legalcode","short":"CC BY-NC-ND (4.0)","image":"/images/cc_by_nc_nd.png"},"oa":1},{"pmid":1,"year":"2014","publisher":"Springer Nature","publication_status":"published","author":[{"first_name":"Tina","last_name":"Lebar","full_name":"Lebar, Tina"},{"full_name":"Bezeljak, Urban","orcid":"0000-0003-1365-5631","id":"2A58201A-F248-11E8-B48F-1D18A9856A87","last_name":"Bezeljak","first_name":"Urban"},{"full_name":"Golob, Anja","first_name":"Anja","last_name":"Golob"},{"full_name":"Jerala, Miha","first_name":"Miha","last_name":"Jerala"},{"full_name":"Kadunc, Lucija","last_name":"Kadunc","first_name":"Lucija"},{"full_name":"Pirš, Boštjan","first_name":"Boštjan","last_name":"Pirš"},{"first_name":"Martin","last_name":"Stražar","full_name":"Stražar, Martin"},{"first_name":"Dušan","last_name":"Vučko","full_name":"Vučko, Dušan"},{"first_name":"Uroš","last_name":"Zupančič","full_name":"Zupančič, Uroš"},{"last_name":"Benčina","first_name":"Mojca","full_name":"Benčina, Mojca"},{"last_name":"Forstnerič","first_name":"Vida","full_name":"Forstnerič, Vida"},{"full_name":"Gaber, Rok","last_name":"Gaber","first_name":"Rok"},{"full_name":"Lonzarić, Jan","last_name":"Lonzarić","first_name":"Jan"},{"first_name":"Andreja","last_name":"Majerle","full_name":"Majerle, Andreja"},{"last_name":"Oblak","first_name":"Alja","full_name":"Oblak, Alja"},{"full_name":"Smole, Anže","last_name":"Smole","first_name":"Anže"},{"full_name":"Jerala, Roman","first_name":"Roman","last_name":"Jerala"}],"volume":5,"date_updated":"2021-01-12T08:13:15Z","date_created":"2020-01-25T15:57:17Z","article_number":"5007","extern":"1","external_id":{"pmid":["25264186"]},"quality_controlled":"1","doi":"10.1038/ncomms6007","language":[{"iso":"eng"}],"publication_identifier":{"issn":["2041-1723"]},"month":"09","_id":"7361","user_id":"2DF688A6-F248-11E8-B48F-1D18A9856A87","intvolume":" 5","title":"A bistable genetic switch based on designable DNA-binding domains","status":"public","oa_version":"None","type":"journal_article","issue":"1","abstract":[{"lang":"eng","text":"Bistable switches are fundamental regulatory elements of complex systems, ranging from electronics to living cells. Designed genetic toggle switches have been constructed from pairs of natural transcriptional repressors wired to inhibit one another. The complexity of the engineered regulatory circuits can be increased using orthogonal transcriptional regulators based on designed DNA-binding domains. However, a mutual repressor-based toggle switch comprising DNA-binding domains of transcription-activator-like effectors (TALEs) did not support bistability in mammalian cells. Here, the challenge of engineering a bistable switch based on monomeric DNA-binding domains is solved via the introduction of a positive feedback loop composed of activators based on the same TALE domains as their opposing repressors and competition for the same DNA operator site. This design introduces nonlinearity and results in epigenetic bistability. This principle could be used to employ other monomeric DNA-binding domains such as CRISPR for applications ranging from reprogramming cells to building digital biological memory."}],"citation":{"short":"T. Lebar, U. Bezeljak, A. Golob, M. Jerala, L. Kadunc, B. Pirš, M. Stražar, D. Vučko, U. Zupančič, M. Benčina, V. Forstnerič, R. Gaber, J. Lonzarić, A. Majerle, A. Oblak, A. Smole, R. Jerala, Nature Communications 5 (2014).","mla":"Lebar, Tina, et al. “A Bistable Genetic Switch Based on Designable DNA-Binding Domains.” Nature Communications, vol. 5, no. 1, 5007, Springer Nature, 2014, doi:10.1038/ncomms6007.","chicago":"Lebar, Tina, Urban Bezeljak, Anja Golob, Miha Jerala, Lucija Kadunc, Boštjan Pirš, Martin Stražar, et al. “A Bistable Genetic Switch Based on Designable DNA-Binding Domains.” Nature Communications. Springer Nature, 2014. https://doi.org/10.1038/ncomms6007.","ama":"Lebar T, Bezeljak U, Golob A, et al. A bistable genetic switch based on designable DNA-binding domains. Nature Communications. 2014;5(1). doi:10.1038/ncomms6007","ieee":"T. Lebar et al., “A bistable genetic switch based on designable DNA-binding domains,” Nature Communications, vol. 5, no. 1. Springer Nature, 2014.","apa":"Lebar, T., Bezeljak, U., Golob, A., Jerala, M., Kadunc, L., Pirš, B., … Jerala, R. (2014). A bistable genetic switch based on designable DNA-binding domains. Nature Communications. Springer Nature. https://doi.org/10.1038/ncomms6007","ista":"Lebar T, Bezeljak U, Golob A, Jerala M, Kadunc L, Pirš B, Stražar M, Vučko D, Zupančič U, Benčina M, Forstnerič V, Gaber R, Lonzarić J, Majerle A, Oblak A, Smole A, Jerala R. 2014. A bistable genetic switch based on designable DNA-binding domains. Nature Communications. 5(1), 5007."},"publication":"Nature Communications","article_type":"original","date_published":"2014-09-29T00:00:00Z","article_processing_charge":"No","day":"29"}]