Proteolytically activated CRAC effectors through designed intramolecular inhibition

Jazbec, Vid (author), Jerala, Roman (author), Benčina, Mojca (author)

Highly regulated intracellular calcium entry affects numerous cellular physiological events. External regulation of intracellular calcium signaling presents a great opportunity for the artificial regulation of cellular activity. Calcium entry can be mediated by STIM proteins interacting with Orai calcium channels; therefore, the STIM1−Orai1 pair has become a tool for artificially modulating calcium entry. We report on an innovative genetically engineered protease-activated Orai activator called PACE. CAD self-dimerization and activation were inhibited with a coiled-coil forming peptide pair linked to CAD via a protease cleavage site. PACE generated sustained calcium entry after its activation with a reconstituted split protease. We also generated PACE, whose transcriptional activation of NFAT was triggered by PPV or TEV protease. Using PACE, we successfully activated the native NFAT signaling pathway and the production of cytokines in a T-cell line. PACE represents a useful tool for generating sustained calcium entry to initiate calcium-dependent protein translation. PACE provides a promising template for the construction of links between various protease activation pathways and calcium signaling.

• Language: English
• Keywords: STIM1, Orai, TEV protease, PPV protease, calcium signaling, coiled-coil peptides, bioluminescent probes, calcium, cell signaling, crystal cleavage, peptides, proteins
• Work type: Article
• Typology: 1.01 - Original Scientific Article
• Organization: MF - Faculty of Medicine
• Publication status: Published
• Publication version: Version of Record
• Year: 2022
• Number of pages: Str. 2756–2765
• Numbering: Vol. 11, iss. 8
• PID: 20.500.12556/RUL-140820
• UDC: 577
• ISSN on article: 2161-5063
• DOI: 10.1021/acssynbio.2c00151
• COBISS.SI-ID: 115643651
• Publication date in RUL: 19.09.2022

Record is a part of a journal

• Title: ACS synthetic biology
• Shortened title: ACS synth. biol.
• Publisher: American Chemical Society
• ISSN: 2161-5063
• COBISS.SI-ID: 519107609

Licences

• License: CC BY 4.0, Creative Commons Attribution 4.0 International
• Link: http://creativecommons.org/licenses/by/4.0/
• Description: This is the standard Creative Commons license that gives others maximum freedom to do what they want with the work as long as they credit the author.

Secondary language

• Language: Slovenian
• Keywords: biokemija, celice, kalcij, beljakovine, proteini