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Rapid secretion system of pre-synthesized proteins with chemical regulation of cleavage of ER retention signal

Praznik et al., Nat.Comm. 2022

Secreted proteins, such as hormones or cytokines, are key mediators in multicellular organisms. Response of protein secretion based on transcriptional control is rather slow, as it requires transcription, translation and transport from the endoplasmic reticulum (ER) to the plasma membrane via the conventional protein secretion pathway and can require several hours before the protein of interest is secreted to a functionally relevant concentration. An alternative regulation to provide faster response would be valuable.

In the research we design and present two genetically encoded orthogonal regulatory secretion systems, which rely on the retention of pre-synthesized proteins on the ER membrane (membER, released by a cytosolic protease) or inside the ER lumen (lumER, released by an ER-luminal protease), respectively, and their release by the chemical signal-regulated proteolytic removal of an ER-retention signal. The lumER and membER system prevented activation of ER stress, which could result in apoptosis.

We demonstrated the system using a design of human insulin adapted for protease processing and secretion through the conventional secretion pathway. The preproinsulin sequence was appended to the lumER secretion system and combined with protease cleavage sites and chemical inducer. The presence of a C-peptide of a preproinsulin in cell media in the case of the lumER system was detected within 45 min, in contrast to several hours, in the case of a system based on the transcriptional induction of insulin expression. Using a similar strategy, the membER system was demonstrated to control the release of the anti-inflammatory cytokine IL-10.

Additionally, we demonstrated a therapeutic applicability of the system to regulate translocation of membrane-associated protein of a chimeric antigen receptor (CAR) targeting cancer antigen to the plasma membrane. Regulation of CAR T cell responsiveness is desired, as the excessive activation of engineered T cells may lead to often fatal cytokine release syndrome. We regulated translocation of CARs to the plasma membrane using a chemically regulated translocation system. In this case, CARs are restricted within the cell until the signal triggers their rapid translocation to the plasma membrane, where they can engage target cells and trigger cell activation.

Regulation of the ER escape represents a platform for the design of fast-responsive and tightly controlled modular and scalable protein secretion system of therapeutically relevant proteins for mammalian cells.

Article: PRAZNIK, Arne, FINK, Tina, FRANKO, Nik, LONZARIĆ, Jan, BENČINA, Mojca, JERALA, Nina, PLAPER, Tjaša, ROŠKAR, Simona, JERALA, Roman. Regulation of protein secretion through chemical regulation of endoplasmic reticulum retention signal cleavage. Nature Communications. 13, Article number: 1323 (2022). 

DOI: 10.1038/s41467-022-28971-9

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