Abstract
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 (CPS) pathway. An alternative regulation to provide faster response would be valuable. Here we 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, without triggering ER stress due to protein aggregates. Design of orthogonal chemically-regulated split proteases enables the combination of signals into logic functions. Its application was demonstrated on a chemically regulated therapeutic protein secretion and regulated membrane translocation of a chimeric antigen receptor (CAR) targeting cancer antigen. Regulation of the ER escape represents a platform for the design of fast-responsive and tightly-controlled modular and scalable protein secretion system for mammalian cells.
Highlights
1,3, Nina Jerala[1,4], Tjaša Plaper[1,2], Secreted proteins, such as hormones or cytokines, are key mediators in multicellular organisms
Examples of such sequences in animals include the C-terminal sequence motif Lys-Asp-Glu-Leu (KDEL), which is recognized by the KDEL receptor (KDELR) and is present on soluble secretory proteins, and the Lys-Lys-X-X sequence (KKXX, where X stands for any amino acid), which interacts with the recycling COPI protein complex and is often present at the cytosolic side of transmembrane proteins[14]
Two types of endoplasmic reticulum (ER)-localized and proteolysis-based protein secretion systems were designed, each featuring a different C-terminal retention signal; the first type, termed membER, was designed for membrane-bound proteins retained in the ER by the addition of a transmembrane domain and a C-terminal KKXX sequence exposed to the cytosol (Fig. 1a)
Summary
1,3, Nina Jerala[1,4], Tjaša Plaper[1,2], Secreted proteins, such as hormones or cytokines, are key mediators in multicellular organisms. Protein secretion is an important cellular mechanism to translocate soluble or membrane-bound proteins in order to respond to the changes in the cellular environment or signals from other cells, influencing a wide range of biological functions. We developed two systems for the controlled release of pre-synthesized proteins localized in the ER-lumen of mammalian cells, by proteolytic removal of an ER-retention signal sequence, based on the chemically regulated reconstitution of split potyviral proteases. Such a system is completely genetically encoded and its activation is considerably faster than systems based on the transcriptional induction of protein synthesis, since it relies on the pre-synthesized pool of proteins. In addition to the secretion of selected proteins, such as hormones or cytokines the described system can regulate translocation of proteins to the plasma membrane, as demonstrated here for the chimeric antigen receptor (CAR) T cells
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