Regulation of protein secretion through chemical regulation of endoplasmic reticulum retention signal cleavage

Praznik, Arne; Fink, Tina; Franko, Nik; Lonzarić, Jan; Benčina, Mojca; Jerala, Nina; Plaper, Tjaša; Roškar, Samo; Jerala, Roman

doi:10.1101/2021.10.19.464966

Cited by 4 publications

(5 citation statements)

References 58 publications

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“…1f). employed similar strategies for engineering gene circuits 47,48 . In this system, the protein of interest (POI) is C' terminally tagged with KDEL, a retention signal that prevents protein emigration from the ER due to its constitutive interaction with ER-dwelling KDEL receptors (Fig.…”

Section: Resultsmentioning

confidence: 99%

Engineering receptors in the secretory pathway for orthogonal signalling control

et al. 2022

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Synthetic receptors targeted to the secretory pathway often fail to exhibit the expected activity due to post-translational modifications (PTMs) and/or improper folding. Here, we engineered synthetic receptors that reside in the cytoplasm, inside the endoplasmic reticulum (ER), or on the plasma membrane through orientation adjustment of the receptor parts and by elimination of dysfunctional PTMs sites. The cytoplasmic receptors consist of split-TEVp domains that reconstitute an active protease through chemically-induced dimerization (CID) that is triggered by rapamycin, abscisic acid, or gibberellin. Inside the ER, however, some of these receptors were non-functional, but their activity was restored by mutagenesis of cysteine and asparagine, residues that are typically associated with PTMs. Finally, we engineered orthogonal chemically activated cell-surface receptors (OCARs) consisting of the Notch1 transmembrane domain fused to cytoplasmic tTA and extracellular CID domains. Mutagenesis of cysteine residues in CID domains afforded functional OCARs which enabled fine-tuning of orthogonal signalling in mammalian cells.

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Section: Resultsmentioning

confidence: 99%

Engineering receptors in the secretory pathway for orthogonal signalling control

et al. 2022

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“…The copyright holder for this this version posted January 18, 2023. ; https://doi.org/10.1101/2023.01.18.524631 doi: bioRxiv preprint One of the prominent features of CCs is their designable orthogonality and tunability, which allows selection of CCs with appropriate affinity 66 to obtain the appropriate response under the selected conditions. Introduction of combinations of several dipartite CCs and use of 4-helix bundles could enable combinations of more than 2 input signals and more versatile complex information processing.…”

Section: Discussionmentioning

confidence: 99%

Engineering a Scalable and Orthogonal Platform for Synthetic Communication in Mammalian Cells

Pistikou

Cremers

Nathalia

et al. 2023

Preprint

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The rational design and implementation of synthetic, orthogonal mammalian communication systems has the potential to unravel fundamental design principles of mammalian cell communication circuits and offer a framework for engineering of designer cell consortia with potential applications in cell therapeutics and artificial tissue engineering. We lay here the foundations for the engineering of an orthogonal, and scalable mammalian synthetic intercellular communication platform that exploits the programmability of synthetic receptors and selective affinity and tunability of diffusing coiled-coil (CC) peptide heterodimers. Leveraging the ability of CCs to exclusively bind to a selected cognate receptor, we demonstrate orthogonal receptor activation, as well as Boolean logic computations. Next, we reveal synthetic intercellular communication based on synthetic receptors and secreted multidomain CC ligands and demonstrate a minimal, three-cell population system that can perform distributed AND gate logic. Our work provides a modular and scalable framework for the engineering of complex cell consortia, with the potential to expand the aptitude of cell therapeutics and diagnostics.

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“…While this system is not currently amenable to parasite proteins that enter the secretory pathway, conditional control of secretory traffic has been achieved in P. falciparum by ligand-mediated alteration of the N-terminal transit peptide that controls apicoplast targeting, enabling redirection of plastid proteins into the default secretory pathway (70). Additionally, conditional ER release systems have been developed in model eukaryotes based on ER retrieval machinery (71). In contrast, knockER provides a simple, DiCre-mediated knock sideways strategy that exploits the ERD2/KDEL receptor (KDELR) system for conditional ER retrieval, providing a generalizable approach for interrogating secreted proteins in genetically tractable eukaryotes.…”

Section: Discussionmentioning

confidence: 99%

“…Additionally, conditional ER release systems have been developed in model eukaryotes based on ER retrieval machinery (71). In contrast, knockER provides a simple, DiCre-mediated knock sideways strategy that exploits the ERD2/KDEL receptor (KDELR) system for conditional ER retrieval, providing a generalizable approach for interrogating secreted proteins in genetically tractable eukaryotes.…”

Section: Discussionmentioning

confidence: 99%

Knock-sideways by inducible ER retrieval enables a novel approach for studyingPlasmodiumsecreted proteins

Fierro

Hussain

Campin

et al. 2022

Preprint

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Obligate intracellular malaria parasites depend on protein secretion to penetrate, subvert, and exit their host cells. Despite this key reliance on the secretory pathway, none of the conditional mutagenesis tools available in Plasmodium spp. exploit the unique trafficking features of secreted proteins. Here we report knockER, a novel DiCre-mediated knock sideways approach that sequesters secreted proteins in the ER via conditional fusion of a KDEL ER-retrieval sequence to the target protein C-terminus. We applied knockER to a diverse set of reporters and endogenous P. falciparum and P. berghei proteins targeted to the rhoptries, dense granules, parasite vacuole and apicoplast and show conditional ER sequestration is not generally toxic, enabling loss-of-function studies. Taking advantage of the unique ability to redistribute secreted protein pools from their terminal destination to the ER, we employed knockER to study components of the PTEX translocon in an attempt to separate HSP101 vacuolar function in protein export from a recently proposed role in cargo recognition at the ER. Strikingly, while KDEL-fusion produced similar levels of ER retrieval for both HSP101 and the translocon adaptor PTEX150, parasite growth and effector export were completely unaffected by HSP101 retention while ER retrieval of PTEX150 produced a lethal export defect, indicating vacuolar HSP101 levels are uniquely maintained in excess. Intriguingly, redistribution of HSP101 to the ER did not further sensitize parasites to TetR-DOZI-mediated knockdown compared to parasites containing normal vacuolar levels of HSP101, suggesting PV-localized function does not fully account for HSP101 contribution to parasite fitness and consistent with an important role for HSP101 in the ER. Collectively, our work provides a novel tool for dissecting secreted protein function with sub-compartmental resolution that should be widely amenable to genetically tractable eukaryotes.

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Regulation of protein secretion through chemical regulation of endoplasmic reticulum retention signal cleavage

Cited by 4 publications

References 58 publications

Engineering receptors in the secretory pathway for orthogonal signalling control

Engineering receptors in the secretory pathway for orthogonal signalling control

Engineering a Scalable and Orthogonal Platform for Synthetic Communication in Mammalian Cells

Knock-sideways by inducible ER retrieval enables a novel approach for studyingPlasmodiumsecreted proteins

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