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Published February 17, 2022 | Supplemental Material + Submitted + Published
Journal Article Open

Protease-controlled secretion and display of intercellular signals


To program intercellular communication for biomedicine, it is crucial to regulate the secretion and surface display of signaling proteins. If such regulations are at the protein level, there are additional advantages, including compact delivery and direct interactions with endogenous signaling pathways. Here we create a modular, generalizable design called Retained Endoplasmic Cleavable Secretion (RELEASE), with engineered proteins retained in the endoplasmic reticulum and displayed/secreted in response to specific proteases. The design allows functional regulation of multiple synthetic and natural proteins by synthetic protease circuits to realize diverse signal processing capabilities, including logic operation and threshold tuning. By linking RELEASE to additional sensing and processing circuits, we can achieve elevated protein secretion in response to "undruggable" oncogene KRAS mutants. RELEASE should enable the local, programmable delivery of intercellular cues for a broad variety of fields such as neurobiology, cancer immunotherapy and cell transplantation.

Additional Information

© The Author(s) 2022. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Received 07 October 2021; Accepted 03 February 2022; Published 17 February 2022. We would like to thank Dr. Lin, and Dr. Leonard for kindly sharing some of their plasmids that were used in this work. We would also like to thank Leo Scheller for providing the protocol for the SEAP assays. This work was funded by NIH (4R00EB027723-02, X.J.G), Stanford Cancer Institute (Cancer Innovation Award #216174, X.J.G.), Stanford SystemX Alliance (Seed Grant, X.J.G.), NSERC (PDF-557516-2021, A.E.V.), the International Human Frontier Science Program Organization (LT000221/2021-L, A.E.V.), and the Stanford Graduate Fellowship (J.K. and C.A.). Data availability: New plasmids used in this study will be made available for distribution from Addgene (https://www.addgene.org/Xiaojing_Gao/). Annotated plasmid sequences used in this study are provided in the Source Data as GeneBank files. Raw.fcs files are available from the corresponding authors upon reasonable request. Raw experimental data and p-values for each figure are provided as Source Data. Source data are provided with this paper. Code availability: EasyFlow MATLAB code used for flow cytometry analysis is available from the GitHub repository at https://github.com/AntebiLab/easyflow.git. Author Contributions: A.E.V. and X.J.G. conceived and directed the study. A.E.V, J.K, and C.A. performed most of the experiments. X.J.G. created the HRAS-sensing protease, and L.S.C and R.Z. created the protease-activated protease under M.B.E.'s supervision. A.E.V, and J.K. analyzed the data for the manuscript. A.E.V., J.K. and X.J.G. wrote the manuscript. All authors provided feedback on the manuscript. Competing interests: The board of trustees of the Leland Stanford Junior University have filed a patent on behalf of the inventors (A.E.V., J.K., and X.J.G.) of the RELEASE platform described (US provisional Application No. 63/282689). The remaining authors declare no competing interests. Peer review information: Nature Communications thanks Yingxiao Wang and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.

Attached Files

Published - s41467-022-28623-y.pdf

Submitted - 2021.10.18.464444v1.full.pdf

Supplemental Material - 41467_2022_28623_MOESM1_ESM.docx

Supplemental Material - 41467_2022_28623_MOESM2_ESM.pdf

Supplemental Material - 41467_2022_28623_MOESM3_ESM.pdf

Supplemental Material - 41467_2022_28623_MOESM4_ESM.pdf

Supplemental Material - 41467_2022_28623_MOESM5_ESM.xlsx

Supplemental Material - 41467_2022_28623_MOESM6_ESM.xlsx

Supplemental Material - 41467_2022_28623_MOESM7_ESM.zip


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August 20, 2023
December 22, 2023