Design and applications of synthetic information processing circuits in mammalian cells

Lonzarić, Jan; Fink, Tina; Jerala, Roman

doi:10.1039/9781782622789-00001

Cited by 5 publications

(4 citation statements)

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“…However, in order to introduce specific, adjustable, and scalable regulation, the information-processing pathways should preferably be designed de novo, as this minimizes the unwanted interactions with the cellular chassis and makes the pathways highly programmable for implementing designed cellular logic. Up to now, most designed cell circuits have been based on transcriptional regulation, drawing on the modular DNA recognition and transcriptional effector domains [7][8][9] . Transcriptional regulation-based cell logic is however inherently slower than protein interaction and modification-based systems.…”

Section: Introductionmentioning

confidence: 99%

Design of fast proteolysis-based signaling and logic circuits in mammalian cells

et al. 2018

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Cellular signal transduction is predominantly based on protein interactions and their posttranslational modifications, which enable a fast response to input signals. Due to difficulties in designing new unique protein-protein interactions, designed cellular logic has focused on transcriptional regulation; however, this has a substantially slower response requiring transcription and translation. Here, we present a de novo design of modular, scalable signaling pathways based on proteolysis and designed coiled-coils (CC) implemented in mammalian cells. A set of split proteases with highly specific orthogonal cleavage motifs was constructed and combined with strategically positioned cleavage sites and designed orthogonal CC dimerizing domains of tunable affinity for competitive displacement after proteolytic cleavage. This enabled implementation of Boolean logic functions and signaling cascades in mammalian cells. Designed split proteasecleavable orthogonal CC-based logic (SPOC logic) circuits enable response to chemical or biological signals within minutes rather than hours, useful for diverse medical and nonmedical applications.

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Section: Introductionmentioning

confidence: 99%

Design of fast proteolysis-based signaling and logic circuits in mammalian cells

et al. 2018

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“…As shown here, this processing can incorporate Boolean logic functions, and could potentially be expanded to feature other protein circuits 16 , feedback loops etc. Furthermore, the inducibility of the input protease could be controlled by a variety of heterodimerization domains 55 . This endows our system with a versatility not found in similar systems, which also rely on the accumulation of pre-synthesized proteins inside of ER, but retain the protein inside the ER through protein aggregation 29 .…”

Section: Discussionmentioning

confidence: 99%

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

Praznik

Fink

Franko

et al. 2021

Preprint

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Secreted proteins, such as hormones or cytokines, are key mediators in multicellular organisms. Protein secretion based on transcriptional control is rather slow, as proteins requires transcription, translation, followed by the transport from the endoplasmic reticulum (ER) through the conventional protein secretion (CPS) pathway towards the plasma membrane. An alternative faster bypass would be valuable. Here we present two genetically encoded orthogonal secretion systems, which rely on the retention of pre-synthesized proteins on the ER membrane (membER, released by cytosolic protease) or inside the ER lumen (lumER, released by 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 precise combination of signals into logic functions and was demonstrated on a chemically regulated insulin secretion. Regulation of ER escape represents a platform for the design of fast responsive and tightly-controlled modular and scalable protein secretion system.Abstract FigureAbstract figure:membER and lumER system.By equipping a protein of interest (POI) with an N-terminal signaling sequence, which initiates the transport of proteins into the endoplasmic reticulum (ER), and a C-terminal KDEL ER retention sequence for luminal proteins or a KKXX sequence for transmembrane proteins, we can retain those proteins inside the ER and cis-Golgi apparatus (GA) through retrograde transport. Insertion of a protease cleavage site adjacent to the retention signal allows for the regulated fast secretion through proteolytic cleavage. The membrane bound, ER membrane (membER) and ER-luminal (lumER) systems allow for the controlled secretion of pre-synthesized protein, stored inside the ER. This platform enables release of target proteins several hours faster than systems relying transcription and translation.

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“…Proteolytically modified effectors can irreversibly initiate signal transduction processes. Meanwhile, proteolytic regulation has been introduced into mammalian cells to control transcription factors and protein interactions [ 211 , 235 , 236 , 237 , 238 ] or even to develop cellular logic by cleaving transcription factors or bound proteins [ 239 , 240 , 241 ]. Since response rates of transcription factors are naturally slow, the application of proteolysis-based signaling systems to protein interactions and modifications is advantageous, as they occur within the range of minutes [ 211 ].…”

Section: Synthetic Biology Application To Crac Channels and Impact On...mentioning

confidence: 99%

Synthetic Biology Meets Ca2+ Release-Activated Ca2+ Channel-Dependent Immunomodulation

Bacsa,

Hopl,

Derler

2024

Cells

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Many essential biological processes are triggered by the proximity of molecules. Meanwhile, diverse approaches in synthetic biology, such as new biological parts or engineered cells, have opened up avenues to precisely control the proximity of molecules and eventually downstream signaling processes. This also applies to a main Ca2+ entry pathway into the cell, the so-called Ca2+ release-activated Ca2+ (CRAC) channel. CRAC channels are among other channels are essential in the immune response and are activated by receptor–ligand binding at the cell membrane. The latter initiates a signaling cascade within the cell, which finally triggers the coupling of the two key molecular components of the CRAC channel, namely the stromal interaction molecule, STIM, in the ER membrane and the plasma membrane Ca2+ ion channel, Orai. Ca2+ entry, established via STIM/Orai coupling, is essential for various immune cell functions, including cytokine release, proliferation, and cytotoxicity. In this review, we summarize the tools of synthetic biology that have been used so far to achieve precise control over the CRAC channel pathway and thus over downstream signaling events related to the immune response.

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Design and applications of synthetic information processing circuits in mammalian cells

Cited by 5 publications

References 0 publications

Design of fast proteolysis-based signaling and logic circuits in mammalian cells

Design of fast proteolysis-based signaling and logic circuits in mammalian cells

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

Synthetic Biology Meets Ca2+ Release-Activated Ca2+ Channel-Dependent Immunomodulation

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