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

Abstract

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.

PERSIST: A programmable RNA regulation platform using CRISPR endoRNases

“…Importantly, while other post-transcriptional platforms, namely protease-mediated regulation [27,21,14], have show repression of another repressor as we have here, none have demonstrated direct activation of a repressor as shown here. Figure 3e shows auto positive feedback where an endoRNase is able to self activate when its recognition site is placed in the 3' ON-switch of its own transcript.…”

“…Such platforms would allow the use of constitutive promoters routinely used in gene therapy, which tend to resist silencing [62,54], and may also find use in modern therapeutic modalities such as mRNA gene therapies. Recently-developed protein-regulation platforms using orthogonal proteases [14,27,21], have begun to address this need by controlling protein degradation. The field would benefit from development of protein-based RNA-regulation platforms with similar properties that enable control of protein production, e.g.…”

Logic‐Gate‐Actuated DNA‐Controlled Receptor Assembly for the Programmable Modulation of Cellular Signal Transduction

“…[20] Typically,c ellular signal transduction is predominantly based on protein interactions, which enable af ast response to input signals. [21] At the nanoscale,the spatial organization of cell surface receptors on living cell membrane is crucial for controlling cellular signal transduction. [22] Aptamer-induced assembly has proved to be an effective way for the design of an artificial ligand that controls the activity of target receptors.…”

A Computational Design of a Programmable Biological Processor

“…Recently, a lot of focus has been devoted to the implementation of logic structures using engineered proteases, which enable us to implement protein-based processing signalling logic [23]. The most recent advances include the construction of split-protease-cleavable orthogonal-coiled coils-based (SPOC) logic circuits [25] and circuits of hacked orthogonal modular proteases (CHOMP) [26]. These can be used to design artificial signalling functions, which operate inde-pendently of the slower cellular processes, such as transcription and translation, and thus have a much faster response time than transcription-based logic.…”

“…These can be used to design artificial signalling functions, which operate inde-pendently of the slower cellular processes, such as transcription and translation, and thus have a much faster response time than transcription-based logic. These circuits have been used to implement Boolean logic functions [25,26] as well as biomolecular switches [27]. However, their capacity to construct more complex circuits and circuits with periodic behaviour has not been investigated yet and might prove to be problematic due to irreversibility of proteolysis.…”