Synthetic Protein Circuits for Signal Transducer Activation Sensing
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Solution Overview
Problem
Current synthetic biology approaches lack effective methods for designing protein circuits that can directly sense the activation level of signal transducers, which is crucial for configuring new functions in living cells.
Innovation Solution
The development of synthetic protein circuits comprising polypeptides with signal transducer binding domains and protease domains that associate weakly, forming a protease active state when signal transducers are in close proximity, allowing for precise regulation through effector proteins that change their activity state based on signal transducer activation levels.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If synthetic biology approaches use traditional gene regulation circuits, then new functions can be designed in living cells, but the ability to directly sense signal transducer activation levels is insufficient
Solution Approach 1:
The protease is divided into two separate polypeptides: one containing the signal transducer binding domain and N-terminal protease domain, and another containing the C-terminal protease domain. These segments only associate when signal transducers are in close proximity, enabling sensing capability while maintaining modular design
Solution Approach 2:
Signal transducers act as intermediaries that bring the two polypeptide segments into close proximity when activated, triggering protease association and subsequent effector activation. This intermediary mechanism enables direct sensing of signal transducer activation levels
2Reliability
If protease domains are designed to associate strongly, then protease activity is stable, but the ability to respond to signal transducer activation levels is reduced
Solution Approach 1:
The association between the two polypeptide segments is designed to be dynamic rather than static. The weak association allows the complex to form and dissociate in response to signal transducer activation levels, providing both stability when inactive and adaptability when activated
Solution Approach 2:
The binding affinity parameter of the protease domains is optimized to be weak, allowing the association constant to change in response to signal transducer activation. This parameter change enables the system to transition between inactive and active states based on cellular signaling conditions
3Measurement precision
If signal transducer binding domains are designed with high affinity, then binding is strong, but the circuit cannot detect changes in activation levels
Solution Approach 1:
The binding domains are designed with moderate rather than maximum affinity, allowing partial binding that can respond to changes in signal transducer activation levels. This partial action enables detection of activation dynamics while maintaining sufficient binding stability
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
These circuits enable direct sensing and regulation of signal transducer activation levels, effectively addressing aberrant signaling and potentially treating associated diseases by inducing appropriate cellular responses.
Implementation Method 1
the first part of the first protease domain and the second part of the first protease domain have weak association affinity, and wherein the first part of the first protease domain and the second part of the first protease domain are capable of associating with each other to constitute a first protease
Implementation Method 2
the first signal transducer binding domain is capable of binding a first signal transducer to form a first signal transducer-bound polypeptide
Implementation Method 3
an effector protein comprising a first cut site the first protease in the first protease active state is capable of cutting
Data Source
AI summary
Disclosed herein include methods, compositions, and kits suitable for use in detecting the activation level of a signal transducer. In some embodiments, there are provided synthetic protein circuits wherein recruitment of synthetic protein circuit components to an association location upon activation of a signal transducer generates an active effector protein. The effector protein can be configured to carry out a variety of functions when in an active state, such as, for example, inducing cell death. Methods of treating a disease or disorder characterized by aberrant signaling are provided in some embodiments.