SspH1 LRR-NEL System for Inducible Protein Degradation in Plant Cells
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Solution Overview
Problem
Current systems for targeted protein degradation in plant cells lack high-temporal resolution and are not suitable for inducible protein depletion, especially for essential proteins, due to the lethality of loss-of-function mutants and interference with endogenous protein degradation pathways.
Innovation Solution
A system utilizing the Leucine-Rich Repeat (LRR) and novel E3 ligase (NEL) domains of SspH1, combined with the Homology Region 1b (HR1b) domain of human PKN1, to specifically and efficiently degrade target proteins in plant cells, utilizing a Dexamethasone-inducible degron system for controlled protein depletion.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If ligand-based systems are used to target proteins for degradation, then protein degradation can be achieved, but the system lacks high-temporal resolution and cannot be easily induced
Solution Approach 1:
The system pre-assembles the degradation machinery by fusing the E3 ligase (NEL) and its binding partner (HR1b) to the target protein of interest before degradation is needed. This preliminary configuration allows rapid induction of degradation when triggered, achieving both high temporal resolution and ease of operation through inducible systems
Solution Approach 2:
The system employs dynamic control of protein degradation through inducible promoters or small molecule triggers that can switch the degradation state on and off as needed. This dynamic capability enables precise temporal control while maintaining ease of operation through external induction signals
2Reliability
If loss-of-function mutants are used to study essential proteins, then protein function can be investigated, but lethality occurs preventing study of dynamic events
Solution Approach 1:
The system prepares the degradation apparatus in advance by expressing the fused E3 ligase-HR1b construct without immediate degradation, allowing the organism to develop normally. When degradation is triggered, it occurs rapidly enough to capture dynamic events while avoiding the lethality of permanent loss-of-function mutants
Solution Approach 2:
The system enables periodic or transient degradation of essential proteins rather than permanent loss, allowing organisms to survive and be studied. This periodic action provides temporal resolution for dynamic events while maintaining organism viability, unlike constitutive loss-of-function mutants
3Productivity
If existing protein degradation systems are used in plants, then protein depletion can be achieved, but off-target effects and interference with endogenous pathways occur
Solution Approach 1:
The system extracts and utilizes a bacterial E3 ligase (NEL from SspH1) that is foreign to plant cells, rather than interfering with endogenous plant degradation pathways. This extraction of a non-endogenous enzyme reduces off-target effects while maintaining efficient protein depletion capability
Solution Approach 2:
The system introduces an intermediary bacterial E3 ligase that mediates target protein degradation through a defined interaction interface (HR1b binding). This intermediary approach allows controlled degradation of specific targets without disrupting native plant protein degradation pathways, minimizing off-target effects
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
The system enables robust and specific degradation of target proteins in plant cells with high temporal resolution, minimizing off-target effects and allowing for the study of dynamic cellular processes without disrupting essential protein functions.
Implementation Method 1
The interaction between the HR1b domain of PKN1 and the LRR domain of SspH1 results in protein binding and ubiquitination by the NEL domain
Implementation Method 2
The HR1b-LRR interaction activates the NEL catalytic domain to transfer ubiquitin to the target protein
Data Source
AI summary
The present disclosure provides components and systems for targeted protein degradation in cells (e.g., in plant cells). In particular, the present disclosure provides a protein containing the Leucine-Rich Repeat (LRR) and novel E3 ligase (NEL) domains of SspH1 for use with Homology Region 1b (HR1b) domain of human PKN1 for degrading target proteins.