pH-Responsive Proteolysis Probe for Autophagy Quantification

Resolve Bottlenecks,
Find Innovative SolutionsGenerate Solutions

Solution Overview

Problem

Current methods for measuring autophagy activity in mammalian cells are hindered by the sensitivity of fluorescent probes to acidic conditions and proteases, leading to irreversible quenching or degradation, and are not suitable for detecting microautophagy and chaperone-mediated autophagy, which do not form autophagosomes.

Innovation Solution

A unimolecular FRET probe is developed, comprising a donor and an acceptor fluorescent protein with different protease sensitivities under acidic conditions, where the acceptor is enzymatically degraded in lysosomes or vacuoles, allowing for a pH-dependent and irreversible change in fluorescence signal, enabling accurate detection of autophagy activity without interference from ambient pH changes.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Adaptability or versatility

If conventional fluorescent probes are used to measure autophagy activity, then the measurement can detect macroautophagy through autophagosome formation, but the probe is degraded by acidic proteases in lysosomes leading to irreversible quenching and inability to detect microautophagy or chaperone-mediated autophagy

Engineering Contradiction:

Improveability to detect different types of autophagyVSAvoidprobe stability in acidic lysosomal environment

Core Design Contradiction:

Adaptability or versatilityVSReliability

Solution Approach 1:

The invention changes the key parameter of probe stability by selecting a fluorescent protein (Keima) with unique properties: its excitation wavelength shifts from 380nm to 586nm in acidic environments, while its emission wavelength remains stable at 620nm. This parameter change allows the probe to remain detectable and quantifiable in the acidic lysosomal environment where conventional probes are degraded, enabling reliable measurement of all autophagy types including microautophagy and chaperone-mediated autophagy.

Inventive Principle:

Principle #35Parameter changes

2Measurement precision

If pH-sensitive fluorescent proteins are used to detect lysosomal transfer, then autophagy activity can be measured, but the fluorescence intensity changes reversibly with pH fluctuations making accurate quantification difficult

Engineering Contradiction:

Improveautophagy activity quantification accuracyVSAvoidfluorescence signal stability

Core Design Contradiction:

Measurement precisionVSStability of the object's composition

Solution Approach 1:

Instead of using fluorescent proteins whose emission intensity changes with pH (conventional approach), the invention inverts the approach by using Keima whose excitation wavelength changes with pH. The excitation spectrum shifts from 380nm (neutral pH) to 586nm (acidic pH), while emission remains constant at 620nm. This inversion allows ratiometric measurement (excitation at 586nm/380nm) that provides stable, reversible, and quantifiable signals that correlate directly with lysosomal acidity and autophagy activity.

Inventive Principle:

Principle #13The other way round (Inversion)

3Ease of operation

If fluorescent probes sensitive to acidic conditions are used, then they can respond to lysosomal environment, but they undergo irreversible quenching or degradation preventing repeated measurements and fixed cell analysis

Engineering Contradiction:

Improvemeasurement procedure simplicityVSAvoidprobe functional lifetime

Core Design Contradiction:

Ease of operationVSDuration of action of stationary object

Solution Approach 1:

The invention ensures continuous useful action of the fluorescent probe by using Keima's unique property of resisting degradation in acidic environments. The probe maintains its fluorescent properties and can be repeatedly excited and measured over extended periods, enabling both real-time monitoring in living cells and post-fixation analysis. This continuity allows researchers to perform multiple measurements, analyze fixed cells, and conduct long-term autophagy studies without probe degradation limiting the experimental duration.

Inventive Principle:

Principle #20Continuity of useful action

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 probe provides a stable and accurate measurement of autophagy activity, unaffected by pH fluctuations, allowing for precise quantification of autophagy in both living and fixed cells, and can detect various types of autophagy, including microautophagy and chaperone-mediated autophagy.

Implementation Method 1

a donor having an amino acid sequence having a sequence identity of 95% or more with respect to an amino acid sequence represented by SEQ ID NO: 1

Methodology Applied

Scientific EffectFluorescence: Fluorescence

Implementation Method 2

an acceptor consisting of a fluorescent protein to be enzymatically degraded in a lysosome or a vacuole

Methodology Applied

Scientific EffectEnzymatic degradation: Enzyme

Data Source

PatentUS11203621B2PH-responsive proteolysis probe

Publication Date: 2021.12.21 RIKEN CO LTD

AI summary

The present invention provides a tool which exhibits excellent properties in the quantification of autophagy activity. A unimolecular FRET probe of the present invention includes an acceptor consisting of a fluorescent protein to be enzymatically degraded inside a lysosome or a vacuole; and a donor having an amino acid sequence having a sequence identity of 95% or more with respect to an amino acid sequence represented by SEQ ID NO: 1.