TanGIBLE: A selective probe for evaluating hydrophobicity-exposed defective proteins in live cells

Area of Science:

• Biochemistry
• Molecular Biology
• Cell Biology

Background:

• Accumulation of defective polypeptides is implicated in numerous diseases.
• Current methods struggle to isolate and study unstable, defective proteins in a soluble state.
• A specific molecular tool is needed to detect structurally defective polypeptides.

Purpose of the Study:

• To develop a novel molecular probe for identifying and analyzing defective polypeptides.
• To create a tool capable of capturing and characterizing unstable protein products.

Main Methods:

• Engineered an artificial protein architecture using tandemly aligned BAG6 Domain I.
• Tested the probe's affinity for model defective polypeptides and endogenous polyubiquitinated proteins.
• Utilized mass spectrometry for identifying endogenous defective proteins.
• Applied the probe for real-time visualization of protein foci in stressed cells.

Main Results:

• The tandem-aligned BAG6 Domain I probe demonstrated enhanced affinity for defective polypeptides.
• The probe successfully identified endogenous defective proteins, including orphaned subunits and misassembled transmembrane proteins.
• Enabled real-time visualization of protein aggregates formed by defective polypeptides in stressed cells.

Conclusions:

• The developed molecular trap is a versatile tool for studying defective polypeptides.
• This probe allows for the evaluation of previously undetectable pools of defective proteins.
• Facilitates understanding of disease mechanisms linked to protein misfolding and aggregation.