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Eukaryotic cells can degrade proteins through several pathways. One of the most important amongst these is the ubiquitin-proteasome pathway. It helps the cell eliminate the misfolded, damaged, or unwarranted cytoplasmic proteins in a highly specific manner.
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Related Experiment Video

Updated: Feb 5, 2026

Quantifying Subcellular Ubiquitin-proteasome Activity in the Rodent Brain

Quantifying Subcellular Ubiquitin-proteasome Activity in the Rodent Brain

Published on: May 21, 2019

Scalable In Vitro Proteasome Activity Assay.

Amit Kumar Singh Gautam¹, Kirby Martinez-Fonts¹, Andreas Matouschek²

¹Department of Molecular Biosciences, The University of Texas at Austin, Austin, TX, USA.

Methods in Molecular Biology (Clifton, N.J.)

|September 23, 2018

Summary

We created a new assay to measure proteasome function using fluorescent protein substrates. This tool helps researchers screen for drugs that modify proteasome activity.

Keywords:

High-throughput degradation assay Proteasome Ubiquitin-proteasome system

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Area of Science:

Biochemistry and Molecular Biology
Drug Discovery

Background:

The proteasome is a crucial cellular machine responsible for protein degradation.
Understanding proteasome function is vital for developing treatments for diseases involving protein homeostasis.
Existing assays for proteasome activity can be limited in scope and throughput.

Purpose of the Study:

To develop a versatile and high-throughput degradation assay for studying proteasome function.
To create a modular fluorescent protein substrate for adaptable proteasome activity measurements.
To enable efficient screening of potential proteasome modulators.

Main Methods:

Development of a novel fluorescent protein-based substrate for proteasome degradation.
The substrate comprises a proteasome-binding tag, a folded domain, and an initiation region.
Assay designed for parallel processing in 384-well plates for high-throughput screening.

Main Results:

The assay effectively measures the recognition, unfolding, translocation, and hydrolysis of substrates by the proteasome.
The modular nature of the substrate allows for customization to modulate degradation rates.
The assay demonstrated suitability for exploring diverse experimental conditions and screening proteasome modulators.

Conclusions:

A robust and adaptable fluorescent protein-based assay for proteasome degradation has been established.
This assay facilitates the investigation of proteasome mechanisms and the discovery of novel therapeutic agents.
The high-throughput capability enables efficient screening for compounds that modulate proteasome activity.