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Protein Dynamics in Living Cells

Protein Dynamics in Living Cells

Different fluorescence-based techniques are used to study the protein dynamics in living cells. These techniques include FRAP, FRET, and PET.
Fluorescent recovery after photobleaching (FRAP) is a fluorescent-protein-based detection technique used to quantify protein movement rates within the cell. This method exposes a small portion of the cell to an intense laser beam. The laser beam causes permanent photobleaching of the fluorophore-tagged proteins in the exposed region. As the bleached...

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Monitoring of Ubiquitin-proteasome Activity in Living Cells Using a Degron (dgn)-destabilized Green Fluorescent Protein (GFP)-based Reporter Protein

Monitoring of Ubiquitin-proteasome Activity in Living Cells Using a Degron (dgn)-destabilized Green Fluorescent Protein (GFP)-based Reporter Protein

Published on: November 10, 2012

A new fluorogenic peptide determines proteasome activity in single cells.

Silvana A M Urru¹, Pietro Veglianese, Ada De Luigi

¹Istituto di Ricerche Farmacologiche Mario Negri, Via La Masa 19, Milan 20156, Italy.

Journal of Medicinal Chemistry

|October 2, 2010

Summary

This summary is machine-generated.

Researchers developed a novel fluorogenic peptide to measure proteasome activity in living cells. This tool enables quantitative assessment of proteasomal function in real-time, aiding disease research.

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Area of Science:

Biochemistry
Cell Biology
Molecular Medicine

Background:

The ubiquitin-proteasome system (UPS) is crucial in cellular processes and disease pathogenesis.
UPS dysfunction is implicated in various diseases, highlighting its potential as a therapeutic target.
Current methods for assessing proteasome activity are limited in living cells, hindering in vivo studies.

Purpose of the Study:

To develop a novel, sensitive, and quantitative method for measuring proteasome activity in living cells.
To create a tool for real-time monitoring of proteasomal function under physiological and pathological conditions.

Main Methods:

Engineering of an internally quenched fluorogenic peptide containing a proteasome-specific cleavage motif.
Fusion of the peptide to the TAT protein transduction domain for cell membrane penetration.
Linking the peptide to DABCYL and EDANS fluorophores to generate a quantitative fluorescent reporter.
Assessment of in vivo proteasome activity using time-lapse and flow cytometry fluorescence analysis.

Main Results:

The engineered peptide efficiently penetrates cell membranes.
The peptide is rapidly cleaved by the proteasome's chymotrypsin-like activity.
The cleavage generates a quantitative fluorescent signal, enabling real-time monitoring of proteasome activity.
The method provides a reliable assessment of in vivo proteasome activity.

Conclusions:

The developed fluorogenic peptide reporter is an innovative tool for assessing proteasome activity in living cells.
This reporter facilitates the study of proteasomal function in both normal and disease states.
The tool has significant potential for biomarker discovery and therapeutic development targeting the ubiquitin-proteasome system.