An Inducible System for Rapid Degradation of Specific Cellular Proteins Using Proteasome Adaptors.

Shameika R Wilmington,Andreas Matouschek,Jeffrey L Brodsky

doi:10.1371/journal.pone.0152679

Shameika R Wilmington, Andreas Matouschek + Show 1 more

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https://doi.org/10.1371/journal.pone.0152679

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Abstract

A common way to study protein function is to deplete the protein of interest from cells and observe the response. Traditional methods involve disrupting gene expression but these techniques are only effective against newly synthesized proteins and leave previously existing and stable proteins untouched. Here, we introduce a technique that induces the rapid degradation of specific proteins in mammalian cells by shuttling the proteins to the proteasome for degradation in a ubiquitin-independent manner. We present two implementations of the system in human culture cells that can be used individually to control protein concentration. Our study presents a simple, robust, and flexible technology platform for manipulating intracellular protein levels.

Highlights

There are many reasons to control protein concentrations artificially, for example to study complex biological systems without genetic manipulation or to elucidate protein function
Most eukaryotic intracellular protein degradation is controlled by the ubiquitin proteasome system (UPS), which tunes the concentrations of hundreds of regulatory proteins [1]
We added three more modifications to the adaptor: we inserted the red fluorescent protein mCherry [40] between the UbL and FKBP12-rapamycin-binding protein (FRB) domains to allow us to monitor abundance of the adaptor protein, we fused a maltose-binding domain to the C terminus of the FRB domain to stabilize the adaptor protein in cells [41], and we introduced a mutation into the FRB domain [16, 42] to allow it to interact with a derivative of rapamycin (AP21967, MaRap or rapalog) [16, 42]

Summary

Introduction

There are many reasons to control protein concentrations artificially, for example to study complex biological systems without genetic manipulation or to elucidate protein function. The most common way to adjust protein concentrations artificially is by regulating protein synthesis. Protein concentrations in the cell are a function of their rates of synthesis and degradation, so another way to manipulate protein abundance is by altering protein degradation. Proteins are targeted to the proteasome by a degradation signal, or degron, that has two components: a proteasomebinding tag in the form of polyubiquitin chains and a proteasomal initiation region [2]. Degradation is regulated mainly by the covalent attachment of polyubiquitin chains, which serves as the proteasome-binding tag. The polyubiquitin chains are recognized by proteasome receptors and degradation initiates at a disordered region in the substrate called an initiation site [2]. The protein is threaded into the proteolytic chamber where it is hydrolyzed into short peptides [1, 3]

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