Ling-chun Chen

Small-molecule displacement of a cryptic degron causes conditional protein degradation

The ability to rapidly regulate the functions of specific proteins in living cells is a valuable tool for biological research. Here we describe a novel technique by which the degradation of a specific protein is induced by a small molecule. A protein of interest is fused to a Ligand-Induced Degradation (LID) domain resulting in the expression of a stable and functional fusion protein. The LID domain is comprised of the FK506- and rapamycin-binding protein (FKBP) and a 19-amino acid degron fused to the C-terminus of FKBP. In the absence of the small molecule Shield-1, the degron binds to the FKBP protein and the fusion protein is stable. Shield-1 binds tightly to FKBP thereby displacing the degron and inducing rapid and processive degradation of the LID domain and any fused partner protein. Structure-function studies of the 19-residue peptide showed that a four-amino acid sequence within the peptide is responsible for degradation.

Destabilizing domains derived from the human estrogen receptor.

Methods to rapidly and reversibly perturb the functions of specific proteins are desirable tools for studies of complex biological processes. We have demonstrated an experimental strategy to regulate the intracellular concentration of any protein of interest by using an engineered destabilizing protein domain and a cell-permeable small molecule. Destabilizing domains have general utility to confer instability to a wide range of proteins including integral transmembrane proteins. This study reports a destabilizing domain system based on the ligand binding domain of the estrogen receptor that can be regulated by one of two synthetic ligands, CMP8 or 4-hydroxytamoxifen.

Recent Progress with FKBP-Derived Destabilizing Domains

The FKBP-derived destabilizing domains are increasingly being used to confer small molecule-dependent stability to many different proteins. The L106P domain confers instability to yellow fluorescent protein when it is fused to the N-terminus, the C-terminus, or spliced into the middle of yellow fluorescent protein, however multiple copies of L106P do not confer greater instability. These engineered destabilizing domains are not dominant to endogenous degrons that regulate protein stability.

The E3 Ubiquitin Ligase UBE3C Enhances Proteasome Processivity by Ubiquitinating Partially Proteolyzed Substrates

Background: An RNAi screen identified UBE3C as a key player in degradation of a model unfolded protein. Results: UBE3C knockdown results in incomplete degradation of relatively stable substrates. Conclusion: UBE3C enhances proteasome processivity to prevent the accumulation of potentially harmful protein fragments. Significance: This advances our understanding of proteasome processivity and the consequences of defects therein. To maintain protein homeostasis, cells must balance protein synthesis with protein degradation. Accumulation of misfolded or partially degraded proteins can lead to the formation of pathological protein aggregates. Here we report the use of destabilizing domains, proteins whose folding state can be reversibly tuned using a high affinity ligand, as model substrates to interrogate cellular protein quality control mechanisms in mammalian cells using a forward genetic screen. Upon knockdown of UBE3C, an E3 ubiquitin ligase, a reporter protein consisting of a destabilizing domain fused to GFP is degraded more slowly and incompletely by the proteasome. Partial proteolysis is also observed when UBE3C is present but cannot ubiquitinate substrates because its active site has been mutated, it is unable to bind to the proteasome, or the substrate lacks lysine residues. UBE3C knockdown also results in less substrate polyubiquitination. Finally, knockdown renders cells more susceptible to the Hsp90 inhibitor 17-AAG, suggesting that UBE3C protects against the harmful accumulation of protein fragments arising from incompletely degraded proteasome substrates.

Rapid and tunable control of protein stability in Caenorhabditis elegans using a small molecule.

Destabilizing domains are conditionally unstable protein domains that can be fused to a protein of interest resulting in degradation of the fusion protein in the absence of stabilizing ligand. These engineered protein domains enable rapid, reversible and dose-dependent control of protein expression levels in cultured cells and in vivo. To broaden the scope of this technology, we have engineered new destabilizing domains that perform well at temperatures of 20–25°C. This raises the possibility that our technology could be adapted for use at any temperature. We further show that these new destabilizing domains can be used to regulate protein concentrations in C. elegans. These data reinforce that DD can function in virtually any organism and temperature.

Intracellular Context Affects Levels of a Chemically Dependent Destabilizing Domain

The ability to regulate protein levels in live cells is crucial to understanding protein function. In the interest of advancing the tool set for protein perturbation, we developed a protein destabilizing domain (DD) that can confer its instability to a fused protein of interest. This destabilization and consequent degradation can be rescued in a reversible and dose-dependent manner with the addition of a small molecule that is specific for the DD, Shield-1. Proteins encounter different local protein quality control (QC) machinery when targeted to cellular compartments such as the mitochondrial matrix or endoplasmic reticulum (ER). These varied environments could have profound effects on the levels and regulation of the cytoplasmically derived DD. Here we show that DD fusions in the cytoplasm or nucleus can be efficiently degraded in mammalian cells; however, targeting fusions to the mitochondrial matrix or ER lumen leads to accumulation even in the absence of Shield-1. Additionally, we characterize the behavior of the DD with perturbants that modulate protein production, degradation, and local protein QC machinery. Chemical induction of the unfolded protein response in the ER results in decreased levels of an ER-targeted DD indicating the sensitivity of the DD to the degradation environment. These data reinforce that DD is an effective tool for protein perturbation, show that the local QC machinery affects levels of the DD, and suggest that the DD may be a useful probe for monitoring protein quality control machinery.

A Novel Destabilizing Domain Based on a Small-Molecule Dependent Fluorophore.

Tools that can directly regulate the activity of any protein-of-interest are valuable in the study of complex biological processes. Herein, we describe the development of a novel protein domain that exhibits small molecule-dependent stability and fluorescence based on the bilirubin-inducible fluorescent protein, UnaG. When genetically fused to any protein-of-interest, this fluorescent destabilizing domain (FDD) confers its instability to the entire fusion protein, facilitating the rapid degradation of the fusion. In the presence of its cognate ligand bilirubin (BR), the FDD fusion becomes stable and fluorescent. This new chemical genetic tool allows for rapid, reversible, and tunable control over the stability and fluorescence of a wide range of protein targets.

Distinct transcriptional responses elicited by unfolded nuclear or cytoplasmic protein in mammalian cells

Eukaryotic cells possess a variety of signaling pathways that prevent accumulation of unfolded and misfolded proteins. Chief among these is the heat shock response (HSR), which is assumed to respond to unfolded proteins in the cytosol and nucleus alike. In this study, we probe this axiom further using engineered proteins called ‘destabilizing domains’, whose folding state we control with a small molecule. The sudden appearance of unfolded protein in mammalian cells elicits a robust transcriptional response, which is distinct from the HSR and other known pathways that respond to unfolded proteins. The cellular response to unfolded protein is strikingly different in the nucleus and the cytosol, although unfolded protein in either compartment engages the p53 network. This response provides cross-protection during subsequent proteotoxic stress, suggesting that it is a central component of protein quality control networks, and like the HSR, is likely to influence the initiation and progression of human pathologies. DOI: http://dx.doi.org/10.7554/eLife.07687.001

A method to rapidly create protein aggregates in living cells.

The accumulation of protein aggregates is a common pathological hallmark of many neurodegenerative diseases. However, we do not fully understand how aggregates are formed or the complex network of chaperones, proteasomes and other regulatory factors involved in their clearance. Here, we report a chemically controllable fluorescent protein that enables us to rapidly produce small aggregates inside living cells on the order of seconds, as well as monitor the movement and coalescence of individual aggregates into larger structures. This method can be applied to diverse experimental systems, including live animals, and may prove valuable for understanding cellular responses and diseases associated with protein aggregates.