An assay for 26S proteasome activity based on fluorescence anisotropy measurements of dye-labeled protein substrates

Abstract

The 26S proteasome is the molecular machine at the center of the ubiquitin proteasome system and is responsible for adjusting the concentrations of many cellular proteins. It is a drug target in several human diseases, and assays for the characterization of modulators of its activity are valuable. The 26S proteasome consists of two components: a core particle, which contains the proteolytic sites, and regulatory caps, which contain substrate receptors and substrate processing enzymes, including six ATPases. Current high-throughput assays of proteasome activity use synthetic fluorogenic peptide substrates that report directly on the proteolytic activity of the proteasome, but not on the activities of the proteasome caps that are responsible for protein recognition and unfolding. Here, we describe a simple and robust assay for the activity of the entire 26S proteasome using fluorescence anisotropy to follow the degradation of fluorescently labeled protein substrates. We describe two implementations of the assay in a high-throughput format and show that it meets the expected requirement of ATP hydrolysis and the presence of a canonical degradation signal or degron in the target protein.

Introduction

The 26S proteasome is a 2.5 MDa molecular machine, composed of at least 33 different proteins, that recognizes and hydrolyzes proteins targeted for degradation by polyubiquitin modifications [1], [2]. The proteasome consists of a 20S proteolytic core particle capped by one or two 19S regulatory particles. The core particle contains six proteolytic sites, which are only accessible through gated pores at either end of the particle [3], [4], [5]. The regulatory caps contain receptors that recognize ubiquitin chains, six ATPase subunits that unfold and translocate substrates through a central channel to the proteolytic sites in the core particle, and enzymatic subunits that remove the ubiquitin chains from substrates as they are translocated to degradation [6]. There are at least three different caps that can associate with the core particle, but the 19S regulatory particle is thought to be the cap primarily responsible for ubiquitin-dependent degradation [1].

The proteasome degrades a large number of regulatory proteins, such as cell cycle regulators and transcription factors; it removes misfolded and damaged proteins from cells; and it produces some of the peptides displayed by MHC complexes [7]. The proteasome has been a target for drug design in several human diseases, and proteasome inhibitors are used successfully in the treatment of cancers, particularly multiple myeloma and mantle cell lymphoma [8], [9], [10]. Cancer drugs currently used in the clinic target the proteolytic active sites in 20S core particles to inhibit proteasomal activity. Recently, proteasome inhibitors targeting the 19S regulatory particle have been developed, including inhibitors of the deubiquitinating activities associated with the proteasome (Usp14/Ubp6 and Uch37/UCHL5) and molecules that disrupt ubiquitin binding to the proteasome [11]. One inhibitor that targets both Usp14 and Uch37 results in the accumulation of polyubiquitinated proteins, which leads to cell apoptosis and has been shown to inhibit tumor growth in mice [12], [13].

In several neurodegenerative disorders, accumulation of aggregation-prone proteins has been implicated in disease progression, and in some cases the proteasome's activity appears to be impaired in affected cells [14], [15], [16], [17]. Thus, it may be beneficial to develop drugs that can activate proteasome activity to improve the clearance of toxic proteins and protein aggregates. Indeed, an inhibitor of the deubiquitinating enzyme Usp14 enhances proteasomal protein degradation and leads to the clearance of protein aggregates in human cells [18].

The search for new modulators of proteasome activity requires assays to measure proteasomal activity that can be carried out in an efficient and reproducible manner. One common proteasomal degradation assay uses peptide substrates that release a fluorescent group upon cleavage, resulting in an increase in fluorescence intensity that is proportional to degradation activity [19], [20]. These substrates are convenient and sensitive reporters of the proteolytic activity of the 20S core of the proteasome and can be used in a high-throughput format. However, the peptide substrates are less well suited to investigate the activity of the 26S proteasome holoenzyme, as their hydrolysis bypasses the steps of substrate recognition by the ubiquitin receptors and does not require unfolding by the ATPase subunits. 26S proteasome activity is generally monitored by following the degradation of ubiquitinated proteins [21], [22]. Ubiquitinated proteins can be produced in vitro using purified enzymes, cell lysates, chemical strategies, or engineered bacteria [21], [23], [24], [25], [26], [27], [28], [29], [30]. The assays typically follow protein degradation by sampling the reaction at discrete time points and measuring the amount of the substrate protein remaining by SDS-PAGE and protein staining, Western blotting, or autoradiography (e.g., [21], [22], [31], [32], [33], [34], [35]). These methods provide information on protein size and thus on the ubiquitination state and can detect the formation of partially degraded protein fragments, but can be time-, labor-, and reagent-consuming and difficult to implement in a high-throughput format. Incorporating a fluorescent protein such as green fluorescent protein (GFP) into the model protein makes it possible to follow protein degradation by measuring fluorescence using in-gel or high-throughput assays. For example, substrates containing a degradation signal (degron)-fused GFP have been used to monitor proteasome activity by following the decay of fluorescence intensity resulting from degradation of the GFP moiety (e.g., [29], [36], [37]). Fluorescent proteins often have complicated maturation kinetics and can be difficult to unfold so that they resist degradation by some ATP-dependent proteases [38]. However, easier-to-unfold circular permutants of GFP have been developed that make it possible to monitor degradation even by proteases less robust than the proteasome [29], [38], [39], [40], [41].

Here, we present a 26S proteasome activity assay based on the measurement of the fluorescence anisotropy of a small molecule dye attached to a substrate protein (Fig. 1a). The fluorescence anisotropy signal depends on the lifetime of the fluorophore and its rotational relaxation time [42], [43], which in turn depends on the size of the dye-labeled molecule. Thus, fluorescence anisotropy can be used to interpret the changes in both the abundance and the molecular size of substrates in the reaction. Indeed, anisotropy has been applied in the studies of proteolysis catalyzed by conventional proteases such as thrombin, enterokinase, Factor Xa, chymotrypsin, trypsin, or more specialized enzymes such as calpain II or botulism neurotoxin metalloproteases often in a high-throughput format [44], [45], [46], [47]. The assay described here measures fluorescence anisotropy to monitor 26S proteasome activity continuously (Fig. 1a). It follows the degradation of a proteasome-targeted protein into short polypeptides and thus reflects all the steps that occur within the 19S cap: substrate binding to the substrate receptors, initiation of degradation, unfolding and translocation into the core particle, and proteolysis. We describe two implementations of the assay in which an Alexa Fluor 546 dye is conjugated to the protein through a SNAP-tag. In one implementation, the substrate is targeted to the proteasome via a polyubiquitin modification that is attached to the protein through an in vitro ubiquitination reaction with purified E1, E2, and E3 enzymes. In the other implementation, the substrate is targeted to the proteasome by a ubiquitin-like (UbL) domain encoded in the substrate's gene. The UbL domain is recognized by the proteasome, thus bypassing the need for ubiquitination. In both cases, the dye is attached to a folded protein that must be unfolded and translocated into the core particle in order to be degraded, which allows probing 26S proteasomal activity. We anticipate that this assay will be a useful tool for high-throughput experiments to probe the effect of inhibitors and activators on proteasome activity, especially those that may act upstream of peptide hydrolysis.