A fluorescence polarization-based competition assay for measuring interactions between unlabeled ubiquitin chains and UCH37•RPN13
Introduction
Protein ubiquitination is a post-translational modification process that orchestrates almost every cellular pathway, including protein degradation, DNA repair, and cell communication [1,2]. Ubiquitin (Ub) is covalently attached to substrate proteins through a series of enzymatic reactions and removed by a class of isopeptidases referred to as deubiquitinases or DUBs. Similar to glycosylation, Ub can modify itself during multiple rounds of conjugation in the form of Ub chains [2]. There are several ways in which chain formation can occur owing to the eight amino groups: Met1, Lys6, Lys11, Lys27, Lys29, Lys33, Lys48, and Lys63. Specific chains are recognized by effector proteins (i.e., proteins containing Ub-binding domains) and DUBs, resulting in precise control over signaling pathways [3,4]. Understanding how the information embedded in different chains is translated into different biological outcomes is key to defining the ‘Ub code’.
Measuring interactions between Ub chains and their binding partners is, however, rather challenging. Common methods for characterizing these interactions include isothermal titration calorimetry (ITC), surface plasmon resonance (SPR), and fluorescence spectroscopy [[5], [6], [7], [8]]. In the context of Ub chains and their interactions with Ub-binding proteins (UBPs), each of these methods has their drawbacks. ITC requires large amounts of material, which is often difficult to achieve with chains longer than two subunits. SPR has the issue of protein immobilization, which can adversely affect native interactions and requires immobilization tags on either the Ub chain or UBP. Similarly, fluorescence-based approaches necessitate the introduction of fluorophore tags, which can also incur non-native binding properties.
To circumvent these issues, we sought to devise a new method for measuring interactions between Ub chains and UBPs. The method described herein is based on a fluorescence polarization competition (FPcomp) assay [9]. FPcomp starts with a high-polarization state in which fluorophore-labeled Ub is bound to a UBP. Titrating in unlabeled Ub then leads to a decrease in polarization as the labeled Ub is forced to dissociate. The resulting titration curve yields an inhibitory constant, which can be treated as an apparent dissociation constant (Ki). Performing the same experiments with unlabeled Ub chains instead of mono-Ub then furnishes a range of Kis whose differences can be correlated with chain length. While this method still utilizes a fluorophore-tagged Ub derivative, the Ub chains themselves are unlabeled. Thus, the FPcomp assay retains the sensitivity of fluorescence measurements and allows comparisons in binding interactions between label-free chains.
To explore the utility of our FPcomp assay, we chose the Ub C-terminal hydrolase UCH37 also known as UCHL5 [10]. UCH37 is a cysteine-dependent DUB involved in regulating several fundamental cellular processes such as cell cycle progression, genome stability, cell migration, and TGF-β signaling [[11], [12], [13], [14]]. Most of these cellular functions have been ascribed to the ability of UCH37 to interact with two distinct multi-subunit complexes: the 26S proteasome and the INO80 chromatin remodeling complex [15]. The UBP RPN13/ADRM1 recruits UCH37 to the proteasome and INO80G/NFRKB interacts with UCH37 in the INO80 complex [[15], [16], [17], [18], [19]]. RPN13 and INO80G have opposing effects on UCH37; RPN13 activates UCH37 toward model mono-Ub substrates and INO80G acts as an inhibitor.
Structural studies have revealed Ub binding sites in both UCH37 and RPN13. UCH37 engages Ub through a large surface exposed region (∼2300 Å2) of the catalytic UCH domain [20,21]. By binding the C-terminal UCH37-like domain (ULD) of UCH37, the C-terminal DEUBAD domain of RPN13 enhances the interaction between Ub and the UCH domain. RPN13 also contains an N-terminal pleckstrin-like receptor for Ub (PRU) domain that preferentially binds Lys48 di-Ub over mono-Ub [22]. Based on the presence of multiple Ub-binding domains, we speculated that the UCH37•RPN13 complex could exhibit high affinity toward Ub chains. Using the FPcomp assay we discovered that a catalytically dead variant of the UCH37•RPN13 complex (UCH37C88A•RPN13) does indeed favor Ub chains over mono-Ub; chains with more than two subunits are preferred. What is surprising, however, is that in the absence of the PRU domain of RPN13, the UCH37C88A•RPN13DEUBAD complex still exhibits a strong preference for longer Ub chains. Indeed, the binding affinities we measured by FPcomp are quite similar for both UCH37-containing complexes. Lastly, we show that the FPcomp assay can be applied in a high-throughput manner for the purpose of identifying inhibitors. This is important considering both UCH37 and RPN13 have emerged as cancer targets [[23], [24], [25]].
Section snippets
Cloning, protein expression and purification
Site directed mutagenesis was performed to generate catalytically inactive UCH37 (UCH37 C88A) and the Ub cysteine variants Ub K6C, Ub K11C, Ub K29C, Ub K48C and Ub K63C. The gene encoding UCH37 C88A was cloned into pVP16 vector with 8xHis MBP tag and TEV protease cleavable site. 6xHis-tagged RPN13 was expressed from a pGXT4 vector. UCH37 C88A and RPN13 were co-purified using amylose resin and Ni2+ affinity column chromatography, followed by size exclusion chromatography with a HiLoad 26/60
Results and discussion
We aimed to develop a method with low material consumption that allows measurement of binding strengths between Ub chains and ubiquitin binding proteins (UBPs). These efforts led to a fluorescence polarization competition (FPcomp) assay in which unlabeled ubiquitin chains compete with fluorophore-labeled mono-Ub (Ub-fluorescein) for binding UBPs (Fig. 1). As our model system, we focused on the proteasome-associated DUB UCH37 and its partner protein RPN13, which has an N-terminal PRU domain
Conclusion
Here, we report on a FPcomp assay for measuring the binding affinity between a deubiquitinase complex, UCH37•RPN13, and unlabeled mono-Ub or Ub chains. The FPcomp assay overcomes many of the limitations of conventional binding measurements; there is low sample consumption, high sensitivity, and fluorophore labeling is only required for the tracer, not the actual Ub derivative of interest. We also demonstrate that this assay can be used for high-throughput screening, which is not feasible with
Acknowledgments
This research was supported by a National Institutes of Health (NIH) grant (GM110543 to E.R.S.). J. D. was supported in part by a NIH Chemistry and Biology Interface Predoctoral Training Grant (GM008515).
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