Observing real-time ubiquitination in high throughput with fluorescence polarization

Abstract

Reconstitution of ubiquitin conjugation and deconjugation in vitro provides access to valuable information on enzyme kinetics, specificity, and structure-function relationships. Classically, these biochemical assays culminate in separation by SDS-PAGE and analysis by immunoblotting, an approach that requires additional time, can be difficult to quantify, and provides granular snapshots of the reaction progression. To address these limitations, we have implemented a fluorescence polarization-based assay that tracks ubiquitin conjugation and deconjugation in real time based upon changes in molecular weight. We find this approach, which we have termed “UbiReal”, to greatly facilitate biochemical studies such as mutational analyses, specificity determination, and inhibitor characterization.

1. INTRODUCTION

Ubiquitination is a versatile post-translational modification that is used to regulate virtually every cellular pathway in eukaryotes with both degradative and non-degradative outcomes (1). Approximately 5% of the human transcriptome encodes ubiquitin (Ub)-regulating proteins, and the dysregulation of even individual proteins in this intricate system can lead to disease states like cancer, neurodegenerative diseases, and autoimmunity in humans (2, 3). In plants, an estimated 6% of the transcriptome is dedicated to the ubiquitin proteasome system (UPS), which crucially regulates plant responses to hormones, stressors, and infectious pathogens (4). The vast number of ubiquitin regulators in humans includes ubiquitin-activating E1s (2), ubiquitin-conjugating E2s (~35), ubiquitin-ligating E3s (>600), deubiquitinases (DUBs, ~100), as well as proteins with ubiquitin-binding domains (UBDs, >100), many of which are incompletely characterized (1, 2). For comparison, the model plant Arabidopsis thaliana expresses 2 E1s, at least 37 E2s, >1400 E3s, and approximately 64 DUBs, with a considerable amount more E3s involved in the UPS relative to humans (4). In a typical ubiquitination event, an ATP-dependent reaction allows the formation of an activated E1~Ub complex, in which the Ub C-terminus is covalently attached to the E1 active site cysteine through a high energy, thioester linkage. Binding of an E2 enables Ub transfer and formation of an activated E2~Ub complex. Next, an E3 ligase will facilitate Ub transfer onto a substrate protein. In the case of E3 ligases from the Really Interesting New Gene (RING) family, Ub is transferred directly from the E2 to a substrate, whereas E3 ligases from the Homologous to the E6AP C-terminus (HECT) and RING-between-RING (RBR) families form one final E3~Ub intermediate before modifying a substrate.

The consequences of ubiquitin dysregulation in humans and plants with respect to the vast number of ubiquitin regulatory proteins make it pertinent to understand how E1s, E2s, E3s, DUBs, and UBDs interact with each other to generate and fine-tune ubiquitin signals. Similarly, therapeutic manipulation of those interactions and ubiquitination events will offer important advances in disease intervention for humans and improved agricultural strategies for plants (3, 4). While many robust assays already exist for monitoring ubiquitin regulation, most are either highly specialized to one network of interactions or are specific to deubiquitination events. Therefore, we sought to develop a versatile assay that could be used in real time to visualize the whole E1-E2-E3-DUB cascade—as well as the effects of inhibitors (or activators) on those regulators—in a high-throughput format.

Using fluorescence polarization (FP), which effectively monitors changes in protein size by virtue of tumbling rate, we developed the “UbiReal” assay that discerns the flow of ubiquitin through the entire E1-E2-E3-DUB signaling cascade. A ubiquitin labeled at the N-terminus with a TAMRA fluorophore (T-Ub) generates a small FP signal because of its small size, but a larger FP signal upon addition of the E1 and subsequent ATP-dependent formation of the E1~Ub complex (Fig. 1). Next, addition of an E2 results in transfer of the T-Ub from the E1 to produce the relatively smaller E2~Ub conjugate, causing a resultant decrease in FP signal (Fig. 1). Addition of a HECT-type E3, followed by excess unlabeled ubiquitin, produces large increases in FP signal as the E3 generates poly-ubiquitin signals and/or adds ubiquitin onto itself (a process known as auto-ubiquitination) (Fig. 1). Finally, addition of a DUB reduces the FP signal over time as the E3-generated poly-ubiquitin signals are hydrolyzed. We have validated this real-time assay in a high-throughput, 384-well format to characterize an E1 inhibitor, monitor amino acid selectivity of E2s, determine specificity of E2 and E3 pairs, and observe the ubiquitin chain-type specificity of E3s and DUBs (5). Here, we provide a detailed description of these and additional applications of UbiReal, demonstrating its utility as a tool for basic and applied ubiquitin research.

Fig. 1.

E1-E2-E3 ubiquitin conjugation and DUB hydrolysis using UbiReal. T-Ub (black) was monitored before addition of E1 to generate E1~T-Ub (red) (5 mM ATP and 10 mM MgCl₂ were already present in the buffer). Next, the E2 UBE2D3 at 300 nM was added to produce E2~T-Ub (green). The E3 NleL at 700 nM was then added to produce NleL~T-Ub (dark blue) (with the possibility of ubiquitin chain formation). Next, unlabeled Ub was added at 25 μM and monitored for several cycles, showing NleL-conjugated poly-ubiquitin substrates which amplified the T-Ub signal (purple). Finally, the non-specific DUB USP21 at 250 nM was added and monitored for several cycles to begin cleaving the poly-ubiquitin signals back into mono-ubiquitin (cyan). The separation in FP signal between each complex presents a potential point at which to explore inhibition/activation by chemical or protein modulators. Other applications include investigating functional mutations of E1s, E2s, E3s, and DUBs, or interactions therein. Raw FP signal is shown. Data represent a single experiment.

2. MATERIALS

2.1. Enzymes required for the ubiquitination reaction

The recombinant enzymes that are required (i.e., E1, E2, etc.) will depend on the specific application and focus of research. These proteins can be produced in-house or many commonly used proteins can be purchased through companies. For the example assays that follow, ubiquitin conjugation is performed with human enzymes including the E1 UBA1, the E2s UBE2D3 or UBE2L3, and the E3 NEDD4L, or with bacterial E3 ligases including NleL from Enterohemorrhagic Escherichia coli or SopA from Salmonella enterica serovar Typhimurium. Ubiquitin deconjugation is performed using the human DUBs OTULIN and USP21, as well as the engineered OTUB1* and AMSH*(6).

2.2. Fluorescent probes

Any ubiquitin with a fluorophore on its N-terminus and an intact C-terminus should function in UbiReal. All experiments herein utilized ubiquitin labeled at the N-terminus with a TAMRA fluorophore (T-Ub) (7) (available from UBPBio). We have also observed equal success using N-terminally labeled fluorescein ubiquitin (5) (available from R&D Systems). Studies that require an available N-terminus, such as the formation of linear poly-ubiquitin, should consider an alternative labeling site such as modification of a Ser20Cys ubiquitin variant (see reference (8) as an example).

2.3. Protein concentrations and buffer conditions

Unless otherwise specified, enzyme concentrations for each assay are as follows: 100 nM T-Ub, 125 nM E1, 2 μM E2 (UBE2D3 or UBE2L3), 2 μM E3 (NleL, SopA, or NEDD4L) (see Note 1).

All experiments were performed in buffer containing 25 mM sodium phosphate (pH 7.4), 150 mM sodium chloride, 0.5 mM dithiothreitol (DTT), 10 mM magnesium chloride, with any augmentations and the specific time of 5 mM ATP addition noted.

2.4. Plate reader and assay parameters

All experiments were performed using a BMG LabTech CLARIOstar plate reader set to a controlled temperature of 20 °C. Data were collected every 30–60 seconds with 20 flashes per well. The instrument was set to read the T-Ub TAMRA fluorophore using an excitation wavelength of 540 nm, an emission wavelength of 590 nm, and an LP 566 nm dichroic mirror. All experiments utilized 384-well small-volume HiBase microplates using total volumes of approximately 20 μL per sample well.

3. METHODS

In this chapter, we provide detailed protocols for selected applications of the UbiReal methodology. Subheading 3.1 details how to use UbiReal for studying functional mutations of E3 ubiquitin ligases. Subheading 3.2 describes how the UbiCRest methodology (9) can be applied to UbiReal to determine chain specificities of E3 ubiquitin ligases. Finally, Subheading 3.3 explains a proof-of-concept application of UbiReal as a screen for ubiquitination inhibitors by quantifying inhibition of the E1~Ub complex by PYR-41 (10).

3.1. Monitoring activity of E3 ubiquitin ligases

1. Prepare a 2X master mix (10 μL × number of samples) containing E1, E2, T-Ub, and 37.5 μM unlabeled wild-type (WT) ubiquitin substrate (see Note 2) in the described buffer lacking ATP. After preparation, allow the master mix to come to room temperature (keeping it in a dark place) for approximately 5–10 minutes (see Notes 3 and 4).

2. Prepare the E3 samples, as well as a ‘no E3’ negative control, at 2X the desired final concentration in buffer containing 10 mM ATP and similarly allow these samples to come to room temperature.

3. Add 10 μL of the 2X master mix from Step 1 to sample wells of a 384-well plate and insert the plate into the plate reader. Begin the FP time-course experiment and record the baseline FP signal of each sample for 5–10 cycles.

4. Pause the FP experiment on the plate reader. Remove the plate from the plate reader and add 10 μL of the E3 samples (or the ‘no E3’ control) from Step 2 to each sample well, mix, and quickly resume the FP experiment in the plate reader (see Note 5). Monitor the experiment for 1–2 hours, or until no further change in FP signal is observed (see Note 6).

5. Analyze and plot the data to compare the kinetics of the E3s (Fig. 2) (see Subheading 3.4).

Fig. 2.

Ubiquitin conjugation assay using the HECT-type E3 ubiquitin ligases NleL and SopA, and some of their functionally defective mutants. The C753A mutants are catalytically inactive forms of both E3s, where the active site cysteine has been mutated to alanine. The NleL F569A mutant lacks a functional phenylalanine residue that supports binding of NleL to the E2 UBE2L3 and subsequent Ub transfer (14). FP data shown are normalized to a ’no E3’ control. Data represent a single experiment.

3.2. Applying UbiCRest to UbiReal to determine poly-ubiquitin linkage types

1. Prepare a 1X master mix (15 μL × number samples) of E1, E2, E3, T-Ub, and 37.5 μM unlabeled WT ubiquitin in the described buffer. Save a portion of the master mix without ATP added, at least 15 μL per DUB to be used later. Finally, add ATP to the remaining master mix.

2. Let the reaction proceed in the dark at 37 °C for 1–2 hours, or more depending on the kinetics of the E2 and E3.

3. Quench the ubiquitin conjugation reaction by adding a solution of high molarity EDTA and DTT to a final concentration of 30 mM and 5 mM, respectively.

4. Prepare the DUBs at 4X the final desired concentration in buffer supplemented with 10 mM DTT (5 μL × number of samples treated by that DUB).

5. Distribute 15 μL of each 1X master mix (including both the +ATP and the ‘no ATP’ control mixtures) from Steps 1–3 to the 384-well plate. Begin the FP time-course experiment and record the baseline FP signal of each sample for 5–10 cycles.

6. Pause the FP experiment on the plate reader. Remove the plate, and add 5 μL of the DUB (or buffer as the negative control), mix, and quickly resume the FP experiment in the plate reader (see Note 4). Each DUB used to cleave the +ATP master mix should also be added to a ’no ATP’ master mix as the positive control (see Note 5). Monitor the experiment for 1–2 hours, or until no further change in FP signal is observed (see Note 6).

7. Analyze and plot the data to compare the kinetics of each DUB treatment (Fig. 3) (see Subheading 3.4).

Fig. 3.

Ubiquitin deconjugation assay using starting material generated by the E3 ubiquitin ligase NEDD4L that is cleaved using several different DUBs with varied linkage specificities. Since K63-specific AMSH cleaves a large amount of substrate, these data support the K63-specificity of NEDD4L (11). AMSH appears unable to cleave the most proximal ubiquitin linkage on the substrate (NEDD4L in this case) and so the difference between the AMSH and nonspecific USP21 may indicate the relative presence of poly-ubiquitin vs. mono-ubiquitinated NEDD4L (see reference (5)). FP data shown are normalized to positive and negative controls (see Subheading 3.4). Data represent a single experiment.

3.3. UbiReal to quantify inhibitor potency

1. Prepare the inhibitor at concentrations at least 40X above the highest desired final value (see Note 7).

2. As starting material, generate the appropriate ubiquitin complex that is the target of the inhibitor. For example, if the inhibitor targets a DUB, generate ubiquitin chains as in Steps 1–3 of Subheading 3.2. In this example, the drug PYR-41 inhibits formation of the E1~Ub complex (Fig. 1), and so a mixture of apo E1 and T-Ub in the absence of ATP is the starting material (Fig. 4) (see Note 8).

3. In the 384-well plate, add the starting material (without inhibitor) from Step 2 and begin the FP time-course experiment, recording the baseline FP signal of each sample for 5–10 cycles.

4. Pause the experiment and add the inhibitor dilutions from Step 1 into the sample wells, mix, and again record the baseline FP signal for 5–10 cycles (see Note 4).

5. Pause the experiment one last time, add the missing substrate to initiate the reaction and mix. For example, if studying a DUB inhibitor, then add the DUB now. In this experiment, the missing substrate is ATP, and so ATP is added now to initiate the reaction (see Note 4).

6. Quickly return the plate to the plate reader, and let the experiment proceed for 1–2 hours, or until no further change FP signal is observed (see Note 6).

7. Analyze and plot the data to observe the inhibitor potency (Fig. 4) (see Subheading 3.4).

Fig. 4.

PYR41-mediated inhibition of E1~Ub complex formation. E1 and T-Ub were incubated with dilutions of PYR-41 and formation of the E1~Ub complex was monitored following addition of ATP. The negative control of ’no ATP’ is shown for reference. Raw FP data are shown. Data represent a single experiment.

3.4. Data analysis

1. Data analysis for each experiment is simple but relies on appropriate positive and negative controls (which will vary for each experiment) to be able to appropriately normalize the data.

2. Subheading 3.1, which explores the effect of functional mutations on ligation activity for an E3 ubiquitin ligase, requires a positive control (WT E3) representing the highest possible signal, as well as a negative control (‘no E3’, or a catalytically inactive form of the E3) representing the lowest possible FP signal. Data can be presented as in Fig. 2, where it is normalized to only the negative control to determine the change in FP over time, or normalized using Equation 1 (see below).

3. Subheading 3.2 requires a negative control (no DUB) representing the highest possible FP signal and a positive control (DUB with the ligation mixture lacking ATP) representing the lowest possible signal and maximum DUB cleavage (this control is specific to each DUB, see Note 5).

4. Each data point may be normalized as in Fig. 3 using the following equation:

   1. $$\left[\raisebox{1ex}{$\left(X_t-L{C}_t\right)$}\!\left/ \!\raisebox{-1ex}{$\left(H{C}_t-L{C}_t\right)$}\right.\right]*100\%$$ Equation 1
   2. Where X represents the sample of interest, HC represents the control with the highest possible signal, and LC represents the control with the lowest possible signal, calculated at each time point t. (see Note 9.)

5. Subheading 3.3, and normalizing inhibitor data in general, requires a control sample with minimal inhibition as well as a control sample with maximum inhibition. For example, in Fig. 4 PYR-41 prevents E1~Ub complex formation, and so maximum inhibition is the lowest possible FP signal (T~Ub alone) and minimal inhibition is the maximum possible FP signal (E1~T-Ub) (Fig. 1). In order to achieve these controls with the equivalent buffer conditions in Subheading 3.3 and Fig. 4, the maximum inhibition control is a sample of E1, T-Ub, and the maximum dose of PYR-41 but lacking ATP, while the minimum inhibition control is a sample of E1, T-Ub and ATP given DMSO alone instead of PYR-41. The exact controls needed to properly normalize the data for a specific experiment will differ on a case-by-case basis.

4. NOTES

1. Enzyme concentrations should be adjusted to suit enzyme kinetics and the desired reaction step. For E1, we have noted a strong dependence on the specific activity of the enzyme preparation, so a higher concentration may be required in order to achieve full activation of the T-Ub substrate. For characterizing E3s, using a concentration of the E3 that consumes the entirety of the ubiquitin substrate in 1–2 hours is generally ideal.

2. Delayed addition of unlabeled Ub produces a higher signal from the early transfer of T-Ub, as done in Fig. 1, while in this example the T-Ub and unlabeled Ub are mixed together prior to initiating the reaction. Ubiquitin mutants can also be utilized to explore chain specificity of a ubiquitin ligase. For example, for a K63-specific ligase like NEDD4L (11), using a panel of ubiquitin mutants where one lysine is mutated to arginine and therefore non-conjugatable at that residue, the K63R mutant should only allow mono-auto-ubiquitination modifications, while the WT ubiquitin substrate or any other K-to-R mutant will allow ubiquitin chain ligation and result in a higher FP signal (see reference (5)).

3. Allowing each master mix/substrate to reach room temperature (the operating temperature of the plate reader in these experiments) will prevent erroneous FP signal changes due to temperature fluctuation.

4. Note that, if screening a large number of samples, use of a multi-channel pipette will improve efficiency and limit the time between addition of ATP/DUB and returning the plate to the plate reader. Simply pipette the reagent into multiple small-volume tubes or a trough prior to addition.

5. The ‘no ATP’ control should resemble the FP signal of the T-Ub substrate alone, and the +ATP samples should be much higher (50–200 mP relative to the control). The ‘no ATP’ control will be important to determine the minimal FP signal of the reaction mixture in the presence of the DUB, as some DUBs have been observed to have moderate affinity for monoubiquitin which may result in an artificially high FP signal (12).

6. The 384-well plates in these experiments are open, and so evaporation will occur over time. We observed that experiments exceeding 2 hours begin to noticeably decrease in volume, which could produce erroneous changes to the observed FP signal. If longer incubations are needed (i.e., if using an enzyme with slow kinetics), the plate should be sealed throughout the experiment, a solution which other groups have utilized successfully (13).

7. Should the drug require DMSO to be dissolved (as is the case for PYR-41), it is best practice to dilute this into the assay as far as possible and include a matched vehicle control, since the DMSO itself may impair enzymatic activity.

8. The difference in FP signals varies between different (E1-E2-E3)~Ub states, but typically a larger FP difference will aid in characterizing the drug and observing FP changes generally. In order to increase this FP difference and improve the Z’ of the inhibition assay, a solubility tag such as GST or SUMO may be added to the target to increase its size and resultant FP signal when conjugated to the fluorescent ubiquitin.

9. This equation can also be used to normalize the data in Fig. 2 and Fig. 4.