Concise machinery for monitoring ubiquitination activities using novel artificial RING fingers

Abstract

Protein ubiquitination is involved in many cellular processes, such as protein degradation, DNA repair, and signal transduction pathways. Ubiquitin‐conjugating (E2) enzymes of the ubiquitination pathway are associated with various cancers, such as leukemia, lung cancer, and gastric cancer. However, to date, detection of E2 activities is not practicable for capturing the pathological conditions of cancers due to complications related to the enzymatic cascade reaction. To overcome this hurdle, we have recently investigated a novel strategy for measuring E2 activities. Artificial RING fingers (ARFs) were developed to conveniently detect E2 activities during the ubiquitination reaction. ARFs were created by grafting the active sites of ubiquitin‐ligating (E3) enzymes onto amino acid sequences with 38 residues. The grafting design downsized E3s to small molecules (ARFs). Such an ARF is a multifunctional molecule that possesses specific E2‐binding capabilities and ubiquitinates itself without a substrate. In this review, we discuss the major findings from recent investigations on a new molecular design for ARFs and their simplified detection system for E2 activities. The use of the ARF allowed us to monitor E2 activities using acute promyelocytic leukemia (APL)‐derived cells following treatment with the anticancer drug bortezomib. The molecular design of ARFs is extremely simple and convenient, and thus, may be a powerful tool for protein engineering. The ARF methodology may reveal a new screening method of E2s that will contribute to diagnostic techniques for cancers.

Introduction

Protein ubiquitination is a post‐translational modification that induces diverse effects in many cellular processes, such as protein degradation, DNA repair, and signal transduction.1, 2, 3 Ubiquitination involves mono‐ubiquitination using a single ubiquitin (Ub), multiubiquitination using several single Ub molecules, or polyubiquitination using polyubiquitin chains on target proteins.4, 5 For example, a polyubiquitin chain covalently attached to a substrate guides it to the Ub/26S proteasome‐dependent degradation pathway. This ubiquitination occurs through an enzymatic cascade reaction consisting of ubiquitin‐activating (E1), ubiquitin‐conjugating (E2), and ubiquitin‐ligating (E3) enzymes.2, 6 Ub is initially activated by an ATP‐dependent reaction with E1, yielding an E1‐Ub thiolester product. Activated Ub is transferred from the E1‐Ub product to the E2 enzyme by a transthiolation reaction. Subsequently, the E3 enzyme transfers activated Ub from the E2 enzyme to the ɛ‐amino groups of a substrate Lys residue.7 The three major classes of E3 enzymes in eukaryotes are known as RING E3, Ubox E3, and HECT E3. The RING and Ubox types do not directly bind to Ub, and they play an indirect catalytic role in protein ubiquitination; however, HECT E3 directly promotes ubiquitination.8 The classical ubiquitination reaction with a RING E3 enzyme, a substrate, and an E2‐Ub thioester conjugate is shown in Figure 1(A). The RING finger of E3 specifically binds to E2, and a substrate‐binding domain (SBD) recognizes a particular substrate. RING fingers consist of ∼50 amino acids and are a zinc‐binding motif with eight ligands of Cys and/or His in a unique cross‐brace arrangement.9, 10 There are approximately 300 RING E3 and 30 E2 enzymes in humans.11, 12 The E2 enzyme transfers Ub to several different substrates, demonstrating the versatility of protein ubiquitination in cells. For example, seven in absentia homolog‐1 (SIAH1) functions as a RING E3 enzyme and is related to leukemogenesis.13 It cooperates with UbcH8 of E2 and ubiquitinates substrates such as AML1‐ETO [acute myeloid leukemia (RUNX1)‐eight–twenty‐one (MTG8)] and PML‐RARα (promyelocytic leukemia‐retinoic acid receptor α).14, 15, 16 E2 and E3 enzymes are associated with a variety of diseases, such as leukemia,17 lung cancer,18 and gastric cancer.19 Thus, there is growing interest in the detection of E2 activities for capturing the pathological conditions of cancers. The E2 activities presented here are equivalent to the amounts of the additional Ub transferred from the E2‐Ub conjugate to the substrates. However, it is difficult to evaluate E2 activities based on the amounts of Ub upon all the substrates. Also, the intermediate complex of RING E3 and E2‐Ub may be implicated in Ub transfer.20 On the other hand, by adding a tag, such as a maltose‐binding protein (MBP), the MBP‐RING E3 fusion construct encodes E3 activity that can transfer Ub to the MBP moiety. The use of the MBP‐RING E3 fusion construct enables the measuring of E2 activities without a substrate.21 However, the molecular design of the MBP‐RING E3 fusion construct is challenging and the steric hindrance of the tag is inevitable. For these reasons, to date, in researching the pathological conditions of cancers, E2 activity has not been practicable for use due to the complications related to its enzymatic cascade reaction. To overcome this hurdle, we have recently investigated a novel strategy for measuring E2 activities. In this paper, we review the major findings from recent investigations on a new molecular design for artificial E3 enzymes and their simplified detection system for E2 activities. The system is applicable to monitoring of E2 activities using acute promyelocytic leukemia (APL)‐derived cells following treatment with the anticancer drug bortezomib. The present methodology may open up novel diagnostic techniques for cancer.

Figure 1.

Schematic representation of the ubiquitination reaction. (A) Typical ubiquitination reaction with E2‐ubiquitin (Ub) conjugate, RING E3, and a substrate (S). RING E3 recognizes the substrate via a substrate‐binding domain (SBD) and indirectly catalyzes Ub transfer. RING does not bind to Ub. (B) Novel ubiquitination machinery with an artificial RING finger (ARF). An ARF directly promotes Ub transfer and ubiquitinates itself.

Molecular Design Strategy of Artificial RING Fingers (ARFs)

ARFs were developed as convenient artificial E3 enzymes to measure E2 activities in the ubiquitination pathway.22, 23 Figure 1(B) shows the novel ubiquitination reaction with an ARF. An ARF binds to the E2‐Ub thioester conjugate and directly catalyzes the transfer of activated Ub from E2 to itself, in contrast to the indirect control by wild‐type RING E3 for Ub transfer to a substrate [Fig. 1(A)]. In the ARF reaction, a substrate is not recruited to promote the Ub chain; instead, the ARF assumes the role of a substrate and ubiquitinates itself. E2 activities are conveniently measured based on ARF reactivity without a substrate and a tag (e.g., MBP), in terms of the amount of Ub transferred to the ARF. The outline of the molecular design method for creating the ARF is shown in Figure 2. The SIAH1 protein consists of 282 amino acids and possesses an N‐terminal RING finger and an SBD domain. It functions as a stable homodimer of 564 residues.24 The RING finger has the active site for E2‐binding and promotes ubiquitination of the substrate.25 The ARF was engineered by inserting an amino acid sequence corresponding to the active site of the RING finger into the PHD finger of Williams–Beuren Syndrome transcription factor (WSTF).22, 23, 26 The WSTF PHD finger was used as a scaffold. The ARF is a RING‐PHD chimeric molecule, designed by replacing the active site in between two different domains.

Figure 2.

Schematic diagram for creating the ARF. SIAH1 belongs to the RING E3 family and consists of a RING and substrate‐binding domain (SBD). The active site, which contributes to specific E2‐binding capabilities, was transplanted into WSTF PHD, leading to the creation of ARF.

Next, we discuss the detailed protocol for the creation of the ARF. Like SIAH1, the EL5 protein possesses a RING finger and belongs to the RING E3 group. The amino acid sequences of the EL5 RING finger and the SIAH1 RING finger are shown in Figure 3(A). The three‐dimensional structures of several RING fingers have been reported by using NMR methods21, 27, 28, 29, 30 and X‐ray diffraction.31, 32 The RING finger structures bind to two zinc atoms and all have the essential active site corresponding to the amino acid sequence between the sixth and the seventh zinc‐binding residues. The formation of a shallow groove for the E2‐binding site was created using the active site, the L1 region, and the L2 region. In particular, the active site controls the specific E2‐E3 binding capabilities, and the RING fingers are classified into four categories: I‐a, I‐b, II‐a, and II‐b.33 Figure 3(B) shows the amino acid sequence of the WSTF PHD finger. The PHD finger always binds to two zinc ions with eight Cys and/or His, and its structure adopts a cross‐braced zinc‐binding arrangement. The structure of the PHD finger is roughly similar to that of a RING finger in that both fingers have the characteristic motif of a cross‐braced structure. However, unlike the RING finger, the PHD finger possesses a randomized, flexible long loop rather than the ordered structure, although it sometimes has a short helix region.34 The PHD finger obviously does not have an active E2‐binding site and, thus, is not involved in the ubiquitination system.35, 36. The PHD finger specifically recognizes histone H3 methylated at Lys4 and plays a role in transcriptional regulation in cells.37, 38 The PHD finger, which is often found in the nucleus, is characterized as a positively charged Lys‐rich motif.39 In our theoretical concept for the molecular design of ARFs [Fig. 3(B)], the long flexible loop of the PHD finger is truncated and, instead, the active site of the RING finger is transplanted into the sequence of the PHD finger. In other words, this substituted region confers specific E2‐binding capabilities on the PHD finger. The ARF exhibits E2‐binding activity and ubiquitinates itself because it has the Lys‐rich motif as an acceptor for Ub transfer. Thus, the ARF is an artificial multifunctional molecule with an auto‐ubiquitination reaction. For designing the ARF, the scaffold sequence should contain Lys residues for the Ub attachment and at least one Lys residue should be solvent‐exposed in the ARF‐E2 complex to accept Ub. In addition, it is imperative that Lys residue secures the space necessary for Ub transfer onto the ARF. The positions and the roles of Lys residues will be discussed later using the ARF structure. The ARFs derived from the EL5 and SIAH1 proteins are ARF_EL5 and ARF_SIAH1, respectively. The ARF methodology may be widely applicable to the molecular design of various ARFs. However, the RING fingers have sequence diversity: the sequence length in between the sixth and the seventh zinc‐binding residues ranges from 4 to 48 residues.7 Experimental evidence will be needed to show whether the ARF methodology can be applied to the majority of the RING fingers. For example, the creation of an ARF based on the RING finger having the longest 48 residues will lend credence to the ARF methodology.

Figure 3.

Molecular design of the ARF. (A) EL5 and SIAH1 RING have active sites for E2‐binding. The sixth and the seventh zinc ligands are boxed. (B) The long flexible loop of WSTF PHD was replaced with the active site of RING. The ARF was designed as a chimeric molecule between RING and WSTF PHD. L1 and L2 indicate short loops between zinc ligands. The five Lys residues replaced with Arg are shown in blue.

Synthesis of ARFs

Although EL5 and SIAH1 are composed of 325 and 564 residues, both ARF_EL5 and ARF_SIAH1 are approximately 50‐mer peptides. In the case of ARF_SIAH1, the molecular size of the ARF is less than one‐tenth of that of the original SIAH1. The insertion of only active sites successfully downsizes E3s into small ARFs. The compact molecular size enables automatic peptide synthesis. ARF_EL5 and ARF_SIAH1 were synthesized by the standard F‐moc solid‐phase method on an automated peptide synthesizer (e.g., PSSM‐8, Shimadzu Corp.).22, 23 Subsequently, after cleavage with trifluoroacetic acid, peptide purification was performed on a reverse‐phase HPLC with a C18 column. The high‐purity ARFs (> 98%) could be stored as a powder in a freezer, providing significant advantages involved in easy handling, transportability, and minimal or no lot‐to‐lot variations, unlike antibody regents. The ARF peptides were dissolved in guanidine‐HCl and completely denatured. The obtained ARF solutions were dialyzed against an appropriate buffer solution containing zinc ions, leading to the refolding of the ARF structures. The confirmation of correct structures was performed by experiments using chemical modification of Cys residues and circular dichroism (CD) spectra, as reported previously.22, 23 ARF forms an ordered structure with a C₄HC₃‐type zinc coordination arrangement in accordance to the molecular design strategy. Therefore, the ARF methodology is very simple and convenient.

In Vitro Substrate‐Independent Ubiquitination with ARFs

The utilization of an ARF enables the ubiquitination reaction without a substrate and a tag,22, 23 although a substrate in traditional ubiquitination is imperative for Ub transfer. The addition of ARFs into ubiquitination buffer containing Ub, E1, and E2 leads to the in vitro accumulation of ARF‐Ub conjugates. The Ub transfer to the ARF was assessed with eleven different E2s (UbcH1, UbcH2, UbcH3, UbcH5a, UbcH5b, UbcH5c, UbcH6, UbcH7, UbcH8, UbcH10, and UbcH13/Mms2). ARF_EL5 was poly‐ubiquitinated in the presence of UbcH1, UbcH2, UbcH3, UbcH5a, UbcH5b, or UbcH5c, but not UbcH6, UbcH7, UbcH8, UbcH10, or UbcH13/Mms2. Also, the mono‐ubiquitination of ARF_EL5 was promoted when ARF_EL5 and UbcH6 or UbcH13/Mms2 were incubated together. Thus, ARF_EL5 possesses specific E2‐binding capabilities and poly‐ or mono‐ubiquitinates itself in the substrate‐independent ubiquitination. As for the ARF_SIAH1, mono‐ubiquitination was developed in the presence of UbcH5a, UbcH5b, UbcH5c, UbcH6, or UbcH8, but not UbcH1, UbcH2, UbcH3, UbcH7, UbcH10, or UbcH13/Mms2. Polyubiquitination of ARF_SIAH1 did not occur if the 11 E2s were incubated together. Taken together, these findings indicated that ARF_EL5 and ARF_SIAH1 specifically recognize E2s and transfer activated Ub from E2s to themselves and develop the Ub chain. The original EL5 protein promotes the polyubiquitination reaction, which cooperates with UbcH5a but not with UbcH7/8.21 The original SIAH1 protein specifically recognizes UbcH5 or UbcH8.16, 40 Therefore, ARF_EL5 and ARF_SIAH1 possess similar E2‐binding capabilities to those of the original EL5 and SIAH1, respectively. This is because ARF_EL5 and ARF_SIAH1 were created by transplanting the active sites derived from the original EL5 and SIAH1. We currently cannot perform the complete manipulation for changing the specific E2‐binding. For instance, it is challenging to create ARFs that bind to only one of the E2s. Future comprehensive research on ARFs by mutational experiments of the active sites, structural studies, and ubiquitination assays will provide a crucial insight into controlling their specific E2‐binding. Some different types of Ub linkages are present in the poly‐ubiquitination reaction. Ubiquitin has seven Lys residues, of which residues 6, 48, and 63 are always used as a linkage position for a Ub chain.41, 42, 43 The Lys6 or Lys48‐linked Ub chain is associated with the target for the 26S proteasome.44 The Lys63‐linked poly‐Ub chain also serves as the target signal for protein degradation, DNA repair, signal transduction, and endocytosis.45 To elucidate the type of the Ub chain on ARFs, the ubiquitination reaction using two biotinylated Ubs (Lys6/Lys48‐dibiotinylated Ub (Lys6/Lys48‐Ub) and Lys6/Lys63‐dibiotinylated Ub (Lys6/Lys63‐Ub)) was assessed in a solution containing E1, E2, and ARF_EL5. Biotin modification of the Lys residues abolishes development of the Ub chain.41 However, the polyubiquitination of ARF_EL5 was observed with Lys6/Lys48‐Ub but not with Lys6/Lys63‐Ub. Therefore, Lys63‐linkages of poly‐Ub chains were facilitated on ARF_EL5.

Next, it has been demonstrated that the ARF has five Lys residues, each of which is possible receptive Lys residue for the Ub transfer. To identify the ubiquitination site of the ARF, five mutants, in which Lys residues were replaced with Arg (K4R, K8R, K9R, K14R, and K23R), were designed and synthesized by the standard F‐moc solid‐phase method [Fig. 3(B)].46 The five mutants were analyzed using CD spectra and in vitro substrate‐independent ubiquitination assays. The secondary structures of wild‐type ARF and its five mutants (K4R, K8R, K9R, K14R, and K23R) had structures roughly similar to one another, although the negative ellipticities of K23R decreased slightly at 225 nm. This is because K23 contributes to the formation of a hydrophobic core in the ARF structure. However, the Arg replacement did not drastically change the secondary structures of the ARFs. Furthermore, in the in vitro ubiquitination assay, the K4R, K8R, and K9R mutants were poly‐ubiquitinated and their ubiquitination activities were similar to that of wild‐type ARF. The ubiquitination of the K23R mutant was slightly decreased compared to that of wild‐type ARF. The ubiquitination of the K14R mutant was not observed in the reaction system, suggesting that the mutation at K14 obstructs the promotion of the Ub chain of the ARF with its cooperating E2 (UbcH5b). Taken together, these data revealed that Lys14 of the ARF is the crucial receptive residue for mono‐ and polyubiquitination. It was concluded that the ARF specifically recognizes E2s and then receives activated Ub from E2s via its Lys14 residue, whereby the ARF ubiquitinates itself. The spatial location and structural features of the five Lys residues on the ARF will be discussed later via structural analyses.

Mechanism of Ubiquitin Transfer for the ARF

For structural analyses for Ub transfer onto the ARF, the ARF structure was proposed by homology modeling. The amino acid sequence of ARF_EL5 was analyzed using the program I‐TASSER server, and its structure was predicted (Fig. 4), because the server is known as the best server for the critical assessment of protein structure prediction.47, 48 The I‐TASSER program consists of multiple threading alignments and iterative template fragment assembly simulations. The quality of the ARF structures calculated was checked by a confidence score (C‐score) and a template modeling score (TM‐score). A C‐score of > −1.5 and TM‐score > 0.5 indicate a correct fold and an accurate calculation of the modeling structures, respectively. The top model structure of the ARF predicted by I‐TASSER had a C‐score of 1.11 and a TM‐score of 0.87. A Ramachandran plot for the dihedral angles of the backbone of ARF_EL5 was validated by PROCHECK (The most favored region was 85.4%, and the additional allowed region was 14.6%.).49 No residue of the structural model of the ARF was located within the disallowed region. The structure of the ARF has one α‐helical region (α1) and two β‐strands (β1 and β2), indicating a compact fold. The α1 region is the active site for specific E2‐binding and corresponds to the amino acid sequence transplanted by the ARF strategy. The Connolly surface of ARF_EL5 was calculated using the program Discovery Studio 2.1 (Accelrys Software Inc.).50 The molecular surface is shown in Figure 4 with the electrostatic potential. α1, L1, and L2 regions contribute to the formation of the hydrophobic groove of ARF_EL5. A cluster of positively charged residues is formed at the molecular surface and thus, the structure of ARF_EL5 finger possesses an amphipathic character.

Figure 4.

Homology modeling structure of ARF. ARF_EL5 was designed by active site substitution based on the sequence derived from EL5. The structure of ARF_EL5 was calculated using I‐TASSER, and its surface representation was calculated using Discovery Studio 2.1. The molecular surface was depicted with the electrostatic potential (blue, positive; red, negative). The α‐helical region, L1, and L2 contribute to the formation of the E2‐recognition site. The turn regions are shown in green.

Next, the structure–function relationships of the ARF provided a clue towards understanding the structural properties responsible for the ubiquitination machinery.46 The structural features of the ARF were analyzed from the viewpoint of its structural similarities with the WSTF PHD and original EL5 RING fingers. The superimposition of ARF_EL5 over the backbone (N, C^α, C’) atoms of the WSTF PHD finger produced rms deviations of 6.16 Å (Ala1–Thr47) and 2.26 Å (Ala1–Leu30), whereas superimposition over the structure of the original EL5 RING finger yielded an rms deviation of 2.81 Å (Ala1–Thr47), as shown in Figure 5. Thus, the overall structure of ARF is similar to that of the original EL5 RING finger, as compared to the WSTF PHD finger. In addition, the shallow groove consisting of α1, L1, and L2 regions of ARF contributes to the specific E2‐binding capabilities. The structural similarities of the ARF_EL5 with the original EL5 RING finger lend credence to their similar, specific ubiquitination activities cooperating with E2s.

Figure 5.

Structural comparison of the ARF with other fingers. ARF_EL5 was superimposed over the backbone atoms for (A) WSTF PHD and (B) EL5 RING. The ARF_EL5 structure has an E2‐binding site similar to that of EL5 RING when compared with WSTF PHD. A ribbon diagram of ARF_EL5 is shown in magenta.

Next, the complex structure of the ARF with the E2‐Ub conjugate provided insight into the functional E2 interaction and the development of ubiquitination at the atomic level.46 The structural docking calculations of the ARF and the E2‐Ub conjugate (PDB code: 3A33) were performed using the program ZDOCK 3.0.1.51 ZDOCK is an initial‐stage rigid‐body molecular docking program and improves the performance for searching in a translational space using a fast Fourier transform algorithm. The ARF_EL5 structure calculated by the I‐TASSER server was used to achieve the docking calculation by ZDOCK. After starting from a randomized 1,000 initial structures using ZDOCK, the complex structures generated were subjected to energy minimization using the Smart Minimizer algorithm (Max steps 200, RMS gradient 0.01) in Discovery Studio 2.1. The resulting top three models of ARF_EL5 in complex with the E2‐Ub conjugate were utilized as appropriate docking poses [Fig. 6(A)]. A best‐fit superposition of the ensemble of all three lowest energy structures produced an rms deviation of 1.58 Å for the backbone (N, C^α, C’) atoms in all residues of the complex. To demonstrate their functional role in ubiquitination, the spatial positions of the five Lys residues of ARF_EL5 are shown on the molecular surface of the complex structure [Fig. 6(B)]. Lys4 and Lys8 bind the residues of UbcH5b: (Pro95 and Ala96) and (Arg5 and Lys8), respectively. In contrast, Lys9 and Lys14 have no contacts with the UbcH5b‐Ub conjugate. Lys9 is located closely in space to the helical region of UbcH5b, as compared with Lys14. Lys23 is buried in the hydrophobic core for proper folding of ARF_EL5. Thus, Lys14 has the space necessary for attachment of Ub to the ARF. The mutation of Lys14 with Arg disturbed the development of mono‐ and polyubiquitination reactions as indicated by the substrate‐independent ubiquitination assays. The helical region of the ARF preferentially plays a crucial role in forming the structure of the ARF‐UbcH5b complex. Taken together, these data illustrate that ARF_EL5 specifically binds UbcH5b via its helical region, and Lys14 serves as scaffolding for activated Ub transfer. Lys63 of Ub is utilized in the linkages for developing the poly‐Ub chains. The I‐TASSER calculation predicted the structure of ARF_EL5, and its structural model with the UbcH5b‐Ub conjugate was proposed at the atomic level using the ZDOCK calculation [Fig. 6(B)]. However, the crystal structure of the ARFs bound to the E2s should provide more precise knowledge about the mechanism of the specific E2‐binding and the Ub transfer in the ubiquitination machinery.

Figure 6.

The mechanism of ubiquitin transfer onto ARF. The docking structure of ARF_EL5 in complex with the UbcH5b–ubiquitin conjugate was calculated using ZDOCK. (A) Docking poses of the three lowest energy structures of ARF_EL5. (B) Structural model for ubiquitin transfer in the lowest energy structure. The five Lys residues of ARF_EL5 and their interacting residues of UbcH5b are shown on the complex structure as well as the interaction of Ser36 from ARF_EL5 and Lys63 from UbcH5b. ARF, UbcH5b, and ubiquitin are drawn in red, blue, and yellow, respectively.

Detection of E2 Activity Using the ARF

For practical use of the ARF system, the simplified detection of E2 activities was successfully achieved in a ubiquitination system. The overexpression of E2 enzymes is associated with various cancer cells.17, 18, 19, 52, 53 APL is a subtype of acute myelogenous leukemia (AML) and is defined as AML‐M3 based on the French–American–British (FAB) classification. In APL, the chromosomal translocation t(15;17) in cells induces the formation of the fusion protein PML‐RARα, which is involved in the ubiquitination system with E2 (UbcH6 and UbcH8) and E3 (SIAH1).15, 54, 55 Expression of the fusion protein PML‐RARα collapses nuclear body structures in promyelocytic cells and increases endoplasmic reticulum stress, which contribute to the genesis of APL.15, 54 Bortezomib is a first‐in‐class proteasome inhibitor and induces cell death in a variety of cancers, such as leukemia, multiple myeloma, and breast cancer.56, 57, 58 Treatment with bortezomib decreases the cell viability of human APL‐derived NB4 cells, where UbcH8 expression was induced, but UbcH5 and UbcH6 expression were hardly affected. In the culture supernatant of bortezomib‐cultured NB4 cells, the abundance of UbcH8‐Ub conjugates was increased in a concentration‐dependent manner by drug treatment, whereas UbcH6‐Ub was detected at a low level, but not UbcH5. It is speculated that the UbcH8‐Ub accumulated by treatment with bortezomib was released from cells into the culture medium.17 UbcH8‐Ub was more strongly detected than other E2s in the culture supernatant. Addition of ARF_SIAH1 (4.8 kDa) into the culture supernatant following treatment with bortezomib promoted its ubiquitination, leading to the accumulation of ARF‐Ub conjugates (13.4 kDa) (Fig. 7). This is because ARF_SIAH1, derived from SIAH1, specifically interacts with UbcH8‐Ub and mono‐ubiquitinates itself. The reactivity of ARF, that is, the amount of mono‐ubiquitinated ARF‐Ub conjugate, is equivalent to the E2 activity. Accordingly, the ARF reaction enables the detection of bortezomib‐induced E2 emigration. Western blotting methods and a signal accumulation ISFET biosensor (AMIS sensor) are available to capture the reactivity of ARF as E2 activities for NB4 cells.55 In the AMIS sensor, free protons are released from the Ub transfer reaction on the ARF, and the resulting proton changes are observed as the alteration of electron polarization in a semiconductor. Therefore, E2 activities are detected as the polarization behavior of electrons in a sensing layer. Using the sensor, E2 activities were measured over a wide range of femtomolar to micromolar concentrations. Furthermore, real‐time monitoring of E2 activities emigrating from NB4 cells (cultured NB4 cells, 1 × 10⁵ cells/ml) was successfully performed in the culture supernatant. Hence, the use of the ARF allows for the detection of E2 activities in crude extracts, such as the culture supernatant of cells. Compared to western blotting, the rapid and accurate monitoring of E2 activities quantitatively succeeded within several minutes. Leukemia is diagnosed by the existence of more than 1 × 10⁹ leukemia cells in the body, which is equivalent to approximately 2 × 10⁵ cells/ml of blood.59 The approach of combining AMIS detection with the ARF could be used for measuring the response to bortezomib for leukemia patients in terms of high detection sensitivity. Therefore, this technique might be useful for arranging appropriate treatment programs for proteasome inhibitors as a new companion diagnostic approach. However, various compounds in blood would disturb the reactivity of ARF. Ubiquitination assays may be required depending on whether the releasing of E2s‐Ub occurs in animal models and whether the highly sensitive detection of E2 activities is achieved in blood or not. On the other hand, some E2s are involved in ubiquitination related to some cancers, such as UbcH10 with lung tumors18 and gastric tumors.19 For example, in gastric cancer patient tumors, 49 out of 59 cases (83%) showed a positive expression of UbcH10, but not in adjacent mesenchymal tissues. Thus, it is considered that UbcH10 is a potential biomarker for gastric cancer.19 Anti‐tumor effects of bortezomib were observed using gastric cancer in vivo.60 It is challenging to evaluate specifically the pathological conditions of cancers, but the ARF strategy could be used as a new screening method for their E2 activities. By contrast, the traditional E2 antibody‐based protein assay is used to measure the expression level of E2; however, it is dissimilar to the capturing of E2 activity related to Ub transfer. Quantitative diGly proteomics enable the identification of substrates for cullin‐RING Ub ligases,61 and Ub remnant profiling is a technique used to estimate ubiquitination events from cell lysates.62 These excellent methods using monoclonal antibodies are useful for researching the ubiquitination of substrates, but not E2 activities. Therefore, the ARF system is a novel technology for E2 detection. Taking advantage of these methods including the ARF strategy will shed light on the relationship between E2 activities and the profiling of protein ubiquitination. Therefore, this work opens up new avenues in the investigation of protein ubiquitination.

Figure 7.

Reaction of ARF in NB4 cells following treatment with bortezomib. Treatment with bortezomib led to the diapedesis of E2‐Ub conjugates from the cells into the culture supernatant. The addition of ARF_SIAH1 into the culture supernatant promoted its ubiquitination, yielding ARF‐Ub conjugates. E2 activities were measured as the reactivity of ARF, which is equivalent to the amount of the mono‐ubiquitinated ARF‐Ub conjugate.

In conclusion, the ARF strategy is extremely simple and convenient, and ARFs can be designed with E2‐binding capabilities for various applications. The use of the ARF enabled the simplified detection of E2s during the ubiquitination reaction. The present system led to the achievement of real‐time monitoring of E2 activities using human APL‐derived cells following treatment with the anticancer drug bortezomib. This technique still requires more precise analyses based on the structure–function relationships. However, the molecular design of ARFs will provide a powerful tool for “protein engineering.” We hope that the ubiquitination machinery of the ARF reveals novel and useful applications for E2 activities, including the diagnosis of cancers.

Author Contributions

K.M. designed this study. K.M. and K.S. wrote the main manuscript text and prepared all the figures.

Conflict of interest

The authors declare no competing financial interests.