Artificial RING Finger Research Articles

Unique E2-binding specificity of artificial RING fingers in cancer cells

Ubiquitin (Ub)-conjugating enzymes (E2s) are involved in various pathways for Ub transfer and deubiquitinating activities. These enzymes are associated with cancers such as breast cancer which is the second deadliest type of malignancy among women. Here, we revealed the unique E2-binding property and the auto-ubiquitination of artificial RING fingers (ARFs). Circular dichroism spectra showed the characteristic structures of ARFs. The proline, lysine, leucine, threonine and cysteine (PKLTC) sequence of ARF was important for E2-recognition and its mutations induced obvious changes in the E2-binding specificity and the auto-ubiquitination activity of ARF. The ARF mutants were applicable to detection of most of E2 activities. Furthermore, adding the ARF mutant C35A to cancer cells promoted its auto-ubiquitination, leading to the preferential detection of E2 UbcH5b activity. The present work opens up a new avenue for investigating intracellular E2 activities for the fatal diseases.

Artificial RING finger reveals unique auto-ubiquitination with E2 specificity.

Ubiquitin (Ub)-conjugating enzymes (E2s) transfer activated Ub from Ub-activating enzymes (E1s) to substrates and are associated with various cancers and neurological disorders. In this study, the unique properties of E2-binding and auto-ubiquitination of artificial RING fingers (ARFs) were demonstrated in ubiquitination assays. Circular dichroism spectra indicated the characteristic structures of ARFs. Point mutations of 31 PKLTC35 in ARF by tryptophan (Trp) resulted in dramatic changes in E2 specificity and the type of Ub chain elongation of mono- and polyubiquitination. The Trp residue was a cue that changed the ubiquitination activity of ARF via E2-binding. Furthermore, the ARF mutants interacted with all 11 E2s and then promoted auto-ubiquitination. Thus, the use of the ARF mutants allowed specific detection of E2 activities during ubiquitination. The present study opens up a new avenue for researching E2 activities related to the fatal diseases.

Solution structure of the zinc finger domain of human RNF144A ubiquitin ligase.

RNF144A is involved in protein ubiquitination and functions as an ubiquitin-protein ligase (E3) via its RING finger domain (RNF144A RING). RNF144A is associated with degradation of heat-shock protein family A member 2 (HSPA2), which leads to the suppression of breast cancer cell proliferation. In this study, the solution structure of RNF144A RING was determined using nuclear magnetic resonance. Moreover, using a metallochromic indicator, we spectrophotometrically determined the stoichiometry of zinc ions and elucidated that RNF144A RING binds two zinc atoms. This structural analysis provided the position and range of the active site of RNF144A RING at the atomic level, which contributes to the creation of artificial RING fingers having the specific ubiquitin-conjugating enzyme (E2)-binding capability.

Zinc finger domain of the human DTX protein adopts a unique RING fold.

The Deltex (DTX) family is involved in ubiquitination and acts as Notch signaling modifiers for controlling cell fate determination. DTX promotes the development of the ubiquitin chain via its RING finger (DTX_RING). In this study, the solution structure of DTX_RING was determined using nuclear magnetic resonance (NMR). Moreover, by experiments with a metallochromic indicator, we spectrophotometrically estimated the stoichiometry of zinc ions and found that DTX_RING possesses zinc-binding capabilities. The Simple Modular Architecture Research Tool database predicted the structure of DTX_RING as a typical RING finger. However, the actual DTX_RING structure adopts a novel RING fold with a unique topology distinct from other RING fingers. We unveiled the position and the range of the DTX_RING active site at the atomic level. Artificial RING fingers (ARFs) are made by grafting active sites of the RING fingers onto cross-brace structure motifs. Therefore, the present structural analysis could be useful for designing a novel ARF.

Unique RING finger structure from the human HRD1 protein

Artificial RING fingers (ARFs) are created by transplanting active sites of RING fingers onto cross-brace structures. Human hydroxymethylglutaryl-coenzyme A reductase degradation protein 1 (HRD1) is involved in the degradation of the endoplasmic reticulum (ER) proteins. HRD1 possesses the RING finger domain (HRD1_RING) that functions as a ubiquitin-ligating (E3) enzyme. Herein, we determined the solution structure of HRD1_RING using nuclear magnetic resonance (NMR). Moreover, using a metallochromic indicator, we determined the stoichiometry of zinc ions spectrophotometrically and found that HRD1_RING binds to two zinc atoms. The Simple Modular Architecture Research Tool database predicted the structure of HRD1_RING as a typical RING finger. However, it was found that the actual structure of HRD1_RING adopts an atypical RING-H2 type RING fold. This structural analysis unveiled the position and range of the active site of HRD1_RING that contribute to its specific ubiquitin-conjugating enzyme (E2)-binding capability.

Unique auto-ubiquitination activities of artificial RING fingers in cancer cells.

Ubiquitin-conjugating (E2) enzymes in protein ubiquitination are associated with various diseases. An artificial RING finger (ARF) is a useful tool, and E2 activities are conveniently estimated based on ARF reactivities. To extend the use of ARF in cells, we constructed a TAT-ARF using a cell-penetrating trans-activator protein (TAT) peptide. An in vitro ubiquitination assay without substrates showed auto-ubiquitination of TAT-ARF via its TAT region. TAT-ARF was translocated into MCF7 breast cancer cells, and then TAT-ARF ubiquitinated itself via its ARF. Experiments using confocal laser-scanning microscopy revealed that FAM-labeled TAT-ARF was readily internalized in cells and it remained encapsulated in vesicles. The Cell Counting Kit-8 assay indicated that the TAT-ARF uptake occurred without cytotoxicity in MCF7 cells at concentrations below 5.0 μM. By taking advantage of TAT-ARF, we, for the first time, succeeded in detecting E2 activities in cells. Thus, the present work opens up new avenues in the investigation of protein ubiquitination.

Concise machinery for monitoring ubiquitination activities using novel artificial RING fingers.

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.

Solution structure of the PHD finger from the human KIAA1045 protein.

Cross-brace structural motifs are required as a scaffold to design artificial RING fingers (ARFs) that function as ubiquitin ligase (E3) in ubiquitination and have specific ubiquitin-conjugating enzyme (E2)-binding capabilities. The Simple Modular Architecture Research Tool database predicted the amino acid sequence 131-190 (KIAA1045ZF) of the human KIAA1045 protein as an unidentified structural region. Herein, the stoichiometry of zinc ions estimated spectrophotometrically by the metallochromic indicator revealed that the KIAA1045ZF motif binds to two zinc atoms. The structure of the KIAA1045ZF motif bound to the zinc atoms was elucidated at the atomic level by nuclear magnetic resonance. The actual structure of the KIAA1045ZF motif adopts a C4 HC3 -type PHD fold belonging to the cross-brace structural family. Therefore, the utilization of the KIAA1045ZF motif as a scaffold may lead to the creation of a novel ARF.

Molecular Design of Artificial Ring Fingers for Detecting Ubiquitination Activities

Molecular Design of Artificial Ring Fingers for Detecting Ubiquitination Activities

Design of aSystem for Monitoring Ubiquitination Activities of E2 Enzymes Using Engineered RING Finger Proteins.

Ubiquitination is a sequential cascade consisting of ubiquitin-activating (E1), ubiquitin-conjugating (E2), and ubiquitin-ligating (E3) enzymes. It controls numerous processes such as protein degradation, DNA repair, and signal transduction pathways. E2 enzymes are associated with a variety of diseases such as leukemia, breast cancer, lung cancer, and colorectal cancer. To date, the monitoring of E2 activity for cancer diagnosis is challenging due to its intricate cascade reaction. To surmount this hurdle, we have recently developed a novel strategy for monitoring E2 activities. Here, we describe the concise machinery of ubiquitination with artificial RING finger proteins (ARFs) functioning as E3 enzymes. This machinery enables the simplified monitoring of E2 activities. Furthermore, our system combines a signal accumulation ion-sensitive field-effect transistor biosensor with ARFs, allowing for real-time monitoring of the pathological conditions of cancer cells. The present methodology may lead to novel diagnostic techniques for cancers.

The unique N-terminal zinc finger of synaptotagmin-like protein 4 reveals FYVE structure.

Synaptotagmin-like protein 4 (Slp4), expressed in human platelets, is associated with dense granule release. Slp4 is comprised of the N-terminal zinc finger, Slp homology domain, and C2 domains. We synthesized a compact construct (the Slp4N peptide) corresponding to the Slp4 N-terminal zinc finger. Herein, we have determined the solution structure of the Slp4N peptide by nuclear magnetic resonance (NMR). Furthermore, experimental, chemical modification of Cys residues revealed that the Slp4N peptide binds two zinc atoms to mediate proper folding. NMR data showed that eight Cys residues coordinate zinc atoms in a cross-brace fashion. The Simple Modular Architecture Research Tool database predicted the structure of Slp4N as a RING finger. However, the actual structure of the Slp4N peptide adopts a unique C4 C4 -type FYVE fold and is distinct from a RING fold. To create an artificial RING finger (ARF) with specific ubiquitin-conjugating enzyme (E2)-binding capability, cross-brace structures with eight zinc-ligating residues are needed as the scaffold. The cross-brace structure of the Slp4N peptide could be utilized as the scaffold for the design of ARFs.

The zinc finger domain of RING finger protein 141 reveals a unique RING fold.

Human RING finger protein 141 (RFP141) is a germ cell-specific transcription factor during spermatogenesis. We synthesized a compact construct encoding the C-terminal zinc finger of RFP141 (RFP141C peptide). Herein we determined the solution structure of the RFP141C peptide by nuclear magnetic resonance (NMR). Moreover, NMR data and the chemical modification of cysteine residues demonstrated that the RFP141C peptide binds to two zinc atoms in a cross-brace arrangement. The Simple Modular Architecture Research Tool database predicted the structure of RFP141C as a RING finger. However, the actual structure of the RFP141C peptide adopts an atypical compact C3 HC4 -type RING fold. The position and range of the helical active site of the RFP141C structure were elucidated at the atomic level. Therefore, structural analysis may allow RFP141C to be used for designing an artificial RING finger possessing specific ubiquitin-conjugating enzyme (E2)-binding capabilities.

Highly sensitive detection of E2 activity in ubiquitination using an artificial RING finger.

The ubiquitin-conjugating (E2) enzymes of protein ubiquitination are associated with various diseases such as leukemia, lung cancer, and breast cancer. Rapid and accurate detection of E2 enzymatic activities remains poor. Here, we described the detection of E2 activity on a signal accumulation ISFET biosensor (AMIS sensor) using an artificial RING finger (ARF). The use of ARF enables the simplified detection of E2 activity without a substrate. The high-sensitivity quantitative detection of E2 activities was demonstrated via real-time monitoring over a response range of femtomolar to micromolar concentrations. Furthermore, the monitoring of E2 activities was successfully achieved using human acute promyelocytic leukemia cells following treatment with the anticancer drug bortezomib, which allowed the assessment of the pathological conditions. This strategy is extremely simple and convenient, and the present detection could be widely applied to specific E2s for various types of cancers. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.

Detection of E2 Activity as a Tumor Marker using an Artificial Ring Finger

Protein ubiquitination is undertaken through an enzymatic cascade comprising ubiquitin-activating (E1), ubiquitin-conjugating (E2), and ubiquitin-ligating (E3) enzymes. The E2 enzymes are associated with various cancers such as leukemia, lung cancer, and breast cancer, and thus, they are attractive targets as tumor markers. In a previous study, we reported the creation of artificial RING fingers that are useful for detecting E2 activity. It was recently reported that a signal accumulation ISFET biosensor (AMIS sensor) allows the detection of proton changes arising during enzyme reactions. In this study, by using the artificial RING fingers and the AMIS sensor, we have succeeded in high-sensitivity quantitative detection of E2 activity. Furthermore, the real-time capturing of E2 activity was successfully achieved using human acute promyelocytic leukemia cells after treatment with the anticancer drug bortezomib. This study allowed the assessment of the pathological conditions of cancer cells. The present method could be applicable to the detection of E2 activity in the blood serum of patients, and therefore, it could be helpful for designing suitable treatment programs for patients. This strategy is extremely simple and convenient, and it opens new possibilities for diagnostic and prognostic methods in tumor development. Patients with cancer could benefit from advances of the quantitative analysis of the E2 activities of the ubiquitination system.

Structural model of ubiquitin transfer onto an artificial RING finger as an E3 ligase.

The artificial WSTF PHD_EL5 RING finger was designed via “α-helical region substitution”, and its structural model for the attachment of activated ubiquitin has been demonstrated. Chemical modifications of Cys residues, the circular dichroism spectra, and substrate-independent ubiquitination assays illustrated that the WSTF PHD_EL5 RING finger has E3 activity, and it is ubiquitinated via Lys14. Homology modeling calculations revealed that the WSTF PHD_EL5 RING finger possesses a classical RING fold for specific E2–E3 binding. The docking poses of the WSTF PHD_EL5 RING finger with the UbcH5b–ubiquitin conjugate provided insight into its functional E2 interaction and development of ubiquitination at the atomic level. The structural model of the artificial WSTF PHD_EL5 RING finger proposed by the present work is useful and may help to extend the strategy of α-helical region substitution.

Ubiquitination of an artificial RING finger without a substrate and a tag

Alpha-helical region substitution was applied to the SIAH1 and EL5 RING fingers. The Williams-Beuren syndrome transcription factor (WSTF) PHD_SIAH1 and WSTF PHD_EL5 RING fingers were created as the artificial ubiquitin-ligating enzyme (E3). These fingers possess E3 activities of mono-ubiquitination and poly-ubiquitination, respectively, with ubiquitin-conjugating enzyme (E2)-binding capabilities. Artificial E3s bind two zinc atoms and adopt a zinc-dependent ordered structure and ubiquitinate upon themselves without a substrate and a tag. Ubiquitination experiments using biotinylated ubiquitin showed that the WSTF PHD_EL5 RING finger is poly-ubiquitinated via residue Lys(63) of ubiquitin. Substitution of alpha-helical region might be applicable to various RING fingers with mono-ubiquitination or poly-ubiquitination.

The creation of the artificial RING finger from the cross-brace zinc finger by α-helical region substitution

The creation of the artificial RING finger as ubiquitin-ligating enzyme (E3) has been demonstrated. In this study, by the α-helical region substitution between the EL5 RING finger and the Williams–Beuren syndrome transcription factor (WSTF) PHD finger, the artificial E3 (WSTF PHD_RING finger) was newly created. The experiments of the chemical modification of residues Cys and the circular dichroism spectra revealed that the WSTF PHD_RING finger binds two zinc atoms and adopts the zinc-dependent ordered-structure. In the substrate-independent ubiquitination assay, the WSTF PHD_RING finger functions as E3 and was poly- or mono-ubiquitinated. The present strategy is very simple and convenient, and consequently it might be widely applicable to the creation of various artificial E3 RING fingers with the specific ubiquitin-conjugating enzyme (E2)-binding capability.