The dwarf neon rainbowfish Melanotaenia praecox, a small spiny-rayed fish with potential as a new Acanthomorpha model fish: II. Establishment of a microinjection procedure for genetic engineering.

BACKGROUND: Rainbowfish is a clade of colorful freshwater fish. Melanotaenia praecox is a small rainbowfish species with biological characteristics that make it potentially useful as an experimental model species. We anticipate that M. praecox could become a new model used in various fields, such as ecology, evolution, and developmental biology. However, few previous studies have described experimental set-ups needed to understand the molecular and genetic mechanisms within this species. RESULTS: We describe detailed procedures for genetic engineering in the rainbowfish M. praecox. By using these procedures, we successfully demonstrated CRISPR/Cas-mediated knockout and Tol2 transposon-mediated transgenesis in this species. Regarding the CRISPR/Cas system, we disrupted the tyrosinase gene and then showed that injected embryos lacked pigmentation over much of their body. We also demonstrated that a Tol2 construct, including a GFP gene driven by a ubiquitous promoter, was efficiently integrated into the genome of M. praecox embryos. CONCLUSIONS: The establishment of procedures for genetic engineering in M. praecox enables investigation of the genetic mechanisms behind a broad range of biological phenomena in this species. Thus, we suggest that M. praecox can be used as a new model species in various experimental biology fields.

The dwarf neon rainbowfish Melanotaenia praecox, a small spiny-rayed fish with potential as a new Acanthomorpha model fish: I. Fin ray ontogeny and postembryonic staging.

BACKGROUND: Fish fins with highly variable color patterns and morphologies have many functions. In Actinopterygii, the free parts of fins are supported by "soft rays" and "spiny rays." Spiny rays have various functions and are extremely modified in some species, but they are lacking in popular model fish such as zebrafish and medaka. Additionally, some model fish with spiny rays are difficult to maintain in ordinary laboratory systems. RESULTS: Characteristics of the small, spiny-rayed rainbowfish Melanotaenia praecox render it useful as an experimental model species. Neither fish age nor body size correlate well with fin development during postembryonic development in this species. A four-stage developmental classification is proposed that is based on fin ray development. CONCLUSIONS: Melanotaenia praecox is an ideal species to rear in laboratories for developmental studies. Our classification allows for postembryonic staging of this species independent of individual age and body size. Development of each fin ray may be synchronized with dorsal fin development. We discuss the differences in mechanisms regulating soft, spiny, and procurrent ray development.

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.

A protein kinase D inhibitor suppresses AKT on T cells and antagonizes cancer immunotherapy by anti-PD-1.

Antibodies that block the interaction between PD-1 and PD-1 ligands (anti-PD-1) are in clinical use for the treatment of cancer, yet their efficacy is limited. Pre-approved therapies that enhance the effect of anti-PD-1 in combination are beneficial. Small-molecule inhibitors that attenuate T cell receptor signaling are reported to prevent T cell exhaustion and induce memory T cells with stem cell potential, resulting in a durable effector T cell response in combination with anti-PD-1. In search of such targets, we focused on protein kinase D (PKD), which is suggested to be suppressive in both tumor growth and TCR signaling. We report that CRT0066101, a PKD inhibitor (PKDi), suppressed the growth of mouse tumors at a sub-micromolar concentration in vitro. Despite its inhibitory effects on tumors, a single treatment of tumor-bearing mice with PKDi did not inhibit, but rather accelerated tumor growth, and reversed the therapeutic effect of anti-PD-1. Mice treated with PKDi showed reduced T cell infiltration and defects in the generation of effector T cells, compared to those treated with anti-PD-1, suggesting that PKDi inhibited ongoing antitumor responses. Mechanistically, PKDi inhibited phosphorylation of AKT, a primary checkpoint that is reactivated by anti-PD-1. In conclusion, PKD is fundamentally required for T cell reactivation by anti-PD-1; therefore, inhibition of PKD is not appropriate for combination therapy with anti-PD-1. On the other hand, a single dose of PKDi was shown to strongly suppress experimental autoimmunity in mice, indicating that PKDi could be useful for the treatment of immune-related adverse events that are frequently reported in anti-PD-1 therapy.

Developmental independence of median fins from the larval fin fold revises their evolutionary origin.

The median fins of modern fish that show discrete forms (dorsal, anal, and caudal fins) are derived from a continuous fold-like structure, both in ontogeny and phylogeny. The median fin fold (MFF) hypothesis assumes that the median fins evolved by reducing some positions in the continuous fin fold of basal chordates, based on the classical morphological observation of developmental reduction in the larval fin folds of living fish. However, the developmental processes of median fins are still unclear at the cellular and molecular levels. Here, we describe the transition from the larval fin fold into the median fins in zebrafish at the cellular and molecular developmental level. We demonstrate that reduction does not play a role in the emergence of the dorsal fin primordium. Instead, the reduction occurs along with body growth after primordium formation, rather than through actively scrapping the non-fin forming region by inducing cell death. We also report that the emergence of specific mesenchymal cells and their proliferation promote dorsal fin primordium formation. Based on these results, we propose a revised hypothesis for median fin evolution in which the acquisition of de novo developmental mechanisms is a crucial evolutionary component of the discrete forms of median fins.

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.

Design of a System 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.

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.

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.

Bortezomib Causes ER Stress-related Death of Acute Promyelocytic Leukemia Cells Through Excessive Accumulation of PML-RARA.

BACKGROUND/AIM: The success of proteasome inhibitors in therapy of multiple myeloma has led to their use for other malignancies. For the proteasome inhibitor bortezomib, combination therapies with histone deacetylase inhibitors, which up-regulate ubiquitin-proteasome system (UPS)-related enzymes, produce a beneficial effect. However, the mechanisms underlying the effect of bortezomib are not completely understood. We hypothesized that bortezomib causes excessive accumulation of aberrant proteins, which augments endoplasmic reticulum (ER) stress, leading to death of malignant cells. MATERIALS AND METHODS: The NB4 cell line established from a patient with acute promyelocytic leukemia (APL) expressing the promyelocytic leukemia/retinoic acid receptor alpha (PML-RARA) fusion protein was used to assess changes in cell viability and apoptosis caused by bortezomib, as well as alterations in PML-RARA and UPS-related enzymes via western blotting and immunoprecipitation assays. RESULTS: Bortezomib time- and dose-dependently reduced cell viability and induced apoptosis. Bortezomib significantly increased the abundance of ubiquitinated-PML-RARA (Ub-PML-RARA), ubiquitin-conjugating human enzyme 8 (UbcH8), and Ub-UbcH8, indicating that UbcH8 is the E2 ubiquitin-conjugating enzyme for PML-RARA. Moreover, UbcH8 abundance was dose-dependently increased in the culture supernatant of bortezomib-treated cells. CONCLUSION: UbcH8 may have a utility as a biomarker of treatment response to bortezomib in patients with APL. Furthermore, bortezomib impairs the UPS that controls normal protein homeostasis by causing excessive accumulation of PML-RARA augmenting ER stress and leading to APL cell death. The study provides a rationale for incorporating proteasome inhibitors in the treatment of diseases expressing aberrant proteins. Furthermore, monitoring of UPS-related enzymes might have use in predicting the treatment response to proteasome inhibitors and in assessing their therapeutic effects.

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.

Oligopeptides derived from autophosphorylation sites of EGF receptor suppress EGF-stimulated responses in human lung carcinoma A549 cells.

Epidermal growth factor (EGF) receptor plays a crucial role in the biology of human cancer, and is a highly appropriate target for anticancer agents. We have previously designed oligopeptides containing the amino acid sequences around autophosphorylation sites of EGF receptor to identify a specific inhibitor of this receptor. We found that Ac-ENAEYLR-NH(2) and Ac-NYQQN-NH(2) suppressed phosphorylation of purified EGF receptor in a non-ATP-competitive manner whereas Ac-QNAQYLR-NH(2) and Ac-DYQQD-NH(2) caused inhibition in an ATP-competitive manner. The aim of this study was to observe the effects of these peptides on the proliferation, cell death, and apoptosis of human lung carcinoma A549 cells. To facilitate transfer of these inhibitory peptides into A549 cells, the cell-penetrating peptide, human immunodeficiency virus type 1-transactivator of transcription (Tat), was linked to the peptides. When A549 cells were treated with each Tat-conjugated peptide, the peptides penetrated the cells and EGF-stimulated tyrosine phosphorylation of EGF receptor was significantly suppressed. These Tat-conjugated peptides played a suppressive role in EGF-stimulated A549 cell responses. In particular, Tat-epsilon-aminocaproic acid (acp)-ENAEYLR-NH(2) significantly inhibited proliferation and showed cytotoxicity, while Tat-acp-NYQQN-NH(2) and Tat-acp-DYQQD-NH(2) suppressed the anti-apoptotic effect of EGF. In addition, we found that Tat-acp-ENAEYLR-NH(2) also inhibited the phosphorylation of epidermal growth factor receptor 2 (ErbB2) as well as EGF receptor in A549 cells. In conclusion, membrane-permeable synthetic peptides derived from EGF receptor autophosphorylation sites have the potential to suppress EGF receptor function in A549 cells and to be developed into novel and useful agents for cancer therapy.

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.