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.     

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.     

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 C₄HC₃‐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.     

Structure of the C-terminal RING finger from a RING-IBR-RING/TRIAD motif reveals a novel zinc-binding domain distinct from a RING

The really interesting new gene (RING) family of proteins contains over 400 members with diverse physiological functions. A subset of these domains is found in the context of the RING-IBR-RING/TRIAD motifs which function as E3 ubiquitin ligases. Our sequence analysis of the C-terminal RING (RING2) from this motif show that several metal ligating and hydrophobic residues critical for the formation of a classical RING cross-brace structure are not present. Thus, we determined the structure of the RING2 from the RING-IBR-RING motif of HHARI and showed that RING2 has a completely distinct topology from classical RINGs. Notably, RING2 binds only one zinc atom per monomer rather than two and uses a different hydrophobic network to that of classical RINGs. Additionally, this RING2 topology is novel, bearing slight resemblance to zinc-ribbon motifs around the zinc site and is different from the topologies of the zinc binding sites found in RING and PHDs. We demonstrate that RING2 acts as an E3 ligase in vitro and using mutational analysis deduce the structural features required for this activity. Further, mutations in the RING-IBR-RING of Parkin cause a rare form of Parkinsonism and these studies provide an explanation for those mutations that occur in its RING2. From a comparison of the RING2 structure with those reported for RINGs, we infer sequence determinants that allow discrimination between RING2 and RING domains at the sequence analysis level.     

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.     

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 C₄C₄‐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.     

NMR assignment of the N-terminal TRAF-like RING zinc finger domain of human FLN29

Resistance of cancer cells to oncotherapeutics designed to trigger programmed cell death (a.k.a. apoptosis) greatly limits clinical efficacy. The human FLN29 protein may play a role in this process via protein-protein interactions. Here we report the NMR spectral assignment of the N-terminal TRAF2/6-RING-zinc finger-like domain of this protein.     

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.     

Estrogen-responsive RING finger protein controls breast cancer growth

Most of the breast cancers initially respond to endocrine therapy that reduces the levels of estrogens or competes with estrogen for binding to its receptor. Most of the patients, however, acquire resistance to endocrine therapy with tamoxifen and aromatase inhibitors later. We assumed that identification of estrogen-responsive genes those regulate the growth of breast cancer is indispensable to develop new strategies targeting the genes and overcome the resistance to current endocrine therapy. Estrogen-responsive finger protein (Efp) is one of the estrogen receptor (ER)-target genes we have cloned using genomic binding site cloning. Efp features a structure of the RING-finger B-box coiled-coil (RBCC) motif. We postulated that Efp is a critical factor in proliferation of breast tumors. In a model system using MCF7 cells grown in xenografts, we showed that inhibition of Efp expression by antisense oligonucleotide reduced the tumor growth. MCF7 cells overexpressing Efp formed tumors in xenografts even in estrogen deprivation environment. By yeast two-hybrid screen, we identified that Efp interacts with 14-3-3σ, which is known as a cell cycle brake that causes G2 arrest and expressed in normal mammary glands. In vitro studies have revealed that Efp functions as a ubiquitin-protein ligase (E3) that targets 14-3-3σ. These data suggest that Efp controls breast cancer growth through ubiquitin-dependent proteolysis of 14-3-3σ. Future studies may provide a new therapy to block breast tumor proliferation by targeting Efp.     

RKP, a RING finger E3 ligase induced by BSCTV C4 protein, affects geminivirus infection by regulation of the plant cell cycle

The C4 protein from Curtovirus is known as a major symptom determinant, but the mode of action of the C4 protein remains unclear. To understand the mechanism of involvement of C4 protein in virus–plant interactions, we introduced the C4 gene from Beet severe curly top virus (BSCTV) into Arabidopsis under a conditional expression promoter; the resulting overexpression of BSCTV C4 led to abnormal host cell division. RKP, a RING finger protein, which is a homolog of the human cell cycle regulator KPC1, was discovered to be induced by BSCTV C4 protein. Mutation of RKP reduced the susceptibility to BSCTV in Arabidopsis and impaired BSCTV replication in plant cells. Callus formation is impaired in rkp mutants, indicating a role of RKP in the plant cell cycle. RKP was demonstrated to be a functional ubiquitin E3 ligase and is able to interact with cell-cycle inhibitor ICK/KRP proteins in vitro . Accumulation of the protein ICK2/KRP2 was found increased in the rkp mutant. The above results strengthen the possibility that RKP might regulate the degradation of ICK/KRP proteins. In addition, the protein level of ICK2/KRP2 was decreased upon BSCTV infection. Overexpression of ICK1/KRP1 in Arabidopsis could reduce the susceptibility to BSCTV. In conclusion, we found that RKP is induced by BSCTV C4 and may affect BSCTV infection by regulating the host cell cycle.     

RNF151, a testis-specific RING finger protein, interacts with dysbindin

RING finger proteins play important roles in spermatogenesis. Here, we report that a novel RING finger protein RNF151, with a C3HC4-type RING finger domain, a putative nuclear localization signal (NLS), and a TRAF-type zinc finger domain, was exclusively expressed in the mouse testis and developmentally regulated during spermatogenesis. While RNF151 mRNA was present in round spermatids, its protein was expressed in elongating spermatids of the stage VIII-IX seminiferous tubules. The NLS together with the RING domain were necessary and sufficient for the nuclear localization of RNF151-EGFP in transfected cells. Yeast two-hybrid screening identified the physical interaction of mouse RNF151 and dysbindin, which was confirmed by the co-immunoprecipitation of the proteins and by their co-localization in intact cells. As dysbindin has lately been shown to be involved in membrane biogenesis and fusion, a key process for acrosome formation, we propose that RNF151 may play a role in acrosome formation.     

Structural basis for role of ring finger protein RNF168 RING domain

Ubiquitin adducts surrounding DNA double-strand breaks (DSBs) have emerged as molecular platforms important for the assembly of DNA damage mediator and repair proteins. Central to these chromatin modifications lies the E2 UBC13, which has been implicated in a bipartite role in priming and amplifying lys63-linked ubiquitin chains on histone molecules through coupling with the E3 RNF8 and RNF168. However, unlike the RNF8-UBC13 holoenyzme, exactly how RNF168 work in concert with UBC13 remains obscure. To provide a structural perspective for the RNF168-UBC13 complex, we solved the crystal structure of the RNF168 RING domain. Interestingly, while the RNF168 RING adopts a typical RING finger fold with two zinc ions coordinated by several conserved cystine and histine residues arranged in a C3HC4 “cross-brace” manner, structural superimposition of RNF168 RING with other UBC13-binding E3 ubiquitin ligases revealed substantial differences at its corresponding UBC13-binding interface. Consistently, and in stark contrast to that between RNF8 and UBC13, RNF168 did not stably associate with UBC13 in vitro or in vivo. Moreover, domain-swapping experiments indicated that the RNF8 and RNF168 RING domains are not functionally interchangeable. We propose that RNF8 and RNF168 operate in different modes with their cognate E2 UBC13 at DSBs.     

Analysis of RING finger genes required for embryogenesis in C. elegans

The RING finger motif exists in E3 ligases of the ubiquitination pathway. These ubiquitin ligases bind to target proteins, leading to their modification by covalent addition of ubiquitin peptides. Current databases contain hundreds of proteins with RING finger motifs. This study investigates the role of RING finger genes in embryogenesis of the nematode, Caenorhabditis elegans. We expand the previous list of RING finger-containing genes and show that there are 103 RING finger-containing genes in the C. elegans genome. DNA microarrays of these 103 genes were probed with various RNA samples to identify 16 RING finger genes whose expression is enriched in the germline. RNA interference (RNAi) analysis was then used to determine the developmental role of these genes. One RING finger gene, C32D5.10, showed a dramatic larval arrest upon RNAi. Three RING finger genes exhibited embryonic lethality after RNAi. These three genes include par-2, and two small RING finger proteins: F35G12.9 (an ortholog of APC11) and ZK287.5 (an ortholog of rbx1). Embryos from RNAi of the APC11 and rbx1 orthologs were arrested in the cell cycle, confirming the role of these particular RING finger proteins in regulation of the cell cycle. genesis 38:1-12, 2004.     

The LIM domain protein UNC-95 is required for the assembly of muscle attachment structures and is regulated by the RING finger protein RNF-5 in C. elegans

Here, we describe a new muscle LIM domain protein, UNC-95, and identify it as a novel target for the RING finger protein RNF-5 in the Caenorhabditis elegans body wall muscle. unc-95(su33) animals have disorganized muscle actin and myosin-containing filaments as a result of a failure to assemble normal muscle adhesion structures. UNC-95 is active downstream of PAT-3/beta-integrin in the assembly pathways of the muscle dense body and M-line attachments, and upstream of DEB-1/vinculin in the dense body assembly pathway. The translational UNC-95::GFP fusion construct is expressed in dense bodies, M-lines, and muscle-muscle cell boundaries as well as in muscle cell bodies. UNC-95 is partially colocalized with RNF-5 in muscle dense bodies and its expression and localization are regulated by RNF-5. rnf-5(RNAi) or a RING domain deleted mutant, rnf-5(tm794), exhibit structural defects of the muscle attachment sites. Together, our data demonstrate that UNC-95 constitutes an essential component of muscle adhesion sites that is regulated by RNF-5.