The RING finger motif of photomorphogenic repressor COP1 specifically interacts with the RING-H2 motif of a novel Arabidopsis protein.

The constitutive photomorphogenic 1 (COP1) protein of Arabidopsis functions as a molecular switch for the seedling developmental fates: photomorphogenesis under light conditions and skotomorphogenesis in darkness. The COP1 protein contains a cysteine-rich zinc-binding RING finger motif found in diverse groups of regulatory proteins. To understand the role of the COP1 RING finger in mediating protein-protein interaction, we have performed a yeast two-hybrid screen and isolated a novel protein with a RING-H2 motif, a variant type of the RING finger. This protein, designated COP1 Interacting Protein 8 (CIP8), is encoded by a single copy gene and localized to cytosol in a transient assay. In addition to the RING-H2 motif, the predicted protein has a C4 zinc finger, an acidic region, a glycine-rich cluster, and a serine-rich cluster. The COP1 RING finger and the CIP8 RING-H2 domains are sufficient for their interaction with each other both in vitro and in yeast, whereas neither motif displayed significant self-association. Moreover, site-directed mutagenesis studies demonstrated that the expected zinc-binding ligands of the RING finger and RING-H2 fingers are essential for their interaction. Our findings indicate that the RING finger motif, in this case, serves as autonomous protein-protein interaction domain. The allele specific effect of cop1 mutations on the CIP8 protein accumulation in seedlings indicates that its stability in vivo is dependent on the COP1 protein.     

Spring, a novel RING finger protein that regulates synaptic vesicle exocytosis

The synaptosome-associated protein of 25 kDa (SNAP-25) interacts with syntaxin 1 and vesicle-associated membrane protein 2 (VAMP2) to form a ternary soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor (SNARE) complex that is essential for synaptic vesicle exocytosis. We report a novel RING finger protein, Spring, that specifically interacts with SNAP-25. Spring is exclusively expressed in brain and is concentrated at synapses. The association of Spring with SNAP-25 abolishes the ability of SNAP-25 to interact with syntaxin 1 and VAMP2 and prevents the assembly of the SNARE complex. Overexpression of Spring or its SNAP-25-interacting domain reduces Ca(2+)-dependent exocytosis from PC12 cells. These results indicate that Spring may act as a regulator of synaptic vesicle exocytosis by controlling the availability of SNAP-25 for the SNARE complex formation.     

NIRF,a novel RING finger protein,is involved in cell-cycle regulation

Through database mining, we found a novel PEST-containing nuclear protein (PCNP). To characterize PCNP, we carried out yeast two-hybrid screening for PCNP-interacting factors. A novel Np95/ICBP90-like RING finger protein (NIRF), which possessed a ubiquitin-like domain, a PHD finger, a YDG/SRA domain and a RING finger, was identified. Interaction between PCNP and NIRF was clarified by mammalian two-hybrid system, GST pull-down assay, and nuclear co-localization. RT-PCR showed that NIRF expression is high in proliferating phase but significantly low in G0/G1 phase in normal TIG-7 and WI-38 cells, while consistently high in tumoral HT-1080 and HepG2 cells, suggesting that NIRF is involved in cell-cycle regulation. The NIRF gene resides in 9p23-24.1 that is altered in numerous types of tumors at the top of frequency. Furthermore, the NIRF gene is just within small amplicons in some tumors, suggesting that PCNP and NIRF might be involved in some aspects of tumorigenesis.     

Isolation and characterization of a novel human gene (HFB30) which encodes a protein with a RING finger motif.

A human cDNA, HFB30, encoding a novel protein that contains a RING finger (C3HC4-type zinc finger) motif was isolated. This cDNA clone consists of 3056 nucleotides and encodes an open reading frame of a 474 amino acid protein. From RT-PCR analysis, the messenger RNA was ubiquitously expressed in various human tissues. The gene was located to the chromosome 5q23.3-q31.1 region by PCR-based analyses with both a human/rodent monochromosomal hybrid cell panel and a radiation hybrid mapping panel. Furthermore, the gene consists of nine exons that span about 20 kb of genome DNA.     

A poxvirus protein with a RING finger motif binds zinc and localizes in virus factories.

Shope fibroma virus (SFV) is a Leporipoxvirus closely related to the highly virulent myxoma virus. The DNA sequence of the BamHI N fragment of the SFV DNA genome was determined, and the single complete open reading frame (N1R) was characterized. The protein encoded by the N1R gene was found to contain a C3HC4 RING finger motif at the C terminus. This C3HC4 motif is the hallmark of a growing family of proteins, many of which are involved in regulation of gene expression, DNA repair, or DNA recombination. Complete homologs of the SFV N1R gene were also detected in variola virus, myxoma virus, and vaccinia virus strain IHD-W. In contrast, the gene is completely absent from vaccinia virus strain Copenhagen, and in vaccinia virus strain WR, the open reading frame is truncated prior to the zinc binding domain because of an 11-bp deletion, thus producing a frameshift and premature stop codon. Recombinant N1R protein from SFV was expressed in Escherichia coli and shown to bind zinc in a specific manner. Using fluorescence microscopy to visualize a peptide epitope tag (derived from ICP27 of herpes simplex virus) fused to the N terminus of the poxvirus proteins, we observed that the N1R protein of SFV and its homologs in myxoma virus and vaccinia virus IHD-W were localized primarily to the virus factories in the cytoplasm of infected cells and, to a lesser degree, the host cell nucleus. The truncated protein of vaccinia virus strain WR failed to localize in this manner but instead was observed throughout the cytoplasm.     

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.     

The Ret finger protein induces apoptosis via its RING finger-B box-coiled-coil motif

The Ret finger protein (RFP) is a member of the tripartite motif family, which is characterized by a conserved RING finger, a B-box, and a coiled-coil domain (together called RBCC). Although RFP is known to become oncogenic when its RBCC moiety is connected to a tyrosine kinase domain by DNA rearrangement, its biological function is not well defined. Here we show that ectopic expression of RFP in human embryonic kidney 293 cells causes extensive apoptosis, as assessed by multiple criteria. RFP expression activates Jun N-terminal kinase and p38 kinase and also increases caspase-3-like activity. However, RFP failed to release cytochrome c and, therefore, to increase caspase-9-like activity. RFP-induced apoptosis could be blocked by the caspase-8 inhibitor crmA and dominant negative ASK1 but not by Bcl-2. These results reveal a novel RFP death pathway that recruits mitogen-activated protein kinase and caspases independently of mitochondrial events. Domain mapping showed that the intact RBCC moiety is necessary for the pro-apoptotic function of RFP. Moreover, expression of the RBCC moiety further potentiated the pro-apoptotic activity and resulted in a 7-fold increase of caspase activation compared with that induced by full-length RFP. This suggests that a large number of tripartite motif family members sharing the RBCC moiety may participate in the control of cell survival.     

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.     

Haprin,a novel haploid germ cell-specific RING finger protein involved in the acrosome reaction

The acrosome reaction (i.e. the exocytosis of the sperm vesicle) is a prerequisite for fertilization, but its molecular mechanism is largely unknown. We have identified a cDNA clone for a gene named haprin, which encodes a haploid germ cell-specific RING finger protein. This protein is a novel member of the RBCC (RING finger, B-box type zinc finger, and coiled-coil domain) motif family that has roles in several cellular processes, such as exocytosis. It is transcribed exclusively in testicular germ cells after meiotic division. Western blot and immunohistochemical analyses showed the molecular weight of Haprin protein to be Mr approximately 82,000. It was localized in the acrosomal region of elongated spermatids and mature sperm and was not present in acrosome-reacted sperm. The specific antibody against the RING finger domain of Haprin inhibited the acrosome reaction in permeabilized sperm. These results indicated that the novel RBCC protein Haprin plays a key role in the acrosome reaction and fertilization.     

A novel human striated muscle RING zinc finger protein, SMRZ, interacts with SMT3b via its RING domain

The RING domain is a conserved zinc finger motif, which serves as a protein-protein interaction interface. Searches of a human heart expressed sequence tag data base for genes encoding the RING domain identified a novel cDNA, named striated muscle RING zinc finger protein (SMRZ). The SMRZ cDNA is 1.9 kilobase pairs in length and encodes a polypeptide of 288 amino acid residues; analysis of the peptide sequence demonstrated an N-terminal RING domain. Fluorescence in situ hybridization localized SMRZ to chromosome 1p33-34. Northern blots demonstrated that SMRZ is expressed exclusively in striated muscle. In the cardiovascular system, SMRZ is more highly expressed in the fetal heart than in the adult heart (slightly higher expression in the ventricle than in the atrium), suggesting that SMRZ is developmentally regulated. SMRZ was found to interact with SMT3b, a ubiquitin-like protein, through the SMRZ-RING domain. This interaction was abolished by mutagenesis of conserved RING domain residues. Transient transfection of SMRZ into C2C12 myoblasts showed localization of SMRZ to the nucleus. These data suggest that SMRZ may play an important role in striated muscle cell embryonic development and perhaps in cell cycle regulation.     

TBC domain family, member 15 is a novel mammalian Rab GTPase-activating protein with substrate preference for Rab7

Ypt/Rabs are Ras-related GTPases that function as key regulators of intracellular vesicular trafficking. Their slow intrinsic rates of GTP hydrolysis are catalyzed by GTPase-activating proteins (GAPs). Ypt/Rab-GAPs constitute a family of proteins that contain a TBC (Tre-2/Bub2/Cdc16) domain. Only three of the 51 family members predicted in the human genome are confirmed Ypt/Rab-GAPs. Here, we report the identification and characterization of a novel mammalian Ypt/Rab-GAP, TBC domain family, member 15 (TBC1D15). TBC1D15 is ubiquitously expressed and localized predominantly to the cytosol. The TBC domain of TBC1D15 exhibits relatively high homology with that of Gyp7p, a yeast Ypt/Rab-GAP. Furthermore, TBC1D15 stimulates the intrinsic GTPase activity of Rab7, and to a lesser extent Rab11, but is essentially inactive towards Rab4 or Rab6. These data increase the number of mammalian TBC domain family members with demonstrated Rab-GAP activity to four, and suggest that TBC1D15 may be involved in Rab7-mediated late endosomal trafficking.     

Cloning and characterization of a novel RING finger protein that interacts with class V myosins.

We have identified a novel protein (BERP) that is a specific partner for the tail domain of myosin V. Class V myosins are a family of molecular motors thought to interact via their unique C-terminal tails with specific proteins for the targeted transport of organelles. BERP is highly expressed in brain and contains an N-terminal RING finger, followed by a B-box zinc finger, a coiled-coil (RBCC domain), and a unique C-terminal beta-propeller domain. A yeast two-hybrid screening indicated that the C-terminal beta-propeller domain mediates binding to the tail of the class V myosin myr6 (myosin Vb). This interaction was confirmed by immunoprecipitation, which also demonstrated that BERP could associate with myosin Va, the product of the dilute gene. Like myosin Va, BERP is expressed in a punctate pattern in the cytoplasm as well as in the neurites and growth cones of PC12 cells. We also found that the RBCC domain of BERP is involved in protein dimerization. Stable expression of a mutant form of BERP lacking the myosin-binding domain but containing the dimerization domain resulted in defective PC12 cell spreading and prevented neurite outgrowth in response to nerve growth factor. Our studies present a novel interaction for the beta-propeller domain and provide evidence for a role for BERP in myosin V-mediated cargo transport.     

Two potato proteins,including a novel RING finger protein (HIP1), interact with the potyviral multifunctional protein HCpro

Potyviral helper-component proteinase (HCpro) is a multifunctional protein exerting its cellular functions in interaction with putative host proteins. In this study, cellular protein partners of the HCpro encoded by Potato virus A (PVA) (genus Potyvirus) were screened in a potato leaf cDNA library using a yeast two-hybrid system. Two cellular proteins were obtained that interact specifically with PVA HCpro in yeast and in the two in vitro binding assays used. Both proteins are encoded by single-copy genes in the potato genome. Analysis of the deduced amino acid sequences revealed that one (HIP1) of the two HCpro interactors is a novel RING finger protein. The sequence of the other protein (HIP2) showed no resemblance to the protein sequences available from databanks and has known biological functions.     

SAG, a novel zinc RING finger protein that protects cells from apoptosis induced by redox agents

SAG (sensitive to apoptosis gene) was cloned as an inducible gene by 1,10-phenanthroline (OP), a redox-sensitive compound and an apoptosis inducer. SAG encodes a novel zinc RING finger protein that consists of 113 amino acids with a calculated molecular mass of 12.6 kDa. SAG is highly conserved during evolution, with identities of 70% between human and Caenorhabditis elegans sequences and 55% between human and yeast sequences. In human tissues, SAG is ubiquitously expressed at high levels in skeletal muscles, heart, and testis. SAG is localized in both the cytoplasm and the nucleus of cells, and its gene was mapped to chromosome 3q22-24. Bacterially expressed and purified human SAG binds to zinc and copper metal ions and prevents lipid peroxidation induced by copper or a free radical generator. When overexpressed in several human cell lines, SAG protects cells from apoptosis induced by redox agents (the metal chelator OP and zinc or copper metal ions). Mechanistically, SAG appears to inhibit and/or delay metal ion-induced cytochrome c release and caspase activation. Thus, SAG is a cellular protective molecule that appears to act as an antioxidant to inhibit apoptosis induced by metal ions and reactive oxygen species.     

RING finger specificity in SCF-driven protein destruction

SCF ubiquitin ligases contain an E3 core composed of Skp1, Cul1, a member of the Rbx1/Roc1 family of RING finger proteins, and a modular F box protein that functions in substrate targeting. Work published in this issue of Developmental Cell indicates that distinct Rbx1/Roc1 family members are used to control ubiquitination of distinct targets, suggesting that the RING finger subunit may contribute to ubiquitination specificity.