Myristoylation of the RING finger Z protein is essential for arenavirus budding

The arenavirus small RING finger Z protein is the main driving force of arenavirus budding. The primary structure of Z is devoid of hydrophobic transmembrane domains, but both lymphocytic choriomeningitis virus (LCMV) and Lassa fever virus Z proteins accumulate near the inner surface of the plasma membrane and are strongly membrane associated. All known arenavirus Z proteins contain a glycine (G) at position 2, which is a potential acceptor site for a myristoyl moiety. Metabolic labeling showed incorporation of [(3)H]myristic acid by wild-type Z protein but not by the G2A mutant. The mutation G2A eliminated Z-mediated budding. Likewise, treatment with the myristoylation inhibitor 2-hydroxymyristic acid inhibited Z-mediated budding, eliminated formation of virus-like particles, and caused a dramatic reduction in virus production in LCMV-infected cells. Budding activity was restored in G2A mutant Z proteins by the addition of the myristoylation domain of the tyrosine protein kinase Src to their N termini. These findings indicate N-terminal myristoylation of Z plays a key role in arenavirus budding.     

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.     

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.     

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.     

Transgenic maize plants expressing the Totivirus antifungal protein, KP4, are highly resistant to corn smut

The corn smut fungus, Ustilago maydis, is a global pathogen responsible for extensive agricultural losses. Control of corn smut using traditional breeding has met with limited success because natural resistance to U. maydis is organ specific and involves numerous maize genes. Here, we present a transgenic approach by constitutively expressing the Totivirus antifungal protein KP4, in maize. Transgenic maize plants expressed high levels of KP4 with no apparent negative impact on plant development and displayed robust resistance to U. maydis challenges to both the stem and ear tissues in the greenhouse. More broadly, these results demonstrate that a high level of organ independent fungal resistance can be afforded by transgenic expression of this family of antifungal proteins.     

An antiviral disulfide compound blocks interaction between arenavirus Z protein and cellular promyelocytic leukemia protein

The promyelocytic leukemia protein (PML) forms nuclear bodies (NB) that can be redistributed by virus infection. In particular, lymphocytic choriomeningitis virus (LCMV) influences disruption of PML NB through the interaction of PML with the arenaviral Z protein. In a previous report, we have shown that the disulfide compound NSC20625 has antiviral and virucidal properties against arenaviruses, inducing unfolding and oligomerization of Z without affecting cellular RING-containing proteins such as the PML. Here, we further studied the effect of the zinc-finger-reactive disulfide NSC20625 on PML-Z interaction. In HepG2 cells infected with LCMV or transiently transfected with Z protein constructs, treatment with NSC20625 restored PML distribution from a diffuse-cytoplasmic pattern to punctate, discrete NB which appeared identical to NB found in control, uninfected cells. Similar results were obtained in cells transfected with a construct expressing a Z mutant in zinc-binding site 2 of the RING domain, confirming that this Z-PML interaction requires the integrity of only one zinc-binding site. Altogether, these results show that the compound NSC20625 suppressed Z-mediated PML NB disruption and may be used as a tool for designing novel antiviral strategies against arenavirus infection.     

Transgenic tobacco plants expressing a coat protein gene of tobacco mosaic virus are resistant to some other tobamoviruses

Transgenic tobacco plants expressing the coat protein (CP) gene of tobacco mosaic virus were tested for resistance against infection by five other tobamoviruses sharing 45-82% homology in CP amino acid sequence with the CP of tobacco mosaic virus. The transgenic plants (CP+) showed significant delays in systemic disease development after inoculation with tomato mosaic virus or tobacco mild green mosaic virus compared to the control (CP-) plants, but showed no resistance against infection by ribgrass mosaic virus. On a transgenic local lesion host, the CP+ plants showed greatly reduced numbers of necrotic lesions compared to the CP- plants after inoculation with tomato mosaic virus, pepper mild mottle virus, tobacco mild green mosaic virus, and Odontoglossum ringspot virus but not ribgrass mosaic virus. The implications of these results are discussed in relation to the possible mechanism(s) of CP-mediated protection.     

How a RING finger protein and a steroid hormone control autophagy

Previous work in our laboratory has indicated that the steroid hormone ecdysone triggers programmed autophagy in the fat body of Drosophila larvae by downregulating the class I phosphoinositide 3-kinase (PI3K) pathway. We recently found evidence that Deep orange (Dor), a Drosophila RING finger protein implicated in late-endosomal trafficking, controls ecdysone signaling as well as autolysosome fusion, thus exerting a dual regulation of autophagy. We found that dor mutants are defective in programmed autophagy. The mutant larvae showed impaired upregulation of ecdysone signaling during development, accompanied by a failure to downregulate the PI3K pathway. Downregulation of the PI3K pathway could be restored by feeding the dor mutants with ecdysone. Even though ecdysone signaling and autophagy were impaired in the dor mutants, we detected an accumulation of autophagosomes in dor mutant fat bodies. This could probably be attributed to the failure of autophagosomes to fuse with lysosomes. In this Addendum we review these findings and provide some speculations about how Dor may control both ecdysone signalling and autolysosomal fusion.     

Transgenic Arabidopsis plants expressing the rice dehydroascorbate reductase gene are resistant to salt stress

Vitamin C (l-ascorbate) is important for antioxidative and metabolic functions in both plants and humans. Ascorbate itself is oxidized to dehydroascorbate during the process of antioxidation, and dehydroascorbate reductase (DHAR, EC 1.8.5.1) re-reduces the oxidized ascorbate. Therefore, this enzyme is assumed to be critical for ascorbate recycling. Here we show that the expression of rice DHAR in transgenic Arabidopsis thaliana enhanced resistance to salt stress. Salt tolerance was remarkably improved despite slight increases in DHAR activity and total ascorbate. This study provides direct evidence for the importance of DHAR in salt tolerance.     

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.     

Novel RING finger protein OIP1 binds to conserved amino acid repeats in sperm tail protein ODF1

Outer dense fibers (ODFs) and the fibrous sheath (FS) are unique structures of the mammalian sperm tail. Recently, progress has been made in the molecular cloning of ODF and FS proteins, and because of this, questions addressing the morphogenesis and underlying protein network that make up sperm tail structures and their function can now be addressed. Using the N-terminal leucine zipper motif of the major ODF protein ODF1, we had previously isolated interacting proteins Odf2, Spag4, and Spag5. We report here a yeast two-hybrid strategy to isolate a novel rat testicular protein, OIP1, that binds to the evolutionarily conserved Cys-Gly-Pro repeats in the C-terminus of ODF1. OIP1 is expressed in round spermatids as well as in spermatocytes and several somatic tissues, albeit at a lower level. No expression was detectable in epididymis, heart, and smooth muscle. OIP1 protein localizes to the sperm tail in a pattern expected for an ODF1-interacting protein. OIP1 belongs to the family of RING finger proteins of the H2 subclass. Deletion of the putative RING motif significantly decreased binding to ODF1. Genomic analysis of rat Oip1 and Oip1 homologs indicates that Oip1 is highly conserved. Oip1 is subject to differential splicing and alternative polyadenylation events. It is interesting that Oip1 mRNAs have been reported that lack the exon encoding the putative RING finger.     

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.