UnpEL/Usp4 is ubiquitinated by Ro52 and deubiquitinated by itself

The autoantigen Ro52 is an E3 ubiquitin ligase that can ubiquitinate itself (self-ubiquitination). Recently, we showed that UnpEL/Usp4 is an isopeptidase that can deconjugate ubiquitin from self-ubiquitinated Ro52. Here, we showed that UnpEL is ubiquitinated by Ro52 in cooperation with UbcH5B in vitro. We also showed that UnpEL is ubiquitinated by Ro52 in HEK293T cells. Interestingly, a catalytically inactive UnpEL mutant was strongly ubiquitinated by Ro52 in HEK293T cells. These results suggest that wild-type UnpEL is ubiquitinated by Ro52 and deubiquitinated by itself (self-deubiquitination), while mutant UnpEL is ubiquitinated by Ro52 but not deubiquitinated by itself. In conclusion, Ro52 and UnpEL transregulate each other by ubiquitination and deubiquitination.     

Oncogenic protein UnpEL/Usp4 deubiquitinates Ro52 by its isopeptidase activity

UnpEL (also known as Usp4 or Unph) is an oncogenic protein, because its expression with a strong promoter results in the tumorigenic transformation of NIH3T3 cells injected into nude mice. Although the structure of UnpEL is that of a deubiquitinating enzyme, neither its precise function in mammalian cells nor the mechanism of UnpEL-mediated tumorigenesis is known. Here, we show that UnpEL functions as a deubiquitinating enzyme in human HEK293T cells and its isopeptidase activity deconjugates ubiquitin specifically from a UnpEL-interacting protein Ro52. We further show that UnpEL translocates to the cytoplasmic rod-like structures and colocalizes with Ro52 when Ro52 is overexpressed in HEK293 cells. These results suggest that UnpEL colocalizes with the unubiquitinated form of Ro52 to the cytoplasmic rod-like structures, where it keeps Ro52 unubiquitinated. The continuous deubiquitination of Ro52 might be involved in tumorigenesis.     

Cytoplasmic relocation of Daxx induced by Ro52 and FLASH

The RING-finger protein Ro52/TRIM21 is known to be an autoantigen and is recognized by anti-Ro/SSA antibodies, which are commonly found in patients with Sjögren’s syndrome and systemic lupus erythematosus. We recently showed that Ro52 is an E3 ubiquitin ligase and localizes to cytoplasmic bodies that are highly motile along the microtubule network. To expand our knowledge of Ro52, we searched partners co-operating with Ro52. We performed a yeast two-hybrid screening of a human brain cDNA library with Ro52 as bait. This screening identified several genes encoding Ro52-interacting proteins, including the apoptosis-related proteins, Daxx and FLASH. Further yeast two-hybrid assays revealed that Daxx binds to the B30.2 domain of Ro52 and that FLASH binds to coiled-coil domains of Ro52 through its death-effector domain-recruiting domain. These results suggest that Ro52, Daxx, and FLASH form heteromeric protein complexes. Indeed, this was supported by results of immunoprecipitation experiments in which we found that Daxx is co-immunoprecipitated with Ro52 in the presence of overexpressed FLASH. Importantly, our fluorescence microscopy revealed that, although Daxx is predominantly located in the nucleus, overexpression of both Ro52 and FLASH leads to relocation of Daxx into the cytoplasm. Thus, Ro52 seems to co-operate with FLASH to induce cytoplasmic localization of Daxx in cells.     

Ubiquitination of Ro52 autoantigen

Anti-Ro/SSA antibodies are antinuclear antibodies most commonly found in patients with Sj?gren's syndrome, a chronic autoimmune disease characterized by dryness of the eyes and mouth. The autoantibodies recognize a RING-finger protein, Ro52/SSA (52 kDa), whose function is still unknown. In this study, the ubiquitination of Ro52 was investigated. We found that Ro52 was strongly conjugated by a single molecule of ubiquitin in cells. Although the biological relevance of this mono-ubiquitination was not defined, the function of Ro52 might be modified by the mono-ubiquitination. We also found that Ro52 was conjugated with poly-ubiquitin chain in cells (poly-ubiquitination), suggesting that Ro52 may be downregulated by the ubiquitin-proteasome pathway in vivo. Interestingly, sera from patients with Sj?gren's syndrome showed heterogeneity in their reactivity to poly-ubiquitinated Ro52, probably because of their differing antigenic determinants. This heterogeneity of the reactivity might be associated with the varying clinical features found in patients with Sj?gren's syndrome.     

The mapping of the human 52-kD Ro/SSA autoantigen gene to human chromosome 11, and its polymorphisms.

Autoantibodies to Ro/SSA occur in nearly half of the patients with systemic lupus erythematosus and are associated with lymphopenia, photosensitive dermatitis, and pulmonary and renal disease, which suggests that they have an immunopathologic role. The majority of Ro/SSA precipitin-positive patients produce serum antibodies that bind to the 60-kD and 52-kD Ro/SSA proteins. We previously isolated and determined the nucleotide sequence of a cDNA clone that encodes the 52-kD form of the human Ro/SSA protein. In the present study, we have determined the chromosomal location of the gene by in situ hybridization to the end of the short arm of chromosome 11. Hybridization of portions of the cDNA probe to restriction enzyme-digested DNA indicated the gene is composed of at least three exons. The exon encoding the putative zinc fingers of this protein was found to be distinct from that which encodes the leucine zipper. An RFLP of this gene was identified and is associated with the presence of lupus, primarily in black Americans.     

The autoantigen Ro52 is an E3 ligase resident in the cytoplasm but enters the nucleus upon cellular exposure to nitric oxide

Patients with the systemic autoimmune diseases Sjögrens's syndrome and systemic lupus erythematosus often have autoantibodies against the intracellular protein Ro52. Ro52 is an E3 ligase dependent on the ubiquitin conjugation enzymes UBE2D1 and UBE2E1. While Ro52 and UBE2D1 are cytoplasmic proteins, UBE2E1 is localized to the nucleus. Here, we investigate how domains of human Ro52 regulate its intracellular localization. By expressing fluorescently labeled Ro52 and Ro52 mutants in HeLa cells, an intact coiled-coil domain was found to be necessary for the cytoplasmic localization of Ro52. The amino acids 381-470 of the B30.2 region were essential for translocation into the nucleus. Furthermore, after exposure of HeLa cells to the inflammatory mediator nitric oxide (NO), Ro52 translocated to the nucleus. A nuclear localization of Ro52 in inflamed tissue expressing inducible NO synthetase (iNOS) from cutaneous lupus patients was observed by immunohistochemistry and verified in NO-treated cultures of patient-derived primary keratinocytes. Our results show that the localization of Ro52 is regulated by endogenous sequences, and that nuclear translocation is induced by an inflammatory mediator. This suggests that Ro52 has both cytoplasmic and nuclear substrates, and that Ro52 mediates ubiquitination through UBE2D1 in the cytoplasm and through UBE2E1 in the nucleus.     

Ral-GTPase interacts with the beta1 subunit of Na+/K+-ATPase and is activated upon inhibition of the Na+/K+ pump

Na+/K+-ATPase functions as both an ion pump and a signal transducer. Cardiac glycosides partially inhibit Na+/K+-ATPase, causing activation of multiple interrelated growth pathways via the Na+/K+-ATPase/c-Src/epidermal growth factor receptor complex. Such pathways include Ras/MEK/ERK and Ral/RalGDS cascades, which can lead to cardiac hypertrophy. In search of novel Ral-GTPase binding proteins, we used RalB as the bait to screen a human testes cDNA expression library using the yeast 2-hybrid system. The results demonstrated that 1 of the RalB interacting clones represented the C-terminal region of the beta1 subunit of Na+/K+-ATPase. Further analysis using the yeast 2-hybrid system and full-length beta1 subunit of Na+/K+-ATPase confirmed the interaction with RalA and RalB. In vitro binding and pull-down assays demonstrated that the beta1 subunit of Na+/K+-ATPase interacts directly with RalA and RalB. Ral-GTP pull-down assays demonstrated that short-term ouabain treatment of A7r5 cells, a rat aorta smooth muscle cell line, caused activation of Ral GTPase. Maximal activation was observed 10 min after ouabain treatment. Ouabain-mediated Ral activation was inhibited upon the stimulation of Na+/K+-ATPase activity by Ang II. We propose that Ral GTPase is involved in the signal transducing function of Na+/K+-ATPase and provides a possible molecular mechanism connecting Ral to cardiac hypertrophy during diseased conditions.     

Identification of PLP2 and RAB5C as novel TPD52 binding partners through yeast two-hybrid screening

Tumor protein D52 (TPD52) is overexpressed in different cancers, but its molecular functions are poorly defined. A large, low-stringency yeast two-hybrid screen using full-length TPD52 bait identified known partners (TPD52, TPD52L1, TPD52L2, MAL2) and four other preys that reproducibly bound TPD52 and TPD52L1 baits (PLP2, RAB5C, GOLGA5, YIF1A). PLP2 and RAB5 interactions with TPD52 were confirmed in pull down assays, with interaction domain mapping experiments indicating that both proteins interact with a novel binding region of TPD52. This study provides insights into TPD52 functions, and ways to maximise the efficiency of low-stringency yeast two-hybrid screens.     

Anti-SSA/Ro and anti-SSB/La autoantibodies bind the surface of apoptotic fetal cardiocytes and promote secretion of TNF-alpha by macrophages

Despite the near universal association of congenital heart block and maternal Abs to SSA/Ro and SSB/La, the intracellular location of these Ags has made it difficult to substantiate their involvement in pathogenicity. To define whether components of the SSA/Ro-SSB/La complex, which translocate during apoptosis, are indeed accessible to extracellular Abs, two approaches were taken: immunoprecipitation of surface biotinylated proteins and scanning electron microscopy. Human fetal cardiocytes from 16-24-wk abortuses were cultured and incubated with staurosporine to induce apoptosis. Surface biotinylated 48-kDa SSB/La was reproducibly immunoprecipitated from apoptotic, but not nonapoptotic cardiocytes. Surface expression of SSA/Ro and SSB/La was further substantiated by scanning electron microscopy. Gold particles (following incubation with gold-labeled sera containing various specificities of anti-SSA/Ro-SSB/La Abs and murine mAb to SSB/La and 60-kDa SSA/Ro) were consistently observed on early and late apoptotic cardiocytes. No particles were seen after incubation with control antisera. To evaluate whether opsonized apoptotic cardiocytes promote inflammation, cells were cocultured with macrophages. Compared with nonapoptotic cardiocytes or apoptotic cardiocytes incubated with normal sera, apoptotic cardiocytes preincubated with affinity-purified Abs to SSB/La, 52-kDa SSA/Ro, or 60-kDa SSA/Ro increased the secretion of TNF-alpha from cocultured macrophages. In summary, apoptosis results in surface accessibility of all SSA/Ro-SSB/La Ags for recognition by circulating maternal Abs. It is speculated that in vivo such opsonized apoptotic cardiocytes promote an inflammatory response by resident macrophages with damage to surrounding conducting tissue.     

Identification of MAL2, a novel member of the mal proteolipid family, though interactions with TPD52-like proteins in the yeast two-hybrid system

The TPD52 (tumor protein D52)-like proteins are small coiled-coil motif-bearing proteins which were first identified though their expression in human breast carcinoma. TPD52-like proteins are known to interact in hetero-and homomeric fashions, but there are no known heterologous binding partners for these proteins. We now report the cloning of a novel member of the MAL proteolipid family, named MAL2, though its interaction with a TPD52L2 bait in a yeast two-hybrid screen. MAL2 is predicted to be 176 residues (19 kDa) with four transmembrane domains and is 35.8% identical to MAL, a proteolipid required in apical vesicle transport. The MAL2 prey bound all TPD52-like baits tested in the yeast two-hybrid system and in vitro translation of MAL2 produced a single 19-kDa (35)S-labeled protein which specifically bound full-length GST-Tpd52 in GST pull-down assays. The gene MAL2, which was localized to human chromosomal band 8q23 and shown to consist of four exons, is predominantly expressed in human kidney, lung, and liver. Our study has therefore identified a novel member of the MAL proteolipid family and potentially implicates TPD52-like proteins in vesicle transport.     

角质细胞生长因子-2与核糖体蛋白L22相互作用的发现及在哺乳动物细胞中的验证

通过聚合酶链式反应从人胎肝cDNA文库中钓取KGF-2 cDNA,构建诱饵蛋白载体pAS2-1-KGF-2并对其自身转录激活活性进行鉴定,利用酵母双杂交系统筛选人胎肝cDNA文库,挑选双阳性克隆.DNA序列分析和同源检索显示,所获侯选蛋白为人核糖体蛋白L22(RPL22).将KGF-2和侯选蛋白分别克隆至哺乳动物细胞双杂交的BD、AD质粒中,共同转染COS-7细胞,通过CAT分析验证了KGF-2和侯选蛋白之间的相互作用.为阐明KGF-2作用的分子机制提供有益线索.     

The Sjogren's syndrome-associated autoantigen Ro52 is an E3 ligase that regulates proliferation and cell death

Patients affected by Sj?gren's syndrome and systemic lupus erythematosus (SLE) carry autoantibodies to an intracellular protein denoted Ro52. Although the serologic presence of Ro52 autoantibodies is used clinically for diagnostic purposes, the function of the protein or why it is targeted as an autoantigen in several rheumatic conditions has not been elucidated. In this study, we show that the expression of Ro52 is significantly increased in PBMC of patients with Sj?gren's syndrome and SLE, and demonstrate that Ro52 is a RING-dependent E3 ligase involved in ubiquitination. Overexpression of Ro52, but not of Ro52 lacking the RING domain, in a mouse B cell line lead to decreased growth in steady state and increased cell death after activation via the CD40 pathway. The role of Ro52 in activation-mediated cell death was further confirmed as a reduction in Ro52 expression restored cell viability. These findings suggest that the increased expression of the Ro52 autoantigen in patients may be directly involved in the reduced cellular proliferation and increased apoptotic cell death observed in Sj?gren's syndrome and SLE, and may thus contribute to the autoantigenic load and induction of autoimmune B and T cell responses observed in rheumatic patients.     

Regulation of p27 degradation and S-phase progression by Ro52 RING finger protein

Ubiquitin-mediated degradation of the cyclin-dependent kinase inhibitor p27 provides a powerful route for enforcing normal progression through the mammalian cell cycle. According to a current model, the ubiquitination of p27 during S-phase progression is mediated by SCF(Skp2) E3 ligase that captures Thr187-phosphorylated p27 by means of the F-box protein Skp2, which in turn couples the bound substrate via Skp1 to a catalytic core complex composed of Cul1 and the Rbx/Roc RING finger protein. Here we identify Skp2 as a component of an Skp1-cullin-F-box complex that is based on a Cul1-Ro52 RING finger B-box coiled-coil motif family protein catalytic core. Ro52-containing complexes display E3 ligase activity and promote the ubiquitination of Thr187-phosphorylated p27 in a RING-dependent manner in vitro. The knockdown of Ro52 expression in human cells with small interfering RNAs causes the accumulation of p27 and the failure of cells to enter S phase. Importantly, these effects are abrogated by the simultaneous removal of p27. Taken together, these data suggest a key role for Ro52 RING finger protein in the regulation of p27 degradation and S-phase progression in mammalian cells and provide evidence for the existence of a Cul1-based catalytic core that utilizes Ro52 RING protein to promote ubiquitination.     

Analysis of protein--RNA interactions within Ro ribonucleoprotein complexes.

The interactions between Ro and La proteins and hY RNAs have been analysed. The binding site for the 60 kDa Ro protein on hY RNAs is shown to be the terminal part of the base paired stem structure, which contains the most highly conserved sequence among hY RNAs. The bulged C-residue within this region plays an important role in the recognition by this protein. The same regions of hY RNAs are essential for the association of the 52 kDa Ro protein with the RNAs, strongly suggesting that the 60 kDa Ro protein is required for the 52 kDa Ro protein to bind, presumably via protein-protein interactions, to Ro RNPs. The binding site for the La protein on hY RNAs is shown to be the oligouridylate stretch near the 3'-end of the RNAs, which is also recognized when additional nucleotides flank this motif at the 3'-side. Additional sequence elements in hY3 and hY5, but not in hY1, are bound by the La protein as well. Deletion mutagenesis showed that the RNP motif, previously identified in many ribonucleoprotein (RNP) proteins and in some cases shown to be almost sufficient for the interaction with RNA, of both the 60 kDa Ro and the La protein are not sufficient for the interaction with hY RNAs. Substantial parts of these proteins flanking the RNP motif are needed as well. It is likely that they stabilize the correct conformation of the RNP motif for RNA binding.