Autoantigen Ro52 is an E3 ubiquitin ligase

Anti-Ro/SSA antibodies are classic autoantibodies 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, whose function is still unknown. Since many RING-finger proteins have been identified as E3 ubiquitin ligases, this study was designed to determine whether Ro52 functions as an E3 ubiquitin ligase. For this purpose, recombinant Ro52 was purified from bacterial lysate and used to investigate its activity of E3 ubiquitin ligase in vitro. Its enzymatic activity was also tested in HEK293T cells using wild-type Ro52 and its RING-finger mutant. Our results indicated that Ro52 ubiquitinates itself in cooperation with E2 ubiquitin-conjugating enzyme UbcH5B, thereby validating that Ro52 is a RING-finger-type E3 ubiquitin ligase. Importantly, this ubiquitin modification is predominantly monoubiquitination, which does not target Ro52 to the proteasome for degradation.     

Function and subcellular location of Ro52beta

Autoantigen Ro52alpha was recently identified as an E3 ubiquitin ligase. Its splicing variant Ro52beta, which lacks a leucine zipper, has not been characterized yet. We therefore characterized Ro52beta in contrast to Ro52alpha. Our biochemical assays revealed that both Ro52alpha and Ro52beta function as E3 ubiquitin ligases and self-ubiquitinate in cooperation with UbcH5B in vitro. In addition, both Ro52alpha and Ro52beta are ubiquitinated when overexpressed with ubiquitin in HEK293T cells, suggesting that both function as E3 ligases and self-ubiquitinate in vivo. However, cytological studies revealed that Ro52alpha mainly localizes to the cytoplasmic rod-like structures, whereas Ro52beta diffusely localizes to both the cytoplasm and the nucleus. Since the leucine zipper plays a role in the homodimerization and heterodimerization of Ro52alpha, the dimerization might be required for the localization of Ro52alpha to the rod-like structures. On the basis of these results, Ro52alpha and Ro52beta appear to ubiquitinate their particular substrates at different locations.     

SSA/Ro52 autoantigen interacts with Dcp2 to enhance its decapping activity

We identified human decapping enzyme 2 (hDCP2) as a binding protein with Ro52, being colocalized in processing bodies (p-bodies). We also showed that the N-terminus and C-terminus of Ro52 bound to hDCP2. Moreover, Ro52 enhanced decapping activity of hDCP2 in a dose-dependent manner. Our data support the novel notion of the association between Ro52 with hDCP2 protein in cytoplasmic p-bodies, playing a role in mRNA metabolism in response to cellular stimulation.     

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.     

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.     

Ro60/SSA levels are increased and promote the progression of pancreatic ductal adenocarcinoma

Ro60/SSA is a vital auto antigen that is targeted in Sjogren's syndrome and systemic lupus erythematosus (SLE). However, its role in solid cancers has rarely been reported. The present study investigated the expression and function of Ro60/SSA in the development of pancreatic ductal adenocarcinoma (PDAC) both in vitro and in vivo. Immunohistochemistry was used to examine the expression of Ro60/SSA in PDAC and normal pancreatic tissues by using tissue microarray chips. The results showed that Ro60/SSA expression was increased in PDAC tissues compared with normal pancreatic tissues. Knockdown of Ro60/SSA by siRNA transfection significantly decreased cell proliferation and invasion in vitro. Furthermore, knockdown of Ro60/SSA inhibited the growth of subcutaneous tumors in vivo. Taken together, the current study provides evidence of new function of Ro60/SSA in the development of cancer. It facilitates pancreatic cancer proliferation, migration and invasion. Therefore, it may represent a novel molecular target for the management of pancreatic cancer.     

Ubiquitination of E3 ligases: self-regulation of the ubiquitin system via proteolytic and non-proteolytic mechanisms

Ubiquitin modification of many cellular proteins targets them for proteasomal degradation, but in addition can also serve non-proteolytic functions. Over the last years, a significant progress has been made in our understanding of how modification of the substrates of the ubiquitin system is regulated. However, little is known on how the ubiquitin system that is comprised of ～1500 components is regulated. Here, we discuss how the biggest subfamily within the system, that of the E3 ubiquitin ligases that endow the system with its high specificity towards the numerous substrates, is regulated and in particular via self-regulation mediated by ubiquitin modification. Ligases can be targeted for degradation in a self-catalyzed manner, or through modification mediated by an external ligase(s). In addition, non-proteolytic functions of self-ubiquitination, for example activation of the ligase, of E3s are discussed.     

Ubiquitination of RhoA by Smurf1 promotes neurite outgrowth

The Rho-family of small GTPases consists of essential regulators of neurite outgrowth, axonal pathfinding, and dendritic arborization. Previous work has demonstrated in non-neuronal cell types that Smurf1, an E3 ubiquitin ligase, regulates cell polarity and protrusive activity via PKCzeta-dependent recruitment to cellular protrusion sites, and subsequent ubiquitination and proteasomal degradation of RhoA. In this study, we show that Smurf1 enhances neurite outgrowth in Neuro2a neuroblastoma cells. We demonstrate that RhoA is ubiquitinated, and that Smurf1 and RhoA physically interact in vivo. Interestingly, Smurf1 overexpression in Neuro2a cells dramatically reduces RhoA protein levels during dibutyric cyclic AMP, but not retinoic acid induced neurite outgrowth. This Smurf1-dependent reduction in RhoA protein levels was abrogated using the general proteasome inhibitor MG132, suggesting that RhoA is targeted for ubiquitination and degradation via Smurf1. Together, our data suggest that localized regulation of different subsets of Rho GTPases by specific guidance signals results in an intracellular asymmetry of RhoA activity, which could regulate neurite outgrowth and guidance.     

RN181, a novel ubiquitin E3 ligase that interacts with the KVGFFKR motif of platelet integrin alpha(IIb)beta3

We previously identified proteins that bind with high affinity to a peptide corresponding to the cytoplasmic regulatory domain (KVGFFKR) of the platelet-specific integrin subunit α_IIb. These included a hypothetical protein termed HSPC238, recently renamed as RING finger protein, RN181. Here, we establish the presence of RN181 in human platelets by RT-PCR, Western blotting and mass spectrometry and confirm its affinity for the platelet integrin. We demonstrate that RN181 has ubiquitin E3 ligase activity and that all other components of the ubiquitination pathway are abundant in platelets, suggesting a novel link of integrin signal transduction pathways with ubiquitin-conjugation events.     

Ubiquitin ligases, critical mediators of endoplasmic reticulum-associated degradation

Endoplasmic reticulum-associated degradation (ERAD) represents the primary means of quality control within the secretory pathway. Critical to this process are ubiquitin protein ligases (E3s) which, together with ubiquitin conjugating enzymes (E2s), mediate the ubiquitylation of proteins targeted for degradation from the ER. In this chapter we review our knowledge of both Saccharomyces cerevisiae and mammalian ERAD ubiquitin ligases. We focus on recent insights into these E3s, their associated proteins and potential mechanisms of action.     

HIV-1 Vpr Loads Uracil DNA Glycosylase-2 onto DCAF1, a Substrate Recognition Subunit of a Cullin 4A-RING E3 Ubiquitin Ligase for Proteasome-dependent Degradation

The human immunodeficiency virus type 1 (HIV-1) accessory protein, Vpr, interacts with several host cellular proteins including uracil DNA glycosylase-2 (UNG2) and a cullin-RING E3 ubiquitin ligase assembly (CRL4^DCAF1). The ligase is composed of cullin 4A (CUL4A), RING H2 finger protein (RBX1), DNA damage-binding protein 1 (DDB1), and a substrate recognition subunit, DDB1- and CUL4-associated factor 1 (DCAF1). Here we show that recombinant UNG2 specifically interacts with Vpr, but not with Vpx of simian immunodeficiency virus, forming a heterotrimeric complex with DCAF1 and Vpr in vitro as well as in vivo. Using reconstituted CRL4^DCAF1 and CRL4^DCAF1-Vpr E3 ubiquitin ligases in vitro reveals that UNG2 ubiquitination (ubiquitylation) is facilitated by Vpr. Co-expression of DCAF1 and Vpr causes down-regulation of UNG2 in a proteasome-dependent manner, with Vpr mutants that are defective in UNG2 or DCAF1 binding abrogating this effect. Taken together, our results show that the CRL4^DCAF1 E3 ubiquitin ligase can be subverted by Vpr to target UNG2 for degradation.     

The<Emphasis Type="Italic"> Arabidopsis KAKTUS</Emphasis> gene encodes a HECT protein and controls the number of endoreduplication cycles

In animals and plants, many cell types switch from mitotic cycles to endoreduplication cycles during differentiation. Little is known about the way in which the number of endoreduplication cycles is controlled in such endopolyploid cells. In this study we have characterized at the molecular level three mutations in the Arabidopsis gene KAKTUS ( KAK), which were previously shown specifically to repress endoreduplication in trichomes. We show that KAK is also involved in the regulation of the number of endoreduplication cycles in various organs that are devoid of trichomes. KAK encodes a protein with sequence similarity to HECT domain proteins. As this class of proteins is known to be involved in ubiquitin-mediated protein degradation, our finding suggests that the number of endoreduplication cycles that occur in several cell types is controlled by this pathway. The KAK gene defines a monophylogenetic subgroup of HECT proteins that also contain Armadillo-like repeats.Communicated by G. Jürgens     

InTRIMsic immunity: Positive and negative regulation of immune signaling by tripartite motif proteins

During the immune response, striking the right balance between positive and negative regulation is critical to effectively mount an anti-microbial defense while preventing detrimental effects from exacerbated immune activation. Intra-cellular immune signaling is tightly regulated by various post-translational modifications, which allow for this dynamic response. One of the post-translational modifiers critical for immune control is ubiquitin, which can be covalently conjugated to lysines in target molecules, thereby altering their functional properties. This is achieved in a process involving E3 ligases which determine ubiquitination target specificity.One of the most prominent E3 ligase families is that of the tripartite motif (TRIM) proteins, which counts over 70 members in humans. Over the last years, various studies have contributed to the notion that many members of this protein family are important immune regulators. Recent studies into the mechanisms by which some of the TRIMs regulate the innate immune system have uncovered important immune regulatory roles of both covalently attached, as well as unanchored poly-ubiquitin chains. This review highlights TRIM evolution, recent findings in TRIM-mediated immune regulation, and provides an outlook to current research hurdles and future directions.     

A Novel Type of E3 Ligase for the Ufm1 Conjugation System

The ubiquitin fold modifier 1 (Ufm1) is the most recently discovered ubiquitin-like modifier whose conjugation (ufmylation) system is conserved in multicellular organisms. Ufm1 is known to covalently attach with cellular protein(s) via a specific E1-activating enzyme (Uba5) and an E2-conjugating enzyme (Ufc1), but its E3-ligating enzyme(s) as well as the target protein(s) remain unknown. Herein, we report both a novel E3 ligase for Ufm1, designated Ufl1, and an Ufm1-specific substrate ligated by Ufl1, C20orf116. Ufm1 was covalently conjugated with C20orf116. Although Ufl1 has no obvious sequence homology to any other known E3s for ubiquitin and ubiquitin-like modifiers, the C20orf116·Ufm1 formation was greatly accelerated by Ufl1. The C20orf116·Ufm1 conjugate was cleaved by Ufm1-specific proteases, implying the reversibility of ufmylation. The conjugation was abundant in the liver and lungs of Ufm1-transgenic mice, fractionated into membrane fraction, and impaired in Uba5 knock-out cells. Intriguingly, immunological analysis revealed localizations of Ufl1 and C20orf116 mainly to the endoplasmic reticulum. Our results provide novel insights into the Ufm1 system involved in cellular regulation of multicellular organisms.     

Dysfunction of the ubiquitin ligase ube3a may be associated with synaptic pathophysiology in a mouse model of huntington disease

Huntington disease (HD) is a hereditary neurodegenerative disorder characterized by progressive cognitive, psychiatric, and motor symptoms. The disease is caused by abnormal expansion of CAG repeats in the gene encoding huntingtin, but how mutant huntingtin leads to early cognitive deficits in HD is poorly understood. Here, we demonstrate that the ubiquitin ligase Ube3a, which is implicated in synaptic plasticity and involved in the clearance of misfolded polyglutamine protein, is strongly recruited to the mutant huntingtin nuclear aggregates, resulting in significant loss of its functional pool in different regions of HD mouse brain. Interestingly, Arc, one of the substrates of Ube3a linked with synaptic plasticity, is also associated with nuclear aggregates, although its synaptic level is increased in the hippocampus and cortex of HD mouse brain. Different regions of HD mouse brain also exhibit decreased levels of AMPA receptors and various pre- and postsynaptic proteins, which could be due to the partial loss of function of Ube3a. Transient expression of mutant huntingtin in mouse primary cortical neurons further demonstrates recruitment of Ube3a into mutant huntingtin aggregates, increased accumulation of Arc, and decreased numbers of GluR1 puncta in the neuronal processes. Altogether, our results suggest that the loss of function of Ube3a might be associated with the synaptic abnormalities observed in HD.     

Dynamic Ubiquitination of the Mitogen-activated Protein Kinase Kinase (MAPKK) Ste7 Determines Mitogen-activated Protein Kinase (MAPK) Specificity

Ubiquitination is a post-translational modification that tags proteins for proteasomal degradation. In addition, there is a growing appreciation that ubiquitination can influence protein activity and localization. Ste7 is a prototype MAPKK in yeast that participates in both the pheromone signaling and nutrient deprivation/invasive growth pathways. We have shown previously that Ste7 is ubiquitinated upon pheromone stimulation. Here, we show that the Skp1/Cullin/F-box ubiquitin ligase SCF^Cdc4 and the ubiquitin protease Ubp3 regulate Ste7 ubiquitination and signal specificity. Using purified components, we demonstrate that SCF^Cdc4 ubiquitinates Ste7 directly. Using gene deletion mutants, we show that SCF^Cdc4 and Ubp3 have opposing effects on Ste7 ubiquitination. Although SCF^Cdc4 is necessary for proper activation of the pheromone MAPK Fus3, Ubp3 is needed to limit activation of the invasive growth MAPK Kss1. Finally, we show that Fus3 phosphorylates Ubp3 directly and that phosphorylation of Ubp3 is necessary to limit Kss1 activation. These results reveal a feedback loop wherein one MAPK limits the ubiquitination of an upstream MAPKK and thereby prevents spurious activation of a second competing MAPK.