Overexpression of a Soybean Ariadne-Like Ubiquitin Ligase Gene GmARI1 Enhances Aluminum Tolerance in Arabidopsis

Ariadne (ARI) subfamily of RBR (Ring Between Ring fingers) proteins have been found as a group of putative E3 ubiquitin ligases containing RING (Really Interesting New Gene) finger domains in fruitfly, mouse, human and Arabidopsis. Recent studies showed several RING-type E3 ubiquitin ligases play important roles in plant response to abiotic stresses, but the function of ARI in plants is largely unknown. In this study, an ariadne-like E3 ubiquitin ligase gene was isolated from soybean, Glycine max (L.) Merr., and designated as GmARI1. It encodes a predicted protein of 586 amino acids with a RBR supra-domain. Subcellular localization studies using Arabidopsis protoplast cells indicated GmARI protein was located in nucleus. The expression of GmARI1 in soybean roots was induced as early as 2–4 h after simulated stress treatments such as aluminum, which coincided with the fact of aluminum toxicity firstly and mainly acting on plant roots. In vitro ubiquitination assay showed GmARI1 protein has E3 ligase activity. Overexpression of GmARI1 significantly enhanced the aluminum tolerance of transgenic Arabidopsis. These findings suggest that GmARI1 encodes a RBR type E3 ligase, which may play important roles in plant tolerance to aluminum stress.     

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.     

Molecular insights into RBR E3 ligase ubiquitin transfer mechanisms

RING‐in‐between‐RING (RBR) ubiquitin (Ub) ligases are a distinct class of E3s, defined by a RING1 domain that binds E2 Ub‐conjugating enzyme and a RING2 domain that contains an active site cysteine similar to HECT‐type E3s. Proposed to function as RING/HECT hybrids, details regarding the Ub transfer mechanism used by RBRs have yet to be defined. When paired with RING‐type E3s, E2s perform the final step of Ub ligation to a substrate. In contrast, when paired with RBR E3s, E2s must transfer Ub onto the E3 to generate a E3~Ub intermediate. We show that RBRs utilize two strategies to ensure transfer of Ub from the E2 onto the E3 active site. First, RING1 domains of HHARI and RNF144 promote open E2~Ubs. Second, we identify a Ub‐binding site on HHARI RING2 important for its recruitment to RING1‐bound E2~Ub. Mutations that ablate Ub binding to HHARI RING2 also decrease RBR ligase activity, consistent with RING2 recruitment being a critical step for the RBR Ub transfer mechanism. Finally, we demonstrate that the mechanism defined here is utilized by a variety of RBRs.     

Parkin mitochondrial translocation is achieved through a novel catalytic activity coupled mechanism

Pink1, a mitochondrial kinase, and Parkin, an E3 ubiquitin ligase, function in mitochondrial maintenance. Pink1 accumulates on depolarized mitochondria, where it recruits Parkin to mainly induce K63-linked chain ubiquitination of outer membrane proteins and eventually mitophagy. Parkin belongs to the RBR E3 ligase family. Recently, it has been proposed that the RBR domain transfers ubiquitin to targets via a cysteine∼ubiquitin enzyme intermediate, in a manner similar to HECT domain E3 ligases. However, direct evidence for a ubiquitin transfer mechanism and its importance for Parkin''s in vivo function is still missing. Here, we report that Parkin E3 activity relies on cysteine-mediated ubiquitin transfer during mitophagy. Mutating the putative catalytic cysteine to serine (Parkin C431S) traps ubiquitin, and surprisingly, also abrogates Parkin mitochondrial translocation, indicating that E3 activity is essential for Parkin translocation. We found that Parkin can bind to K63-linked ubiquitin chains, and that targeting K63-mimicking ubiquitin chains to mitochondria restores Parkin C431S localization. We propose that Parkin translocation is achieved through a novel catalytic activity coupled mechanism.     

Structure of the HHARI Catalytic Domain Shows Glimpses of a HECT E3 Ligase

The ubiquitin-signaling pathway utilizes E1 activating, E2 conjugating, and E3 ligase enzymes to sequentially transfer the small modifier protein ubiquitin to a substrate protein. During the last step of this cascade different types of E3 ligases either act as scaffolds to recruit an E2 enzyme and substrate (RING), or form an ubiquitin-thioester intermediate prior to transferring ubiquitin to a substrate (HECT). The RING-inBetweenRING-RING (RBR) proteins constitute a unique group of E3 ubiquitin ligases that includes the Human Homologue of Drosophila Ariadne (HHARI). These E3 ligases are proposed to use a hybrid RING/HECT mechanism whereby the enzyme uses facets of both the RING and HECT enzymes to transfer ubiquitin to a substrate. We now present the solution structure of the HHARI RING2 domain, the key portion of this E3 ligase required for the RING/HECT hybrid mechanism. The structure shows the domain possesses two Zn²⁺-binding sites and a single exposed cysteine used for ubiquitin catalysis. A structural comparison of the RING2 domain with the HECT E3 ligase NEDD4 reveals a near mirror image of the cysteine and histidine residues in the catalytic site. Further, a tandem pair of aromatic residues exists near the C-terminus of the HHARI RING2 domain that is conserved in other RBR E3 ligases. One of these aromatic residues is remotely located from the catalytic site that is reminiscent of the location found in HECT E3 enzymes where it is used for ubiquitin catalysis. These observations provide an initial structural rationale for the RING/HECT hybrid mechanism for ubiquitination used by the RBR E3 ligases.     

RNF185, a novel mitochondrial ubiquitin E3 ligase, regulates autophagy through interaction with BNIP1

Autophagy is an evolutionarily conserved catabolic process that allows recycling of cytoplasmic organelles, such as mitochondria, to offer a bioenergetically efficient pathway for cell survival. Considerable progress has been made in characterizing mitochondrial autophagy. However, the dedicated ubiquitin E3 ligases targeting mitochondria for autophagy have not been revealed. Here we show that human RNF185 is a mitochondrial ubiquitin E3 ligase that regulates selective mitochondrial autophagy in cultured cells. The two C-terminal transmembrane domains of human RNF185 mediate its localization to mitochondrial outer membrane. RNF185 stimulates LC3II accumulation and the formation of autophagolysosomes in human cell lines. We further identified the Bcl-2 family protein BNIP1 as one of the substrates for RNF185. Human BNIP1 colocalizes with RNF185 at mitochondria and is polyubiquitinated by RNF185 through K63-based ubiquitin linkage in vivo. The polyubiquitinated BNIP1 is capable of recruiting autophagy receptor p62, which simultaneously binds both ubiquitin and LC3 to link ubiquitination and autophagy. Our study might reveal a novel RNF185-mediated mechanism for modulating mitochondrial homeostasis through autophagy.     

New classes of E3 ligases illuminated by chemical probes

Specificity in the ubiquitin system depends on E3 ligases, largely belonging to a handful of families discovered more than a decade ago. However, the last two years brought a quantum leap in the identification and/or mechanistic characterization of eukaryotic ubiquitin ligases, in part through implementation of activity-based chemical probes and cryo-EM. Here, we survey recent discoveries of RING-Cys-Relay, RZ-finger, and neddylated cullin–RING–ARIH RBR E3–E3 ubiquitin ligase mechanisms. These ligases transfer ubiquitin through unprecedented mechanisms—via novel catalytic domains or domain combinations—and collectively modify unconventional amino acids, non-proteinaceous bacterial lipid targets, and structurally-diverse substrates recruited to numerous cullin–RING ligases. We anticipate major expansion of the types, features, and mechanisms of E3 ligases will emerge from such chemical and structural approaches in the coming years.     

Nuclear Targeting of an Endosomal E3 Ubiquitin Ligase

Ring finger protein 13 (RNF13) is an E3 ubiquitin ligase embedded in endosome membranes. The protein undergoes constitutive post‐translational proteolysis, making its detection difficult unless cells are incubated with a proteasome inhibitor to allow biosynthetic forms to accumulate. When cells were treated with phorbol 12‐myristate 13‐acetate (PMA), RNF13 avoided proteolysis. A similar stabilization was seen on ionomycin treatment of cells. Drug treatment stabilized both the full‐length protein and a membrane‐embedded C‐terminal fragment generated following ectodomain shedding. Immunofluorescence staining revealed that PMA treatment caused the protein to accumulate in recycling endosomes, where it colocalized with transferrin receptor, and on the inner nuclear membrane, where it colocalized with lamin B. Expression of dominant‐negative Rab11 inhibited nuclear localization, suggesting RNF13 was targeted to the inner nuclear membrane through recycling endosomes. New protein synthesis was necessary for this targeting. Nuclear localization was confirmed by immunoelectron microscopy and by purification of the inner nuclear membrane. Stress‐induced transport of an endosomal protein to the inner nuclear membrane is a novel mechanism for introduction of regulatory proteins to the DNA environment. RNF13, with its ubiquitin ligase‐active RING domain, has the potential to turn over key nuclear proteins in response to signals received at the plasma membrane.     

Mim1 functions in an oligomeric form to facilitate the integration of Tom20 into the mitochondrial outer membrane

The translocase of the outer mitochondrial membrane (TOM) complex is the general entry site into the organelle for newly synthesized proteins. Despite its central role in the biogenesis of mitochondria, the assembly process of this complex is not completely understood. Mim1 (mitochondrial import protein 1) is a mitochondrial outer membrane protein with an undefined role in the assembly of the TOM complex. The protein is composed of an N-terminal cytosolic domain, a central putative transmembrane segment (TMS) and a C-terminal domain facing the intermembrane space. Here we show that Mim1 is required for the integration of the import receptor Tom20 into the outer membrane. We further investigated what the structural characteristics allowing Mim1 to fulfil its function are. The N- and C-terminal domains of Mim1 are crucial neither for the function of the protein nor for its biogenesis. Thus, the TMS of Mim1 is the minimal functional domain of the protein. We show that Mim1 forms homo-oligomeric structures via its TMS, which contains two helix-dimerization GXXXG motifs. Mim1 with mutated GXXXG motifs did not form oligomeric structures and was inactive. With all these data taken together, we propose that the homo-oligomerization of Mim1 allows it to fulfil its function in promoting the integration of Tom20 into the mitochondrial outer membrane.     

Human RNF169 is a negative regulator of the ubiquitin-dependent response to DNA double-strand breaks

Nonproteolytic ubiquitylation of chromatin surrounding deoxyribonucleic acid double-strand breaks (DSBs), mediated by the RNF8/RNF168 ubiquitin ligases, plays a key role in recruiting repair factors, including 53BP1 and BRCA1, to reestablish genome integrity. In this paper, we show that human RNF169, an uncharacterized E3 ubiquitin ligase paralogous to RNF168, accumulated in DSB repair foci through recognition of RNF168-catalyzed ubiquitylation products by its motif interacting with ubiquitin domain. Unexpectedly, RNF169 was dispensable for chromatin ubiquitylation and ubiquitin-dependent accumulation of repair factors at DSB sites. Instead, RNF169 functionally competed with 53BP1 and RAP80-BRCA1 for association with RNF168-modified chromatin independent of its catalytic activity, limiting the magnitude of their recruitment to DSB sites. By delaying accumulation of 53BP1 and RAP80 at damaged chromatin, RNF169 stimulated homologous recombination and restrained nonhomologous end joining, affecting cell survival after DSB infliction. Our results show that RNF169 functions in a noncanonical fashion to harness RNF168-mediated protein recruitment to DSB-containing chromatin, thereby contributing to regulation of DSB repair pathway utilization.     

A Small Molecule Inhibitor of Polycomb Repressive Complex 1 Inhibits Ubiquitin Signaling at DNA Double-strand Breaks

Polycomb-repressive complex 1 (PRC1)-mediated histone ubiquitylation plays an important role in aberrant gene silencing in human cancers and is a potential target for cancer therapy. Here we show that 2-pyridine-3-yl-methylene-indan-1,3-dione (PRT4165) is a potent inhibitor of PRC1-mediated H2A ubiquitylation in vivo and in vitro. The drug also inhibits the accumulation of all detectable ubiquitin at sites of DNA double-strand breaks (DSBs), the retention of several DNA damage response proteins in foci that form around DSBs, and the repair of the DSBs. In vitro E3 ubiquitin ligase activity assays revealed that PRT4165 inhibits both RNF2 and RING 1A, which are partially redundant paralogues that together account for the E3 ubiquitin ligase activity found in PRC1 complexes, but not RNF8 nor RNF168. Because ubiquitylation is completely inhibited despite the efficient recruitment of RNF8 to DSBs, our results suggest that PRC1-mediated monoubiquitylation is required for subsequent RNF8- and/or RNF168-mediated polyubiquitylation. Our results demonstrate the unique feature of PRT4165 as a novel chromatin-remodeling compound and provide a new tool for the inhibition of ubiquitylation signaling at DNA double-strand breaks.     

Transmembrane Segment 11 Appears to Line the Purine Permeation Pathway of the Plasmodium falciparum Equilibrative Nucleoside Transporter 1 (PfENT1)

Purine transport is essential for malaria parasites to grow because they lack the enzymes necessary for de novo purine biosynthesis. The Plasmodium falciparum Equilibrative Nucleoside Transporter 1 (PfENT1) is a member of the equilibrative nucleoside transporter (ENT) gene family. PfENT1 is a primary purine transport pathway across the P. falciparum plasma membrane because PfENT1 knock-out parasites are not viable at physiologic extracellular purine concentrations. Topology predictions and experimental data indicate that ENT family members have eleven transmembrane (TM) segments although their tertiary structure is unknown. In the current work, we showed that a naturally occurring polymorphism, F394L, in TM11 affects transport substrate K_m. We investigated the structure and function of the TM11 segment using the substituted cysteine accessibility method. We showed that mutation to Cys of two highly conserved glycine residues in a GXXXG motif significantly reduces PfENT1 protein expression levels. We speculate that the conserved TM11 GXXXG glycines may be critical for folding and/or assembly. Small, cysteine-specific methanethiosulfonate (MTS) reagents reacted with four TM11 Cys substitution mutants, L393C, I397C, T400C, and Y403C. Larger MTS reagents do not react with the more cytoplasmic positions. Hypoxanthine, a transported substrate, protected L393C, I397C, and T400C from covalent modification by the MTS reagents. Plotted on an α-helical wheel, Leu-393, Ile-397, and Thr-400 lie on one face of the helix in a 60° arc suggesting that TM11 is largely α helical. We infer that they line a water-accessible surface, possibly the purine permeation pathway. These results advance our understanding of the ENT structure.     

A new non-catalytic role for ubiquitin ligase RNF8 in unfolding higher-order chromatin structure

The ubiquitin ligases RNF8 and RNF168 orchestrate DNA damage signalling through the ubiquitylation of histone H2A and the recruitment of downstream repair factors. Here, we demonstrate that RNF8, but not RNF168 or the canonical H2A ubiquitin ligase RNF2, mediates extensive chromatin decondensation. Our data show that CHD4, the catalytic subunit of the NuRD complex, interacts with RNF8 and is essential for RNF8-mediated chromatin unfolding. The chromatin remodelling activity of CHD4 promotes efficient ubiquitin conjugation and assembly of RNF168 and BRCA1 at DNA double-strand breaks. Interestingly, RNF8-mediated recruitment of CHD4 and subsequent chromatin remodelling were independent of the ubiquitin-ligase activity of RNF8, but involved a non-canonical interaction with the forkhead-associated (FHA) domain. Our study reveals a new mechanism of chromatin remodelling-assisted ubiquitylation, which involves the cooperation between CHD4 and RNF8 to create a local chromatin environment that is permissive to the assembly of checkpoint and repair machineries at DNA lesions.     

TRIAD1 and HHARI bind to and are activated by distinct neddylated Cullin‐RING ligase complexes

RING (Really Interesting New Gene)‐in‐between‐RING (RBR) enzymes are a distinct class of E3 ubiquitin ligases possessing a cluster of three zinc‐binding domains that cooperate to catalyse ubiquitin transfer. The regulation and biological function for most members of the RBR ligases is not known, and all RBR E3s characterized to date are auto‐inhibited for in vitro ubiquitylation. Here, we show that TRIAD1 and HHARI, two members of the Ariadne subfamily ligases, associate with distinct neddylated Cullin‐RING ligase (CRL) complexes. In comparison to the modest E3 ligase activity displayed by isolated TRIAD1 or HHARI, binding of the cognate neddylated CRL to TRIAD1 or HHARI greatly stimulates RBR ligase activity in vitro, as determined by auto‐ubiquitylation, their ability to stimulate dissociation of a thioester‐linked UBCH7～ubiquitin intermediate, and reactivity with ubiquitin‐vinyl methyl ester. Moreover, genetic evidence shows that RBR ligase activity impacts both the levels and activities of neddylated CRLs in vivo. Cumulatively, our work proposes a conserved mechanism of CRL‐induced Ariadne RBR ligase activation and further suggests a reciprocal role of this special class of RBRs as regulators of distinct CRLs.     

Ring finger protein 146/Iduna is a Poly (ADP-ribose) polymer binding and PARsylation dependent E3 ubiquitin ligase

Recent findings suggest that Ring finger protein 146 (RNF146), also called Iduna, have neuroprotective property due to its inhibition of Parthanatos via binding with Poly(ADP-ribose) (PAR). The Parthanatos is a PAR dependent cell death that has been implicated in many human diseases. RNF146/Iduna acts as a PARsylation-directed E3 ubquitin ligase to mediate tankyrase-dependent degradation of axin, thereby positively regulates Wnt signaling. RNF146/Iduna can also facilitate DNA repair and protect against cell death induced by DNA damaging agents or γ-irradiation. It can translocate to the nucleus after cellular injury and promote the ubiquitination and degradation of various nuclear proteins involved in DNA damage repair. The PARsylation-directed ubquitination mediated by RNF146/Iduna is analogous to the phosphorylation-directed ubquitination catalyzed by Skp1-Cul1-F-box (SCF) E3 ubiquitin complex. RNF146/Iduna has been found to be implicated in neurodegenerative disease and cancer development. Therefore modulation of the PAR-binding and PARsylation dependent E3 ligase activity of RNF146/Iduna could have therapeutic significance for diseases, in which PAR and PAR-binding proteins play key pathophysiologic roles.Key words: E3 ubquitin ligase, ring finger protein 146, PAR-binding proteins, PARsylation, Parthanatos inhibitors, ubiquitinationRing finger proteins contain ring fingers, which are considered to be the functional module for E3 ubiquitin ligase activity.^1,2 Ring finger protein 146 (RNF146), a novel PARsylation-directed ring finger E3 ubiquitin ligase, is located at 6q22.1-q22.33 of human chromosome.³ RNF146 encodes a protein of 359 amino acids with a predicted molecular weight of 39.8 kDa.³ The molecular structure of RNF146 contains one N-terminus C₃HC₄ ring finger domain (35–77 aa) as well as one WWE domain (91–167 aa).³ The poly(ADP-ribose) (PAR) binding motif (144–167 aa) at the tail of WWE domain of RNF146 is involved in various important functions. The detailed molecular structure of RNF146 is illustrated in Figure 1. This PAR-binding protein was recently demonstrated to protect against Parthanatos and function as PARsylation-directed E3 ubquitin ligase to ubiquitinate PARsylated substrates.^3–5 Here, we provide a concise summary of these novel functions of RNF146 and discuss the potential pathophysiological and therapeutic significance of novel functions of RNF146 for human diseases.Open in a separate windowFigure 1Molecular structure of RNF146. The RNF146 is a novel ring finger E3 ubiquitin ligase with 359 amino acids. The molecular structure of RNF146 contains one N-terminus C₃HC₄ ring finger domain (35–77 aa, 7 cysteine and 1 histidine residues involved in ring finger formation is highlighted in red) as well as one WWE domain (91–167 aa, the highly conserved 2 tryptophan and 1 glutamic acid, which WWE domain is named after, are highlighted in red). The PAR binding motif (144–167 aa) is at the C-terminus tail of WWE domain of RNF146 (highlighted in blue at the end of WWE sequence).     

RNF152, a novel lysosome localized E3 ligase with pro-apoptotic activities

RING finger protein 152 (RNF152) is a novel RING finger protein and has not been well characterized. We report here that RNF152 is a canonical RING finger protein and has E3 ligase activity. It is polyubiqitinated partly through Lys-48-linked ubiquitin chains in vivo and this phenomenon is dependent on its RING finger domain and transmembrane domain. RNF152 is localized in lysosomes and co-localized with LAMP3, a lysosome marker. Moreover, over-expression of RNF152 in Hela cells induces apoptosis. These results suggest that RNF152 is a lysosome localized E3 ligase with pro-apoptotic activities. It is the first E3 ligase identified so far that is involved in lysosome-related apoptosis.     

RNF13: a novel RING-type ubiquitin ligase over-expressed in pancreatic cancer

Protein ubiquitination by E3 ubiquitin ligases plays an important role in cancer development. In this study, we provide experimental evidence that a RING-finger-containing protein RNF13 is an ER/Golgi membrane-associated E3 ubiquitin ligase and its RING finger domain is required for the ubiquitin ligase activity. Immunohistochemical analysis of pancreatic ductal adenocarcinoma (PDAC) and paracancerous normal tissues from 72 patients documented RNF13 over-expression in 30 tumor samples (41.7%, 30/72), and its expression was significantly associated with histological grading (P = 0.024). In addition, RNF13 was detected in precancerous lesions: tubular complexes in chronic pancreatitis (CP) and pancreatic intraepithelial neoplasia (PanIN) (79.3%, 23/29 and 62.8%, 22/35, respectively). Moreover, RNF13 staining was significantly correlated with Tenascin-C expression (P = 0.004) in PDAC samples, further supporting the role of RNF13 in cancer progression. Over-expression of wild type but not RING domain-mutant RNF13 in pancreatic MiaPaca-2 cancer cells increased invasive potential and gelatinolytic activity by matrix metalloproteinase-9. Taken together, these findings reveal that RNF13 is a novel E3 ubiquitin ligase involved in pancreatic carcinogenesis; ubiquitin-mediated modification of proteins by RNF13 may participate in pancreatic cancer development.     

LUBAC synthesizes linear ubiquitin chains via a thioester intermediate

The linear ubiquitin chain assembly complex (LUBAC) is a RING E3 ligase that regulates immune and inflammatory signalling pathways. Unlike classical RING E3 ligases, LUBAC determines the type of ubiquitin chain being formed, an activity normally associated with the E2 enzyme. We show that the RING-in-between-RING (RBR)-containing region of HOIP-the catalytic subunit of LUBAC-is sufficient to generate linear ubiquitin chains. However, this activity is inhibited by the N-terminal portion of the molecule, an inhibition that is released upon complex formation with HOIL-1L or SHARPIN. Furthermore, we demonstrate that HOIP transfers ubiquitin to the substrate through a thioester intermediate formed by a conserved cysteine in the RING2 domain, supporting the notion that RBR ligases act as RING/HECT hybrids.