Retraction: Residues 240–250 in the C-Terminus of the Pirh2 Protein Complement the Function of the RING Domain in Self-Ubiquitination of the Pirh2 Protein

The recently identified RNF125 [RING (really interesting new gene) finger protein 125], or TRAC-1 (T-cell RING protein in activation 1), is unique among ubiquitin ligases in being a positive regulator of T-cell activation. In addition, TRAC-1 has been shown to down-modulate HIV replication and to inhibit pathogen-induced cytokine production. However, apart from the presence of an N-terminal C3HC4 (Cys3-His-Cys4) RING domain, the TRAC-1 protein remains uncharacterized. In the present paper, we report novel interactions and modifications for TRAC-1, and elucidate its domain organization. Specifically, we determine that TRAC-1 associates with membranes and is excluded from the nucleus through myristoylation. Our data are further consistent with a crucial role for the C-terminus in TRAC-1 function. In this region, novel domains were recognized through the identification of three closely related proteins: RNF114, RNF138 and RNF166. TRAC-1 and its relatives were found to contain, apart from the RING domain, a C2HC (Cys2-His-Cys)- and two C2H2 (Cys2-His2)-type zinc fingers, as well as a UIM (ubiquitin-interacting motif). The UIM of TRAC-1 binds Lys48-linked polyubiquitin chains and is, together with the RING domain, required for auto-ubiquitination. As a consequence of auto-ubiquitination, the half-life of TRAC-1 is shorter than 30 min. The identification of these novel modifications, interactions, domains and relatives significantly widens the contexts for investigating TRAC-1 activity and regulation.

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Zinc Binding Properties of Engineered RING Finger Domain of Arkadia E3 Ubiquitin Ligase

Bioinorganic Chemistry and Applications ◽

10.1155/2010/323152 ◽

2010 ◽

Vol 2010 ◽

pp. 1-7 ◽

Cited By ~ 9

Author(s):

Christos T. Chasapis ◽

Ariadni K. Loutsidou ◽

Malvina G. Orkoula ◽

Georgios A. Spyroulias

Keyword(s):

Ubiquitin Ligase ◽

Structural Integrity ◽

Recombinant Expression ◽

E3 Ubiquitin Ligase ◽

Ring Finger ◽

Ring Domain ◽

Site Directed Mutagenesis ◽

Zinc Binding ◽

Ring Finger Domain ◽

C Terminus

Human Arkadia is a nuclear protein consisted of 989 amino acid residues, with a characteristic RING domain in its C-terminus. The RING domain harbours the E3 ubiquitin ligase activity needed by Arkadia to ubiquitinate its substrates such as negative regulators of TGF- signaling. The RING finger domain of Arkadia is a RING-H2 type and its structure and stability is strongly dependent on the presence of two bound Zn(II) ions attached to the protein frame through a defined Cys3-His2-Cys3 motif. In the present paper we transform the RING-H2 type of Arkadia finger domain to nonnative RING sequence, substituting the zinc-binding residues or to Arginine, through site-directed mutagenesis. The recombinant expression, inEscherichia coli, of the mutants C955R and H960R reveal significant lower yield in respect with the native polypeptide of Arkadia RING-H2 finger domain. In particular, only the C955R mutant exhibits expression yield sufficient for recombinant protein isolation and preliminary studies. Atomic absorption measurements and preliminary NMR data analysis reveal that the C955R point mutation in the RING Finger domain of Arkadia diminishes dramatically the zinc binding affinity, leading to the breakdown of the global structural integrity of the RING construct.

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The Mdm2 RING domain C-terminus is required for supramolecular assembly and ubiquitin ligase activity

The EMBO Journal ◽

10.1038/sj.emboj.7601465 ◽

2006 ◽

Vol 26 (1) ◽

pp. 90-101 ◽

Cited By ~ 130

Author(s):

Masha V Poyurovsky ◽

Christina Priest ◽

Alex Kentsis ◽

Katherine L B Borden ◽

Zhen-Qiang Pan ◽

...

Keyword(s):

Ubiquitin Ligase ◽

Supramolecular Assembly ◽

Ring Domain ◽

C Terminus ◽

Ubiquitin Ligase Activity

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Regulation of Mitochondrial Antiviral Signaling (MAVS) Expression and Signaling by the Mitochondria-associated Endoplasmic Reticulum Membrane (MAM) Protein Gp78

Journal of Biological Chemistry ◽

10.1074/jbc.m113.520254 ◽

2013 ◽

Vol 289 (3) ◽

pp. 1604-1616 ◽

Cited By ~ 28

Author(s):

Jana L. Jacobs ◽

Jianzhong Zhu ◽

Saumendra N. Sarkar ◽

Carolyn B. Coyne

Keyword(s):

Ubiquitin Ligase ◽

High Throughput Screening ◽

E3 Ubiquitin Ligase ◽

Ring Domain ◽

Endoplasmic Reticulum Membrane ◽

Type I ◽

Antiviral Signaling ◽

C Terminus ◽

Vesicular Stomatitis ◽

Mitochondrial Antiviral Signaling

In a previous study, we identified the E3 ubiquitin ligase Gp78 by RNAi high-throughput screening as a gene whose depletion restricted enterovirus infection. In the current study, we show that Gp78, which localizes to the ER-mitochondria interface, is a regulator of RIG-I-like receptor (RLR) antiviral signaling. We show that depletion of Gp78 results in a robust decrease of vesicular stomatitis virus (VSV) infection and a corresponding enhancement of type I interferon (IFN) signaling. Mechanistically, we show that Gp78 modulates type I IFN induction by altering both the expression and signaling of the mitochondria-localized RLR adaptor mitochondrial antiviral signaling (MAVS). Expression of mutants of Gp78 that abolish its E3 ubiquitin ligase and its participation in ER-associated degradation (ERAD) lost their ability to degrade MAVS, but surprisingly maintained their ability to repress RLR signaling. In contrast, Gp78 lacking its entire C terminus lost both its ability to degrade MAVS and repress RLR signaling. We show that Gp78 interacts with both the N- and C-terminal domains of MAVS via its C-terminal RING domain, and that this interaction is required to abrogate Gp78-mediated attenuation of MAVS signaling. Our data thus implicate two parallel pathways by which Gp78 regulates MAVS signaling; one pathway requires its E3 ubiquitin ligase and ERAD activity to directly degrade MAVS, whereas the other pathway occurs independently of these activities, but requires the Gp78 RING domain and occurs via a direct association between this region and MAVS.

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Sub-Cellular Localization Determines the Delicate Balance between the Anti and Proapoptotic Activity of Livin.

Blood ◽

10.1182/blood.v104.11.1531.1531 ◽

2004 ◽

Vol 104 (11) ◽

pp. 1531-1531

Author(s):

Dina Ben-Yehuda ◽

Boaz Nachmias ◽

Yaqoub Ashhab ◽

Itay Lazar ◽

Rebeca Perlman

Keyword(s):

Nuclear Localization ◽

Cellular Localization ◽

Family Members ◽

Ring Finger ◽

Ring Domain ◽

Nuclear Accumulation ◽

Cytoplasmic Localization ◽

Truncated Form ◽

C Terminus ◽

Localization Signal

Abstract Inhibitor of Apoptosis proteins (IAP) comprise a family of intracellular proteins that play an essential role in the regulation of apoptosis. IAP family members are defined by one or more repeats of a highly conserved anti-apoptotic domain termed the baculovirus IAP repeat (BIR), located at the amino-terminus. In addition, some IAPs contain a conserved domain termed RING finger at the carboxy-terminus. In a previous study, we identified an IAP family member termed Livin, which contains a single BIR domain and a carobxy-terminal RING finger motif. Livin inhibits apoptosis induced by a variety of stimuli. We further demonstrated that following apoptotic stimuli, Livin is cleaved by effector caspases 3 and 7, to produce a truncated form with paradoxical proapoptotic activity. In our current study, we reveal that while the full-length Livin shows a cytoplasmic distribution, the truncated form is readily detected in the nucleus. Using mutation analysis, we identified two domains outside the BIR sequence that are strongly engaged in determining the subcellular localization as well as the proapoptotic activity of Livin. These two domains are the N-terminus post-cleavage exposed area and the C-terminus RING domain. Remarkably, a single substitution of the first exposed amino acid Glycine, is sufficient to significantly diminish nuclear localization as well as the proapoptotic activity, thus linking the nuclear accumulation of the cleavage fragment with the proapoptotic activity. We demonstrate that the cytoplasmic localization of Livin is mediated through the RING domain, as a mutation that disables Livin from forming the zinc finger results in both a cytoplasmic and nuclear localization. Moreover, RING-mutated Livin subunit also loses its proapoptotic activity. Thus we suggest a model in which the exposed N-terminal region counteracts the cytoplasmic localization signal of the RING domain, promoting nuclear localization of the truncated form. The change in sub-cellular localization allows the RING domain to mediate the proapoptotic effect. To the best of our knowledge, this is the first example of such a regulatory mechanism in IAP family members.

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Immunocytochemical localization of β1-4 galactosyltransferase in endometrial carcinoma cells

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100162284 ◽

1990 ◽

Vol 48 (3) ◽

pp. 944-945

Author(s):

Ichiro Yamamoto ◽

Toshiaki Tachibana ◽

Hiroko Maruyama ◽

Noriyuki Komatsu ◽

Hiroyuki Kuramoto ◽

...

Keyword(s):

Endometrial Carcinoma ◽

Cell Lines ◽

Endometrial Adenocarcinoma ◽

Protein A ◽

Rabbit Antiserum ◽

Carcinoma Cells ◽

Affinity Column ◽

Antibody Technique ◽

Galactosyl Transferase ◽

C Terminus

We have paid attention to the alteration of glycosyltransferase in carcinoma cells, because it might be related to the malignancy of the cells. In this connection, localization of β1-4 galactosyl transferase (β1-4 Gal T) in human endometrial carcinoma cells was examined immunocytochemically using two kinds of cell lines, each of which showed different degree of differentiation.An antibody was purified from the rabbit antiserum against the synthetic peptide, IFNRLVFRGMSC (W89) of human β1-4 Gal T coupled with KLH (keyhole limpet hemocyanine) by protein A column and peptide-affinity column chromatography. The anti-W89 serum reacts to the C-terminus of human β 1-4 Gal T and to both membrane-bound and soluble forms of the enzyme. Cell line of well differentiated endometrial adenocarcinoma (I) and that of poorly differentiated endometrial adenocarcinoma (50B) were cultivated respectively in MEM medium containing 15% FCS and 2 mM glutamine for 4 d at 37°C under 5% CO2. The cells were fixed in a mixture of 4% paraformaldehyde and 0.1% glutaraldehyde in 0.1 M Soerensen’s phosphate buffer (pH 7.4) at 4°C for 30 min, washed with PBS, then freezed and thawed. The indirect method of the peroxidase- labeled antibody technique was used for immunocytochemistry of both LM and TEM on the cell lines. The cells were dehydrated in ethanol and embedded in TAAB 812. Ultrathin sections were observed under a TEM, JEM-100S.

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SAC3B is a target of CML19, the centrin 2 of Arabidopsis thaliana

Biochemical Journal ◽

10.1042/bcj20190674 ◽

2020 ◽

Vol 477 (1) ◽

pp. 173-189 ◽

Cited By ~ 1

Author(s):

Marco Pedretti ◽

Carolina Conter ◽

Paola Dominici ◽

Alessandra Astegno

Keyword(s):

Excision Repair ◽

Complex Structure ◽

Binding Motif ◽

C Terminus ◽

Repair Protein ◽

Nucleotide Excision ◽

Ef Hand ◽

Molecular Determinants ◽

Structure In Solution

Arabidopsis centrin 2, also known as calmodulin-like protein 19 (CML19), is a member of the EF-hand superfamily of calcium (Ca2+)-binding proteins. In addition to the notion that CML19 interacts with the nucleotide excision repair protein RAD4, CML19 was suggested to be a component of the transcription export complex 2 (TREX-2) by interacting with SAC3B. However, the molecular determinants of this interaction have remained largely unknown. Herein, we identified a CML19-binding site within the C-terminus of SAC3B and characterized the binding properties of the corresponding 26-residue peptide (SAC3Bp), which exhibits the hydrophobic triad centrin-binding motif in a reversed orientation (I8W4W1). Using a combination of spectroscopic and calorimetric experiments, we shed light on the SAC3Bp–CML19 complex structure in solution. We demonstrated that the peptide interacts not only with Ca2+-saturated CML19, but also with apo-CML19 to form a protein–peptide complex with a 1 : 1 stoichiometry. Both interactions involve hydrophobic and electrostatic contributions and include the burial of Trp residues of SAC3Bp. However, the peptide likely assumes different conformations upon binding to apo-CML19 or Ca2+-CML19. Importantly, the peptide dramatically increases the affinity for Ca2+ of CML19, especially of the C-lobe, suggesting that in vivo the protein would be Ca2+-saturated and bound to SAC3B even at resting Ca2+-levels. Our results, providing direct evidence that Arabidopsis SAC3B is a CML19 target and proposing that CML19 can bind to SAC3B through its C-lobe independent of a Ca2+ stimulus, support a functional role for these proteins in TREX-2 complex and mRNA export.

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E3 ubiquitin ligases

Essays in Biochemistry ◽

10.1042/bse0410015 ◽

2005 ◽

Vol 41 ◽

pp. 15-30 ◽

Cited By ~ 123

Author(s):

Helen C. Ardley ◽

Philip A. Robinson

Keyword(s):

Protein Complexes ◽

Loss Of Function ◽

Direct Role ◽

Cellular Processes ◽

Substrate Protein ◽

Protein Ubiquitination ◽

C Terminus ◽

Full Complement ◽

Spatio Temporal ◽

Eukaryotic Organisms

The selectivity of the ubiquitin–26 S proteasome system (UPS) for a particular substrate protein relies on the interaction between a ubiquitin-conjugating enzyme (E2, of which a cell contains relatively few) and a ubiquitin–protein ligase (E3, of which there are possibly hundreds). Post-translational modifications of the protein substrate, such as phosphorylation or hydroxylation, are often required prior to its selection. In this way, the precise spatio-temporal targeting and degradation of a given substrate can be achieved. The E3s are a large, diverse group of proteins, characterized by one of several defining motifs. These include a HECT (homologous to E6-associated protein C-terminus), RING (really interesting new gene) or U-box (a modified RING motif without the full complement of Zn2+-binding ligands) domain. Whereas HECT E3s have a direct role in catalysis during ubiquitination, RING and U-box E3s facilitate protein ubiquitination. These latter two E3 types act as adaptor-like molecules. They bring an E2 and a substrate into sufficiently close proximity to promote the substrate's ubiquitination. Although many RING-type E3s, such as MDM2 (murine double minute clone 2 oncoprotein) and c-Cbl, can apparently act alone, others are found as components of much larger multi-protein complexes, such as the anaphase-promoting complex. Taken together, these multifaceted properties and interactions enable E3s to provide a powerful, and specific, mechanism for protein clearance within all cells of eukaryotic organisms. The importance of E3s is highlighted by the number of normal cellular processes they regulate, and the number of diseases associated with their loss of function or inappropriate targeting.

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Pharmacokinetics of Full Length and Two-Domain Tissue Factor Pathway Inhibitor in Combination with Heparin in Rabbits

Thrombosis and Haemostasis ◽

10.1055/s-0038-1649604 ◽

1993 ◽

Vol 70 (03) ◽

pp. 454-457 ◽

Cited By ~ 14

Author(s):

Claus Bregengaard ◽

Ole Nordfang ◽

Per Østergaard ◽

Jens G L Petersen ◽

Giorgio Meyn ◽

...

Keyword(s):

Tissue Factor ◽

Tissue Factor Pathway Inhibitor ◽

Anticoagulant Activity ◽

Fold Increase ◽

Volume Of Distribution ◽

Full Length ◽

Heparin Binding ◽

Extrinsic Pathway ◽

C Terminus ◽

Tissue Factor Pathway

SummaryTissue factor pathway inhibitor (TFPI) is a feed back inhibitor of the initial activation of the extrinsic pathway of coagulation. In humans, injection of heparin results in a 2-6 fold increase in plasma TFPI and recent studies suggest that TFPI may be important for the anticoagulant activity of heparin. Full length (FL) TFPI, but not recombinant two-domain (2D) TFPI, has a poly cationic C-terminus showing very strong heparin binding. Therefore, we have investigated if heparin affects the pharmacokinetics of TFPI with and without this C-terminus.FL-TFPI (608 U/kg) and 2D-TFPI (337 U/kg) were injected intravenously in rabbits with and without simultaneous intravenous injections of low molecular weight heparin (450 anti-XaU/kg).Heparin decreased the volume of distribution and the clearance of FL-TFPI by a factor 10-15, whereas the pharmacokinetics of 2D-TFPI were unaffected by heparin. When heparin was administered 2 h following TFPI the recovery of FL-TFPI was similar to that found in the group receiving the two compounds simultaneously, suggesting that the releasable pool of FL-TFPI is removed very slowly in the absence of circulating heparin.

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