SAG (Sensitive to Apoptosis Gene), also known as RBX2 (RING box protein 2), ROC2 (Regulator of Cullins 2), or RNF7 (RING Finger Protein 7), was originally cloned in our laboratory as a redox inducible antioxidant protein and later characterized as the second member of the RBX/ROC RING component of the SCF (SKP1-CUL-F-box Proteins) E3 ubiquitin ligase. When acting alone, SAG scavenges oxygen radicals by forming inter- and intra-molecular disulfide bonds, whereas by forming a complex with other components of the SCF E3 ligase, SAG promotes ubiquitination and degradation of a number of protein substrates, including c-JUN, DEPTOR, HIF-1α, IκBα, NF1, NOXA, p27, and procaspase-3, thus regulating various signaling pathways and biological processes. Specifically, SAG protects cells from apoptosis, confers radioresistance, and plays an essential and non-redundant role in mouse embryogenesis and vasculogenesis. Furthermore, stress-inducible SAG is overexpressed in a number of human cancers and SAG overexpression correlates with poor patient prognosis. Finally, SAG transgenic expression in epidermis causes an early stage inhibition, but later stage promotion, of skin tumorigenesis triggered by DMBA/TPA. Given its major role in promoting targeted degradation of tumor suppressive proteins, leading to apoptosis suppression and accelerated tumorigenesis, SAG E3 ligase appears to be an attractive anticancer target.
Animals ; Antioxidants ; metabolism ; Apoptosis ; Carrier Proteins ; chemistry ; genetics ; metabolism ; Cell Transformation, Neoplastic ; Humans ; RING Finger Domains ; Substrate Specificity ; Ubiquitin-Protein Ligases ; chemistry ; metabolism ; Ubiquitination

Clonorchis sinensis habitating in the bile duct of mammals causes clonorchiasis endemic in East Asian countries. Parkin is a RING-between-RING protein and has E3-ubiquitin ligase activity catalyzing ubiquitination and degradation of substrate proteins. A cDNA clone of C. sinensis was predicted to encode a polypeptide homologous to parkin (CsParkin) including 5 domains (Ubl, RING0, RING1, IBR, and RING2). The cysteine and histidine residues binding to Zn2+ were all conserved and participated in formation of tertiary structural RINGs. Conserved residues were also an E2-binding site in RING1 domain and a catalytic cysteine residue in the RING2 domain. Native CsParkin was determined to have an estimated molecular weight of 45.7 kDa from C. sinensis adults by immunoblotting. CsParkin revealed E3-ubiquitin ligase activity and higher expression in metacercariae than in adults. CsParkin was localized in the locomotive and male reproductive organs of C. sinensis adults, and extensively in metacercariae. Parkin has been found to participate in regulating mitochondrial function and energy metabolism in mammalian cells. From these results, it is suggested that CsParkin play roles in energy metabolism of the locomotive organs, and possibly in protein metabolism of the reproductive organs of C. sinensis.
Amino Acid Sequence ; Animals ; Clonorchis sinensis/*enzymology ; Cluster Analysis ; Conserved Sequence ; DNA, Complementary/genetics ; Energy Metabolism ; Gene Expression Profiling ; Mitochondria/metabolism ; Models, Molecular ; Molecular Weight ; Phylogeny ; Protein Conformation ; Sequence Homology, Amino Acid ; Ubiquitin-Protein Ligases/chemistry/*genetics/*metabolism

Clonorchis sinensis habitating in the bile duct of mammals causes clonorchiasis endemic in East Asian countries. Parkin is a RING-between-RING protein and has E3-ubiquitin ligase activity catalyzing ubiquitination and degradation of substrate proteins. A cDNA clone of C. sinensis was predicted to encode a polypeptide homologous to parkin (CsParkin) including 5 domains (Ubl, RING0, RING1, IBR, and RING2). The cysteine and histidine residues binding to Zn2+ were all conserved and participated in formation of tertiary structural RINGs. Conserved residues were also an E2-binding site in RING1 domain and a catalytic cysteine residue in the RING2 domain. Native CsParkin was determined to have an estimated molecular weight of 45.7 kDa from C. sinensis adults by immunoblotting. CsParkin revealed E3-ubiquitin ligase activity and higher expression in metacercariae than in adults. CsParkin was localized in the locomotive and male reproductive organs of C. sinensis adults, and extensively in metacercariae. Parkin has been found to participate in regulating mitochondrial function and energy metabolism in mammalian cells. From these results, it is suggested that CsParkin play roles in energy metabolism of the locomotive organs, and possibly in protein metabolism of the reproductive organs of C. sinensis.
Amino Acid Sequence ; Animals ; Clonorchis sinensis/*enzymology ; Cluster Analysis ; Conserved Sequence ; DNA, Complementary/genetics ; Energy Metabolism ; Gene Expression Profiling ; Mitochondria/metabolism ; Models, Molecular ; Molecular Weight ; Phylogeny ; Protein Conformation ; Sequence Homology, Amino Acid ; Ubiquitin-Protein Ligases/chemistry/*genetics/*metabolism

Retinoic acid-inducible gene I (RIG-I) is an important pattern recognition receptor that detects viral RNA and triggers the production of type-I interferons through the downstream adaptor MAVS (also called IPS-1, CARDIF, or VISA). A series of structural studies have elaborated some of the mechanisms of dsRNA recognition and activation of RIG-I. Recent studies have proposed that K63-linked ubiquitination of, or unanchored K63-linked polyubiquitin binding to RIG-I positively regulates MAVS-mediated antiviral signaling. Conversely phosphorylation of RIG-I appears to play an inhibitory role in controlling RIG-I antiviral signal transduction. Here we performed a combined structural and biochemical study to further define the regulatory features of RIG-I signaling. ATP and dsRNA binding triggered dimerization of RIG-I with conformational rearrangements of the tandem CARD domains. Full length RIG-I appeared to form a complex with dsRNA in a 2:2 molar ratio. Compared with the previously reported crystal structures of RIG-I in inactive state, our electron microscopic structure of full length RIG-I in complex with blunt-ended dsRNA, for the first time, revealed an exposed active conformation of the CARD domains. Moreover, we found that purified recombinant RIG-I proteins could bind to the CARD domain of MAVS independently of dsRNA, while S8E and T170E phosphorylation-mimicking mutants of RIG-I were defective in binding E3 ligase TRIM25, unanchored K63-linked polyubiquitin, and MAVS regardless of dsRNA. These findings suggested that phosphorylation of RIG inhibited downstream signaling by impairing RIG-I binding with polyubiquitin and its interaction with MAVS.
Adaptor Proteins, Signal Transducing ; metabolism ; Adenosine Triphosphate ; metabolism ; DEAD Box Protein 58 ; DEAD-box RNA Helicases ; chemistry ; genetics ; metabolism ; Dimerization ; Humans ; Mutagenesis, Site-Directed ; Phosphorylation ; Polyubiquitin ; metabolism ; Protein Binding ; Protein Structure, Tertiary ; RNA, Double-Stranded ; metabolism ; Recombinant Proteins ; biosynthesis ; chemistry ; genetics ; Signal Transduction ; Transcription Factors ; metabolism ; Tripartite Motif Proteins ; Ubiquitin-Protein Ligases ; metabolism ; Ubiquitination

Fe65 has been characterized as an adaptor protein, originally identified as an expressed sequence tag (EST) corresponding to an mRNA expressed at high levels in the rat brain. It contains one WW domain and two phosphotyrosine interaction/phosphotyrosine binding domains (PID1/PID2). As the neuronal precursor cell expressed developmentally down regulated 4-2 (Nedd4-2) has a putative WW domain binding motif (72PPLP75) in the N-terminal domain, we hypothesized that Fe65 associates with Nedd4-2 through a WW domain interaction, which has the characteristics of E3 ubiquitin-protein ligase. In this paper, we present evidence for the interaction between Fe65 WW domain and Nedd4-2 through its specific motif, using a pull down approach and co-immunoprecipitation. Additionally, the co-localization of Fe65 and Nedd4-2 were observed via confocal microscopy. Co-localization of Fe65 and Nedd4-2 was disrupted by either the mutation of Fe65 WW domain or its putative binding motif of Nedd4-2. When the ubiquitin assay was performed, the interaction of Nedd4-2 (wt) with Fe65 is required for the cell apoptosis and the ubiquitylation of Fe65. We also observed that the ubiquitylation of Fe65 (wt) was augmented depending on Nedd4-2 expression levels, whereas the Fe65 WW domain mutant (W243KP245K) or the Nedd4-2 AL mutant (72PPLP75 was changed to 72APLA75) was under-ubiquitinated significantly. Thus, our observations implicated that the protein-protein interaction between the WW domain of Fe65 and the putative binding motif of Nedd4-2 down-regulates Fe65 protein stability and subcellular localization through its ubiquitylation, to contribute cell apoptosis.
Adaptor Proteins, Signal Transducing/chemistry/genetics/*metabolism ; Cell Line ; *Down-Regulation ; Endosomal Sorting Complexes Required for Transport/genetics/*metabolism ; *Gene Expression Regulation, Developmental ; Humans ; Immunoprecipitation ; Microscopy, Confocal ; Mutation ; Protein Interaction Mapping ; Protein Structure, Tertiary/*physiology ; Transfection ; Ubiquitin-Protein Ligases/genetics/*metabolism ; Ubiquitination

OBJECTIVETo evaluate the significance of somatic mutations of VHL gene and hypoxia-inducible factor-1alpha (HIF-1alpha) expression in primary renal clear cell carcinoma (RCC).

METHODSMutation of VHL gene and HIF-1alpha expression were detected by means of PCR, denaturing high-performance liquid chromatography (DHPLC), direct sequencing and immunohistochemistry in 32 samples from primary renal clear cell carcinoma patients.

RESULTSIn 32 RCC samples, 17 samples (53.1%) had and 32 samples of adjacent nonmalignant renal tissue had not mutations of VHL gene expression. Twelve RCC samples (70.6%) which had mutations of VHL gene expressed HIF-1alpha, and it had significant difference to 4 RCC (26.7%) samples which didn't have mutations of VHL gene (P < 0.05).

CONCLUSIONMutations of VHL gene may play a significant role in the tumorigenesis of RCC, and HIF-1alpha expression correlates with it.

Adenocarcinoma, Clear Cell ; genetics ; pathology ; Adult ; Aged ; Carcinoma, Renal Cell ; genetics ; pathology ; Chromatography, Liquid ; Female ; Gene Expression Regulation, Neoplastic ; Humans ; Hypoxia-Inducible Factor 1, alpha Subunit ; Immunohistochemistry ; Kidney ; chemistry ; metabolism ; pathology ; Kidney Neoplasms ; genetics ; pathology ; Male ; Middle Aged ; Mutation ; genetics ; Polymerase Chain Reaction ; Transcription Factors ; analysis ; genetics ; Tumor Suppressor Proteins ; analysis ; genetics ; Ubiquitin-Protein Ligases ; analysis ; genetics ; Von Hippel-Lindau Tumor Suppressor Protein