RING-box Protein Research Articles

RBX1 prompts degradation of EXO1 to limit the homologous recombination pathway of DNA double-strand break repair in G1 phase

End resection of DNA double-strand breaks (DSBs) to form 3′ single-strand DNA (ssDNA) is critical to initiate the homologous recombination (HR) pathway of DSB repair. HR pathway is strictly limited in the G1-phase cells because of lack of homologous DNA as the templates. Exonuclease 1 (EXO1) is the key molecule responsible for 3′ ssDNA formation of DSB end resection. We revealed that EXO1 is inactivated in G1-phase cells via ubiquitination-mediated degradation, resulting from an elevated expression level of RING-box protein 1 (RBX1) in G1 phase. The increased RBX1 significantly prompted the neddylation of Cullin1 and contributed to the G1 phase-specific degradation of EXO1. Knockdown of RBX1 remarkedly attenuated the degradation of EXO1 and increased the end resection and HR activity in γ-irradiated G1-phase cells, as demonstrated by the increased formation of RPA32, BrdU, and RAD51 foci. And EXO1 depletion mitigated DNA repair defects due to RBX1 reduction. Moreover, increased autophosphorylation of DNA-PKcs at S2056 was found to be responsible for the higher expression level of the RBX1 in the G1 phase. Inactivation of DNA-PKcs decreased RBX1 expression, and simultaneously increased EXO1 expression and DSB end resection in G1-phase cells. This study demonstrates a new mechanism for restraining the HR pathway of DNA DSB repair in G1 phase via RBX1-prompted inactivation of EXO1.

CENP-A Ubiquitylation Contributes to Maintaining the Chromosomal Location of the Centromere.

The centromere plays an essential role in accurate chromosome segregation, and the chromosomal location of the centromere is determined by the presence of a histone H3 variant, centromere protein A (CENP-A), in centromeric nucleosomes. However, the precise mechanisms of deposition, maintenance, and inheritance of CENP-A at centromeres are unclear. We have reported that CENP-A deposition requires ubiquitylation of CENP-A lysine 124 mediated by the E3 ligase activity of Cullin 4A (CUL4A)—RING-box protein 1 (RBX1)—COP9 signalsome complex subunit 8 (COPS8). We have proposed a model of inheritance for CENP-A ubiquitylation, through dimerization between rounds of cell divisions, that maintains the position of centromeres.

Expression pattern and functional analysis of the two RING box protein RBX in spermatogenesis of Chinese mitten crab Eriocheir sinensis

Studies in E. sinensis have shown that ubiquitination mediated by Cullin-RING E3 ligases (CRLs) plays important roles in spermatogenesis. In other species, CRLs are also essential in cell cycle progression, DNA replication, signal transduction, gene transcription, and development. The catalytic RING component, the RING box protein, is an important part of CRLs. However, there have been few studies on CRLs in crustaceans. In this study, we cloned two RING box protein genes from the Chinese mitten crab, Eriocheir sinensis, termed Es-RBX1 and Es-RBX2 The full length Es-RBX1 cDNA comprises 741 nucleotides, and encodes a protein of 124 amino acid residues, whereas the Es-RBX2 cDNA comprises 1325 nucleotides, and encodes a protein of 110 amino acid residues. Bioinformatics analysis showed that the domains and structure of the RBX proteins have been highly conserved during evolution. Quantitative real-time polymerase chain reaction and western blotting showed that Es-RBX1 is highly expressed in the testis, particularly during the spermatocyte stage, whereas Es-RBX2 did not show specific expression in the male reproductive system. Furthermore, Es-RBX1 is mainly distributed in the nucleus, and changed its location with the development of the nucleus. Co-immunoprecipitation showed that Es-RBX1 could bind Cullin4. These results suggested that Es-RBX1 plays a key role in spermatogenesis of E. sinensis though forming a complex with Cullin4.

The Dynamic Self-regulation of Modular Cullin-Ring Ligases

E3 Ubiquitin (Ub) Ligases (UBLs) trigger cellular signaling through the covalent attachment of Ub (ubiquitylation) to lysine residues on bound proteins. The multi-subunit Cullin-RING ligase (CRL) represents the largest family of E3 Ligases and is responsible for protein degradation through the proteasome. CRLs contain a Cullin protein (CUL 1-7) with a RING-box (RBX1 or 2) protein bound at its C-terminus. Adapter proteins link a substrate receptor to the N-terminus of CUL and a ubiquitin-conjugating protein (E2) binds to RBX. An active CRL facilitates the transfer of ubiquitin from E2 across a gap of more than 50 Å to a substrate at the other end of the ligase. The Ankyrin Repeat and SOCS-box (ASB) substrate recognition proteins associate with CUL5 and RBX2 through the Elongin B and C (EBC) adapter proteins. Modification of CUL by Nedd8 (neddylation) regulates substrate-receptor exchange and the E2 specificity of RBX. We have expressed and purified all of the proteins in the ASB9-containng CRL, which ubiquitylates creatine kinase (CK). We show that the purified complex becomes neddylated at K724 of CUL5, and creatine kinase is ubiquitylated at several lysines on the protein surface. We are pursuing cryoEM and hydrogen-deuterium exchange mass spectrometry (HDX-MS) experiments to elucidate the structure and dynamics of the complex. Initial cryo-electron microscopy reveals the ASB9 protein is highly structured, consistent with its low incorporation of deuterium in HDXMS. HDXMS also revealed that binding of EBC alters exchange within CK bound on the other end of ASB9.

RBX1-mediated ubiquitination of SESN2 promotes cell death upon prolonged mitochondrial damage in SH-SY5Y neuroblastoma cells.

Sestrins are evolutionary conserved stress-inducible genes which regulate the axis of cell survival and cell death. Suppression of Sestrin 2 (SESN2) has been linked with increase in oxidative stress and cell death but mechanistic details related to regulation of SESN2 during mitochondrial damage remain unknown. Our study shows that prolonged CCCP-induced mitochondrial damage decreases SESN2 levels and viability of SH-SY5Y cells while overexpression of SESN2 significantly rescues the viability of cells. Further, we demonstrate that Ring box protein 1 (RBX1) is a novel interactive partner and E3 ligase for SESN2 which mediates its K-48-linked ubiquitination upon extensive mitochondrial damage. Downregulation of RBX1 causes stabilization in levels of SESN2. Notably, silencing of RBX1 expression substantially declines cell death and generation of mitochondrial ROS in response to prolonged mitochondrial damage. Taken together, we suggest that SESN2 is critical to protect cells against detrimental effect of mitochondrial damage and RBX1 is a negative regulator of SESN2 which hampers its stabilization.

The relation between Ring Box-1 protein overexpression and tumor grade and stage in bladder urothelial cell carcinoma.

Ring Box Protein-1 (RBX-1), a component of SCF E3 ubiquitin ligases, has a crucial role in bladder urothelial cell carcinoma (UCC) carcinogenesis and progression. In the present study, it is aimed to determine the expression of RBX-1 protein in bladder UCC and the association between tumor grade, stage and RBX-1 expression. Ninety UCC samples and 20 samples containing foci of normal bladder urothelium were recruited and analyzed immunohistochemically in terms of RBX-1 expression. Immuno-reactivity scoring system (IRS) was used to determine RBX-1 expression levels. RBX-1 overexpression was associated with high tumor grade (p= 0.001) and advanced stage (p= 0.001). pT1 tumors showed higher RBX-1 expression than pTa tumors. pT2 tumors showed not only higher expression than pTa tumors but also higher expression than the total of pTa and pT1 groups combined. There was no statistically significant relation between RBX-1 expression and patient gender (p= 0.116) or age (p= 0.191). In bladder UCC, RBX-1 overexpression is associated with high tumor grade and advanced stage and represents biological potential of invasiveness and aggressive disease. Results of the present study have to be supported with further studies to reveal clinical and therapeutic implications of RBX-1 overexpression in bladder UCC.

Cullin neddylation may allosterically tune polyubiquitin chain length and topology.

Conjugation of Nedd8 (neddylation) to Cullins (Cul) in Cul-RING E3 ligases (CRLs) stimulates ubiquitination and polyubiquitination of protein substrates. CRL is made up of two Cul-flanked arms: one consists of the substrate-binding and adaptor proteins and the other consists of E2 and Ring-box protein (Rbx). Polyubiquitin chain length and topology determine the substrate fate. Here, we ask how polyubiquitin chains are accommodated in the limited space available between the two arms and what determines the polyubiquitin linkage topology. We focus on Cul5 and Rbx1 in three states: before Cul5 neddylation (closed state), after neddylation (open state), and after deneddylation, exploiting molecular dynamics simulations and the Gaussian Network Model. We observe that regulation of substrate ubiquitination and polyubiquitination takes place through Rbx1 rotations, which are controlled by Nedd8-Rbx1 allosteric communication. Allosteric propagation proceeds from Nedd8 via Cul5 dynamic hinges and hydrogen bonds between the C-terminal domain of Cul5 (Cul5CTD) and Rbx1 (Cul5CTD residues R538/R569 and Rbx1 residue E67, or Cul5CTD E474/E478/N491 and Rbx1 K105). Importantly, at each ubiquitination step (homogeneous or heterogeneous, linear or branched), the polyubiquitin linkages fit into the distances between the two arms, and these match the inherent CRL conformational tendencies. Hinge sites may constitute drug targets.

Dynamics and Assembly of ASB-Containing E3 Ubiquitin Ligases

E3 Ubiquitin Ligases (UBLs) facilitate the highly-specific covalent attachment of activated ubiquitin to bound substrate proteins through an isopeptide bond on an exposed lysine residue. Ubiquitin signaling regulates cellular protein degradation which is essential for proper cell functioning. The multi-subunit Cullin-RING ligase (CRL) represents the most prominent form of E3 Ligases, and it is responsible for a substantial percentage of ubiquitin-directed protein degradation through the K48 ubiquitin linkage. CRLs share a common structure, composed (in order of arrangement) of a substrate receptor with a suppressor of cytokine signaling (SOCS) domain, one or two adapter proteins, a Cullin protein, and a RING-box (RBX) protein that recruits E2 enzymes charged with activated ubiquitin. Among the substrate receptors belonging to this class of UBLs, all 18 proteins in the Ankyrin Repeat and SOCS-box (ASB) family of proteins associate with Cullin 5 and RBX2 through the Elongin B and C (EB/C) adapter proteins. ASB9 has previously been shown to tightly and specifically bind to Creatine Kinase (CK). I am building the entire seven-protein ASB9-containing E3 ligase complex in order to characterize its dynamics. I have expressed and purified all components of the ASB9 UBL, along with the necessary Ubiquitin and Nedd8 conjugating enzymes. I have obtained crystals of CK-ASB9-EB/C for structural determination, and I performed hydrogen-deuterium exchange (HDX) on the same complex to characterize how protein complex formation influences the dynamics of each subunit in the complex. CK binding to ASB9 reduced exchange across both of the first two Ankyrin repeats, while the removal of the SOCS box increased deuterium exchange in the final Ankyrin repeats in ASB9. This demonstrates the complex relationship between the components of the E3 ligase and the sophisticated substrate recognition by ASB proteins.

RING box protein-1 gene involved in flagellar disassembly of Dunaliella salina.

Ring box protein-1 (RBX1), also called Regulator of Cullins-1 (ROC1), is a key component of SCF (Skp-1, cullins, F-box proteins) E3 ubiquitin ligases, which regulate diverse cellular processes by targeting protein substrates for degradation. Although RBX1 plays an important role in ubiquitination machinery of both prokaryotes and eukaryotes, studies on the RBX1 have not been involved in the unicellular green alga Dunaliella salina. In this study, a full-length RBX1 cDNA fragment of 817bp was cloned using rapid amplification of cDNA end (RACE) technique. The full-length sequence contained an open reading frame of 411bp encoding 136 amino acids. The predicted protein had a molecular molar mass of 14.8kDa and pI of 5.9 with a high degree of homology to RBX1 from Chlamydomonas reinhardtii (92%). Recombinant RBX1 was expressed in Escherichia coli BL21 and was purified and characterized. The apparent molecular mass of the recombinant protein was approximately 17kDa, and the optimal induction time and concentration were 3h and 0.1mmol/L IPTG, respectively. The predicted 3D structures of RBX1 proteins contained RING-H2 finger domain including "Cys59-X2-Cys62-X30-Cys93-X1-His95-X2-His98-X2-Cys101-X10-Cys112-X2-Cys115." The expression of RBX1 protein was increased by 132% during flagellar disassembly and decreased by 76% during flagellar assembly of D. salina. The expression of RBX1 mRNA had a similar tendency with the expression of RBX1 protein. The results indicated that RBX1 responded to flagellar disassembly of D. salina.

Simultaneous expression of regulatory genes associated with specific drought-adaptive traits improves drought adaptation in peanut.

Adaptation of crops to drought-prone rain-fed conditions can be achieved by improving plant traits such as efficient water mining (by superior root characters) and cellular-level tolerance mechanisms. Pyramiding these drought-adaptive traits by simultaneous expression of genes regulating drought-adaptive mechanisms has phenomenal relevance in improving stress tolerance. In this study, we provide evidence that peanut transgenic plants expressing Alfalfa zinc finger 1 (Alfin1), a root growth-associated transcription factor gene, Pennisetum glaucum heat-shock factor (PgHSF4) and Pea DNA helicase (PDH45) involved in protein turnover and protection showed improved tolerance, higher growth and productivity under drought stress conditions. Stable integration of all the transgenes was noticed in transgenic lines. The transgenic lines showed higher root growth, cooler crop canopy air temperature difference (less CCATD) and higher relative water content (RWC) under drought stress. Low proline levels in transgenic lines substantiate the maintenance of higher water status. The survival and recovery of transgenic lines was significantly higher under gradual moisture stress conditions with higher biomass. Transgenic lines also showed significant tolerance to ethrel-induced senescence and methyl viologen-induced oxidative stress. Several stress-responsive genes such as heat-shock proteins (HSPs), RING box protein-1 (RBX1), Aldose reductase, late embryogenesis abundant-5 (LEA5) and proline-rich protein-2 (PRP2), a gene involved in root growth, showed enhanced expression under stress in transgenic lines. Thus, the simultaneous expression of regulatory genes contributing for drought-adaptive traits can improve crop adaptation and productivity under water-limited conditions.

Next-generation sequencing (NGS) transcriptomes reveal association of multiple genes and pathways contributing to secondary metabolites accumulation in tuberous roots of Aconitum heterophyllum Wall.

The transcriptomes of Aconitum heterophyllum were assembled and characterized for the first time to decipher molecular components contributing to biosynthesis and accumulation of metabolites in tuberous roots. Aconitum heterophyllum Wall., popularly known as Atis, is a high-value medicinal herb of North-Western Himalayas. No information exists as of today on genetic factors contributing to the biosynthesis of secondary metabolites accumulating in tuberous roots, thereby, limiting genetic interventions towards genetic improvement of A. heterophyllum. Illumina paired-end sequencing followed by de novo assembly yielded 75,548 transcripts for root transcriptome and 39,100 transcripts for shoot transcriptome with minimum length of 200 bp. Biological role analysis of root versus shoot transcriptomes assigned 27,596 and 16,604 root transcripts; 12,340 and 9398 shoot transcripts into gene ontology and clusters of orthologous group, respectively. KEGG pathway mapping assigned 37 and 31 transcripts onto starch-sucrose metabolism while 329 and 341 KEGG orthologies associated with transcripts were found to be involved in biosynthesis of various secondary metabolites for root and shoot transcriptomes, respectively. In silico expression profiling of the mevalonate/2-C-methyl-D-erythritol 4-phosphate (non-mevalonate) pathway genes for aconites biosynthesis revealed 4 genes HMGR (3-hydroxy-3-methylglutaryl-CoA reductase), MVK (mevalonate kinase), MVDD (mevalonate diphosphate decarboxylase) and HDS (1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate synthase) with higher expression in root transcriptome compared to shoot transcriptome suggesting their key role in biosynthesis of aconite alkaloids. Five genes, GMPase (geranyl diphosphate mannose pyrophosphorylase), SHAGGY, RBX1 (RING-box protein 1), SRF receptor kinases and β-amylase, implicated in tuberous root formation in other plant species showed higher levels of expression in tuberous roots compared to shoots. A total of 15,487 transcription factors belonging to bHLH, MYB, bZIP families and 399 ABC transporters which regulate biosynthesis and accumulation of bioactive compounds were identified in root and shoot transcriptomes. The expression of 5 ABC transporters involved in tuberous root development was validated by quantitative PCR analysis. Network connectivity diagrams were drawn for starch-sucrose metabolism and isoquinoline alkaloid biosynthesis associated with tuberous root growth and secondary metabolism, respectively, in root transcriptome of A. heterophyllum. The current endeavor will be of practical importance in planning a suitable genetic intervention strategy for the improvement of A. heterophyllum.

MiR-194 targets RBX1 gene to modulate proliferation and migration of gastric cancer cells.

RING box protein1 (RBX1), an essential component of SCF E3 ubiquitin ligases, plays an important role in gastric cancer. In the study, miR-194 and RBX1 expression was evaluated in 76 pairs of gastric tumor and non-tumor tissue samples by qRT-PCR, and clinicopathological characteristics were analyzed. CCK8, transwell assay, wound healing assay, and flow cytometry assay were performed to evaluate the effect of miR-194 on gastric cancer (GC) cellular proliferation, invasion, migration, apoptosis, and cell cycle, respectively. Luciferase reporter assays and Western blotting were used to evaluate whether RBX1 is a direct target of miR-194. The Kaplan-Meier method and log-rank test were used to evaluate the correlation between miR-194 or RBX1 expression and patient survival. Then, we found that miR-194 was significantly downregulated and RBX1 upregulated in GC tissues; both of which showed significant association with tumor size, location, invasion, and tumor node metastasis. Cell proliferation, invasion, and migration were significantly restricted with miR-194 overexpression. miR-194 downregulated RBX1 protein expression, and luciferase assays showed that binding sites in the RBX1 3'UTR were required for miR-194-mediated repression of RBX1, indicating that RBX1 was a direct target of miR-194. Transfection of RBX1 without the 3'UTR restored the miR-194-inhibiting migration function. miR-194 overexpression or RBX1 lowexpression was associated with prolonged survival of GC patients. In conclusion, upregulation of miR-194 can inhibit proliferation, migration, and invasion of GC cells, possibly by targeting RBX1. Aberrant expression of miR-194 and RBX1 is correlated to GC patient survival time.

An integrated computational approach can classify VHL missense mutations according to risk of clear cell renal carcinoma.

Mutations in the von Hippel–Lindau (VHL) gene are pathogenic in VHL disease, congenital polycythaemia and clear cell renal carcinoma (ccRCC). pVHL forms a ternary complex with elongin C and elongin B, critical for pVHL stability and function, which interacts with Cullin-2 and RING-box protein 1 to target hypoxia-inducible factor for polyubiquitination and proteasomal degradation. We describe a comprehensive database of missense VHL mutations linked to experimental and clinical data. We use predictions from in silico tools to link the functional effects of missense VHL mutations to phenotype. The risk of ccRCC in VHL disease is linked to the degree of destabilization resulting from missense mutations. An optimized binary classification system (symphony), which integrates predictions from five in silico methods, can predict the risk of ccRCC associated with VHL missense mutations with high sensitivity and specificity. We use symphony to generate predictions for risk of ccRCC for all possible VHL missense mutations and present these predictions, in association with clinical and experimental data, in a publically available, searchable web server.

HIV-1 Vif N-terminal Motif is required for recruitment of Cul5 to Suppress APOBEC3

BackgroundHIV-1 Vif promotes the degradation of host anti-retroviral factor family, APOBEC3 proteins via the recruitment of a multi-subunit E3 ubiquitin ligase complex. The complex is composed of a scaffold protein, Cullin 5 (Cul5), RING-box protein (Rbx), a SOCS box binding protein complex, Elongins B/C (Elo B/C), as well as newly identified host co-factor, core binding factor beta (CBF-β). Cul5 has previously been shown to bind amino acids within an HCCH domain as well as a PPLP motif at the C–terminus of Vif; however, it is unclear whether Cul5 binding requires additional regions of the Vif polypeptide.ResultsHere, we provide evidence that an amino terminal region of full length Vif is necessary for the Vif-Cul5 interaction. Single alanine replacement of select amino acids spanning residues 25–30 (25VXHXMY30) reduced the ability for Vif to bind Cul5, but not CBF-β or Elo B/C in pull-down experiments. In addition, recombinant Vif mutants had a reduced binding affinity for Cul5 compared to wild-type as measured by isothermal titration calorimetry. N-terminal mutants that demonstrated reduced Cul5 binding were also unable to degrade APOBEC3G as well as APOBEC3F and were unable to restore HIV infectivity, in the presence of APOBEC3G. Although the Vif N-terminal amino acids were necessary for Cul5 interaction, the mutation of each residue to alanine induced a change in the secondary structure of the Vif-CBF-β-Elo B/C complex as suggested by results from circular dichroism spectroscopy and size-exclusion chromatography experiments. Surprisingly, the replacement of His108 to alanine also contributed to the Vif structure. Thus, it is unclear whether the amino acids contribute to a direct interaction with Cul5 or whether the amino acids are responsible for the structural organization of the Vif protein that promotes Cul5 binding.ConclusionsTaken together, we propose a novel Vif N-terminal motif that is responsible for Vif recruitment of Cul5. Motifs in Vif that are absent from cellular proteins represent attractive targets for future HIV pharmaceutical design.

Structure and Mechanism of the E3 Ligase Rbx1 in Complex with the E2 Enzyme CDC34 Charged with Ubiquitin

Cells grow and divide through a delicate balance between the synthesis and breakdown of proteins. Many of these proteins are involved in cell cycle progression and require tight regulation through the ubiquitylation-signaling pathway. Ubiquitylation involves the transfer of ubiquitin between a series of enzymes (E1, E2, E3) until it labels a lysine residue of the substrate protein. When repeated several times, the E1-E2-E3 cascade forms a K48-linked polyubiquitin chain targeting the substrate protein for 26S proteasomal degradation. RING-box protein-1 (Rbx1) is a RING domain protein found in the Skp1/Cullin-1/F-box (SCF) E3 ubiquitin ligase complex that recruits and facilitates the efficient transfer of ubiquitin from the E2 enzyme CDC34 to a substrate protein or an elongating ubiquitin chain. We previously demonstrated a 50-fold increase in affinity between CDC34 conjugated to ubiquitin and Rbx1 when compared to naked CDC34 [Spratt et al. (2012) J. Biol. Chem. 287, 17374-85]. To expand on these findings, the ternary complex composed of CDC34∼ubiquitin/Rbx1 was examined by NMR spectroscopy. To determine the complete triple resonance assignments of CDC34, ubiquitin and Rbx1, 13C15N- and 2H13C15N-labeling methods were required. The sites of protein-protein interactions within the complex were identified using chemical shift perturbations. Distance restraints between the proteins were determined using 15N and 13C NOE assignments that were used to build a structural model of this multi-protein complex. These studies provide the first detailed structural information for the CDC34∼ubiquitin/Rbx1 complex and clarify its unique mechanism for efficient polyubiquitin synthesis.

Prognostic impact of RING box protein-1 (RBX1) expression in gastric cancer

RING box protein-1 (RBX1) is an essential component of the E3 ubiquitin ligase Skp1/Cullin/RBX1/F-box protein complex. Although an altered expression of RBX1 has been reported in several human cancers, the role of RBX1 in gastric cancer remains unknown. We investigated the RBX1 expression in primary gastric cancer tissues from 145 patients by immunohistochemistry, and explored its clinical relevance and prognostic value. Furthermore, the effect of RBX1 expression on cancer cell proliferation was analyzed in vitro using a siRNA silencing technique. The RBX1 expression was abundant in gastric cancer tissues. There was a significant difference in the expression level of RBX1 in terms of the tumor depth (P = 0.008), presence of distant metastasis (P = 0.016) and venous invasion (P = 0.005). The postoperative overall (P < 0.001) and relapse-free survival (P < 0.001) rates were significantly poorer in patients with RBX1-high tumors than in patients with RBX1-low tumors. There was a significant correlation of the RBX1 status with postoperative hematogenous recurrence (P = 0.013). Importantly, the RBX1 status was identified as an independent prognostic factor for gastric cancer (P = 0.002). Furthermore, RBX1 gene silencing significantly inhibited the proliferation of gastric cancer cells in vitro. The RBX1 expression has a significant prognostic value in gastric cancer. RBX1 might play an important role in regulating the proliferation of gastric cancer cells and promoting the development of postoperative recurrence. Our data provide a rationale for developing a novel therapy targeting RBX1 for gastric cancer.

Cullin, a component of the SCF complex, interacts with TaRMD5 during wheat spike development

Cullin, a major component of the SKP1-cullin-F box protein (SCF) complex, is a scaffold protein that binds to both SKP1 and RBX1 for selective protein degradation through the ubiquitin proteasome system. In order to study the role of cullin in common wheat, we isolated TaCullin (Cullin gene from Triticum aestivum) from wheat spike cDNA. TaCullin was expressed during all spike/grain developmental stages and in high amounts during early spike/grain development. The TaCullin gene is located on the chromosome arm 2DL. Our results suggest that unneddylated TaCullin is located in the nucleus. Based on previous proposals of Cullin-SKP1 interactions, we examined the interaction between TaCullin and SKP1-like protein (TaSKP) families by using a yeast two-hybrid approach. Yeast cotransformation demonstrated that the N-terminus of TaCullin physically interacts with TaSKP proteins. Using the yeast two-hybrid screen, we identified potential TaCullin-interacting proteins in a wheat spike library. Among the 9 clones that were identified as potential interacting partners of TaCullin, we identified E3-like ubiquitin ligase, targeting fructose-1,6-bisphosphatase (RMD5) homolog A-like protein. The interaction between TaCullin and the TaRMD5 homolog A-like protein was specifically mediated through the C-terminus of TaCullin. The results of bimolecular fluorescence complementation assay indicated that TaCullin-TaRMD5 is localized in the plasma membrane and cytoplasm. In this study, we present that TaRMD5, such as RING box protein 1 (RBX1), has the potential to interact with TaCullin, depending on the developmental stage and particular organ tissues analyzed.

The Role of RING Box Protein 1 in Mouse Oocyte Meiotic Maturation

RING box protein-1 (RBX1) is an essential component of Skp1-cullin-F-box protein (SCF) E3 ubiquitin ligase and participates in diverse cellular processes by targeting various substrates for degradation. However, the physiological function of RBX1 in mouse oocyte maturation remains unknown. Here, we examined the expression, localization and function of RBX1 during mouse oocyte meiotic maturation. Immunofluorescence analysis showed that RBX1 displayed dynamic distribution during the maturation process: it localized around and migrated along with the spindle and condensed chromosomes. Rbx1 knockdown with the appropriate siRNAs led to a decreased rate of first polar body extrusion and most oocytes were arrested at metaphase I. Moreover, downregulation of Rbx1 caused accumulation of Emi1, an inhibitor of the anaphase-promoting complex/cyclosome (APC/C), which is required for mouse meiotic maturation. In addition, we found apparently increased expression of the homologue disjunction-associated protein securin and cyclin B1, which are substrates of APC/C E3 ligase and need to be degraded for meiotic progression. These results indicate the essential role of the SCFβTrCP-EMI1-APC/C axis in mouse oocyte meiotic maturation. In conclusion, we provide evidence for the indispensable role of RBX1 in mouse oocyte meiotic maturation.

Overexpression of RING box protein‐1 (RBX1) associated with poor prognosis of non‐muscle‐invasive bladder transitional cell carcinoma

RING box protein-1 (RBX1) is a key subunit of the ubiquitin E3 ligase Skp1/Cullin1/Rbx1/F-box protein complex. Altered expression RBX1 is shown to associate with tumorigenesis and tumor progression. This study detected RBX1 expression for association with clinical significance (such as clinicopathological data and survival of the patients) in non-muscle-invasive bladder transitional cell carcinoma (NMIBC). A total of 70 primary NMIBC tissue specimens and 24 normal tissue specimens were recruited and analyzed immunohistochemically for expression of RBX1 protein and associated with clinicopathological data and survival of the patients. RBX1 was highly expressed in NMIBC, but was lowly expressed in the normal tissue. RBX1 expression was associated with high tumor grade and advanced clinical stage (P < 0.01 and P < 0.05, respectively). Moreover, patients with high RBX1 expression had shorter recurrence-free survival and progression-free survival rates (P < 0.001 and P < 0.01, respectively). Multivariate analysis demonstrated that RBX1 expression is an independent prognostic factor for tumor recurrence and progression of NMIBC (P < 0.05). Overexpression of RBX1 protein contributes to tumor progression and poor prognosis of NMIBC.

Functional characterization of SAG/RBX2/ROC2/RNF7, an antioxidant protein and an E3 ubiquitin ligase

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.