Ras-related Nuclear Protein Research Articles

Differential Display Analysis of 2,3,7,8-Tetrachlorodibenzo-p-dioxin Identified Induction of Ras-related Nuclear Protein Binding Protein2 (RanBP2) Gene.

TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin) and related halogenated aromatic hydrocarbons elicit a diverse spectrum of biochemical and toxic responses in laboratory animals and mammalian cells in culture. Toxicity and carcinogenicity of TCDD is well established but the molecular mechanism is still poorly understood. Here, we found the noble responsive genes to TCDD using the differential display analysis. Treatment of HepG2 cells with TCDD showed a significantly different mRNA expression pattern from the untreated cells in differential display analysis. The differentially displayed bands were isolated and used as probes in dot blot and Northern blot analyses. Of thirty-five isolated differentially displayed bands, only two bands were confirmed as positive in dot blot and Northern blot analyses. The nucleotides sequences of these clones were analyzed and the search of Genebank database revealed that one clone is highly homologous with RanBP2 (Ras-related nuclear protein binding protein2; 92%) and the other is an unknown gene. RanBP2 is a nucleoporin with SUMO E3 ligase activity that functions in both nucleocytoplasmic transport and mitosis and its role as a novel tumor suppressor has been recently proposed. Thus, these results may suggest the clue elucidating the toxic mechanism of TCDD through RanBP2.

Comparative Proteomic Analysis of Osteosarcoma Cell and Human Primary Cultured Osteoblastic Cell

To identify new biomarkers that facilitate the accurate early diagnosis of osteosarcoma and that may possibly include novel therapeutic candidates, we performed a proteomic approach to compare osteosarcoma cells and human primary cultured osteoblastic cells. Image analysis of silver-stained 2-DE gels revealed that the level of 12 protein spots was significantly different between the two groups of samples (p <. 004). After mass spectroscopic identification and database searches, we found that in osteosarcoma cells, the level of HSP70, actin capping protein, ATP synthase, Mthsp75, UQCRC1, Ras-related nuclear protein, UCH-L1, and PRDX4 was elevated. However, the level of pyruvate dehydrogenase E1, Prohibitin, and Annexin V was decreased. Subsequent Western blot analyses of UQCRC1, UCH-L1, and PRDX4 in osteosarcoma tissues confirmed the results obtained by the proteomic analyses. These identified proteins may be potential molecular targets for osteosarcomatous diagnostics and therapeutics.

Functional analysis of key nuclear trafficking components reveals an atypical Ran network required for parasite pathogenesis

Protozoan parasites represent major public health challenges. Many aspects of their cell biology are distinct from their animal hosts, providing potential therapeutic targets. Toxoplasma gondii is a protozoan parasite that contains a divergent regulator of chromosome condensation 1 (TgRCC1) that is required for virulence and efficient nuclear trafficking. RCC1 proteins function as a guanine exchange factor for Ras-related nuclear protein (Ran), an abundant GTPase responsible for the majority of nucleocytoplasmic transport. Here we show that while there are dramatic differences from well-conserved RCC1 proteins, TgRCC1 associates with chromatin, interacts with Ran and complements a mammalian temperature-sensitive RCC1 mutant cell line. During the investigation of TgRCC1, we observed several unprecedented phenotypes for TgRan, despite a high level of sequence conservation. The cellular distribution of TgRan is found throughout the parasite cell, whereas Ran in late branching eukaryotes is predominantly nuclear. Additionally, T. gondii tolerates at least low-level expression of dominant lethal Ran mutants. Wild type parasites expressing dominant negative TgRan grew similarly to wild type in standard tissue culture conditions, but were attenuated in serum-starved host cells and mice. These growth characteristics paralleled the TgRCC1 mutant and highlight the importance of the nuclear transport pathway for virulence of eukaryotic pathogens.

Ran suppresses paclitaxel-induced apoptosis in human glioblastoma cells

Yeast-based functional screening of a human glioblastoma cDNA library identified ras-related nuclear protein (Ran) as a novel suppressor of Bcl-2-associated X protein (Bax), a pro-apoptotic member of the Bcl-2 family of proteins. Yeast cells that expressed human Ran were resistant to Bax-induced cell death. In U373MG glioblastoma cells, stable overexpression of Ran significantly attenuated apoptotic cell death induced by the chemotherapeutic agent paclitaxel. FACS analysis demonstrated that Ran is involved in paclitaxel-induced cell cycle arrest. Stable overexpression of Ran also markedly inhibited the phosphorylation of Bcl-2 by paclitaxel, and inhibited the translocation of Bax, the release of cytochrome c and activation of caspase-3. Paclitaxel-induced phosphorylation of c-JUN N-terminal kinase (JNK), but not p38, extracellular signal-regulated kinase and Akt, was markedly suppressed in U373MG cells that stably expressed Ran. These results suggest that Ran suppresses paclitaxel-induced cell death through the downregulation of JNK-mediated signal pathways.

Identification of 5-fluorouracil response proteins in colorectal carcinoma cell line SW480 by two-dimensional electrophoresis and MALDI-TOF mass spectrometry

Colorectal cancer (CRC) is the second most prevalent cause of cancer-related deaths in the Western world. 5-Fluorouracil (5-FU) is a standard chemotherapeutic drug to treat CRC. However, the response rate is less than 20% and patients who have responded to 5-FU may become resistant. Therefore there is an urgent need to examine the 5-FU response proteins so that patients with no response to 5-FU can change to other treatment strategies promptly. In this study, the proteomic expression profile in a CRC cell line SW480 before and after 5-FU treatment was examined using 2-dimensional electrophoresis technology. Fourteen proteins with differential expression were identified using mass spectrometry and 7 of them were validated using immunocytochemical (ICC) staining. Protein identification indicated that cyclophilin A, cytokeratin 19 (CK19), cytokeratin 8 (CK8), ras-related nuclear protein, heat shock protein 27 (hsp27) and peroxiredoxin 6 (Prx 6) were upregulated whereas heat shock protein 60 (hsp60), cytokeratin 18 (CK18), cytokeratin 9 (CK9), carbamoylphosphate synthetase I, alpha-enolase, heat shock protein 70 (hsp70), nm23 and beta-actin were down-regulated. Seven of the 14 proteins detected were validated by ICC staining, which showed that the expression of hsp27, Prx 6 and hsp70 correlated with that from proteomics profiling. Our results suggest that hsp27, Prx 6 and hsp70 are potential 5-FU response proteins and they may represent potential targets for further evaluation in other 5-FU-sensitive and -resistant CRC cell lines.

Induction of Apoptosis in Murine Neuroblastoma by Systemic Delivery of Transferrin-Shielded siRNA Polyplexes for Downregulation of Ran

The polymer, OEI-HD, based on beta-propionamide-cross-linked oligoethylenimine and its chemical transferrin conjugate were evaluated for siRNA delivery into murine Neuro2A neuroblastoma cells in vitro and in vivo. An 80% silencing of luciferase expression in neuroblastoma cells, stably transfected with a luciferase gene, was obtained using standard OEI-HD polyplexes or transferrin-conjugated shielded OEI-HD polyplexes. The Ras-related nuclear protein Ran was selected as a therapeutically relevant target protein. Systemic delivery of transferrin-conjugated OEI-HD/RAN siRNA formulations (three intravenous applications at 3 days interval) resulted in >80% reduced Ran protein expression, apoptosis, and a reduced tumor growth in Neuro2A tumors of treated mice. The treatment was not associated with signs of acute toxicity or significant changes in weight, hematology parameters, or liver enzymes (AST, ALT, or AP) of mice. All our results demonstrate that OEI-HD/siRNA formulations can knockdown genes in tumor cells in vitro and in vivo in mice in the absence of unspecific toxicity.

Regulation of Wnt Signaling by the Nuclear Pore Complex

The function of beta-catenin as a transcriptional coactivator of T-cell factor-4 (TCF-4) is crucial for colorectal carcinogenesis. However, beta-catenin has no nuclear localization signal, and the mechanisms by which beta-catenin is imported into the nucleus and forms a complex with the TCF-4 nuclear protein are poorly understood. Proteins of 2 colorectal cancer cell lines, HCT-116 and DLD1, were immunoprecipitated with anti-TCF-4 antibody and analyzed directly by nanoflow liquid chromatography and mass spectrometry. The functional significance of nuclear pore complex (NPC) proteins in Wnt signaling was evaluated by in vitro and in vivo sumoylation, luciferase reporter, and colony formation assays. TCF-4 interacted with a large variety of NPC proteins including ras-related nuclear protein (Ran), Ran binding protein-2 (RanBP2), and Ran GTPase-activating protein-1 (RanGAP1). The NPC protein RanBP2 functioned as the small ubiquitin-related modifier (SUMO) E3 ligase of TCF-4, and sumoylation of TCF-4 enhanced the interaction between TCF-4 and beta-catenin. The overexpression of NPC proteins increased the nuclear import of the TCF-4 and beta-catenin proteins and enhanced the transcriptional activity. NPC proteins increased the growth of colorectal cancer cells, whereas sentrin-specific protease-2 inhibited it. Through a comprehensive proteomics approach, we revealed that NPC functions as a novel regulator of Wnt signaling and is possibly involved in colorectal carcinogenesis. A new drug targeting the interactions of TCF-4 with NPC proteins as well as their sumoylation activity might be effective for suppressing aberrant Wnt signaling and the proliferation of colorectal cancer cells.

Proof concept for clinical justification of network mapping for personalized cancer therapeutics

To identify signature targets associated with patient-specific cancer lesions based on tumor versus normal tissue differential protein and mRNA coexpression patterns for the purpose of synthesizing cancer-specific customized RNA interference knockdown therapeutics. Analysis of biopsied tissue involved two-dimensional difference in-gel electrophoresis (2D-DIGE) analysis coupled with MALDI-TOF/TOF mass spectrometry for proteomic assessment. Standard microarray techniques were utilized for mRNA analysis. Priority was assigned to overexpressed protein targets with co-overexpressed genes with a high likelihood of functional nodal centrality in the cancer network as defined by the interactive databases BIND, HPRD and ResNet. HPLC-grade small interfering RNA (siRNA) duplexes were utilized to assess knockdown of target proteins in expressive cell lines as measured by western blot. Seven patients with metastatic cancer underwent biopsy. One patient (RW001) had biopsies from two disease sites 10 months apart. Seven priority proteins were identified, one for each patient (RACK 1, Ras related nuclear protein, heat-shock 27 kDa protein 1, superoxide dismutase, enolase1, stathmin1 and cofilin1). Prioritized proteins in RW001 from the two disease sites over time were the same. We demonstrated >80% siRNA inhibition of RACK 1 and stathmin1 of inexpressive malignant cell lines with correlated cell kill. Identification of functionally relevant target gene fingerprints, unique to an individual's cancer, is feasible 'at the bedside' and can be utilized to synthesize siRNA knockdown therapeutics. Further animal safety testing followed by clinical study is recommended.

Identification of Ras-Related Nuclear Protein, Targeting Protein for Xenopus Kinesin-like Protein 2, and Stearoyl-CoA Desaturase 1 as Promising Cancer Targets from an RNAi-Based Screen

To identify new candidate cancer drug targets, we used RNAi as a tool to functionally evaluate genes that play a role in maintaining human tumor cell survival. We screened a small interfering RNA (siRNA) library directed against approximately 3,700 individual genes to assess the ability of siRNAs to induce cell death in an in vitro cell cytotoxicity assay. We found that siRNAs specifically targeting ras-related nuclear protein (Ran), targeting protein for Xenopus kinesin-like protein 2 (TPX2), and stearoyl-CoA desaturase 1 (SCD1), significantly reduced the survival of multiple human tumor cell lines. Further target validation studies revealed that treatment with Ran and TPX2 siRNAs differentially reduced the survival of activated K-Ras-transformed cells compared with their normal isogenic counterparts in which the mutant K-Ras gene had been disrupted (DKS-8). Knockdown of Ran and TPX2 in activated mutant K-Ras cells selectively induced S-phase arrest or transient G(2)-M arrest phenotypes, respectively, that preceded apoptotic cell death. Given our observations that Ran and TPX2 depletion preferentially reduces the survival of activated K-Ras-transformed cells, these two proteins may serve as useful anticancer targets in tumors expressing the activated K-Ras oncogene.

Characterization of a ras-related nuclear protein (Ran protein) up-regulated in shrimp antiviral immunity

Diseases caused by viruses, especially white spot syndrome virus (WSSV), are the greatest challenge to worldwide shrimp aquaculture. Therefore, the innate immunity of shrimp has attracted extensive attentions these years. To date, however, no mechanism of immuno-related signal transduction pathway has been reported. In this investigation, an important signal transduction factor—Ran gene encoding ras-related nuclear protein (Ran protein) was characterized in shrimp. The shrimp Ran gene, without introns when compared with genomic DNA, was 645 bp in length. The GTP-binding assay showed that the Ran protein had GTP-binding activity. The results of RT-PCR and Western blot indicated that the transcript and protein of Ran were detected in every tissue of shrimp including hepatopancreas, haemolymph, gill, intestine, heart and muscle. In the WSSV-resistant and WSSV-infected shrimp at 4 h postinfection, the Ran gene was obviously up-regulated, indicating that it played a role in shrimp immunity against virus infection. This study, therefore, might provide a clue to elucidate shrimp innate immunity.

Comparison of intestinal gene expression in Atlantic cod ( Gadus morhua) fed standard fish meal or soybean meal by means of suppression subtractive hybridization and real-time PCR

Gene expression was studied in Atlantic cod fed two different diets, fish meal (FM) and dehulled and extracted soybean meal (SBM). RNA was isolated from the distal part of the mid-intestine of Atlantic cod and suppression subtractive hybridization (SSH) was employed to screen for genes that showed changes in expression in response to the two dietary treatments. We made a cDNA subtracted library, isolated and sequenced 192 clones. Identification of 157 clones was predicted by BLAST. Most of the clones were previously unidentified in cod. Expression of 12 selected clones was further studied by quantitative PCR. Expression of four clones showing similarity to aminopeptidase N, transcobalamin I precursor, cytochrome P450 3A40, and ras-related nuclear protein was significantly up regulated in intestine of cod fed SBM compared to cod fed FM. A trend towards up regulation of a clone with similarity to fatty acid binding protein in SBM-fed cod was also observed. No significant differences in expression were observed for: transmembrane 4 superfamily protein member, polypeptide N-acetylgalactosaminyltransferase, glutathione peroxidase, peroxiredoxin 4, SEC61, F-BOX, and 14-3-3.

Identification of Genes Related to Nasopharyngeal Carcinoma with the Help of Pathway-based Networks

cDNA microarray is a powerful tool to analyze simultaneously the expression levels of tens of thousands of genes. Compared with normal nasopharynx (NP) tissues, 2210 genes were highly differentially expressed in nasopharyngeal carcinoma (NPC) tissues detected by cDNA microarray. Since signal pathway is widely used to describe the complex relationship between genes, a pathway-based network was constructed to visualize the connection between the genes obtained from microarray data in this report. We analyzed the targeted genes that may have more important influence on this gene network with statistical methods and found that some genes might have significant influence on this network, especially Ras-related nuclear protein (RAN), carboxyl ester lipase (CEL), v-rel reticuloendotheliosis viral oncogene homolog A (RELA) genes. To verify the results from pathway-based selection, reverse transcription-polymerase chain reaction (RT-PCR) and real-time RT-PCR were performed to detect the expression levels of RAN, CEL and RELA genes and it was found that the RAN and CEL genes were significantly up-regulated in more than 80% of NPC tissues. To further elucidate the function of the RAN gene, RAN expression was specifically suppressed in a 5-8F NPC cell line by RNA interference (RNAi). As expected, the depletion of RAN could effectively block the proliferation of tumor cells. Therefore, our study may open up a new way to analyze the vast microarray data.

Importin α/β Mediates Nuclear Transport of a Mammalian Circadian Clock Component, mCRY2, Together with mPER2, through a Bipartite Nuclear Localization Signal

Circadian rhythms, which period is approximately one day, are generated by endogenous biological clocks. These clocks are found throughout the animal kingdom, as well as in plants and even in prokaryotes. Molecular mechanisms for circadian rhythms are based on transcriptional oscillation of clock component genes, consisting of interwoven autoregulatory feedback loops. Among the loops, the nuclear transport of clock proteins is a crucial step for transcriptional regulation. In the present study, we showed that the nuclear entry of mCRY2, a mammalian clock component, is mediated by the importin alpha/beta system through a bipartite nuclear localization signal in its carboxyl end. In vitro transport assay using digitonin-permeabilized cells demonstrated that all three importin alphas, alpha1 (Rch1), alpha3 (Qip-1), and alpha7 (NPI-2), can mediate mCRY2 import. mCRY2 with the mutant nuclear localization signal failed to transport mPER2 into the nucleus of mammalian cultured cells, indicating that the nuclear localization signal identified in mCRY2 is physiologically significant. These results suggest that the importin alpha/beta system is involved in nuclear entry of mammalian clock components, which is indispensable to transcriptional oscillation of clock genes.

383 Preclinical antitumor activity of second generation analogs of SDX-101

Background & Aims: The function of β-catenin as a transcriptional coactivator of T-cell factor-4 (TCF-4) is crucial for colorectal carcinogenesis. However, β-catenin has no nuclear localization signal, and the mechanisms by which β-catenin is imported into the nucleus and forms a complex with the TCF-4 nuclear protein are poorly understood. Methods: Proteins of 2 colorectal cancer cell lines, HCT-116 and DLD1, were immunoprecipitated with anti-TCF-4 antibody and analyzed directly by nanoflow liquid chromatography and mass spectrometry. The functional significance of nuclear pore complex (NPC) proteins in Wnt signaling was evaluated by in vitro and in vivo sumoylation, luciferase reporter, and colony formation assays. Results: TCF-4 interacted with a large variety of NPC proteins including ras-related nuclear protein (Ran), Ran binding protein-2 (RanBP2), and Ran GTPase-activating protein-1 (RanGAP1). The NPC protein RanBP2 functioned as the small ubiquitin-related modifier (SUMO) E3 ligase of TCF-4, and sumoylation of TCF-4 enhanced the interaction between TCF-4 and β-catenin. The overexpression of NPC proteins increased the nuclear import of the TCF-4 and β-catenin proteins and enhanced the transcriptional activity. NPC proteins increased the growth of colorectal cancer cells, whereas sentrin-specific protease-2 inhibited it. Conclusions: Through a comprehensive proteomics approach, we revealed that NPC functions as a novel regulator of Wnt signaling and is possibly involved in colorectal carcinogenesis. A new drug targeting the interactions of TCF-4 with NPC proteins as well as their sumoylation activity might be effective for suppressing aberrant Wnt signaling and the proliferation of colorectal cancer cells.

Identification of genes involved in the response of haemocytes of Penaeus japonicus by suppression subtractive hybridization (SSH) following microbial challenge

Penaeus japonicus were injected with a heat-killed microorganism suspension and 291 randomly selected cDNA fragments generated by suppression subtractive hybridization (SSH) were sequenced. A total of 71 cDNA clones corresponding to 25 genes were found to have enhanced expression, of which eight are found for the first time in shrimp. The most abundant gene in the subtractive library was Kunitz-type protease inhibitor, clearly indicating this protease inhibitor in the response. A number of genes encoding signaling molecules, such as Ras-related nuclear protein (Ran), growth factor receptor bound protein (Grb), TGF-β receptor interacting protein, integrin binding protein and interferon receptor bound protein were found for the first time in the shrimp, and they may be involved in the regulation of the host defense against the injected microbes. Furthermore, cDNAs of chaperonin, proteasome, antioxidant as well as genes associated with actin reorganization, which may be necessary for phagocytosis and encapsulation, were also expressed at a higher level after the challenge. These results may facilitate the understanding of shrimp immune responses.

An Arabidopsis Ran-binding protein, AtRanBP1c, is a co-activator of Ran GTPase-activating protein and requires the C-terminus for its cytoplasmic localization.

Ran-binding proteins (RanBPs) are a group of proteins that bind to Ran (Ras-related nuclear small GTP-binding protein), and thus either control the GTP/GDP-bound states of Ran or help couple the Ran GTPase cycle to a cellular process. AtRanBP1c is a Ran-binding protein from Arabidopsis thaliana (L.) Heynh. that was recently shown to be critically involved in the regulation of auxin-induced mitotic progression [S.-H. Kim et al. (2001) Plant Cell 13:2619-2630]. Here we report that AtRanBP1c inhibits the EDTA-induced release of GTP from Ran and serves as a co-activator of Ran-GTPase-activating protein (RanGAP) in vitro. Transient expression of AtRanBP1c fused to a beta-glucuronidase (GUS) reporter reveals that the protein localizes primarily to the cytosol. Neither the N- nor C-terminus of AtRanBP1c, which flank the Ran-binding domain (RanBD), is necessary for the binding of PsRan1-GTP to the protein, but both are needed for the cytosolic localization of GUS-fused AtRanBP1c. These findings, together with a previous report that AtRanBP1c is critically involved in root growth and development, imply that the promotion of GTP hydrolysis by the Ran/RanGAP/AtRanBP1c complex in the cytoplasm, and the resulting concentration gradient of Ran-GDP to Ran-GTP across the nuclear membrane could be important in the regulation of auxin-induced mitotic progression in root tips of A. thaliana.

How Ran is regulated.

Ran, the Ras-related nuclear protein, is a regulatory GTP-Binding protein belonging to a large family of small GTPases. Many aspects of cell behavior are controlled by these proteins, including cell shape and cell movement, cell polarity, intracellular transport, and the decision for proliferation or differentiation. Ran is by far the most abundant of these GTPases, and in human HeLa cells it comprises some 0.4% of cellular protein. It regulates import and export of proteins and RNA through the nuclear pores and is involved in the assembly of the mitotic spindle and of the nuclear membrane.

Putative Reaction Intermediates in Crm1-mediated Nuclear Protein Export

We discovered several novel interactions between proteins involved in Crm1-mediated nuclear export of the nuclear export signal containing human immunodeficiency virus type 1 protein Rev. First, a Rev/Crm1/RanGTP complex (where Ran is Ras-related nuclear protein) reacts with some nucleoporins (Nup42 and Nup159) but not others (NSP1, Nup116, and Nup1), forming a Nup/Crm1/RanGTP complex and concomitantly releasing Rev. Second, RanBP1 (or homologous proteins) can displace Nup and form a ternary RanBP1/RanGTP/Crm1 complex that can be disassembled by RanGAP via GTP hydrolysis. Third, and most surprisingly, RanBP1/RanGTP/Crm1 can be disassembled without GTP hydrolysis by the nucleotide exchange factor RanGEF. Recycling of a Ran/RanGEF complex by GTP and Mg2+ is stimulated by both Crm1 and Rev, allowing reformation of a Rev/Crm1/RanGTP complex. Based on these reactions we propose a model for Crm1-mediated export.

Effects of Overexpression of Ran/TC4 on Mammalian Cellsin Vitro

We investigated the effect of overexpression of Ran/TC4 on cell cycle progression. Ran/TC4 (ras-related nuclear protein) is a highly conserved 25-kDa GTP-binding protein that, in concert with its guanine-nucleotide-exchange factor RCC1, is involved in signal transduction. Ran and RCC1 act on nuclear transport of RNA and protein, cell cycle regulation at the G1/S interphase, chromatin decondensation after mitosis, and chromosome stability. These two proteins are essential for the coupling of DNA synthesis with the onset of mitosis. The cDNA for rabbit Ran/TC4 was identified in a cDNA library using degenerate oligonucleotide probes devised on the basis of deduced protein sequence data. This cDNA was cloned into pCDM8 expression vector to yield a plasmid, pTC4, in which Ran/TC4 expression is driven by the cytomegalovirus intermediate early promoter. Both a human tumor cell line, MCF7, and a normal rabbit fibroblast line, RK-13, were tested. Following transfection with pTC4 we observed an increase in Ran/TC4 transcript levels. Transfection with pTC4 prolonged the duration of S phase in both MCF7 and RK-13 cells and led to reduced cell proliferation and decreased total cell numbers. DNA fragmentation was seen in pTC4-transfected cultures but not in control cultures. These findings underscore the function of Ran/TC4 as a molecular switch that guides the cell to completion of DNA synthesis before it enters mitosis and suggest that its overexpression may greatly alter cell cycle kinetics and cell viability.

An abundant zebrafish cDNA clone encodes a ras-like protein which is expressed ubiquitously.

An abundant clone from a zebrafish embryonic cDNA library was identified and sequenced. It encodes a ras-like protein, being 94-95% identical to vertebrate Ran, ras-related nuclear protein. The zebrafish Ran mRNA is expressed in all tissues examined and throughout embryogenesis; therefore, it is likely that the Ran protein functions in all types of cells.