Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0027819 (neuroblastoma)
 27,800 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Human insulin-like growth factor-1 (hIGF-1) is a growth factor with clinical significance in medicine. The therapeutic potential of recombinant hIGF-1 (rthIGF-1) stems from the fact that hIGF-1 resembles insulin in many aspects of physiology. The expression of hIGF-1 in transgenic tobacco and rice plants using different expression cassettes is reported here. In the present study, two coding sequences were tested, one with the original human sequence, but partially optimized for expression in E. coli and the other with a plant-codon-optimized sequence that was expected to give a higher level of expression in plant systems. Three different hIGF-1 recombinant expression constructs were generated. All expression constructs utilized the maize ubiquitin 1 promoter with or without a signal sequence. Analyses conducted using a hIGF-1 specific ELISA kit showed all transgenic plants produced hIGF-1 and the accumulated hIGF-1 increased from the E. coli codon bias to higher levels when the hIGF-1 coding sequence was codon-optimized to match that of the maize zeamatin protein--the most transcribed gene in maize endosperm suspension cells. Further analyses that compared the functionality of the bacterial signal peptide Lam B in plants showed that this leader peptide led to lower expression levels when compared to transgenic plants that did not contain this sequence. This indicated that this expression construct was functional without removal of the bacterial signal sequence. The maize ubiquitin 1 promoter was found to be more active in rice plants than tobacco plants indicating that in this case, there was a class preference that was biased towards a monocot host. Biological analyses conducted using protein extracts from transgenic plants showed that the rthIGF-1 was effective in stimulating the in vitro growth and proliferation of human SH-SY5Y neuroblastoma cells. This indicated that the plant-produced rthIGF-1 was stable and biologically active. As some plants have been reported to express an endogenous insulin-like protein, we also looked for any effect of the human growth factor in transgenic plants, but no developmental or morphological differences with wild type tobacco or rice plants were detected. Since insulin and hIGF-1 share some overlapping roles, hIGF-1 may become a substitute therapeutic agent in subjects with certain defects in their insulin receptor signaling. Hence, if the full beneficial potential of rthIGF-1 is achieved, it is expected that in the future the demand will likely increase significantly.
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PMID:Recombinant protein expression plasmids optimized for industrial E. coli fermentation and plant systems produce biologically active human insulin-like growth factor-1 in transgenic rice and tobacco plants. 1535 2

Mutations of parkin, a protein-ubiquitin E3 ligase, are linked to Parkinson's disease (PD). Although a variety of parkin substrates have been identified, none of these is selectively expressed in dopaminergic neurons, whose degeneration plays a critical role in PD. Here we show that parkin significantly increased dopamine uptake in the human dopaminergic neuroblastoma cell line SH-SY5Y. This effect was accompanied by increased V(max) of dopamine uptake and unchanged K(m). Consistent with this, increased binding sites for dopamine transporter (DAT) ligand were observed in SH-SY5Y cells overexpressing parkin. The results were confirmed when parkin was transfected in HEK293 cells stably expressing DAT. In these cells, parkin enhanced the ubiquitination and degradation of DAT, increased its cell surface expression, and augmented dopamine uptake. The effects of parkin were significantly abrogated by its PD-causing mutations. Because the cell surface expression of functional DAT requires its oligomerization, misfolded DAT, induced either by the protein glycosylation inhibitor tunicamycin or by its C-terminal truncation, significantly attenuated cell surface expression of native DAT and reduced dopamine uptake. Expression of parkin, but not its T240R mutant, significantly alleviated these detrimental effects of misfolded DAT. Thus, our studies suggest that parkin increases dopamine uptake by enhancing the ubiquitination and degradation of misfolded DAT, so as to prevent it from interfering with the oligomerization and cell surface expression of native DAT. This function of parkin would enhance the precision of dopaminergic transmission, increase the efficiency of dopamine utilization, and reduce dopamine toxicity on neighboring cells.
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PMID:Parkin increases dopamine uptake by enhancing the cell surface expression of dopamine transporter. 1549 1

The basic helix-loop-helix (bHLH) transcription factor mammalian achaete-scute homologue-1 (MASH-1 in mouse and HASH-1 in humans) is expressed in specific subsets of embryonic neuronal precursors of both the peripheral and central nervous systems. This gene is essential for development of olfactory and most peripheral autonomic neurons. Neuro-blastoma is a pediatric malignancy derived from sympathetic nervous system precursors and HASH-1 is expressed in a majority of neuroblastoma tumors and cell lines, indicating the immature phenotype of these cells. Using a human neuroblastoma cDNA library and the yeast two-hybrid system to identify novel HASH-1-interacting proteins, we isolated ubiquilin-1 (DA41, hPLIC-1), a gene that contains multiple ubiquitin-related domains. Further analyses showed that ubiquilin-1 interacts not only with HASH-1, but also with other tissue-specific bHLH proteins, including HES-1. Overexpression of ubiquilin-1 led to accumulation of HASH-1 and HES-1. Moreover, ubiquilin-1 was translocated from the cytoplasm to the nucleus upon co-expression with HASH-1. These results indicate that ubiquilin-1 plays an active role in the precise regulation of HASH-1 and of other tissue-specific bHLH proteins.
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PMID:Ubiquilin-1 is a novel HASH-1-complexing protein that regulates levels of neuronal bHLH transcription factors in human neuroblastoma cells. 1549 8

To seek a novel therapeutic approach to neuroblastoma (NBL), we used three NBL cell lines (SK-N-DZ, NH12, and SK-N-SH) to examine the underlining molecular mechanisms of cellular reactions and sensitivity to all-trans-retinoic acid (ATRA). SK-N-DZ cells expressed relatively high levels of retinoic acid receptor alpha (RAR-alpha) and underwent ATRA-induced cell death that was blocked by an RAR-alpha antagonist. By contrast, RAR-alpha expression gradually decreased in NH12 and SK-N-SH cells, which did not experience increased cell death in response to ATRA. We report here the ubiquitin-dependent down-regulation of RAR-alpha expression during ATRA treatment. Our data suggest that SK-N-DZ cells have a defect in RAR-alpha down-regulation, resulting in sustained high expression of RAR-alpha that confers high sensitivity to ATRA. Accordingly, treatment with a proteasome inhibitor dramatically increased ATRA-induced cell death in NH12 and SK-N-SH cell lines. Our results reveal the crucial involvement of the RAR-alpha signaling pathway in NBL cell death and show that three NBL cell lines are differentially sensitive to ATRA. These data suggest a potential novel therapy for NBL involving retinoic acid treatment combined with the inhibition of RAR-alpha degradation.
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PMID:Retinoic acid induces neuroblastoma cell death by inhibiting proteasomal degradation of retinoic acid receptor alpha. 1552 Jan 97

Recent studies indicate that NF-E2 related factor 2 (Nrf2) is a substrate for the ubiquitin-proteasome pathway. The present study is aimed to determine whether increased protein stability is a mechanism by which quinone compounds, like tert-butylhydroquinone (tBHQ), may enhance Nrf2-mediated transcriptional activation and subsequent antioxidant protection. H2O2-induced necrotic cell death, evidenced by transmission electronic microscope (TEM) imaging with no caspase 3 activation and PARP cleavage, was significantly attenuated by pretreatment with tBHQ or overexpression of Nrf2 through advenovirus-mediated infection in human neural stem cells (hNSCs). Microarray analysis showed that those identified antioxidant genes, responsible for antiapoptotic action in IMR-32 cells (J. Li et al., 2002, J. Biol. Chem. 277, 388-394), were also coordinately upregulated through Nrf2-dependent antioxidant responsive element (ARE) activation in hNSC. The stabilization of Nrf2 by tBHQ in IMR-32 cells was evidenced by a pulse-chase assay showing no significant increase in Nrf2 protein synthesis after tBHQ treatment, and by ubiquitin immunoprecipitation showing that tBHQ stabilized ubiquitinated Nrf2. An in vitro proteasomal activity assay showed that tBHQ did not act as a 20S/26S proteasome inhibitor. Nrf2 stabilization by tBHQ also was observed in hNSCs. Taken together, this study suggests that identified antioxidant genes, which were upregulated through tBHQ induced Nrf2 stabilization, confer protection on target cells against H2O2-induced apoptotic cell death in neuroblastoma cells as well as the necrotic cell death in the hNSC. Nrf2 stabilization by pharmacological modulation or adenovirus-mediated Nrf2 overexpression, therefore, might be viable strategies to prevent a wide-spectrum of oxidative stress-related neuronal cell injuries.
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PMID:Stabilization of Nrf2 by tBHQ confers protection against oxidative stress-induced cell death in human neural stem cells. 1552 90

Mutations in the PARKIN gene are the most common cause of hereditary parkinsonism. The parkin protein comprises an N-terminal ubiquitin-like domain, a linker region containing caspase cleavage sites, a unique domain in the central portion, and a special zinc finger configuration termed RING-IBR-RING. Parkin has E3 ubiquitin-protein ligase activity and is believed to mediate proteasomal degradation of aggregation-prone proteins. Whereas the effects of mutations on the structure and function of parkin have been intensely studied, post-translational modifications of parkin and the regulation of its enzymatic activity are poorly understood. Here we report that parkin is phosphorylated both in human embryonic kidney HEK293 cells and human neuroblastoma SH-SY5Y cells. The turnover of parkin phosphorylation was rapid, because inhibition of phosphatases with okadaic acid was necessary to stabilize phosphoparkin. Phosphoamino acid analysis revealed that phosphorylation occurred mainly on serine residues under these conditions. At least five phosphorylation sites were identified, including Ser101, Ser131, and Ser136 (located in the linker region) as well as Ser296 and Ser378 (located in the RING-IBR-RING motif). Casein kinase-1, protein kinase A, and protein kinase C phosphorylated parkin in vitro, and inhibition of casein kinase-1 caused a dramatic reduction of parkin phosphorylation in cell lysates. Induction of protein folding stress in cells reduced parkin phosphorylation, and unphosphorylated parkin had slightly but significantly elevated autoubiquitination activity. Thus, complex regulation of the phosphorylation state of parkin may contribute to the unfolded protein response in stressed cells.
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PMID:Parkin phosphorylation and modulation of its E3 ubiquitin ligase activity. 1555 40

Amyloid-beta peptide 42 (Abeta42) mediates neuronal degeneration in Alzheimer's disease (AD). We sought to produce recombinant Abeta42 as an ubiquitin extension. A synthetic oligonucleotide encoding Abeta42 was constructed and cloned as an extended polypeptide of hexahistidine-tagged ubiquitin (H(6)Ub) using the pET vector. Isopropyl-beta-D-thiogalactopyranoside induction of transformed Escherichia coli resulted in the production of large amounts of insoluble H(6)Ub-Abeta42 fusion protein. H(6)Ub-Abeta42 was solubilized in 8 M urea and applied to a nickel-nitrilotriacetic acid affinity column for purification. Column washing removed the urea and soluble H(6)Ub-Abeta42 was eluted, indicating that covalently attached ubiquitin prevented Abeta42 from aggregating. Abeta42 was cleaved from H(6)Ub using recombinant yeast ubiquitin hydrolase-1 (YUH-1) and purified using reverse-phase chromatography. The recombinant Abeta42 prepared in this study has the same toxic effect on human neuroblastoma SH-SY5Y cells comparing with chemically synthesized, commercial one. The peptide yield was more than 4 mg/L culture, indicating this ubiquitin fusion technique is an attractive method for production of aggregation-prone peptides such as Abeta42.
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PMID:Production of recombinant amyloid-beta peptide 42 as an ubiquitin extension. 1572 87

Tau-positive inclusions in neurons are consistent neuropathologic features of the most common causes of dementias such Alzheimer's disease and frontotemporal dementia. Ubiquitinated tau-positive inclusions have been reported in brains of Alzheimer's disease patients, but involvement of the ubiquitin-dependent proteasomal system in tau degradation remains controversial. Before considering the tau degradation in pathologic conditions, it is important to determine whether or not endogenous tau is normally degraded by the proteasome pathway. We therefore investigated this question using two complementary approaches in vitro and in vivo. Firstly, SH-SY5Y human neuroblastoma cells were treated with different proteasome inhibitors, MG132, lactacystin, and epoxomicin. Under these conditions, neither total nor phosphorylated endogenous tau protein levels were increased. Instead, an unexpected decrease of tau protein was observed. Secondly, we took advantage of a temperature-sensitive mutant allele of the 20S proteasome in Drosophila. Genetic inactivation of the proteasome also resulted in a decrease of tau levels in Drosophila. These results obtained in vitro and in vivo demonstrate that endogenous tau is not normally degraded by the proteasome.
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PMID:Tau is not normally degraded by the proteasome. 1579 29

The initial aim of this study was to identify protein changes associated with long-term morphine treatment in a recombinant human neuroblastoma SH-SY5Y clone (sc2) stably overexpressing the human mu-opioid (MOP) receptor. In MOP receptor-overexpressing sc2 cells, short-term morphine exposure was found to be much more potent and efficacious in inhibiting forskolin-elicited production of cAMP, and long-term morphine exposure was shown to induce a substantially higher degree of opiate dependence, as reflected by adenylate cyclase sensitization, than it did in wild-type neuroblastoma cells. Differential proteomic analysis of detergent-resistant membrane rafts isolated from untreated and chronically morphine-treated sc2 cells revealed long-term morphine exposure to have reliably induced a 30 to 40% decrease in the abundance of five proteins, subsequently identified by mass spectrometry as G protein subunits alphai(2), alphai(3), beta(1), and beta(2), and prohibitin. Quantitative Western blot analyses of whole-cell extracts showed that long-term morphine treatment-induced down-regulation of Gbeta but not of the other proteins is highly correlated (r(2) = 0.96) with sensitization of adenylate cyclase. Down-regulation of Gbeta and adenylate cyclase sensitization elicited by long-term morphine treatment were suppressed in the presence of carbobenzoxy-l-leucyl-l-leucyl-l-norvalinal (MG-115) or lactacystin. Thus, sustained activation of the MOP receptor by morphine in sc2 cells seems to promote proteasomal degradation of Gbeta to sensitize adenylate cyclase. Together, our data suggest that the long-term administration of opiates may elicit dependence by altering the neuronal balance of heterotrimeric G proteins and adenylate cyclases, with the ubiquitin-proteasome pathway playing a pivotal role.
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PMID:Long-term morphine treatment enhances proteasome-dependent degradation of G beta in human neuroblastoma SH-SY5Y cells: correlation with onset of adenylate cyclase sensitization. 1590 46

The mechanisms implicated in the aggregation of ubiquitinated proteins detected in neurodegenerative disorders remain elusive. We report that prostaglandin J2 (PGJ2), an endogenous product of inflammation, up-regulates sequestosome 1/p62 in a time- and dose-dependent manner in human neuroblastoma SK-N-SH cells. We previously demonstrated that prostaglandins of the J2 series inhibit ubiquitin hydrolases, such as UCH-L1. Herein, we show that sequestosome 1/p62 is co-localized with ubiquitinated proteins and the ubiquitin hydrolase UCH-L1 in cytoplasmic aggregates induced by PGJ2. Preventing sequestosome 1/p62 up-regulation by RNA interference abolishes the aggregation but not the accumulation of ubiquitinated proteins or PGJ2 cytotoxicity. Sequestosome 1/p62 is known to bind poly-ubiquitinated proteins through its ubiquitin-associated domain. Our data support the notion that sequestosome 1/p62 up-regulation under stress conditions contributes to the "sequestration" of poly-ubiquitinated proteins into aggregates. However, the overwhelming accumulation of ubiquitinated proteins, rather than their aggregation, is likely to be an important contributor to PGJ2 cytotoxicity.
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PMID:Inhibition of sequestosome 1/p62 up-regulation prevents aggregation of ubiquitinated proteins induced by prostaglandin J2 without reducing its neurotoxicity. 1591 46


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