Glypican-3 induces oncogenicity by preventing IGF-1R degradation, a process that can be blocked by Grb10.

Hepatocellular carcinoma (HCC) is the most common primary liver malignancy and is a major cause of cancer-related death worldwide. Previously, we demonstrated that glypican-3 (GPC3) is highly expressed in HCC, and that GPC3 induces oncogenicity and promotes the growth of cancer cells through IGF-1 receptor (IGF-1R). In the present study, we investigated the mechanisms of GPC3-mediated enhancement of IGF-1R signaling. We demonstrated that GPC3 decreased IGF-1-induced IGF-1R ubiquitination and degradation and increased c-Myc protein levels. GPC3 bound to Grb10, a mediator of ligand-induced receptor ubiquitination, and the overexpression of Grb10 blocked GPC3-enhanced IGF-1-induced ERK phosphorylation. GPC3 promoted the growth of NIH3T3 and PLC-PRF-5 cells in serum-free medium but did not promote the growth of IGF-1R negative R- cells. Grb10 overexpression decreased GPC3-promoted cell growth. Therefore, the present study elucidates the mechanisms of GPC3-induced oncogenicity, which may highlight new strategies for the treatment of HCC.

Gene therapy with modified U1 small nuclear RNA.

INTRODUCTION: More than 15% of all disease-causing mutations result in mRNA splicing defects. U1 snRNA binds to the 5' splice site (5'ss) through base pairing. Mutation-adapted U1 snRNA (with compensatory U1 snRNA changes) and exon-specific U1 snRNA (complementary to intronic sequences) have been shown to suppress 5'ss mutations in cellular and animal models. Areas covered: The history, mechanism of action, and efficacy of U1 snRNA-mediated gene therapy are covered. The clinical utility of this technology and its limitations will be discussed. Expert commentary: Recently, gene therapies with mutation-adapted U1 snRNAs have been conducted on animal models, including aromatic l-amino acid decarboxylase deficiency and spinal muscular atrophy. However, although U1-mediated therapy has the advantage of maintaining the regulated expression of defective genes, its accuracy and efficacy needs to be improved before clinical application of this technique is possible.

Mutation-adapted U1 snRNA corrects a splicing error of the dopa decarboxylase gene.

Aromatic l-amino acid decarboxylase (AADC) deficiency is an inborn error of monoamine neurotransmitter synthesis, which results in dopamine, serotonin, epinephrine and norepinephrine deficiencies. The DDC gene founder mutation IVS6 + 4A > T is highly prevalent in Chinese patients with AADC deficiency. In this study, we designed several U1 snRNA vectors to adapt U1 snRNA binding sequences of the mutated DDC gene. We found that only the modified U1 snRNA (IVS-AAA) that completely matched both the intronic and exonic U1 binding sequences of the mutated DDC gene could correct splicing errors of either the mutated human DDC minigene or the mouse artificial splicing construct in vitro. We further injected an adeno-associated viral (AAV) vector to express IVS-AAA in the brain of a knock-in mouse model. This treatment was well tolerated and improved both the survival and brain dopamine and serotonin levels of mice with AADC deficiency. Therefore, mutation-adapted U1 snRNA gene therapy can be a promising method to treat genetic diseases caused by splicing errors, but the efficiency of such a treatment still needs improvements.

The Novel VEGF121-VEGF165 Fusion Attenuates Angiogenesis and Drug Resistance via Targeting VEGFR2-HIF-1α-VEGF165/Lon Signaling Through PI3K-AKT-mTOR Pathway.

Anti-angiogenesis therapy is one major approach of cancer therapies nowadays. Unfortunately, anti-angiogenesis therapy targeting VEGF-A was recently stumbled by the drugresistance that results from adaptive mechanisms, such as intratumor hypoxia. To obtain a more efficient therapeutic response, we created and identified a novel chimeric fusion of VEGF121 and VEGF165, which was connected by Fc region of human IgG1 to enhance dimerization. We found that the treatment of VEGF121-VEGF165 chimeric protein reduces proliferation, migration, invasion, and tube formation in endothelial and/or cancer cells through competing VEGF165 homodimer in a paracrine and an autocrine manner. Furthermore, the fusion protein attenuated autocrine VEGFR2-HIF-1α-VEGF165/Lon signaling through PI3KAKT- mTOR pathway in cancer cells. In conclusion, our data demonstrated that the chimeric VEGF121-VEGF165 arrests the tube formation of endothelial cells and interferes with tumor cell growth, migration and invasion, suggesting that it could be a potential drug as an angiogenesis antagonist in cancer therapy. The VEGF121-VEGF165 targets not only paracrine angiogenic cascade of endothelial cells but also autocrine PI3K-AKT-mTOR-mediated VEGFR2-HIF-1α- VEGF165/Lon signaling that drives drug resistance in tumor cells. Our study will open up the patient opportunities to combat drug resistance to antiangiogenic therapy.

Exchange of active site residues alters substrate specificity in extremely thermostable ß-glycosidase from Thermococcus kodakarensis KOD1.

ß-Glycosidase from Thermococcus kodakarensis KOD1 is a hyperthermophilic enzyme with ß-glucosidase, ß-mannosidase, ß-fucosidase and ß-galactosidase activities. Sequence alignment with other ß-glycosidases from hyperthermophilic archaea showed two unique active site residues, Gln77 and Asp206. These residues were represented by Arg and Asp in all other hyperthermophilic ß-glycosidases. The two active site residues were mutated to Q77R, D206N and D206Q, to study the role of these unique active site residues in catalytic activity and to alter the substrate specificity to enhance its ß-glucosidase activity. The secondary structure analysis of all the mutants showed no change in their structure and exhibited in similar conformation like wild-type as they all existed in dimer form in an SDS-PAGE under non-reducing conditions. Q77R and D206Q affected the catalytic activity of the enzyme whereas the D206N altered the catalytic turn-over rate for glucosidase and mannosidase activities with fucosidase activity remain unchanged. Gln77 is reported to interact with catalytic nucleophile and Asp206 with axial C2-hydroxyl group of substrates. Q77R might have made some changes in three dimensional structure due to its electrostatic effect and lost its catalytic activity. The extended side chains of D206Q is predicted to affect the substrate binding during catalysis. The high-catalytic turn-over rate by D206N for ß-glucosidase activity makes it a useful enzyme in cellulose degradation at high temperatures.

An intermolecular disulfide bond is required for thermostability and thermoactivity of ß-glycosidase from Thermococcus kodakarensis KOD1.

Scientists are interested in understanding the molecular origin of protein thermostability and thermoactivity for possible biotechnological applications. The enzymes from extremophilic organisms have been of particular interest in the last two decades. ß-glycosidase, Tkßgly is a hyperthermophilic enzyme from Thermococcus kodakarensis KOD1. Tkßgly contains two conserved cysteine residues, C88 and C376. The protein tertiary structure obtained through homology modeling suggests that the C88 residue is located on the surface whereas C376 is inside the protein. To study the role of these cysteine residues, we substituted C88 and C376 with serine residues through site-directed mutagenesis. The wild-type and C376S protein existed in dimeric form and C88S in monomeric form, in an SDS-PAGE gel under non-reducing conditions. Optimal temperature experiments revealed that the wild-type was active at 100 °C whereas the C88S mutant exhibited optimal activity at 70 °C. The half-life of the enzyme at 70 °C was drastically reduced from 266 h to less than 1 h. Although C88 was not present in the active site region, the kcat/Km of C88S was reduced by 2-fold. Based on the structural model and biochemical properties, we propose that C88 is crucial in maintaining the thermostability and thermoactivity of the Tkßgly enzyme.

Inhibition of glutaminyl cyclase attenuates cell migration modulated by monocyte chemoattractant proteins.

QC (glutaminyl cyclase) catalyses the formation of N-terminal pGlu (pyroglutamate) in peptides and proteins. pGlu formation in chemoattractants may participate in the regulation of macrophage activation and migration. However, a clear molecular mechanism for the regulation is lacking. The present study examines the role of QC-mediated pGlu formation on MCPs (monocyte chemoattractant proteins) in inflammation. We demonstrated in vitro the pGlu formation on MCPs by QC using MS. A potent QC inhibitor, PBD150, significantly reduced the N-terminal uncyclized-MCP-stimulated monocyte migration, whereas pGlu-containing MCP-induced cell migration was unaffected. QC small interfering RNA revealed a similar inhibitory effect. Lastly, we demonstrated that inhibiting QC can attenuate cell migration by lipopolysaccharide. These results strongly suggest that QC-catalysed N-terminal pGlu formation of MCPs is required for monocyte migration and provide new insights into the role of QC in the inflammation process. Our results also suggest that QC could be a drug target for some inflammatory disorders.

White spot syndrome virus protein ICP11: A histone-binding DNA mimic that disrupts nucleosome assembly.

White spot syndrome virus (WSSV) is a large ( approximately 300 kbp), double-stranded DNA eukaryotic virus that has caused serious disease in crustaceans worldwide. ICP11 is the most highly expressed WSSV nonstructural gene/protein, which strongly suggests its importance in WSSV infection; but until now, its function has remained obscure. We show here that ICP11 acts as a DNA mimic. In crystal, ICP11 formed a polymer of dimers with 2 rows of negatively charged spots that approximated the duplex arrangement of the phosphate groups in DNA. Functionally, ICP11 prevented DNA from binding to histone proteins H2A, H2B, H3, and H2A.x, and in hemocytes from WSSV-infected shrimp, ICP11 colocalized with histone H3 and activated-H2A.x. These observations together suggest that ICP11 might interfere with nucleosome assembly and prevent H2A.x from fulfilling its critical function of repairing DNA double strand breaks. Therefore, ICP11 possesses a functionality that is unique among the handful of presently known DNA mimic proteins.

Glypican-3-mediated oncogenesis involves the Insulin-like growth factor-signaling pathway.

Glypican-3 (gpc3) is the gene responsible for Simpson-Golabi-Behmel overgrowth syndrome. Previously, we have shown that GPC3 is overexpressed in hepatocellular carcinoma (HCC). In this study, we demonstrated the mechanisms for GPC3-mediated oncogenesis. Firstly, GPC3 overexpression in NIH3T3 cells gave to cancer cell phenotypes including growing in serum-free medium and forming colonies in soft agar, or on the other way, GPC3 knockdown in HuH-7 cells decreased oncogenecity. We further demonstrated that GPC3 bound specifically through its N-terminal proline-rich region to both Insulin-like growth factor (IGF)-II and IGF-1R. GPC3 stimulated the phosphorylation of IGF-1R and the downstream signaling molecule extracellular signal-regulated kinase (ERK) in an IGF-II-dependent way. Also, GPC3 knockdown in HCC cells decreased the phosphorylation of both IGF-1R and ERK. Therefore, GPC3 confers oncogenecity through the interaction between IGF-II and its receptor, and the subsequent activation of the IGF-signaling pathway. This data are novel to the current understanding of the role of GPC3 in HCC and will be important in future developments of cancer therapy.

Hsp27 decreases inclusion body formation from mutated GTP-cyclohydrolase I protein.

GTP cyclohydrolase I (GCH), an oligomeric protein composed of 10 identical subunits, is required for the synthesis of neurotransmitters; mutations in GCH are associated with dopa-responsive dystonia (DRD) and hyperphenylalaninemia. Mutated GCH proteins are unstable and prone to dominant-negative effect. We show herein that expression of the GCH mutant GCH-201E or the splicing variant GCH-II caused intracellular inclusion bodies. When Hsp27 was expressed together with the GCH mutants, Hsp27 expression decreased the formation of inclusion bodies by GCH (as assessed by immunofluorescence) and decreased the amount of insoluble GCH mutant proteins (as assessed by Western blot). Transfection of pcDNA-Hsp27-S3D, a phosphorylation-mimicry Hsp27 mutant, was more effective at the mutated GCH proteins than transfection with pcDNA-Hsp27, but okadaic acid, a phosphatase inhibitor, enhanced the effect of pcDNA-Hsp27. Hsp27-S3D also abolished the dominant-negative action of GCH-II. The mutated GCH proteins interacted with the wild-type GCH protein; the inclusion bodies were positive for lysosomal marker LAMP1, soluble in 2% SDS, and were not ubiquitinated. Phophorlyated Hsp27 also decreased the inclusion body formation by the huntingtin polyglutamines. Therefore, diseases involving mutated oligomeric proteins would be manageable by chaperone therapies.

Increased oxidative damage and mitochondrial abnormalities in the peripheral blood of Huntington's disease patients.

Increased oxidative stress and mitochondrial abnormalities contribute to neuronal dysfunction in Huntington's disease (HD). We investigated whether these pathological changes in HD brains may also be present in peripheral tissues. Leukocyte 8-hydroxydeoxyguanosine (8-OHdG) and plasma malondialdehyde (MDA) were elevated, and activities of erythrocyte Cu/Zn-superoxide dismutase (Cu/Zn-SOD) and glutathione peroxidase (GPx) reduced in 16 HD patients when compared to 36 age- and gender-matched controls. Deleted and total mitochondrial DNA (mtDNA) copy numbers were increased, whereas the mRNA expression levels of mtDNA-encoded mitochondrial enzymes are not elevated in HD leukocytes compared to the normal controls. Plasma MDA levels also significantly correlated with HD disease severity. These results indicate means to suppress oxidative damage or to restore mitochondrial functions may be beneficial to HD patients. Plasma MDA may be used as a potential biomarker to test treatment efficacy in the future, if confirmed in a larger, longitudinal study.

Differential expression of mu-opioid receptor gene in CXBK and B6 mice by Sp1.

It is well known that there are individual differences in the sensitivity to analgesics. The CXBK mice are characterized by reduced sensitivity to morphine and by partial deficiency in mu-opioid receptor (MOR) expression. The sequences of MOR genes in CXBK and B6 mice are identical in their coding regions but differ at 5'-untranslated region (UTR) nucleotide -202 (C nucleotide in CXBK, but A nucleotide in B6). In this report, we identified an Sp1 element (-211 to -204) immediately before the polymorphic nucleotide. In electrophoretic mobility shift assay, nuclear protein binding to the B6-Sp1 sequence was more efficient than to the CXBK-Sp1 sequence, and anti-Sp1 but not anti-CREB antibody interfered with the formation of the DNA-protein complex. In MOR-expressing cell lines SH-SY5Y, P19, and PC12, B6 MOR promoter possessed high transcription activity than the CXBK promoter, and Sp1 inhibitor PDTC reduced the promoter activities. In SL2 cells that lack endogenous Sp1 expression, B6 and CXBK MOR promoters demonstrated equal activity, whereas overexpression of Sp1 in SL2 cells enhanced B6 MOR promoter activity better than the CXBK promoter. Together, the A-to-C change at MOR 5'-UTR decreases Sp1 binding and MOR gene transcription, which could underlie the reduced morphine expression in CXBK mice.

RNF4 is a coactivator for nuclear factor Y on GTP cyclohydrolase I proximal promoter.

GTP cyclohydrolase I (GCH) is the rate-controlling enzyme in the production of tetrahydrobiopterin (BH4) that is essential for the synthesis of nitric oxide and catecholamines including dopamine and serotonin. Therefore, the regulation of GCH expression is important in determining the catecholamine levels in the brain under pathophysiological conditions. During the study of human disease dopa-responsive dystonia, we found that coactivator RNF4 is involved in the GCH gene expression. Through serial deletion and mutagenesis studies of the GCH promoter, we defined the RNF4-responsive element on GCH proximal promoter as a CCAAT box. RNF4 did not possess specific DNA binding activity toward this CCAAT box, which suggests that RNF4 may be a coactivator of the CCAAT boxbinding protein nuclear factor Y (NF-Y). Cotransfection of a dominant-negative mutant of NF-Y resulted in a significant reduction in RNF4-mediated CCAAT box activation. In addition, overexpression of RNF4 could not activate the CCAAT box in Drosophila melanogaster SL2 cells, which are devoid of endogenous NF-Y, whereas overexpression of RNF4 and NF-Y could. Furthermore, immunoprecipitation experiments revealed the physical association between RNF4 and the NF-Y complex. These data indicate that RNF4 imposes functional importance on GCH promoter.

Molecular chaperones affect GTP cyclohydrolase I mutations in dopa-responsive dystonia.

Unstable GTP cyclohydrolase I (GCH) mutations in dopa-responsive dystonia (DRD) can exert a dominant-negative effect in the HeLa cell model, but in a batch of cells this effect could not be shown. Through differential display, we found a higher Hsc70 expression in the non-dominant-negative cells. We further demonstrated that ectopic expression of Hsp40/Hsp70 stabilized the GCH mutant G201E. Moreover, Hsp90 inhibitor geldanamycin destroyed the wild-type GCH level, and heat shock increased the synthesis of GCH protein. Therefore, the dominant-negative effect produced by unstable proteins would be susceptible to the status of molecular chaperones, which could be the modifying genes and therapeutic targets for DRD and other genetic diseases.

Transcriptional regulation of mu opioid receptor gene by cAMP pathway.

The utility of morphine for the treatment of chronic pain is hindered by the development of tolerance. Fentanyl has been shown to be a potent analgesic with a lower propensity to produce tolerance and physical dependence in the clinical setting. Previous finding has shown that fentanyl induces mu opioid receptor gene expression in PC-12 cells (Brain Res 859:217-223, 2000). In this report, we aim to identify the molecular mechanism of mu-opioid receptor (MOR) gene regulation by fentanyl. We demonstrated that the 4.7-kilobase MOR promoter could be induced by fentanyl in PC-12 cells, and we defined a partial cAMP response element (CRE) located at -106/-111 in 5'-untranslated region of the MOR gene. In electrophoretic mobility shift assay, cAMP response element-binding protein (CREB) was found in the protein-DNA complex formed on the CRE box. CREB was phosphorylated after forskolin induction, and both CREB and CREB-binding protein (CBP) binding to the endogenous MOR promoter was increased by forskolin in chromatin immunoprecipitation assay. The functional role of CREB in the induction of MOR gene was further elucidated by an experiment in which a dominant-negative mutant CREB, CREB-S133A, abolished the forskolin-mediated MOR induction. Moreover, we found that this CRE box is conserved in mouse, rat, and human MOR gene, implying physiological relevance in different species. Collectively, this study demonstrated that fentanyl-triggered MOR gene induction was mediated by the sequential activation of CREB and the binding of CREB and CBP to MOR promoter, thus provides direct evidence for lower propensity of fentanyl to produce tolerance.

Differential activation of a C/EBP beta isoform by a novel redox switch may confer the lipopolysaccharide-inducible expression of interleukin-6 gene.

C/EBP beta, a member of the CCAAT/enhancer binding protein (C/EBP) family, is one of the key transcription factors responsible for the induction of a wide array of genes, some of which play important roles in innate immunity, inflammatory response, adipocyte and myeloid cell differentiation, and the acute phase response. Three C/EBP beta isoforms (i.e. LAP*, LAP, and LIP) were known to arise from differential translation initiation and display different functions in gene regulation. C/EBP beta is known to induce interleukin (IL)-6 gene when P388D1 cells are treated with lipopolysaccharide (LPS). Exactly how the transcriptional activities of C/EBP beta isoforms are involved in the regulation of the IL-6 gene remains unclear. Here we report that LPS-induced expression of IL-6 gene in P388D1 cells is mediated by a redox switch-activated LAP*. The intramolecular disulfide bonds of LAP* and LAP have been determined. Among the cysteine residues, amino acid 11 (Cys11) of LAP* plays key roles for determining the overall intramolecular disulfide bonds that form the basis for redox switch regulation. The DNA binding activity and transcriptional activity of LAP* are enhanced under reducing condition. LAP and LIP, lacking 21 and 151 amino acids, respectively, in the N-terminal region, are not regulated in a similar redox-responsive manner. Our results indicate that LAP* is the primary isoform of C/EBP beta that regulates, through a redox switch, the LPS-induced expression of the IL-6 gene.

Regulation of GTP cyclohydrolase I by alternative splicing in mononuclear cells.

GTP cyclohydrolase I (GCH, EC 3.5.4.16) regulates the level of tetrahydrobiopterin and in turn the activities of nitric oxide synthase and aromatic amino acid hydroxylases. Type II GCH mRNA, an alternatively spliced species abundant in blood cells, encodes a truncated and nonfunctional protein. When we stimulate peripheral blood mononuclear cells by PHA, the transcription of full-length GCH mRNA increased, but that of type II mRNA decreased transiently. We further demonstrated that the type II cDNA exerted a dominant-negative effect on the wild-type cDNA, similar to the effect of some GCH mutants. Therefore, type II mRNA may regulate GCH and then contribute to the regulation of NO production by BH4-dependent iNOS in mononuclear cells. Selection of the splicing sites may be coupled with transcriptional activation of the GCH gene.