[Show abstract][Hide abstract] ABSTRACT: Matrix metalloproteinase-7 (MMP-7) secreted by cancer cells has been implicated classically in the basement membrane destruction associated with tumor cell invasion and metastasis. Recent epidemiologic studies have established a correlation between high levels of circulating insulin-like growth factor (IGF) and low levels of IGF binding protein 3 (IGFBP-3), and relative risk of developing colon, breast, prostate, and lung cancer, which are known to produce MMP-7. In this study, IGFBP-3 was assessed as a candidate for the physiologic substrate of MMP-7. MMP-7 proteolysis generated four major fragments (26 kDa, 17 kDa, 15.5 kDa, and 15.5 kDa), and two cleavage sites were identified: one at the site of hydrolysis of the K(144)-I(145) peptide bond and one at the R(95)-L(96) peptide bond. The former site is different from the previously reported site of cleavage of IGFBP-3 by other proteases. Addition of IGFBP-3 inhibited IGF-I-mediated IGF type 1 receptor (IGF-IR) phosphorylation and activation of the downstream molecule Akt in BALB/c 3T3 fibroblasts overexpressing human IGF-IR (3T3-IGF-IR) and in two human colon cancer cell lines (COLO201 and HT29). Coincubation of the IGF-I/IGFBP-3 complex with MMP-7 restored IGF-I-mediated IGF-IR phosphorylation and activation of Akt in these cell lines. The IGF-I signal recovered by MMP-7 protected against apoptosis induced by anoikis in 3T3-IGF-IR cells. These results indicate that MMP-7 proteolysis of IGFBP-3 plays a crucial role in regulating IGF-I bioavailability, thereby promoting cell survival. This mechanism may contribute to the tumorigenesis of MMP-7-producing IGF-IR-expressing tumors in the primary site and to organ-specific metastasis in a paracrine manner.
Cancer Research 02/2004; 64(2):665-71. DOI:10.1158/0008-5472.CAN-03-1916 · 9.33 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The multisubunit transcription elongation factor NELF (for negative elongation factor) acts together with DRB (5,6-dichloro-1-β-d-ribofuranosylbenzimidazole) sensitivity-inducing factor (DSIF)/human Spt4-Spt5 to cause transcriptional pausing of RNA polymerase
II (RNAPII). NELF activity is associated with five polypeptides, A to E. NELF-A has sequence similarity to hepatitis delta
antigen (HDAg), the viral protein that binds to and activates RNAPII, whereas NELF-E is an RNA-binding protein whose RNA-binding
activity is critical for NELF function. To understand the interactions of DSIF, NELF, and RNAPII at a molecular level, we
identified the B, C, and D proteins of human NELF. NELF-B is identical to COBRA1, recently reported to associate with the
product of breast cancer susceptibility gene BRCA1. NELF-C and NELF-D are highly related or identical to the protein called
TH1, of unknown function. NELF-B and NELF-C or NELF-D are integral subunits that bring NELF-A and NELF-E together, and coexpression
of these four proteins in insect cells resulted in the reconstitution of functionally active NELF. Detailed analyses using
mutated recombinant complexes indicated that the small region of NELF-A with similarity to HDAg is critical for RNAPII binding
and for transcriptional pausing. This study defines several important protein-protein interactions and opens the way for understanding
the mechanism of DSIF- and NELF-induced transcriptional pausing.
[Show abstract][Hide abstract] ABSTRACT: Efficient transcription and replication of the influenza virus genome are dependent upon host-derived factors. Using an in vitro RNA synthesis system, we have purified and identified Hsp90 as one of the host factors that stimulate viral RNA polymerase activity. Hsp90 interacted with the PB2 subunit of the viral RNA polymerase through the amino-terminal chaperone domain and the middle region containing a highly acidic domain. The acidic middle region was also responsible for its stimulatory activity. We found that a portion of Hsp90 is re-localized to the cell nucleus after viral infection. A PB2 fragment containing a Hsp90 binding domain inhibited viral gene expression in a dominant-negative manner. These results suggest that Hsp90 is a host factor for the influenza virus RNA polymerase.
[Show abstract][Hide abstract] ABSTRACT: Formation of oligosaccharides occurs both in the cytosol and in the lumen of the endoplasmic reticulum (ER). Luminal oligosaccharides are transported into the cytosol to ensure that they do not interfere with proper functioning of the glycan-dependent quality control machinery in the lumen of the ER for newly synthesized glycoproteins. Once in the cytosol, free oligosaccharides are catabolized, possibly to maximize the reutilization of the component sugars. An endo-beta-N-acetylglucosaminidase (ENGase) is a key enzyme involved in the processing of free oligosaccharides in the cytosol. This enzyme activity has been widely described in animal cells, but the gene encoding this enzyme activity has not been reported. Here, we report the identification of the gene encoding human cytosolic ENGase. After 11 steps, the enzyme was purified 150,000-fold to homogeneity from hen oviduct, and several internal amino acid sequences were analyzed. Based on the internal sequence and examination of expressed sequence tag (EST) databases, we identified the human orthologue of the purified protein. The human protein consists of 743 aa and has no apparent signal sequence, supporting the idea that this enzyme is localized in the cytosol. By expressing the cDNA of the putative human ENGase in COS-7 cells, the enzyme activity in the soluble fraction was enhanced 100-fold over the basal level, confirming that the human gene identified indeed encodes for ENGase. Careful gene database surveys revealed the occurrence of ENGase homologues in Drosophila melanogaster, Caenorhabditis elegans, and Arabidopsis thaliana, indicating the broad occurrence of ENGase in higher eukaryotes. This gene was expressed in a variety of human tissues, suggesting that this enzyme is involved in basic biological processes in eukaryotic cells.
Proceedings of the National Academy of Sciences 08/2002; 99(15):9691-6. DOI:10.1073/pnas.152333599 · 9.67 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A new glucagon-like peptide was isolated from the intestine of the eel Anguilla japonica. The primary structure was determined by sequence analysis after cleavage with lysyl endopeptidase, quantitative amino acid analysis and fast atom bombardment mass spectrometry as HSQGTFTNDY(10)SKYLETRRAQ(20)DFVQWLMNSK(30)RSGGPT. Since its structure is similar to that of oxyntomodulins (OXMs) reported in various vertebrates, we named this peptide eel oxyntomodulin (eOXM). We found that eOXM enhanced the contractile force and the beating rate of the eel atrium in a dose-dependent manner. These effects of eOXM were not inhibited by betaxolol, a beta(1)-adrenoceptor antagonist, indicating that the actions of eOXM were independent of those of adrenaline. eOXM enhanced the intracellular Ca(2+) concentration of the myocardium. The contractility of the eel atrium was greatly reduced after omitting Ca(2+) from the bathing medium or after treatment with verapamil, a Ca(2+) channel blocker. After inhibiting Ca(2+) entry under these conditions, the inotropic effect of eOXM was markedly reduced, but the chronotropic effect was not altered significantly. These results indicate that the inotropic effect of eOXM is via a stimulation of Ca(2+) influx but that the chronotropic effect may be independent of extracellular Ca(2+).
[Show abstract][Hide abstract] ABSTRACT: Transcription elongation by RNA polymerase II (RNAPII) is negatively regulated by the human factors DRB-sensitivity inducing factor (DSIF) and negative elongation factor (NELF). A 66-kilodalton subunit of NELF (NELF-A) shows limited sequence similarity to hepatitis delta antigen (HDAg), the viral protein required for replication of hepatitis delta virus (HDV). The host RNAPII has been implicated in HDV replication, but the detailed mechanism and the role of HDAg in this process are not understood. We show that HDAg binds RNAPII directly and stimulates transcription by displacing NELF and promoting RNAPII elongation. These results suggest that HDAg may regulate RNAPII elongation during both cellular messenger RNA synthesis and HDV RNA replication.
[Show abstract][Hide abstract] ABSTRACT: A specific binding protein for 12-O-tetradecanoylphorbol 13-acetate (TPA), different from protein kinase C (PKC) and histone H1, was purified from HeLa cell extract by the use of affinity gel pendanted with phorbol ester (TPA-GEL). The purified binding protein was identified as protein disulfide isomerase (PDI, EC 220.127.116.11) by peptide sequence analysis. The dissociation constants (Kd's) of TPA to PDI, histone H1 and PKCalpha were determined to be 1.03 x 10(-6) M, 5.70 x 10(-7) M, and 4.00 x 10(-7) m, respectively, by the surface plasmon resonance (SPR) method. TPA moderately inhibited PDI activity assessed in terms of reactivation of denatured RNase A.
[Show abstract][Hide abstract] ABSTRACT: DRB is a classic inhibitor of transcription elongation by RNA polymerase II (pol II). Since DRB generally affects class II genes, factors involved in this process must play fundamental roles in pol II elongation. Recently, two elongation factors essential for DRB action were identified, namely DSIF and P-TEFb. Here we describe the identification and purification from HeLa nuclear extract of a third protein factor required for DRB-sensitive transcription. This factor, termed negative elongation factor (NELF), cooperates with DSIF and strongly represses pol II elongation. This repression is reversed by P-TEFb-dependent phosphorylation of the pol II C-terminal domain. NELF is composed of five polypeptides, the smallest of which is identical to RD, a putative RNA-binding protein of unknown function. This study reveals a molecular mechanism for DRB action and a regulatory network of positive and negative elongation factors.
[Show abstract][Hide abstract] ABSTRACT: Synthetic urea derivatives such as N-phenyl-N'-(4-pyridyl)urea (4PU) and N-(2-chloro-4-pyridyl)-N'-phenylurea (4PU30) have strong cytokinin activities. Using tritiated 4PU30 as a probe, we previously established the presence of a cytokinin-specific binding protein (CSBP) of high affinity (Ka for 4PU30 = 4x10(10) M(-1)) in the soluble fraction of etiolated mung bean seedlings [Nagata, R., Kawachi, E., Hashimoto, Y. & Shudo, K. (1993) Biochem. Biophys. Res. Commun. 191, 543-549]. In this report, we purified CSBP by the use of 4PU-Sepharose 4B, an affinity gel liganded with 4PU. We determined partial amino acid sequences of CSBP and isolated its cDNA by reverse-transcription (RT) PCR. The cDNA encoded a protein with a calculated molecular mass of 17 kDa. A data base homology search revealed that CSBP is a novel member of a major pollen allergen/pathogenesis-related protein family. Recombinant CSBP was expressed in Escherichia coli and was confirmed to bind specifically to cytokinins.
European Journal of Biochemistry 12/1998; 258(2):794-802. DOI:10.1046/j.1432-1327.1998.2580794.x · 3.58 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We report the identification of a transcription elongation factor from HeLa cell nuclear extracts that causes pausing of RNA polymerase II (Pol II) in conjunction with the transcription inhibitor 5,6-dichloro-1-β-d-ribofuranosylbenzimidazole (DRB). This factor, termed DRB sensitivity-inducing factor (DSIF), is also required for transcription inhibition by H8. DSIF has been purified and is composed of 160-kD (p160) and 14-kD (p14) subunits. Isolation of a cDNA encoding DSIF p160 shows it to be a homolog of the Saccharomyces cerevisiae transcription factor Spt5. Recombinant Supt4h protein, the human homolog of yeast Spt4, is functionally equivalent to DSIF p14, indicating that DSIF is composed of the human homologs of Spt4 and Spt5. In addition to its negative role in elongation, DSIF is able to stimulate the rate of elongation by RNA Pol II in a reaction containing limiting concentrations of ribonucleoside triphosphates. A role for DSIF in transcription elongation is further supported by the fact that p160 has a region homologous to the bacterial elongation factor NusG. The combination of biochemical studies on DSIF and genetic analysis of Spt4 and Spt5 in yeast, also in this issue, indicates that DSIF associates with RNA Pol II and regulates its processivity in vitro and in vivo.
Genes & Development 02/1998; 12:343-356. DOI:10.1101/gad.12.3.343 · 10.80 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A neuropeptide Y (eNPY) was isolated from the intestinal extract of eels. This peptide enhanced significantly the serosa-negative transepithelial potential difference (PD) and short-circuit current (Isc) across the intestine of the seawater eel after pretreatment with isobutylmethylxanthine, serotonin and methacholine. The effects of eNPY on the Isc were concentration-dependent with a threshold concentration of 3 x 10(-9) M and a maximal effect at 3 x 10(-7) M. Similar concentration-response curve was obtained by porcine peptide YY (pPYY). Since 9 amino acid residues are replaced in the pPYY, this result indicates that these substitutions do not change the potency and the efficacy. These stimulatory actions of eNPY were not blocked by tetrodotoxin, an inhibitor of neural firing, or yohimbine, an alpha 2-adrenoceptor antagonist, indicating that eNPY acts without enteric neural firing or catecholamine release. When eNPY and adrenaline (AD) were applied simultaneously, the effects were additive only at lower dosage (3 x 10(-8) M for eNPY, 3 x 10(-8) M for AD), but not at high dosage (10(-6) M eNPY, 10(-7) M AD). The ceiling effect at high dosage suggests that these two regulators act through common signal transduction systems and affect the Na(+)-K(+)-Cl- cotransport system, since both effects were completely blocked by bumetanide, a specific inhibitor of Na(+)-K(+)-Cl- cotransporter.