Human Liver cDNA Library Research Articles

Induction of G1 cell cycle arrest and P15INK4b expression by ECRG1 through interaction with Miz-1.

ECRG1 is a novel candidate of tumor suppressor gene identified from human esophagus. To study the biological role of ECRG1 gene, we performed a GAL4-based yeast two-hybrid screen of a human fetal liver cDNA library. Using the ECRG1 cDNA as bait, we identified two putative clones as associated proteins, Miz-1 and FLNA (Filamin A). The interaction of ECRG1 and Miz-1 was confirmed by glutathione-S-transferase (GST)-pull-down assays in vitro and co-immunoprecipitation experiments in vivo. ECRG1 was co-localized with Miz-1 in nucleus, as shown by confocal microscopy. Transfection of ECRG1 gene into the esophageal cancer (EC) cells inhibited cell proliferation and induced G1 phase arrest of cell cycle. In the co-transfection of ECRG1 and Miz-1 assays, we found inhibition of cell proliferation and G1/S phase in EC cells, but the levels of cell proliferation inhibition and G1/S phase arrest were more strongly compared with the transfection of ECRG1 or Miz-1 alone. In addition, the interaction of ECRG1 and Miz-1 could induce expression of P15(INK4b) gene in esophageal cancer 9706 (EC9706) cells. However, the transfection of ECRG1 or Miz-1 alone was not revealed the expressions of P15(INK4b) gene. When antisense ECRG1 interdicted expression of endogenous ECRG1 in Balb/c-3T3 cells, Transfection of Miz-1 couldn't induce P15(INK4b) expression. The results provide evidences that ECRG1 and Miz-1 in EC cells may be acting as a co-functional protein associated with regulation of cell cycle and induction of P15(INK4b) expression. It suggests that ECRG1 may inhibit tumor cell growth by affecting cell cycle, and that expression of P15(INK4b) may be likely to enhance G1 cell cycle arrest during the interaction of ECRG1 and Miz-1. The physical interaction of ECRG1 and Miz-1 may play an important role in carcinogenesis of EC.

Gene transfer into human hepatoma cells by receptor-associated protein/polylysine conjugates.

Receptor-associated protein (RAP) is a ligand for all members of low-density lipoprotein (LDL) receptor families. RAP is internalized into cells via receptor-mediated endocytic trafficking, making it an attractive mechanism for efficient gene delivery. In this study, we have developed a gene delivery system using RAP as a targeting ligand. A RAP cDNA lacking a C-terminal heparin-binding domain was amplified by polymerase chain reaction (PCR) from a human liver cDNA library and was reamplified by using a primer containing a cysteine codon at its carboxyl end to facilitate its conjugation to polylysine (polyK). RAP was purified using a bacterial expression system and coupled to poly-D-lysine (PDL) or poly-L-lysine (PLL) of average MW 50 kDa via the heterobifunctional cross-linker SPDP. Using fluorescence-labeled RAP ligand, cellular uptake of the transfection complexes into HepG2 cells was shown to be highly efficient and more specific to PDL-conjugated RAP compared with PLL-conjugated one. Plasmid DNA containing a luciferase reporter gene was condensed with either RAP-PDL or RAP-PLL. In vitro transfection into HepG2 cells with RAP-PDL conjugate resulted in significantly higher luciferase expression levels in comparison to either nonconjugated PDL, or RAP-PLL, or LipofecAMINE/DNA complexes in the presence of 10% fetal bovine serum. Luciferase expression was inhibited by the addition of excess RAP. Treatment of the cells with Lovastatin, which inhibits HMG-Co reductase and increases expression of LDL receptor, stimulates luciferase expression, suggesting that the gene delivery is specifically mediated by LDL receptor. Thus, RAP-PDL conjugates have the potential to be used as a new nonviral gene delivery vector.

LFIRE-1/HFREP-1, a liver-specific gene, is frequently downregulated and has growth suppressor activity in hepatocellular carcinoma.

We have previously identified several novel genes, which are differentially expressed among human normal liver and hepatocellular carcinomas (HCCs). The full-length liver fibrinogen-related gene-1 (LFIRE-1) cDNA was cloned from the human normal liver cDNA library. LFIRE-1 is highly homologous to HFREP-1 with discrepancy at 5' untranslated region (UTR) and encodes the same fibrinogen-related protein, which suggest that these two sequences might be alternative splicing forms of the same gene, LFIRE-1/HFREP-1, located at human chromosome 8p22. The LFIRE-1 and HFREP-1 are specifically expressed in normal human liver tissue, but reduced or undetectable in most of HCC specimens at both RNA and protein level. Furthermore, the reduction or nonexpression of LFIRE-1/HFREP-1 is significantly associated with the degree of tumor differentiation. Loss of heterozygosity (LOH) analysis revealed allelic loss of LFIRE-1/HFREP-1 on chromosome 8p22 in 57.1% (24/42) of HCC specimens. We detected three inactivation mutations among 45 cases of HCC specimens examined, two of which lost the remaining allele and the third had a replacement of conserved cysteine residue with glycine residue. Notably, the downregulation of LFIRE-1/HFREP-1 expression is frequently associated with allelic loss. The reduction of LFIRE-1/HFREP-1 expression by antisense approach enhances cancer cell proliferation and colony formation in soft agar. Moreover, restoration of exogenous wild-type LFIRE-1/HFREP-1 expression but not LFIRE-1/HFREP-1 missense mutations in human HCC cells inhibited their anchorage-dependent or -independent growth in vitro, and suppressed their tumorigenicity in nude mice. In conclusion, our data demonstrated that liver-specific gene LFIRE-1/HFREP-1 was frequently downregulated and might possess growth suppressor activity in HCC.

Expression of Human Serum Albumin in Milk of Transgenic Mice Using Goat β-casein/Human Serum Albumin Fusion Gene

The gene encoding human serum albumin (HSA) was cloned from human liver cDNA library by PCR. The HSA cDNA in size of 2,176 bp, including 1,830 bp of open reading frame, was cloned into the plasmid carried with the 5' flanking sequence of goat (3-casein gene (-4,044 to +2,025 bp) to get a tissue specific expression vector in mammary gland named pGB562/HSA (12.5 kb). A 9.6 kb DNA fragment in which the sequence is in order of goat β-casein gene regulatory sequence, HSA cDNA and SV40 polyadenylation signals was isolated from the pGB562/HSA by SacI and DraIII cutting, and used to microinject into the pronuclei of mouse fertilized eggs to produce transgenic mice. Three transgenic mice (2 female and I male) were identified by PCR and dot Southern blot analysis. The copy numbers of integrated transgene were more than 10 copies in line #21 and #26 as well as over 50 copies in line #31 of transgenic mice. HSA protein collected from the milk of lactating transgenic mice was confirmed by immuno-detection of Western and slot blot. The concentrations of HSA in the milk were from 0.05 to 0.4 mg/ml. An obvious antigen and antibody conjugate could be observed in immunohistochemical stain of mammary gland tissue from lactating day 11 of HSA transgenic mice. The transmission of transgene and its expression was recognized according to the results of RT-PCR and sequences analyses of their progeny.

Generation of Embryonic Stem Cell-derived Transgenic Mice by Using Tetraploid Complementation

The standard protocol for the production of transgenic mouse from ES-injected embryo has to process via chimera producing and several times breeding steps, In contrast, tetraploid-ES cell complementation method allows the immediate generation of targeted murine mutants from genetically modified ES cell clones. The advantage of this advanced technique is a simple and efficient without chimeric intermediates. Recently, this method has been significantly improved through the discovery that ES cells derived from hybrid strains support the development of viable ES mice more efficiently than inbred ES cells do. Therefore, the objective of this study was to generate transgenic mice overexpressing human resistin gene by using tetrapioid-ES cell complementation method. Human resistin gene was amplified from human fetal liver cDNA library by PCR and cloned into pCR 2.1 TOPO T-vector and constructed in pCMV-Tag4C vector. Human resistin mammalian expression plasmid was transfected into D3-GL ES cells by lipofectamine 2000, and then after 8~10 days of transfection, the human resistin-expressing cells were selected with G418. In order to produce tetraploid embryos, blastomeres of diploid embryos at the two-cell stage were fused with two times of electric pulse using 60 V 30 sec. (fusion rate : 93.5%) and cultured upto the blastocyst stage (development rate : 94.6%). The 15~20 previously G418-selected ES cells were injected into tetraploid blastocysts, and then transferred into the uterus of E2.5d pseudopregnant recipient mice. To investigate the gestation progress, two El9.5d fetus were recovered by Casarean section and one fetus was confirmed to contain human resistin gene by genomic DNA-PCR. Therefore, this finding demonstrates that tetraploid-ES mouse technology can be considered as a useful tool to produce transgenic mouse for the rapid analysis of gene function in vivo.

Protein product encoded by a human novel gene E9730 enhances AP-1 activity through interacting with Jab1.

A novel human gene, named E9730 (a clone number of fetal liver cDNA library), has been identified from more than 14,000 expressed sequence tags (ESTs) based on our large scale sequencing of human fetal liver cDNA libraries. Although sequencing of this novel human gene indicates that it is a leucine zipper protein, the function of E9730 and its homongous genes among species is unknown yet. To find out physiological functional clue of E9730, the yeast two-hybrid system was used to screen the E9730-interacting protein(s), and one clone containing a cDNA insert with almost the entire coding sequence (amino acids 39 C335) of human Jab1 (Jun-activating domain binding protein 1) that interacted specifically with E9730 was identified. A specific association between Jab1 and E9730 was shown by co-immunoprecipitation and co-localization experiments. Furthermore, the data indicated that E9730 appeared to enhance Jab1-induced AP-1 activity in a concentration-dependent manner and Jab1 may be involved in the intracellular signaling transduction from E9730 to AP-1.

Cytochrome C oxidase III interacts with hepatitis B virus X protein in vivo by yeast two-hybrid system.

To screen and identify the proteins which interact with hepatitis B virus (HBV) X protein in hepatocytes by yeast two-hybrid system and to explore the effects of X protein in the development of hepatocellular carcinoma (HCC). With HBV X gene amplified by polymerase chain reaction (PCR), HBV X bait plasmid, named pAS2-1-X, was constructed by yeast-two hybridization system3 and verified by auto-sequencing assay. pAS2-1-X was transformed into the yeast AH109, and X-BD fusion protein expressed in the yeast cells was detected by Western blotting. The yeast cells cotransformed with pAS2-1-X and normal human liver cDNA library were grown in selective SC/-trp-leu-his-ade medium. The second screen was performed with beta-gal activity detection, and false positive clones were eliminated by segregation analysis, true positive clones were amplified, sequenced and analyzed with bioinformatics. Mating experiment was performed to confirm the binding of putative proteins to X protein in the yeast cells. Bait plasmid pAS2-1-X was successfully constructed and pAS2-1-X correctly expressed BD-X fusion protein in yeast AH109. One hundred and three clones grew in the selective SC/-trp-leu-his-ade medium, and only one clone passed through beta-gal activity detection and segregation analysis. The inserted cDNA fragment showed high homology with Homo sapiens cytochrome C oxidase III (COXIII). Furthermore, mating experiment identified that the binding of COXIII to X protein was specific. COXIII protein is a novel protein that can interact with X protein in vivo by yeast two-hybrid system, and may contribute to the development of HCC through the interaction with X protein.

Molecular cloning and immunologic characterization of a novel cDNA coding for progesterone-induced blocking factor.

Previous studies from our laboratory showed that the immunomodulatory effects of progesterone are mediated by a 34-kDa protein, named the progesterone-induced blocking factor (PIBF). Lymphocytes of women with threatened abortion fail to produce this factor. Via inducing a Th2 biased cytokine production and blocking of NK activity, PIBF prevents induced pregnancy loss in mice, suggesting that substitution therapy with PIBF could be useful as an alternative treatment of certain forms of recurrent spontaneous abortions. Our study was aimed at mapping the sequence and structure of PIBF coding cDNA and characterizing the encoded protein product. Screening of a human liver cDNA library revealed a 2765-bp clone with a 2271-bp open reading frame. The PIBF1 cDNA encodes a protein of 757 amino acid residues with an 89-kDa predicted molecular mass, which shows no significant amino acid sequence homology with any known protein. PIBF produced via recombinant technique is recognized by the Ab specific for the secreted lymphocyte PIBF Ab, and possesses the biological activities of the secreted lymphocyte PIBF. The full-length PIBF is associated with the nucleus, whereas secretion of shorter forms, such a 34-kDa protein is induced by activation of the cell. The 48-kDa N-terminal part of PIBF is biologically active, and the part of the molecule, responsible for modulating NK activity is encoded by exons 2-4. These data provide an initial step for exploiting the possible diagnostic and therapeutic potential of this immunomodulatory molecule.

Ligand-binding domain of farnesoid X receptor (FXR) had the highest sensitivity and activity among FXR variants in a fluorescence-based assay.

The farnesoid X receptor (FXR, NR1H4) has been recognized as an attractive therapeutic target because it is a nuclear hormone receptor that controls the expression level of cholesterol-7alpha-hydroxylase, which in turn regulates bile acid production and cholesterol excretion. To compare receptor activity between each domain and the full-length protein, human FXR cDNA was cloned from a human liver cDNA library. Three human FXR cDNA, designated FXR20, FXR33, and FXR53 cDNA, were subcloned and ligated into a pET28a expression vector. Each protein was expressed in Escherichia coli (BL21) and purified by nickel-nitrilotriacetic acid column chromatography. Approximately 5 mg of FXR33 (1-182 amino acids deleted from FXR, 37 kDa) and 2 mg of FXR53 (the full-length protein of FXR, 59 kDa) was purified from 1 L of Luria-Bertani culture, achieving at least 90% purity. The coactivator recruitment assay for FXR activation was carried out with the three variants of the FXR protein by using dissociation-enhanced lanthanide fluoroimmunoassay-europium-N1-labeled anti-His antibody. From an optimized assay, a saturated hyperbolic fluorescence signal curve was produced when 250 nM of FXR33 and 100 nM of steroid receptor coactivator-1 peptide, a coactivator of FXR consisting of 26 amino acids, were used with a concentration dependence on chenodeoxycholic acid (from 0 to 200 microM). The ligand-binding domain of FXR (FXR33) was the most suitable protein for studying the activation of FXR with a fluorescence-based assay, because it showed better structural stability than either the full length of FXR (FXR53) or the DNA-binding domain of FXR (FXR20).

Identification of a novel liver-specific expressed gene, TCP10L, encoding a human leucine zipper protein with transcription inhibition activity.

The incidence of hepatoma is high in the Chinese population. Searching for genes involved in the functions of the liver, especially genes specifically expressed in the liver, will facilitate an insight into the molecular basis of normal and abnormal liver functions. Based on a differentially displayed cDNA fragment, which was down regulated in hepatoma tissues, we cloned a novel cDNA of 957 bp, TCP10L (T-complex protein 10 like), from the human liver cDNA library. Northern hybridization of this novel gene in 30 adult human tissues was examined. The result revealed that TCP10L expressed specifically in the human liver and testis. The TCP10L contains a 645-bp open reading frame encoding a deduced protein of 215 amino acids. As the deduced protein was analyzed further, a typical leucine zipper motif was found. We firstly examined the transcriptional function of the TCP10L protein by transfecting recombinant pM-TCP10L into mammalian cells. The subsequent analysis based on the dual luciferase assay system showed that TCP10L significantly inhibited the expression of reporter genes. Compared with that of the negative control, the luciferase activity were down regulated in HEK293 and SK-HEP-1, CHO cells by about 2.6, 9.8, and 5.5 folds respectively. A mutated type of TCP10L was also constructed. It showed that the repression of TCP10L to the expression of the reporter gene almost completely decreased, suggesting that the leucine zipper structure is critical for TCP10L to play its role in regulation function. Then we transfected the recombinant TCP10L-EGFP into cells. The results indicated that TCP10L subcellularly located in nuclei, either in HEK 293 or SK-HEP-1 cells. In addition, human TCP10L was found comprised of five exons and four introns, and mapped to chromosome 21q22.11.

Direct interaction between α-actinin and hepatitis C virus NS5B

It has been suggested that cellular proteins are involved in hepatitis C virus (HCV) RNA replication. By using the yeast two-hybrid system, we isolated seven cDNA clones encoding proteins interacting with HCV RNA polymerase (NS5B) from a human liver cDNA library. For one of these, α-actinin, we confirmed the interaction by coimmunoprecipitation, immunofluorescent staining and confocal microscopic analysis. Experiments with deletion mutants showed that domains NS5B 84–95, NS5B 466–478, and α-actinin 621–733 are responsible for the interaction. Studies of the HCV subgenomic replicon system with small interference RNA indicate that α-actinin is essential for HCV RNA replication. Our results suggest α-actinin may be a component of the HCV replication complex.

Suppression of cell migration in ovarian cancer cells mediated by PTEN overexpression

Peritoneal dissemination is the major progression pathway of ovarian cancer, and its control is important for improvement of the prognosis. PTEN is a tumor suppressor gene, and is known to inhibit cancer cell growth and migration. To investigate the possibility of gene therapy using PTEN for ovarian cancer, we introduced PTEN cDNA into an ovarian cancer cell line HRA carrying wild-type PTEN, and examined the effects in vitro and in vivo. Using PTEN cDNA cloned from a human liver cDNA library, a PTEN expression vector was constructed. This vector was introduced into HRA cells by the standard calcium phosphate precipitation method, and an HRA cell line overexpressing PTEN (HRA/PTEN) was established. On the cell migration test by in vitro scratch wound healing assay, the number of migrating cells was 6.3+/-0.9 cells/mm(2) in HRA/PTEN, which was significantly smaller than that in the control (39.7+/-3.2 cells/mm(2)) (p<0.01). No significant differences were observed in the in vitro cell growth or in vivo tumor growth between HRA/PTEN and the control. The findings described above, show that enhanced expression of PTEN inhibits ovarian cancer cell migration, suggesting that gene therapy approaches using PTEN for control of peritoneal dissemination of ovarian cancer are possible.

The Hepatitis C Virus NS2 Protein Is an Inhibitor of CIDE-B-induced Apoptosis

Chronic hepatitis C virus (HCV) infection frequently leads to liver cancer. To determine the viral factor(s) potentially involved in viral persistence, we focused our work on NS2, a viral protein of unknown function. To assign a role for NS2, we searched for cellular proteins that interact with NS2. Performing a two-hybrid screen on a human liver cDNA library, we found that NS2 interacted with the liver-specific pro-apoptotic CIDE-B protein. Binding specificity of NS2 for CIDE-B was confirmed by cell-free assays associated with colocalization studies and coprecipitation experiments on human endogenous CIDE-B. CIDE-B, a member of the novel CIDE family of apoptosis-inducing factors, has been reported to show strong cell death-inducing activity in its C-terminal domain. We show that this CIDE-B killing domain is involved in the NS2 interaction. NS2 binding was sufficient to inhibit CIDE-B-induced apoptosis because an NS2 deletion mutant unable to interact with CIDE-B in vitro lost its capacity to interfere with CIDE-B cell death activity. Although it has been reported that CIDE-B-induced apoptosis is characterized by mitochondrial localization, the precise apoptotic mechanism remained unknown. Here, we show that CIDE-B induced cell death in a caspase-dependent manner through cytochrome c release from mitochondria. Furthermore, we found that NS2 counteracted the cytochrome c release induced by CIDE-B. In vivo, the CIDE-B protein level was extremely low in adenovirus-infected transgenic mice expressing the HCV polyprotein compared with that in wild-type mice. We suggest that NS2 interferes with the CIDE-B-induced death pathway and participates in HCV strategies to subvert host cell defense.

The C-terminal Kinase Domain of the p34cdc2-related PITSLRE Protein Kinase (p110C) Associates with p21-activated Kinase 1 and Inhibits Its Activity during Anoikis

The PITSLRE protein kinases are parts of the large family of p34cdc2-related kinases. During apoptosis induced by some stimuli, specific PITSLRE isoforms are cleaved by caspase to produce a protein that contains the C-terminal kinase domain of the PITSLRE proteins (p110C). The p110C induces apoptosis when it is ectopically expressed in Chinese hamster ovary cells. In our study, similar induction of this p110C was observed during anoikis in NIH3T3 cells. To investigate the molecular mechanism of apoptosis mediated by p110C, we used the yeast two-hybrid system to screen a human fetal liver cDNA library and identified p21-activated kinase 1 (PAK1) as an interacting partner of p110C. The association of p110C with PAK1 was further confirmed by in vitro binding assay, in vivo coimmunoprecipitation, and confocal microscope analysis. The interaction of p110C with PAK1 occurred within the residues 210-332 of PAK1. Neither association between p58PITSLRE or p110PITSLRE and PAK1 nor association between p110C and PAK2 or PAK3 was observed. Anoikis was increased and PAK1 activity was inhibited when NIH3T3 cells were transfected with p110C. Furthermore, the binding of p110C with PAK1 and inhibition of PAK1 activity were also observed during anoikis. Taken together, these data suggested that PAK1 might participate in the apoptotic pathway mediated by p110C.

ECRG2, a novel candidate of tumor suppressor gene in the esophageal carcinoma, interacts directly with metallothionein 2A and links to apoptosis

Esophageal cancer related gene 2 (ECRG2) is a novel candidate of the tumor suppressor gene identified from human esophagus. To study the biological role of the ECRG2 gene, we performed a GAL4-based yeast two-hybrid screening of a human fetal liver cDNA library. Using the ECRG2 cDNA as bait, we identified nine putative clones as associated proteins. The interaction of ECRG2 and metallothionein 2A (MT2A) was confirmed by glutathione S-transferase pull-down assays in vitro and co-immunoprecipitation experiments in vivo. ECRG2 co-localized with MT2A mostly to nuclei and slightly to cytoplasm, as shown by confocal microscopy. Transfection of ECRG2 gene inhibited cell proliferation and induced apoptosis in esophageal cancer cells. In the co-transfection of ECRG2 and MT2A assays, cell proliferation was inhibited and apoptosis was slightly induced compared with control groups. When we used antisense MT2A to interdict the effect of MT2A, the inhibition of cell proliferation and induction of apoptosis were significantly enhanced. When we used antisense ECRG2 to interdict the effect of ECRG2 in the group of Bel7402 cells co-transfected with ECRG2 and MT2A, the inhibition of cell proliferation and induction of apoptosis disappeared. The results provide evidence for ECRG2 in esophageal cancer cells acting as a bifunctional protein associated with the regulation of cell proliferation and induction of apoptosis. ECRG2 might reduce the function of MT2A on the regulation of cell proliferation and induction of apoptosis. The physical interaction of ECRG2 and MT2A may play an important role in the carcinogenesis of esophageal cancer.

Using yeast two-hybrid system to identify ECRG2 associated proteins and their possible interactions with ECRG2 gene.

To identify esophageal cancer related gene2 (ECRG2) associated proteins and their possible interactions with ECRG2 gene. In the yeast forward two-hybrid system, ECRG2 was fused with the DNA-binding domain (DBD) of Gal4 and human fetal liver cDNA library was fused with the transcriptional activation domain (AD) of Gal4. We performed a high-stringency scale procedure to screen ECRG2 against human fetal liver cDNA library and characterized positives by sequence analysis. We found the following 9 putatively associated proteins. They were metallothionein2A, metallothionein1H, metallothionein1G, ferritin, erythrocyte membrane protein band4.2, mitochondrial ribosomal protein S12, hypothetical protein FLJ10101, and a novel gene whose cDNA was found to have no strong homology to any other previously characterized gene whose DDBJ/EMBL/GenBank accession number is AF422192 mapped to human chromosome 14q31. MT, a potential interaction partner for ECRG2, might be involved in the regulation of cell proliferation and apoptosis, and in various physiological processes. Determination of a reliability score for each single protein-protein interaction, especially interaction of ECRG2 and MT, permits the assignment of ECRG2 and unannotated proteins to biological pathways. A further understanding of the association between ECRG2 and MT should facilitate the functions of ECRG2 gene.

Isolation, sequencing, and expression of a cDNA for the HXM-A form of xenobiotic/medium-chain fatty acid:CoA ligase from human liver mitochondria.

The purification of xenobiotic/medium-chain fatty acid:CoA ligases (XM-ligases) from human liver mitochondria resulted in the isolation of two chromatographically separable forms (HXM-A and HXM-B). These two forms were purified to near homogeneity, cleaved with cyanogen bromide, the resulting peptides separated, and the N-terminus of two of the peptides partially sequenced. Identical sequences were obtained for HXM-A and HXM-B for the two peptides. These sequences were used to design probes for screening a human liver cDNA library. This resulted in the isolation of two overlapping cDNAs. Using these sequences we were able to design PCR primers that resulted in the isolation of a full-length cDNA from a human cDNA library. The cDNA contained 1731 bp of open reading frame and coded for a 64230-Da protein. This protein bears 56.2% amino acid homology to the MACS1 (medium-chain acyl-CoA synthetase) enzyme, 58.7% homology to the bovine XL-III XM-ligase, and 81.5% homology to the bovine XL-I XM-ligase. The cDNA could be expressed in COS cells, and the expressed enzyme had greater benzoate activity than phenylacetate activity, which is consistent with the known substrate specificity of HXM-A.

Absence of functional insulin receptor substrate-3 (IRS-3) gene in humans.

Insulin receptor substrate (IRS) proteins play important roles in insulin action and pancreatic beta-cell function. At least four mammalian IRS molecules have been identified. Although genes and cDNAs encoding human IRS-1, IRS-2, and IRS-4 have been cloned, IRS-3 has been identified only in rodents. Thus, we have attempted to clone the human IRS-3 gene. Insulin-stimulated rat or human adipocytes were subjected to Western blot analysis to assess IRS-3 tyrosine phosphorylation. Human liver and adipose cDNA libraries were screened in an effort to clone IRS-3 cDNA. A PCR-based approach was designed to amplify IRS-3 cDNA. Reverse transcription PCR was carried out using mRNA from adipose tissue, liver, and skeletal muscle as templates in combination with an in silico screen using mouse IRS-1, IRS-2 and IRS-3 in a tblastn search of the draft public human genome. In human adipocytes we did not detect a M(r) 60 000 phosphoprotein corresponding to IRS-3, whereas in rat adipocytes IRS-3 protein and insulin-stimulated tyrosine phosphorylation was readily observed. None of the molecular approaches provided evidence for a functional IRS-3gene in human tissue. Two deletions in human IRS-3 gene were identified using bioinformatics. The human IRS-3 gene product is predicted to lack a phosphotyrosine binding domain and also the sequence corresponding amino acid 353-407 of murine IRS-3. The contiguous sequence of genomic DNA between these two homologous regions does not have the coding information for human IRS-3. In silico screening of the human IRS-3 genome region, combined with further biological and molecular validation, provides evidence against a functional IRS-3 in humans.

Interaction of p58PITSLRE, a G2/M-specific Protein Kinase, with Cyclin D3

The p58(PITSLRE) is a p34(cdc2)-related protein kinase that plays an important role in normal cell cycle progression. Elevated expression of p58(PITSLRE) in eukaryotic cells prevents them from undergoing normal cytokinesis and appears to delay them in late telophase. To investigate the molecular mechanism of p58(PITSLRE) action, we used the yeast two-hybrid system, screened a human fetal liver cDNA library, and identified cyclin D3 as an interacting partner of p58(PITSLRE). In vitro binding assay, in vivo coimmunoprecipitation, and immunofluorescence cell staining further confirmed the association of p58(PITSLRE) with cyclin D3. This binding was observed only in the G(2)/M phase but not in the G(1)/S phase of the cell cycle; meanwhile, no interaction between p110(PITSLRE) and cyclin D3 was observed in all the cell cycle. The overexpression of cyclin D3 in 7721 cells leads to an exclusively accumulation of p58(PITSLRE) in the nuclear region, affecting its cellular distribution. Histone H1 kinase activity of p58(PITSLRE) was greatly enhanced upon interaction with cyclin D3. Furthermore, kinase activity of p58(PITSLRE) was found to increase greatly in the presence of cyclin D3 using a specific substrate, beta-1,4-galactosyltransferase 1. These data provide a new clue to our understanding of the cellular function of p58(PITSLRE) and cyclin D3.

Identification of hepatopoietin dimerization, its interacting regions and alternative splicing of its transcription.

Hepatopoietin (HPO) is a novel human hepatotrophic growth factor. Recently, we demonstrated that the extracellular ligand form of HPO can stimulate proliferation of hepatic cells via its specific receptor, which is on the surface of these cells. Also, the intracellular form of HPO potentiates the transcriptional factor AP-1. Intriguingly, a variety of HPO complexes with different molecular masses were detected in hepatocytes. In this study, we screened a human fetal liver cDNA library using the yeast two hybrid system with HPO as bait, hoping to find an HPO-binding protein. Surprisingly, HPO, itself, and a previously unidentified alternatively spliced form of HPO (named HPO23) were identified as interacting with HPO, indicating that HPO may exist as a homodimer or heterodimer. These results were further confirmed in vitro and in vivo. First, mass spectrometry analysis demonstrated that recombinant human HPO exist as a homodimer and that disulfide bonds were involved in the formation of rhHPO dimer. Secondly, a pull-down experiment confirmed that GST-HPO and HA-HPO, could bind together in vitro. Using HPO and various HPO deletion mutants, we identified the extreme 30 amino acids at both N- and C-termini are essential for HPO dimerization. Finally, Western blotting analysis demonstrated that HPO exists as two isoforms at 15 kDa and 23 kDa (HPO23) in liver cells, the 15 kDa species is located in nucleus, and the 23 kDa species mainly in the cytoplasm. These results implicated that the capacity of HPO to form both homodimers and heterodimers with the alternatively spliced forms might contribute to the existence of various HPO complexes in hepatic cells.