A large-scale functional approach to uncover human genes and pathways in Drosophila.

We demonstrate the feasibility of performing a systematic screen for human gene functions in Drosophila by assaying for their ability to induce overexpression phenotypes. Over 1 500 transgenic fly lines corresponding to 236 human genes have been established. In all, 51 lines are capable of eliciting a phenotype suggesting that the human genes are functional. These heterologous genes are functionally relevant as we have found a similar mutant phenotype caused either by a dominant negative mutant form of the human ribosomal protein L8 gene or by RNAi downregulation of the Drosophila RPL8. Significantly, the Drosophila RPL8 mutant can be rescued by wild-type human RPL8. We also provide genetic evidence that Drosophila RPL8 is a new member of the insulin signaling pathway. In summary, the functions of many human genes appear to be highly conserved, and the ability to identify them in Drosophila represents a powerful genetic tool for large-scale analysis of human transcripts in vivo.

Cyclophilin A is upregulated in small cell lung cancer and activates ERK1/2 signal.

Cyclophilin A (CypA), a peptidyl-prolyl cis-trans isomerase (PPIase), was originally identified as the intracellular receptor for cyclosporin A (CsA). Recently, correlations of CypA with tumor pathogenesis have been studied. Here, we studied the expression of CypA and its receptor CD147 in several kinds of lung cancer cells as well as a normal lung cell and found that in H446 cell, a kind of small cell lung cancer cell, the expression are the highest. The exogeneous CypA protein can substantially stimulate H446 cell growth in dependence on its PPIase activity. We also showed that CypA protein can stimulate ERK1/2 signal in dose and time dependent manners and almost has no effect to p38 and JNK signals. Elucidation of the precise role of CypA in these pathways may lead to new targeted therapies for small cell lung cancer.

Identification of the auto-inhibitory domains of Aurora-A kinase.

Aurora-A is a centrosome-localized serine/threonine kinase that is overexpressed in multiple human cancers. Here, we report an intramolecular inhibitory regulation in Aurora-A between its N-terminal regulatory domain (aa 1-128, Nt) and the C-terminal catalytic domain (aa 129-403, Cd). Removal of Nt results in a significant increase in kinase activity. Nt inhibited the activity of the single C-terminal kinase domain, but had little effect on the activity of the full-length of Aurora-A. PP1 is not involved in this regulation, instead, Nt interacts Cd directly in vitro and in vivo. The non-Aurora box (aa 64-128) in the N-terminal negatively regulated the kinase activity of the C-terminal kinase domain by intramolecular interaction with aa 240-300 within the C-terminal.

Natural antisense transcripts of Alzheimer's disease associated genes.

Natural antisense transcripts (NATs), also named endogenous antisense transcripts, are a class of genes whose role in controlling gene expression is becoming more and more relevant. NATs might play important roles in gene expression and translation regulation. Present work investigated the presence of NATs of Alzheimer's disease associated genes including PRESENILIN1, PRESENILIN2, BACE1, BACE2, APP, APOE, TAU (MAPT), PRION, alpha-SYNUCLEIN (SNCA), NICASTRIN, PEN2, APH1A, APH1B as well as CD147 (BASIGIN), and the results revealed that APP, BACE2, APH1A, TAU, CD147 and alpha-SYNUCLEIN contain natural antisense transcripts. These NATs were characterized according to the sense-antisense overlapping information and potential functional mechanisms were proposed. Present findings provide preliminary but important information about transcription regulation of AD associated genes, which would further our understanding of the gene expression regulation of AD, and also suggest a novel potential strategy for the therapy of AD.

Decreased expression of the human carbonyl reductase 2 gene HCR2 in hepatocellular carcinoma.

Altered gene expression was associated with the induction and maintenance of hepatocellular carcinoma (HCC). To determine the significance of HCR2 in HCC, here we compare the expression levels of HCR2 in carcinoma and in paired non-carcinoma tissues using semiquantitative reverse-transcription polymerase chain reaction (RT-PCR), Western blot analysis, and immunohistochemical staining. The expression ratio (ER) of HCR2 between the tumor and paired tumor-free tissues was calculated for each case and the data was clinicopathologically analyzed. The expression of HCR2 mRNA was found to be significantly decreased in HCC tissues compared with paired normal tissues (P < 0.001). HCR2 was downregulated in 58% (n = 22) of 38 HCC patients. The ER of HCR2 was higher in Edmondson's grade I/II carcinomas than that in Edmondson's grade III/IV carcinomas (P < 0.05). Western blot analysis showed HCR2 to be notably depressed in carcinoma tissues in 3 out of 4 HCC patients. Immunohistochemical staining indicated most HCR2 protein accumulated in non-carcinoma cells. These results suggested that altered HCR2 expression might play roles in the carcinogenesis and progression of HCC, and it could be a clinical marker for prognosis, and a molecular target for screening potential anti-HCC drugs.

Isolation and characterization of the human D-glyceric acidemia related glycerate kinase gene GLYCTK1 and its alternatively splicing variant GLYCTK2.

Deficiency of human glycerate kinase leads to D-glycerate acidemia/D-glyceric aciduria. Through PCR cloning assisted by in silico approach, we isolated the human glycerate kinase genes--Glycerate Kinase 1 (GLYCTK1) and its alternatively splicing variant--Glycerate Kinase 2 (GLYCTK2), which might be associated with D-glycerate acidemia/D-glyceric aciduria. The locus of GLYCTK gene is mapped to 3p21. PCR amplification in seventeen human tissue cDNAs revealed that both GLYCTK1 and GLYCTK2 are expressed widely almost in all these tissues. The expression of mouse Glyctk in various tissues was demonstrated by in situ hybridization. Both GLYCTK1 and GLYCTK2 proteins are localized in cytosol, and GLYCTK2 proteins are specifically localized in mitochondria. Present results revealed the characteristic expression pattern of murine Glyctk in neural system, skeleton muscle, supporting that glycerate kinase is implicated in D-glycerate acidemia/D-glyceric aciduria.

GG: a domain involved in phage LTF apparatus and implicated in human MEB and non-syndromic hearing loss diseases.

Here, we report the identification of a novel domain--GG (domain in KIAA1199, FAM3, POMGnT1 and Tmem2 proteins, with two well-conserved glycine residues), present in eukaryotic FAM3 superfamily (FAM3A, FAM3B, FAM3C and FAM3D), POMGnT1 (protein O-linked mannose beta-1,2-N-acetylglucosaminyltransferase), TEM2 proteins as well as phage gp35 proteins. GG domain has been revealed to be implicated in muscle-eye-brain disease and non-syndromic hearing loss. The presence of GG domain in Bacteriophage gp35 hinge connector of long tail fiber might reflect the horizontal gene transfer from organisms. And we proposed that GG domain might function as important structural element in phage LTF.

Cloning, expression and characterization of the human NOB1 gene.

The yeast Nob1p (Nin one binding protein) gene is required for proteasome function and RNA metabolism. We report here the cloning and characterization of the human orthologue NOB1 gene and its products. The human NOB1 gene is composed of nine exons and eight introns and is localized on human chromosome 16q22.1. The NOB1 cDNA is 1749 bp long and contains a putative open reading frame of 1239 bp. The predicted NOB1 protein comprises a PIN (PilT amino terminus) domain and a zinc ribbon domain. Western blot analysis showed that the molecular weight of NOB1 is about 50 KDa. RT-PCR analysis of mRNA from human adult tissues showed that NOB1 is expressed mainly in liver, lung and spleen. Expression of NOB1 in mammalian culture cells indicated that the NOB1 protein is mainly localized in the nucleus. Our data provides important information for further study of the function of the NOB1 gene and its products.

Septin1, a new interaction partner for human serine/threonine kinase aurora-B.

Several families of kinases work together to ensure the rate and precision of mitosis. Aurora-B is an important serine/threonine kinase required for chromosome segregation and cytokinesis. Identification of Aurora-B substrates will help to enhance our understanding of the molecular mechanism of mitosis. Through a yeast two-hybrid screen, we found a novel partner of Aurora-B, Septin1, belonging to a conserved family of GTPase proteins that localize to the cleavage furrow and are involved in cytokinesis. We confirmed this interaction using Co-immunoprecipitation experiments in mammalian cells and GST-pull-down analysis in vitro. Moreover, Aurora-B can phosphorylate Septin1 in vitro. We identified that Ser248, Ser307, and Ser315 are the main phosphorylation sites in Septin1. These two proteins partially co-localize to the midbody during cytokinesis. So, it is possible that Septin1's role in the regulation of cytokinesis is related to its phosphorylation by Aurora-B. Unlike previous reports that Septins function in cytokinesis and localize to the cleavage furrow, we found that Septin1 localizes to the spindle pole throughout mitosis, indicating that Septin1 may function in chromosome segregation as well.

Human THROMBOSPONDIN-1 gene contains a natural antisense transcript, and characterization of its expression in human multiple tissues and cells.

Human THROMBOSPONDIN-1 play versatile roles in platelet aggregation, angiogenesis, and tumorigenesis, which forms a disulfide-linked homotrimeric complex. Here we reported that the genomic lotus of TSP1 also transcribes a natural antisense transcript (NAT) with an overlapping and reverse complement region against TSP1. The NAT of TSP1 was expressed in human testis, lung, thymus, colon, placenta, kidney and skeleton muscle, revealed by PCR amplification. It was also expressed in some tumor cell lines. The identification of NAT of TSP1 would be of significant importance to understand the functions of TSP1, and it would also suggest the potential attempt of using RNA interference for related tumor therapy, for instance, breast cancer.

Molecular cloning and characterization of three novel lysozyme-like genes, predominantly expressed in the male reproductive system of humans, belonging to the c-type lysozyme/alpha-lactalbumin family.

Lysozymes, especially c-type lysozymes, are well-recognized bacteriolytic factors widely distributed in the animal kingdom and play a mainly protective role in host defense. The relatives of c-type lysozymes, alpha-lactalbumins, however, are only found in mammalian milk and possess a distinct biological function. These two proteins, having similar amino acid sequences, gene structure, and dimensional conformation, belong to the c-type lysozyme/alpha-lactalbumin family. Using human lysozyme as an information probe, we cloned four human cDNAs encoding homologues of human lysozyme; these were named LYZL2, LYZL4, LYZL6, and SPACA3 by the HUGO Gene Nomenclature Committee. Of these four, SPACA3 has been reported to code an intra-acrosomal sperm protein SLLP1. To our knowledge, the other three are reported here for the first time. Using Northern blot hybridization, including 16 different human tissues, we found that these four lysozyme-like genes were all highly expressed in the testis/epididymis. Further analysis of one, LYZL4, by in situ hybridization revealed that its mRNA was only detected in the epithelium of human epididymis, most abundantly in the caput, suggesting that LYZL4 plays a physiological role in male reproduction. By sequence analysis, we found that two essential catalytic residues of the human lysozyme were conserved in LYZL2 and LYZL6, whereas one site in LYZL4 and two sites in SPACA3 were replaced. The LYZL2, LYZL4, LYZL6, and SPACA3 genes were mapped to human chromosome 10p11.23, 3p21.33, 17q11.2, and 17q12, respectively, and displayed a similar genomic structure. Our data suggest that these four lysozyme-like genes, which have arisen from a common progenitor gene, play a major role in human reproduction.

ADAM22 plays an important role in cell adhesion and spreading with the assistance of 14-3-3.

Cellular adhesion plays important roles in a variety of biological processes. The ADAM family contains disintegrin-like and metalloproteinase-like domains which potentially have cell adhesion and protease activities. Recent studies suggest that the interaction between 14-3-3zeta and ADAM22cyt can regulate cell adhesion and spreading, therefore it has a potential role in neural development and function. 14-3-3 family has seven highly conserved members that regulate various cellular functions. Using yeast two-hybrid method, we identified that ADAM22cyt bound some other 14-3-3 family members. The interaction was further confirmed by in vitro protein pull-down assay and co-immunoprecipitation. We also found that the overexpression of exogenous ADAM22 in HEK293 cells could significantly enhance cell adhesion and spreading, compared with the truncated ADAM22 lack of 14-3-3 binding motifs. These results strongly demonstrated a functional role for ADAM22/14-3-3 in cell adhesion and spreading.

Identification and characterization of D8C, a novel domain present in liver-specific LZP, uromodulin and glycoprotein 2, mutated in familial juvenile hyperuricaemic nephropathy.

Present work reported a novel domain--D8C (domain with conserved eight cysteines in liver-specific ZP domain-containing protein, glycoprotein 2 (GP-2) and uromodulin (UMOD)), present in liver-specific LZP, UMOD, GP-2 and some uncharacterized proteins, most of which are membrane proteins, extracellular proteins or nuclear membrane proteins. D8C contains eight well-conserved cysteine residues, which were predicted to form four pairs of disulfide bridges. D8C is composed mainly of beta-strands. Mutation in the D8C at Cys217 in human UMOD is associated with familial juvenile hyperuricaemic nephropathy, which might be due to the disruption of the disulfide bridge. Identification of D8C would further the understandings of related proteins.

Human liver specific transcriptional factor TCP10L binds to MAD4.

A human gene T-complex protein 10 like (TCP10L) was cloned in our lab. A previous study showed that it expressed specifically in the liver and testis. A transcription experiment revealed that TCP10L was a transcription factor with transcription inhibition activity. In this study, the human MAD4 was identified to interact with TCP10L by a yeast two-hybrid screen. This finding was confirmed by immunoprecipitation and subcellular localization experiments. As MAD4 is a member of the MAD family, which antagonizes the functions of MYC and promotes cell differentiation, the biological function of the interaction between TCP10L and MAD4 may be to maintain the differentiation state in liver cells. Also, we propose that the up-regulation of Myc is caused by the down-regulation of TCP10L in human hepatocarcinomas.

Cloning, expression and subcellular localization of HN1 and HN1L genes, as well as characterization of their orthologs, defining an evolutionarily conserved gene family.

The present work reported the cloning and characterization of two novel human genes--HN1 (hematopoietic- and neurologic-expressed sequence 1) and HN1L (HN1-like gene) which are proposed to be involved in embryo development. HN1 is mapped on chromosome 17q25.2, with two transcripts (1.0 and 1.6 kb in length, respectively) due to alternative splicing. HN1 is expressed abundantly in testis and skeletal muscle among 16 human tissues, and it is localized in the nucleus indicated by GFP fusion expression. Western blot confirmed that HN1 encodes a 16.5-kDa protein. HN1L is on chromosome 16p13.3, with three splicing in the length of 2.0, 4.0 and 4.2 kb, respectively. HN1L is expressed in a variety of tissues such as liver, kidney, prostate, testis and uterus at varying levels. HN1L gene encodes a 20-kDa protein, which is localized in both the nucleus and cytoplasm. Fourteen of HN1 and sixteen of HN1L homologous genes in different species were determined and analyzed by BLAST searches. Silicon analyses of the 14 orthologous proteins of HN1 and 16 orthologous proteins of HN1L revealed that they share great conservation in vertebrate. Additionally, we identified nine pseudogenes of HN1 (six) and HN1L (three) in the genomes of the human, mouse and rat. Based on sequence alignments and phylogenetic analysis, all these homologous genes and pseudogenes were defined as a HN1 gene family.

Two novel isoforms of Adam23 expressed in the developmental process of mouse and human brains.

A metalloprotease and disintegrin (ADAM) is a family of membrane-anchored proteins and all family members have a multi-domain structure containing a zinc metalloprotease domain and a disintegrin domain that may serve as an integrin ligand. Here we reported two novel mammalian transcripts of Adam23, named Adam23 beta and Adam23 gamma, to be involved in the development and functional activities of mammalian brains. Adam23 gamma was isolated from a 22-week human fetal brain cDNA library, using an EST homologous to Adam as a probe, and is 100% homologous to human Adam23 (Adam23 alpha) except that it lacks a fragment of 91 bp near the C-terminal, thus it could not form obvious transmembrane domain. Adam23 beta was discovered while the diversity at the transmembrane domain (TM) was analyzed. Adam23 beta has a different sequence in the 91 nucleotides and thus encode different transmembrane domain. Adam23 beta and Adam23 gamma are mainly expressed in brain like Adam23 alpha. RT-PCR experiments in mouse brain also detected the two isoforms, consistent with observation of Northern analysis of human RNAs. Furthermore, results of RT-PCR amplification of Adam23 gamma in mouse brains of different developmental stages revealed a developmentally regulated expression pattern: Adam23 gamma is expressed in embryonic and infant brain, and disappeared after the 10th postnatal day. This temporally changing expression pattern of Adam23 gamma suggests that ADAM23 gamma likely plays an important role in brain development.

GADD45gamma, down-regulated in 65% hepatocellular carcinoma (HCC) from 23 chinese patients, inhibits cell growth and induces cell cycle G2/M arrest for hepatoma Hep-G2 cell lines.

Growth-arrest and DNA-damage inducible (GADD) genes and Myeloid differentiation primary response (MyD) genes represent a family of genes that play a key role in negative control of cell growth. In the present study, following clone and location of human GADD45 gamma (MyDL) gene, we have found that its mRNA expression level was down-regulated in 15/23 cases of clinic hepatocellular carcinoma (HCC) by comparing the northern hybridization results between the tumor tissues and adjacent normal tissues. Transient transfection of GADD45 gamma cDNA with intact open reading frame sequence into the human hepatoma cells Hep-G2 resulted in dramatic growth suppression in colony formation assays. Furthermore, flow cytometry analysis indicated that GADD45 y caused cell cycle arrest at G2/M transition when transfected into Hep-G2 cells. Therefore, the possible role of GADD45 gamma in cell growth control was further confirmed in this paper.