Yu-Yang Li

Cathepsin B and its interacting proteins, bikunin and TSRC1, correlate with TNF‐induced apoptosis of ovarian cancer cells OV‐90

Increasing evidence suggests that lysosomal cysteine proteases cathepsins contribute to the progression of cell apoptosis. Here we found that apoptosis of ovarian cancer cells OV‐90 triggered by TNF was cathepsin B‐depended. Two cathepsin B binding proteins, bikunin and TSRC1, were identified by yeast two‐hybrid method and the interactions were confirmed in vitro and in vivo. Overexpression of bikunin could suppress TNF‐induced apoptosis of OV‐90 cells, and TSRC1 overexpression had an opposite effect on apoptosis. The presented results suggest that cathepsin B and its interacting proteins, bikunin and TSRC1, are involved in the apoptotic pathway of ovarian cancer cells.

The apoptosis-associated protein BNIPL interacts with two cell proliferation-related proteins, MIF and GFER.

Bcl‐2/adenovirus E1B 19 kDa interacting protein 2‐like, BNIP‐2‐like (BNIPL) is a recently cloned and characterized apoptosis‐associated protein that shares 72% homology with BNIP‐2. It is highly expressed in human placenta and lung. A yeast two‐hybrid system was used to obtain two BNIPL‐interacting proteins, MIF (macrophage migration inhibitory factor) and GFER (growth factor erv1 (Saccharomyces cerevisiae)‐like). The interactions were confirmed by glutathione S‐transferase pull‐down assay in vitro and co‐immunoprecipitation assay in vivo. Colony formation assay and cell proliferation test suggest that overexpression of BNIPL could inhibit the growth of BEL‐7402 cells. These findings suggest that BNIPL may physically bind to cell proliferation‐related proteins, MIF and GFER.

A zinc finger HIT domain-containing protein, ZNHIT-1, interacts with orphan nuclear hormone receptor Rev-erbβ and removes Rev-erbβ-induced inhibition of apoCIII transcription

The orphan receptors, Rev‐erbα and Rev‐erbβ, are members of the nuclear receptor superfamily and specifically repress apolipoprotein CIII (apoCIII) gene expression in rats and humans. Moreover, Rev‐erbα null mutant mice have elevated very low density lipoprotein triacylglycerol and apoCIII levels. However, ligands for Rev‐erb are unknown and the regulatory mechanism of Rev‐erb is poorly understood. Conceivably, cofactors for Rev‐erb may play an important role in the regulation of lipid metabolism. In this study, a zinc finger HIT domain‐containing protein, ZNHIT‐1, interacted with Rev‐erbβ. ZNHIT‐1 was found to be a conserved protein in eukaryotes and was highly abundant in human liver. Furthermore, ZNHIT‐1 was identified as a nuclear protein. Serial truncated fragments and substitution mutations established a putative nuclear localization signal at amino acids 38–47 of ZNHIT‐1. A putative ligand‐binding domain of Rev‐erbβ and the FxxLL motif of ZNHIT‐1 were required for their interaction. Finally, ZNHIT‐1 was recruited by Rev‐erbβ to the apoCIII promoter and removed the Rev‐erbβ‐induced inhibition of apoCIII transcription. These findings demonstrate that ZNHIT‐1 functions as a cofactor to regulate the activity of Rev‐erbβ, and may play a role in lipid metabolism.

Molecular cloning and characterization of CT120, a novel membrane-associated gene involved in amino acid transport and glutathione metabolism

Within the minimum LOH region on chromosome 17p13.3 deleted in hepatocellular carcinoma, a novel human plasma membrane-associated gene, named CT120, was isolated from a human kidney cDNA library using electronical cloning and RACE. The novel gene CT120 consists of 2145bp and encodes a protein with 257 amino acids. Database search revealed that homologs of CT120 exist in different organisms from plant to animal kingdoms, which suggests that CT120 is a highly conserved gene during biological evolution. Different expression patterns of CT120 were observed in many different human normal tissues and in various human tumor cell lines. Transcript of CT120 was not detectable in normal lung tissue, but was abundant in SPC-A-1 (human epithelial-like lung adenocarcinoma) cell line, suggesting that CT120 may be involved in lung cancer development. Subcellular localization analysis showed that CT120 is a novel membrane-associated protein. CT120 can interact with SLC3A2 (member 2 of solute carrier family 3) and GGTL3B (isoform of gamma-glutamyltranspeptidase-like 3) in eukaryotic cells by yeast two-hybrid screen and co-immunoprecipitation assay, which suggested that CT120 may assume very essential physiological functions involved in amino acid transport and glutathione metabolism.

Human Homologue of Seta Binding Protein 1 Interacts with Cathepsin B and Participates in TNF-Induced Apoptosis in Ovarian Cancer Cells

Lysosomal cysteine protease cathepsin B has been reported to play an important role in apoptosis of many different cancer cells, but the regulation of cathepsin B in apoptosis is poorly understood. Human homologue of SETA binding protein 1 (hSB1) was identified to interact with cathepsin B by yeast-two hybrid method, and the interaction was confirmed in vitro GST pull-down assay and in vivo coimmunoprecipitation experiment. hSB1 was co-localized with cathepsin B in cellular lysosomes. Our previous study has shown that TNF can induce ovarian cancer cells OV-90 apoptosis and the apoptosis process is cathepsin B-depended. Here we provide evidence that overexpression of cathepsin B-interacting protein hSB1 could suppress TNF-triggered apoptosis in OV-90 cells, but has no effect on cellular cathepsin B activity. hSB1 may function as a regulator of cathepsin B-mediated apoptosis.

Characterization of human dopamine responsive protein DRG-1 that binds to p75NTR-associated cell death executor NADE

Expression of human dopamine responsive gene-1 (DRG-1) is up-regulated in response to treatment of dopamine in the rat astrocytes. However, its functions are not clear up to now. In the presented studies, DRG-1 was identified to be a conserved gene in the vertebrate and expressed abundantly in human testis, brain and skeletal muscle. DRG-1 was shown to interact with human p75NTR-associated cell death executor (NADE) in vivo and in vitro, and the interaction occurred in cytoplasm. The regions required for the interaction were subsequently mapped to the N-terminal of DRG-1 and the C-terminal of NADE. Furthermore, MTT assay showed that stable expression of DRG-1 in 293 cells could promote cell proliferation, and this promotion was suppressed by overexpression of NADE. In flow cytometry cell cycle analysis, overexpression of DRG-1 in 293 or PC12 cells increased the population of cells in the S phase with a concomitant decrease in G0/G1 population. These findings suggest that DRG-1 may contribute to the dopamine-induced cell growth, which is negatively regulated by NADE.

pp5644 Interacts with phosphatidylinositol-4-phosphate adaptor protein-1 associated protein-1

The human novel gene pp5644 (GeneBank Accession No. AF289559) coding for 124 amino acids was recently cloned. Overexpression of pp5644 in Hela cells significantly inhibited the growth and colony formation. The pp5644-interacting protein FAPP1 (phosphatidylinositol-four-phosphate adaptor protein1) associated protein-1, called FASP1, was obtained by using yeast two-hybrid system. The interaction between pp5644 and FASP1 was experimentally confirmed by GST pull-down assay In vitro and co-immunoprecipitation assay in vivo. Co-localization of pp5644 and FASP1 in cytoplasm in Hela cells could further support the interaction. Based on the experimental results, it is suggested that pp5644 physically bind to FASP1 and the biological significance of this kind of interaction in vivo is discussed. (Mol Cell Biochem 271: 151–158, 2005)

Identification of LZP Gene from Mus musculus and Rattus norvegicus Coding for a Novel Liver-specific ZP Domain-Containing Secretory Protein

Zona pellucida (ZP) domain has been recognized in a number of receptor-like eukaryotic glycoproteins, which involved in many important biological processes, such as signal transduction, development, differentiation and so on. Here we report the identification of Mus musculus and Rattus norvegicus orthologues of Homo sapiens LZP gene which codes for a novel ZP domain-containing protein. Sequence analysis revealed that human, rat and mouse LZP proteins are highly conserved. Mouse LZP gene has two transcripts, 2.4 and 2.8 KB long respectively, coding for identical protein. Mouse LZP mRNA is expressed specifically in hepatocytes. Our data also showed that mouse LZP localizes mostly on nuclear envelope, and at the same time, it can be secreted into blood in a truncated form.