Biomedical subjects

Bing-Ren Huang

Publications and source records attributed to Bing-Ren Huang.

6 recordsLinked to original sources

PubMed2006

The Ran binding protein RanBPM interacts with TrkA receptor.

RanBPM as a novel binding protein can interact with neurotrophin receptor p75NTR and tyrosine kinase receptor Met which has a similar tyrosine kinase structure as receptor TrkA has. Whether RanBPM interacts with neurotrophin receptor TrkA has not been established to date. In this study, using yeast two-hybrid system, it was identified that RanBPM bound to the intracellular domain (ICD) of neurotrophin receptor TrkA through its SPRY motif. We confirmed the formation of complexes between RanBPM and TrkA by co-immunoprecipitation studies and GST pull-down assays. The region of TrkA interacted with the SPRY domain of RanBPM was located in its tyrosine kinase domain. Furthermore, coimmunoprecipitaiton revealed endogenous RanBPM and receptors TrkA did interact in several mammalian cell lines. It was found that the overexpression of RanBPM could inhibit NGF-induced increase of nuclear factor of activated T cells (NFAT) dependent luciferase expression through its interaction with receptor TrkA, and NFAT transcriptional activity plays an important role in neuronal signal transduction. These data suggested that RanBPM could participate in neurotrophin-mediated gene transcription and expression by its binding to TrkA.

Adaptor Proteins, Signal Transducing↗

PubMed2006

Cytotoxicity of a recombinant fusion protein of adenovirus early region 4 open reading frame 4 (E4orf4) and human epidermal growth factor on p53-deficient tumor cells.

Adenovirus early region 4 open reading frame 4 (E4orf4) protein is a novel cell death factor that selectively induces p53-independent apoptosis in cancer cells, but not in normal human cells. This study presents an approach for inhibiting p53-deficient tumor cell growth by using protein-based E4orf4 that had been genetically fused to epidermal growth factor (EGF) to ensure selective targeting of EGF receptor-overexpressing tumor cells. EGF-E4orf4 enables binding onto the cell surface and is then internalized into Saos-2 cells. The success of the process had been demonstrated by immunofluorescence assay and confocal laser microscopy. After prolonged exposure, E4orf4 remained mostly in the nuclei. EGF-E4orf4 treatment of Saos-2 cells showed dose-dependent cytotoxicity. Nearly 50% of the Saos-2 cells were killed at a concentration of 250 nmol/l. In contrast, EGF-E4orf4 showed no significant inhibitory effect iresn primary cells of human umbilical vein endothelial cells. To confirm the ability of EGF-E4orf4 to induce apoptosis, DNA fragmentation was detected using BrdUTP end-labeling. Flow cytometric analysis revealed a significant increase of apoptotic cells in Saos-2 cells treated with EGF-E4orf4, but not in the case of cells cultured in plain medium (t=0.028, P<0.05). In conclusion, these preliminary results indicate that EGF-E4orf4 could show promise as a new reagent that is more efficient and less toxic in anti-cancer therapy.

Cell Death↗

PubMed2005

[Expression of epidermal growth factor-adenovirus E4orf4 fusion protein in tumor cells and its cytotoxicity].

BACKGROUND & OBJECTIVE: Human epidermal growth factor (EGF), an important growth factor, may stimulate cell growth and proliferation. EGF receptor (EGFR) expresses on the surface of normal cells, and abnormally over-expresses on many kinds of tumor cells, especially on solid tumor cells. Adenovirus early region 4 open reading frame 4 protein (E4orf4) is a novel cytotoxin that can specifically induce p53-independent apoptosis in tumor cells. Based on the targeting of EGF and cytotoxicity of E4orf4, we proposed to design a novel fusion protein at molecular level by recombining EGF and E4orf4 to target and then kill tumor cells. METHODS: EGF and E4orf4 coding sequences were amplified by polymerase chain reaction (PCR), and then genetically fused by overlapping PCR. EGF-E4orf4 fragment was cloned into the yeast expression vector. Recombinant plasmid DNA was transformed into the yeast Pichia pastoris. Fusion proteins were purified by SP Sepharose ion exchange chromatography. Cytotoxicity of EGF-E4orf4 on cultured BT325 and MDA-MB-231 cells was detected by MTT assay, and cell apoptosis was measured by flow cytometry. RESULTS: The fusion fragment has 805 base pairs, which consists of Kozak consensus sequence, and the sequences encoding alpha-factor signal peptide, EGF, flexible linker, and E4orf4. Recombinant plasmids pAO-EGF-E4orf4, and pAO-3EGF-E4orf4 were obtained, the latter contained 3 expression cassettes. Apparent molecular weight of fusion protein was 20 ku. Immunoblot analysis showed that the fusion protein was immunoreactive with rabbit-anti-human EGF polyclonal antibody. EGF-E4orf4 in high concentrations (5, and 0.5 microg/ml) inhibited growth of BT325 and MDA-MB-231 tumor cells as compared with controls. Apoptosis was induced in 15.4%-28.2% of MDA-MB-231 cells by EGF-E4orf4 at the dosage of 10-25 microg/3 x 10(5) cells. CONCLUSIONS: Fusion protein EGF-E4orf4 may enter cells mediated by EGFR, and thus inhibit growth of tumor cells.

Adenovirus E4 Proteins↗

PubMed2005

[The structure, classification and function of RGS proteins].

Regulators of G protein signaling (RGS) are a family of highly diverse, multifunctional signaling proteins that bind directly to activated Galpha subunits and markedly stimulates the GTPase activity of Galpha subunits leading to their deactivation and termination of downstream signals. RGS family members share a conserved 130-amino acid core domain which is responsible for binding Galpha subunits and negatively regulate G protein signal. Many RGS proteins possess additional non RGS domains and motifs. The non RGS domains are involved to integrate different G protein signaling pathways and as a saffolding protein that links G protein to related signaling proteins.

Protein Interaction Mapping↗

PubMed2003

RanBPM is a novel binding protein for p75NTR.

The neurotrophin receptor p75NTR can induce signal transduction both in vivo and in vitro. The mechanisms by which p75NTR transduces signals have remained mostly unknown. Using yeast two-hybrid system, we identified the Ran-binding protein (RanBPM) as an interactor with the intracytoplasmic domain of p75NTR (p75ICD). The interaction was then validated by immunoprecipitation in mammalian cells and immunoblotting analysis. The domain in p75ICD interacting with RanBPM was mapped to the death domain.

Adaptor Proteins, Signal Transducing↗

PubMed1997

Expression of Human Low-affinity Nerve Growth Factor Receptor in Mammanlian Neural Cells and its Induction of Apoptosis.

Human low-affinity nerve growth factor receptor (p75NGFR) was amplified from human fetal brain by RT-PCR and ligated into the retrovirus expression vector pXT-1. The cloned pXT-1/p75NGFR was packaged by PA37 cell line and the collected pseudovirus was used to infect neural cell line R2. The expression of p75NGFR on R2 cells at the RNA transcriptional and protein translation levels were demonstrated by Northern bloting and FITC-labeled immunological analysis. Further studies indicated that p75NGFR could induce apoptosis of neural cells in culture medium deprived of serum. Such cell death could be prevented by inhibitors of macromolecular syntheses such as actinomycin D and cycloheximidine.

Journal Article↗