Keke Huo

Cloning and characterization of a novel gene which encodes a protein interacting with the mitosis-associated kinase-like protein NTKL

AbstractNTKL is an evolutionarily conserved kinase-like protein. The cell-cycle-dependent centrosomal localization of NTKL suggested that it was involved in centrosome-related cellular function. The mouse NTKL protein is highly homologous with human NTKL. A novel mouse protein was identified as an NTKL-binding protein (NTKL-BP1) by yeast two-hybrid screening, and the full-length cDNA was amplified based on the result of a sequence data analysis cloning strategy. The full-length cDNA sequence of the NTKL-BP1 gene consists of 2,537 bp, which encode 368 amino acids. A database search revealed that homologues of NTKL-BP1 exist in different organisms, including Arabidopsis thaliana, Drosophila melanogaster, Plasmodium falciparum, Geobacter metallireducens, Anopheles gambiae and human. It suggests that NTKL-BP1 is an evolutionarily conserved protein. The expression of NTKL-BP1 was observed in multiple normal mouse tissues. The interaction of the two proteins was confirmed by co-immunoprecipitation. Moreover, immunofluorescent staining indicated that NTKL and NTKL-BP1 were all localized in the cytoplasm.

NUMBL interacts with TRAF6 and promotes the degradation of TRAF6

Tumor necrosis factor-associated factor 6 (TRAF6) is an essential adaptor protein for IL-1R or TLR-mediated NF-kappaB signaling pathway activation. In previous work we have found NUMBL interacts with TAB2 and negatively regulates NF-kappaB signaling pathway. Here, we report that NUMBL directly binds to TRAF6 in vivo and in vitro. NUMBL down-regulates TRAF6 protein level and shortens its half-life. Furthermore, knockdown of NUMBL significantly increases endogenous TRAF6 protein level in the cultured cortical neurons. In vivo ubiquitination assays indicate that NUMBL promotes the assembly of K48-linked polyubiquitination chains on TRAF6, but has no significant effect on its K63-linked polyubiquitination. Our results collectively reveal that NUMBL interacts with TRAF6 and promotes the degradation of TRAF6 in vivo, leading to the inhibition of NF-kappaB signaling pathway.

NUMBL interacts with TAB2 and inhibits TNFα and IL-1β-induced NF-κB activation

The cytokines TNFalpha and IL-1beta induce inflammation through activation of transcription factors NF-kappaB. TAB2 is an adapter protein that facilitates TNFalpha and IL-1beta-mediated NF-kappaB activation. In this work, using yeast two-hybrid system TAB2 was identified to interact with NUMBL. The interaction was further confirmed in vitro and in vivo. PTB domain of NUMBL and C-terminal region are required for their interaction. Overexpression of NUMBL inhibited TNFalpha, IL-1beta-induced activation of NF-kappaB signaling pathway. NUMBL also inhibited TAB2, TAK1, TRAF6 and RIP-induced activation of NF-kappaB in a dose-dependent manner. We found that NUMBL can impair TAB2 binding to TRAF6 or RIP and inhibit ubiquitination of TRAF6 enhanced by TAB2. Taken together, our data suggest that NUMBL is involved in negative regulation of NF-kappaB signaling through its interaction with TAB2. These findings also reveal the new functions of NUMBL and implicate that NUMBL potentially links Notch pathway to NF-kappaB pathway.

RIOK3 interacts with caspase-10 and negatively regulates the NF-κB signaling pathway

RIOK3 was initially characterized as a homolog of Aspergillus nidulans sudD and showed down-regulation at the invasive front of malignant melanomas, but the molecular mechanism remains elusive. Here, we report that overexpression of RIOK3 inhibits TNFα-induced NF-κB activation, but down-regulation of endogenous RIOK3 expression by siRNA potentiates it. A yeast two-hybrid experiment revealed that RIOK3 interacted with caspase-10, and further, a GST pull-down assay and endogenous coimmunoprecipitation validated the interaction. We subsequently showed that the interaction was mediated by the RIO domain of RIOK3 and each death effector domain of caspase-10. Interestingly, our data demonstrated that RIOK3 suppressed caspase-10-mediated NF-κB activation by competing RIP1 and NIK to bind to caspase-10. Importantly, the kinase activity of RIOK3 was confirmed to be relevant to NF-κB signaling. Taken together, our findings strongly suggest that RIOK3 negatively regulates NF-κB signaling pathway activated by TNFα dependent on its kinase activity and NF-κB signaling pathway activated by caspase-10 independent of its kinase activity.

SSX2IP promotes metastasis and chemotherapeutic resistance of hepatocellular carcinoma

BackgroundSynovial sarcoma, X breakpoint 2 interacting protein (SSX2IP), which has been identified as an acute myeloid leukemia associated antigen, is a potential target for leukemia immunotherapy. In rodents, its homologous gene, ADIP, plays an important role in the regulation of cell adhesion and migration, underlying its potential role in promoting metastasis of other cancers.MethodsTo investigate the correlation between the expression level of SSX2IP and the clinicopathologic factors of hepatocellular carcinoma (HCC), 53 cases were studied by qPCR and statisted. To directly testing SSX2IP’s contribution to HCC in animal models, 45 nude mice were enrolled in peritoneal spreading and liver metastasis models. For the migration and invasion assays, cell culture experiments were performed using QCMTM 24-Well Colorimetric Migration Assay Kit and Cell Invasion Assay Kit (Millipore). Moreover we examined the influence of SSX2IP overexpression on the chemosensitivity of hepatocellular carcinoma cells to two most common chemotherapy drugs (5-Fu and CDDP) using Cell counting kit-8 (CCK-8). The chemotherapeutic drugs sensitivity was evaluated by IC50 parameter.ResultsStatistical analysis of clinical cases revealed that the SSX2IP high expression group had inclinations towards larger tumor size, more tumor thrombus and shorter survival period, implying a strong correlation between the expression level of SSX2IP and HCC tumorigenesis. Consistently in abdominal cavity metastasis and liver metastasis models of immune-deficient mice, SSX2IP was able to promote the metastasis of hepatoma cells. At the cytological level, SSX2IP stimulates the wound healing, metastasis and invasion of hepatoma cells, and reduces the sensitivity of hepatoma cells to 5-Fu and CDDP.ConclusionsOur results showed that SSX2IP promotes the development and metastasis of hepatocellular carcinoma and contributes to the drug resistance of hepatoma cells, suggesting that SSX2IP is expected to become a new diagnostic and prognostic marker and a new target of the treatment of hepatocellular carcinoma.

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.

C12ORF39, a novel secreted protein with a typical amidation processing signal

In the present study we describe a novel secreted protein, named C12ORF39 (chromosome 12 open-reading framework 39), which contains a typical amidation/proteolytic processing signal (Gly-Arg-Arg motif). Interestingly, C12ORF39 protein is not hydrolysed, but is a full-length protein without signal peptides. Western blotting indicated that c-Myc-tagged C12ORF39 is secreted into culture medium in transfected HeLa cells. Quantitative RT-PCR (reverse transcription-PCR) analysis revealed that c12orf39 is mainly expressed in placenta and brain. Immunohistochemistry on formalin-fixed paraffin-embedded human term placenta using a rabbit antibody against human C12ORF39 demonstrated that the protein was localized extracellularly, surrounding the trophoblastic cells. In addition, C12ORF39 secretion could be blocked by brefeldin A, suggesting that the secretion of C12ORF39 is dependent on the Golgi apparatus. Furthermore, laser-scanning confocal microscopy also confirmed that the C12ORF39 protein co-localized with the Golgi apparatus. Taken together, although C12ORF39 is not a secreted small peptide, it can also be secreted to play a role in the biological functions of the placenta.

Eukaryotic translation elongation factor 1 delta inhibits the ubiquitin ligase activity of SIAH-1

SIAH-1, an E3 ubiquitin ligase, plays an important role in regulating cell cycle, tumorigenesis and several neurodegenerative diseases. In this study, we found a novel SIAH-1-interacting protein, EEF1D (Eukaryotic translation elongation factor 1 delta). The interaction was confirmed in vitro and in vivo, and both proteins were co-localized in the cytoplasm. The Cys-rich domain of SIAH-1 was essential for its interaction with EEF1D. Overexpressing SIAH-1 had no effect on the protein level of EEF1D, implying that EFF1D is not the substrate of SIAH-1. In contrast, the protein level of SIAH-1 increased significantly in the cells overexpressing EEF1D. Increased amount of SIAH-1 was caused by the EEF1D-mediated inhibition of auto-ubiquitination and degradation of SIAH-1. Furthermore, EEF1D was able to inhibit the degradation of HPH2, a known substrate of SIAH-1. Taken together, our data suggest EFF1D functions as a novel negative regulator of SIAH-1.

Overexpression of SCYL1-BP1 stabilizes functional p53 by suppressing MDM2-mediated ubiquitination.

Previously, we defined SCY1‐like 1 binding protein 1 (SCYL1‐BP1) to be a substrate of Pirh2 that binds to mouse double minute gene number 2 (MDM2). In the current study, we found that an increase in SCYL1‐BP1 protein levels caused a parallel change in the amount of p53 protein due to the inhibition by SCYL‐BP1 of MDM2‐mediated p53 ubiquitination. SCYL1‐BP1 was not able to alter the ubiquitination of p53 by human papillomavirus protein E6, indicating that the effect was specific for MDM2. Increases in the level of SCYL1‐BP1 protein in cells led to the greater transcriptional activation of p21 and gadd45, reduced rate of cellular proliferation, increased levels of apoptosis and inhibition of tumorigenicity. Thus, we propose that SCYL1‐BP1 is a novel regulator of the MDM2‐p53 feedback loop and that it may be a potential tumor suppressor.

Cloning of the KcURA3 gene and development of a transformation system for Kluyveromyces cicerisporus

KcURA3 was cloned from Kluyveromyces cicerisporus CBS4857 by complementation of the ura3 mutation in Saccharomyces cerevisiae. KcURA3 encodes a 267-amino-acid protein with 80% sequence identity to that of S. cerevisiae. An ura3 mutant strain from K. cicerisporus CBS4857, named Y179U, was obtained by selection on 5-fluoroorotic acid plates. Sequence analysis of this mutated gene revealed that it contained a point mutation at nucleotide position +277. Two vectors, pUK1 and pUKD, bearing KcURA3 were constructed. Either the lithium acetate method or electroporation could be used to transform pUK1 and pUKD intoY179U. The transformation efficiency using electroporation was higher than that using the lithium acetate method.