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Xiaohuan Guo,
Yanfei Zhang, Pingzhang Wang,
Ting Li,
Weiwei Fu,
Xiaoning Mo,
Taiping Shi,
Zhixin Zhang,
Yingyu Chen,
Dalong Ma,
Wenling Han
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ABSTRACT: Cytokines are soluble proteins that mediate immune reactions and are responsible for communication among immune cells. CD4(+) T cells are the principle sources of cytokines of adaptive immunity. Cytokines play critical roles in the differentiation and effector function of CD4(+) T cells. They also play key roles in diseases, and some of them have been developed into drugs in the forms of recombinant cytokines, soluble receptors and neutralizing antibodies. Therefore, identifying novel potential cytokines is necessary and beneficial for better understanding immunology and enhancing human health. To find novel potential cytokines, we carried out an integrated bioinformatics analysis on the whole human genome. Cytokine candidates were selected for cDNA cloning, sub-cloning, secretion verification, expression profile analysis and functional study. Here, we report a novel soluble protein, VSTM1-v2, which is a classical secretory glycoprotein mainly expressed in immune tissues, and can promote the differentiation and activation of Th17 cells.
Cellular Immunology 08/2012; 278(1-2):136-142. · 1.97 Impact Factor
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ABSTRACT: MAP kinase-interacting kinase-2 (Mnk2) is one of the downstream kinases activated by MAP kinases. It phosphorylates the eukaryotic
initiation factor 4E (elF4E), although the role of elF4E phosphorylation and the role of Mnk2 in the process of protein translation
are not well understood. Except for elF4E, other physiological substrates of Mnk2 are still unidentified. To look for these
unidentified substrates and to reveal the physiological function of Mnk2, we performed a yeast two-hybrid screening with Mnk2
as the bait. The results demonstrated Mnk2 could interact with VHL (von Hippel-Lindau tumor suppressor), Rbx1 (ring-box 1)
and Cul2 (Cullin2) proteins in yeast cells. Furthermore, we validated the interaction between Mnk2 and VHL proteins in mammalian
cells by co-immunoprecipitation analysis. Because the three proteins VHL, Rbx1 and Cul2 are all components of the CBCVHL ubiquitin ligase E3 complex, it has been shown that Mnk2 can interact with CBCVHL complex, and is probably one of the new substrates of the CBCVHL complex. Furthermore, during the interaction of Mnk2 with von Hippel-Lindau (VHL) tumor suppressor-binding protein 1 (VBP1),
it appears that Mnk2 also joins to modulate cell shape as VBP1 plays an important role in the process of the maturation of
the cytoskeleton and in the process of morphogenesis.
Science in China Series C Life Sciences 04/2012; 49(3):265-273. · 1.61 Impact Factor
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ABSTRACT: Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) has many transcript variants, but whether they possess distinct function is not completely known. In the present study, we compared the function of these TRAIL variants.
A bioinformatics analysis was performed to examine potential TRAIL variants. For the functional study, over-expression of TRAIL isoforms was used to examine their NF-κB inducing and apoptotic activities in both cancer and normal cells. Moreover, soluble TRAIL E4 variant protein was expressed and purified in prokaryotic cells, and was used for apoptotic assay.
We cloned seven truncated TRAIL variants, designated as AK, E2, E3, E4, DA, BX424, and BX439. In comparison with the wild type TRAIL protein expressed from full-length RefSeq, over-expression of all these TRAIL variants activated NF-κB and its targeting genes in human cells at varying degrees. Some isoforms including BX424, DA and E4 even showed NF-κB, IL8, CCL4 and CCL20 promoter activating activity stronger than the wild type protein. All truncated variant proteins had no toxicity to normal human cells, similar to the wild type protein; however, they all failed to induce apoptosis in cancer cells that are sensitive to TRAIL. Recombinant soluble TRAIL E4 protein also failed to antagonize TRAIL-induced apoptosis in cancer cells.
Truncated TRAIL variant proteins lost apoptotic activity but retained or even enhanced the NF-κB activating potentials, these results suggest that TRAIL variants may play roles in non-apoptotic cellular processes that are more important than we previously thought.
Life sciences 09/2011; 89(23-24):839-46. · 2.56 Impact Factor
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ABSTRACT: The c-Jun NH(2)-terminal kinase (JNK) signaling pathway participates in many physiological functions. In the current study we reported the cloning and characterization of five novel JNK2 transcript variants, which were designated as JNK2α3, JNK2α4, JNK2β3, JNK2γ1 and JNK2γ2, respectively. Among them, JNK2α4 and JNK2γ2 are potential non-coding RNA because they contain pre-mature stop codons. Both JNK2α3 and JNK2β3 contain an intact kinase domain, and both encode a protein product of 46 kDa, the same as those of JNK2α1 and JNK2β1. JNK2γ1 contains a disrupted kinase domain and it showed a disable function. When over-expressed in mammalian cells, JNK2α3 showed higher activity on AP-1 than that of JNK2β3 and JNK2γ1. Furthermore, JNK2α3 and JNK2β3 showed different levels of substrate phosphorylation, although they both could promote the proliferation of 293T cells. Our results further demonstrate that JNK2 isoforms preferentially target different substrates and may regulate the expression of various target genes.
BMB reports 11/2010; 43(11):738-43. · 1.72 Impact Factor
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ABSTRACT: Protein kinases are involved in comprehensive cellular processes and also implicated in many human diseases. SH3-binding domain kinase 1 (SBK1) was first cloned and characterized in rat and the human cDNA was cloned in our lab in 2006, but the expression and function of endogenous protein have not been well studied in human. In this follow up study, we screened a panel of cell lines and tissues, as well as a tumor tissue array for SBK1 expression at both RNA and protein levels. To detect the protein, we generated the first rabbit polyclonal antibody against human SBK1. We show that the SBK1 is expressed in most of the cells and tissues examined, and the protein is highly up-regulated in ovarian serous adenocarcinoma while down-regulated in esophagus squamous cell carcinoma and stomach adenocarcinoma. When over-expressed in an ovarian cancer cells SK-OV-3 by adenovirus infection, SBK1 protected the cells from apoptosis induced by the viral infection, therefore promoting cancer cell survival. Given that a missense mutation K92E in human SBK1 was identified recently from ovarian mucinous carcinoma, together, these results suggest that the wide-spread expression pattern of human SBK1 may predict a broad cellular function, and its dysregulated in certain cancers suggests an involvement of the protein in the pathogenesis of human cancers.
Molecular Biology Reports 11/2010; 38(5):3551-9. · 2.93 Impact Factor
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ABSTRACT: Mitogen-activated protein kinase (MAPK) cascades play an important role in regulation of AP-1 activity through the phosphorylation of distinct substrates. In the present study, we identified a novel protein, TMEM174, whose RNA transcripts are highly expressed in human kidney tissue. TMEM174 is comprised of 243 amino acids, and contains two predicted transmembrane helices which determine its subcellular localization in endoplasmic reticulum and influences its functions. Over-expression of TMME174 enhanced the transcriptional activity of AP-1 and promoted cell proliferation, whereas the truncated mutant TMEM174DeltaTM without the transmembrane regions did not retain these functions. The possible mechanism of activation of AP-1 by TMEM174 was further examined. Our results suggest the potential role of TMEM174 in renal development and physiological function.
Biochemical and Biophysical Research Communications 03/2010; 394(4):993-9. · 2.48 Impact Factor
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ABSTRACT: Alternative polyadenylation sites within a gene can lead to alternative transcript variants. Although bioinformatic analysis has been conducted to detect polyadenylation sites using nucleic acid sequences (EST/mRNA) in the public databases, one special type, single-block EST is much less emphasized. This bias leaves a large space to discover novel transcript variants.
In the present study, we identified novel transcript variants in the human genome by detecting intronic polyadenylation sites. Poly(A/T)-tailed ESTs were obtained from single-block ESTs and clustered into 10,844 groups standing for 5,670 genes. Most sites were not found in other alternative splicing databases. To verify that these sites are from expressed transcripts, we analyzed the supporting EST number of each site, blasted representative ESTs against known mRNA sequences, traced terminal sequences from cDNA clones, and compared with the data of Affymetrix tiling array. These analyses confirmed about 84% (9,118/10,844) of the novel alternative transcripts, especially, 33% (3,575/10,844) of the transcripts from 2,704 genes were taken as high-reliability. Additionally, RT-PCR confirmed 38% (10/26) of predicted novel transcript variants.
Our results provide evidence for novel transcript variants with intronic poly(A) sites. The expression of these novel variants was confirmed with computational and experimental tools. Our data provide a genome-wide resource for identification of novel human transcript variants with intronic polyadenylation sites, and offer a new view into the mystery of the human transcriptome.
BMC Genomics 11/2009; 10:518. · 4.07 Impact Factor
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ABSTRACT: 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 TNFalpha-induced NF-kappaB 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-kappaB activation by competing RIP1 and NIK to bind to caspase-10. Importantly, the kinase activity of RIOK3 was confirmed to be relevant to NF-kappaB signaling. Taken together, our findings strongly suggest that RIOK3 negatively regulates NF-kappaB signaling pathway activated by TNFalpha dependent on its kinase activity and NF-kappaB signaling pathway activated by caspase-10 independent of its kinase activity.
Molecular and Cellular Biochemistry 07/2009; 332(1-2):113-20. · 2.06 Impact Factor
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ABSTRACT: Caspase-10 (also known as Mch4 and FLICE2) is an initiator caspase in the death receptor (DR)-dependent apoptotic pathway. So far six splice variants (caspase-10a-f) have been identified. Here we describe a novel isoform of the caspase-10 family named caspase-10g that is widely expressed in normal human tissues and various cell lines. Caspase-10g consists of 247 amino acids and does not contain the large or small subunit. A caspase-10g-specific exon is present between exon 5 and exon 6, which results in a protein product truncated shortly after the death-effector domain (DED)-containing prodomain. We further show that overexpression of caspase-10g dramatically enhances NF-kappaB activity in a dose- and time-dependent manner. Moreover, caspase-10g, unlike the protease-active caspase-10a, only promotes slight apoptosis when overexpressed in mammalian cells and it has no effect on caspase-10a-mediated apoptosis. Taken together, these results suggest that caspase-10g, as a novel prodomain-only isoform of caspase-10, may play a regulatory role preferentially in the NF-kappaB pathways.
Biochimica et Biophysica Acta 12/2007; 1770(11):1528-37. · 4.66 Impact Factor
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ABSTRACT: In this study, cell microarray technology is used to identify novel human genes associated with CRE pathway activation. By reverse transfection, expression plasmids containing full-length cDNAs were cotransfected with the reporter plasmid pCRE-d2EGFP to monitor the activation of the CRE pathway via enhanced green fluorescence protein (EGFP) expression. Of the 575 predominantly novel genes screened, 22 exhibited relatively higher EGFP fluorescence compared with a negative control. After a functional validation with a dual luciferase reporter system that included both cis- and trans-luciferase assays, 4 of the 22 genes (RNF41, C8orf32, C6orf208, and MEIS3P1) were confirmed as CRE-pathway activators. Western blot analysis revealed that RNF41 can promote CREB phosphorylation. These results demonstrate the successful combination of cell microarray technology with this reporting system and the potential of this tool to characterize functions of novel genes in a highly parallel format.
Genomics 08/2007; 90(1):28-34. · 3.02 Impact Factor
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ABSTRACT: Caspase-9 plays a key role in the intrinsic apoptotic pathway and currently two splice variants (caspase-9-alpha and -beta) have been identified. The present study cloned and characterized a novel caspase-9 splice variant, hereby designated Casp9-gamma. Casp9-gamma is generated from an additional alternative 3' splice site in the fourth exon of caspase-9, resulting in a 58-nucleotide fragment insertion compared with the full-length caspase-9-alpha. The fragment introduces an in-frame stop codon, and the resulting open reading frame (ORF) is preterminated. The Casp9-gamma comprises the deduced 154 amino acid residues containing only the caspase recruitment domain (CARD) and does not contain the large and small subunits. The Casp9-gamma does not promote apoptosis when overexpressed in mammalian cells. Moreover, it inhibits the cleavage of procaspase-3 mediated by proapoptotic member Bax or apoptosis inductor staurosporine. Therefore, Casp9-gamma may function as an endogenous apoptotic inhibitor by interfering with the CARD-CARD interaction between Apaf-1 (apoptotic protease activating factor-1) and procaspase-9. In addition, Casp9-gamma does not enhance NF-kappaB activation in transfected 293T cells, conflicting with previous evidence that the isolated CARD of caspase-9 activates NF-kappaB in ND7 cells. This suggests that the procaspase-9-mediated NF-kappaB activation in response to cellular stresses is cell type-specific through an unidentified mechanism.
Life Sciences 09/2006; 79(10):934-40. · 2.53 Impact Factor
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ABSTRACT: MAP kinase-interacting kinase-2 (Mnk2) is one of the downstream kinases activated by MAP kinases. It phosphorylates the eukaryotic initiation factor 4E (eIF4E), although the role of eIF4E phosphorylation and the role of Mnk2 in the process of protein translation are not well understood. Except for eIF4E, other physiological substrates of Mnk2 are still unidentified. To look for these unidentified substrates and to reveal the physiological function of Mnk2, we performed a yeast two-hybrid screening with Mnk2 as the bait. The results demonstrated Mnk2 could interact with VHL (von Hippel-Lindau tumor suppressor), Rbx1 (ring-box 1) and Cul2 (Cullin2) proteins in yeast cells. Furthermore, we validated the interaction between Mnk2 and VHL proteins in mammalian cells by co-immunoprecipitation analysis. Because the three proteins VHL, Rbx1 and Cul2 are all components of the CBC(VHL) ubiquitin ligase E3 complex, it has been shown that Mnk2 can interact with CBC(VHL) complex, and is probably one of the new substrates of the CBC(VHL) complex. Furthermore, during the interaction of Mnk2 with von Hippel-Lindau (VHL) tumor suppressor- binding protein 1 (VBP1), it appears that Mnk2 also joins to modulate cell shape as VBP1 plays an important role in the process of the maturation of the cytoskeleton and in the process of morphogenesis.
Science in China Series C Life Sciences 07/2006; 49(3):265-73. · 1.61 Impact Factor
