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Jian Wang,
Keke Huo,
Lixin Ma,
Liujun Tang,
Dong Li,
Xiaobi Huang,
Yanzhi Yuan,
Chunhua Li,
Wei Wang,
Wei Guan, [......],
Xiang Gan,
Xiaolan Yu,
Qi Ma,
Jing Yan,
Li Zhou,
Zhongyang Liu,
Yunping Zhu,
Tao Zhou,
Fuchu He,
Xiaoming Yang
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ABSTRACT: Proteome-scale protein interaction maps are available for many organisms, ranging from bacteria, yeast, worms and flies to humans. These maps provide substantial new insights into systems biology, disease research and drug discovery. However, only a small fraction of the total number of human protein-protein interactions has been identified. In this study, we map the interactions of an unbiased selection of 5026 human liver expression proteins by yeast two-hybrid technology and establish a human liver protein interaction network (HLPN) composed of 3484 interactions among 2582 proteins. The data set has a validation rate of over 72% as determined by three independent biochemical or cellular assays. The network includes metabolic enzymes and liver-specific, liver-phenotype and liver-disease proteins that are individually critical for the maintenance of liver functions. The liver enriched proteins had significantly different topological properties and increased our understanding of the functional relationships among proteins in a liver-specific manner. Our data represent the first comprehensive description of a HLPN, which could be a valuable tool for understanding the functioning of the protein interaction network of the human liver.
Molecular Systems Biology 01/2011; 7:536. · 8.63 Impact Factor
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ABSTRACT: Analysis of protein-protein interactions (PPIs) is a valuable approach for the characterization of huge networks of protein complexes or proteins of unknown function. Co-immunoprecipitation (coIP) using affinity resins coupled to protein A/G is the most widely used method for PPI detection. However, this traditional large scale resin-based coIP is too laborious and time consuming. To overcome this problem, we developed a miniaturized sandwich immunoassay platform (MSIP) by combining antibody array technology and coIP methods.
Based on anti-FLAG antibody spotted aldehyde slides, MSIP enables simple, rapid and large scale detection of PPIs by fluorescent labeling anti-myc antibody. By analyzing well-known interacting and non-interacting protein pairs, MSIP was demonstrated to be highly accurate and reproducible. Compared to traditional resin-based coIP, MSIP results in higher sensitivity and enhanced throughput, with the additional benefit of digital read-outs. In addition, MSIP was shown to be a highly useful validation platform to confirm PPI candidates that have been identified from yeast two hybrid systems.
In conclusion, MSIP is proved to be a simple, cost-saving and highly efficient technique for the comprehensive study of PPIs.
BMC Biotechnology 10/2010; 10:78. · 2.35 Impact Factor
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ABSTRACT: The Ras-MAPK and PI3K-AKT pathways are conserved in metazoan organisms, which involve a series of signaling cascades and form the basis for numerous physiological and pathological processes. Here we report on yeast two hybrid screening results of a protein interaction network around the known components of human Ras-MAPK/PI3K pathways. A total of 42 independent cDNA library screenings resulted in 200 protein-protein interaction (PPI) pairs among 180 molecules. Most of the proteins formed a large cluster that contains 193 PPIs between 169 proteins. Seventy-four interactions indicate high-confidence according to bioinformatics analysis. The prey list contains high enrichment genes with specific Gene Ontology (GO) terms such as response to stress and response to external stimulus. Most interactions link the Ras signaling pathway with various cellular processes. Five interactions were validated by coimmunoprecipitation and colocalization assays in mammalian cells to confirm their in vivo interactions. This protein interaction network provides further insights into the molecular mechanism of Ras-MAPK/PI3K signaling pathways.
Journal of Proteome Research 08/2008; 7(9):3879-89. · 5.11 Impact Factor
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Jing Sun,
Jing Nie,
Bingtao Hao,
Lu Li,
Guichun Xing,
Zhaoqing Wang, Ying Zhou,
Qihong Sun,
Guiyuan Li,
Lingqiang Zhang,
Fuchu He
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ABSTRACT: The centrosome associated protein Ceap-16 (also termed BLOS2) can accelerate the proliferation of mouse fibroblast NIH3T3 cells, which mechanism remains unclear. Here we identified tumor suppressor candidate BRD7 (bromodomain containing protein 7), which could negatively regulate cell proliferation and growth, as a novel Ceap-16-interacting protein. Ceap-16 and BRD7 interacted with each other both in vitro and in vivo. The C-terminus of BRD7 and the central region of Ceap-16 mediated the interaction. Through this binding, Ceap-16 could translocate from cytoplasm to the nucleus where it selectively inhibited the transcriptional suppression activity of BRD7 towards certain target genes including E2F3 and cyclin A. Moreover, Ceap-16, BRD7 and histone H3/H4 could form a complex and Ceap-16 did not compete with BRD7 binding to histones. These findings suggest a novel function for Ceap-16 in the transcriptional regulation through associating with BRD7.
Cellular Signalling 07/2008; 20(6):1151-8. · 4.06 Impact Factor
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ABSTRACT: Tribbles 3 homolog (TRB3) is recently identified as a scaffold-like regulator of various signal transducers and has been implicated in several processes including insulin signaling, NF-kappaB signaling, lipid metabolism and BMP signaling. To further understand cellular mechanisms of TRB3 regulation, we performed a yeast two-hybrid screen to identify novel TRB3 interacting proteins and totally obtained ten in-frame fused preys. Candidate interactions were validated by co-immunoprecipitation assays in mammalian cells. We further characterized the identified proteins sorted by Gene Ontology Annotation. Its interaction with the E3 ubiquitin ligase SIAH1 was further investigated. SIAH1 could interact with TRB3 both in vitro and in vivo. Importantly, SIAH1 targeted TRB3 for proteasome-dependent degradation. Cotransfection of SIAH1 could withdraw up-regulation of TGF-beta signaling by TRB3, suggesting SIAH1-induced degradation of TRB3 represents a potential regulatory mechanism for TGF-beta signaling.
Cellular Signalling 06/2008; 20(5):942-8. · 4.06 Impact Factor
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ABSTRACT: Microtubule associated proteins are involved in regulation of microtubule dynamics. Its mutation and dysregulation result in severe consequences such as mitotic block and apoptosis. NuSAP has been reported as a microtubule associated protein, depletion of which by RNAi results in spindle deficiency and cytokinesis failure. However, its role in regulation of cell cycle and how NuSAP protein is controlled during cell cycle progression still remains unclear. Here we show that NuSAP can be ubiquitinated and degraded by APC/C-hCdh1 E3 ligase. Evolutionally conserved KEN box functions as the degron of NuSAP. Overexpression of NuSAP induces mitotic arrest and the microtubule associated domain and nuclear localization are both required for NuSAP to induce mitotic arrest. Furthermore, overexpression of NuSAP results in cells accumulation with microtubule bundling and spindle deficiency. Thus, our results give evidence for the first time that NuSAP protein level is tightly regulated by the APC/C ubiquitin ligase complex and NuSAP induces mitotic arrest dependent of its microtubule affinity.
Cellular Signalling 11/2007; 19(10):2046-55. · 4.06 Impact Factor
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Zhaoqing Wang,
Handong Wei,
Yongtao Yu,
Jing Sun,
Yi Yang,
Guichun Xing,
Songfeng Wu, Ying Zhou,
Yunping Zhu,
Chenggang Zhang,
Tao Zhou,
Xiaohang Zhao,
Qihong Sun,
Fuchu He
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ABSTRACT: A novel human gene, encoding two polypeptide-isoforms, has been identified from human fetal liver cDNA library. These two alternatively spliced polypeptide-variants are associated with centrosomes, and are designated Ceap-11 and Ceap-16, respectively, according to the acronym Ceap for centrosomal-associated protein and the approximate relative molecular mass. The high degree of sequence similarity between Ceap proteins of divergent species indicates that the Ceap homologous genes are significantly conserved in evolution and constitute a new gene family without any functional information until now. Human Ceap gene is mapped on 10q24.2. These two Ceap cDNA isoforms are generated by RNA alternative splicing on the 5' terminus of the Ceap gene, and are composed of four and five exons, respectively. Ceap-11 and Ceap-16 are co-immunoprecipitated and co-located with gamma-tubulin; ectopic overexpression of these two proteins in NIH3T3 cells induces microtubule aggregation and cell proliferation; the protein level of Ceap in certain tumors is significantly higher than that in corresponding normal tissues. Taken together, our data provide the first evidence for the function of Ceap-11 and Ceap-16, the two novel human proteins, namely, association with centrosome, microtubule aggregation and cell proliferation.
Journal of Molecular Biology 11/2004; 343(1):71-82. · 4.00 Impact Factor
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ABSTRACT: Microtubule associated proteins are involved in regulation of microtubule dynamics. Its mutation and dysregulation result in severe consequences such as mitotic block and apoptosis. NuSAP has been reported as a microtubule associated protein, depletion of which by RNAi results in spindle deficiency and cytokinesis failure. However, its role in regulation of cell cycle and how NuSAP protein is controlled during cell cycle progression still remains unclear. Here we show that NuSAP can be ubiquitinated and degraded by APC/C–hCdh1 E3 ligase. Evolutionally conserved KEN box functions as the degron of NuSAP. Overexpression of NuSAP induces mitotic arrest and the microtubule associated domain and nuclear localization are both required for NuSAP to induce mitotic arrest. Furthermore, overexpression of NuSAP results in cells accumulation with microtubule bundling and spindle deficiency. Thus, our results give evidence for the first time that NuSAP protein level is tightly regulated by the APC/C ubiquitin ligase complex and NuSAP induces mitotic arrest dependent of its microtubule affinity.
Cellular Signalling.
