• Journal of Life Science
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• v.23 no.11
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• pp.1311-1316
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• 2013

Heterogeneous nuclear ribonucleoprotein L (hnRNP L) is a major pre-mRNA binding protein and it is an abundant nuclear protein that shuttles between the nucleus and the cytoplasm. hnRNP L is known to be related to many cellular processes, including chromatin modification, pre-mRNA splicing, mRNA export of intronless genes, internal ribosomal entry site (IRES)-mediated translation, mRNA stability, and spermatogenesis. In order to identify the cellular proteins interacting with hnRNP L, this study performed a yeast two-hybrid screening, using a human liver cDNA library. The study identified insulin-like growth factor binding protein-4 (IGFBP-4) as a novel interaction partner of hnRNP L in the human liver. It then discovered, for the first time, that hnRNP L interacts specifically with IGFBP-4 in a yeast two-hybrid system. The authenticity of this two-hybrid interaction of hnRNP L and IGFBP-4 was confirmed by an in vitro pull-down assay.

• Microbiology and Biotechnology Letters
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• v.33 no.4
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• pp.260-266
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• 2005

IgE-dependent histamine releasing factor (HRF), previously known as P23/P21 or translationally controlled tumor protein (TCTP), induces the degranulation of histamine in mast cell and basophil. Yeast two hybrid results showed that HRF interacts with the alpha subunit of Na, K-ATPase, suggesting that HRF is a regulator for governing the activity of Na, K-ATPase. In this study, we examined the interaction of HRF and Wa,K-ATPase after treatments of various PKC isotype inhibitors. Membrane fractionation, pull-down assay and immunoprecipitation results showed that PKC $\alpha,\;PKC\;\beta,\;\delta$ subunits are involved in the phosphorylation of HRF. However, these results did not correlate with the results of histamine release assay since histamine release assay results suggested that some PKC isotype inhibitors induced the histamine release in RBL-2H3 cell.

• Biomedical Science Letters
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• v.12 no.4
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• pp.303-310
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• 2006

Ankyrins are a ubiquitously expressed family of intracellular adaptor proteins involved in targeting diverse proteins to specialized membrane domains in both the plasma membrane and the endoplasmic reticulum. We described here that the C-terminal domain of ankyrin-B interact specifically with Z-line portion of titin in yeast two-hybrid analysis, in vitro pull-down assays and localization experiments in COS-7 cells. In this study we provide the first experimental evidence that Z-line portion of titin is necessary for the localization of ankyrin-B and ankyrin-B links between the sarcolemma and the myofibril in costameres.

• Animal cells and systems
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• v.13 no.3
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• pp.283-288
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• 2009

The ubiquitous plasma membrane $Na^+/H^+$ exchanger 1 (NHE1) is rapidly activated in response to various extracellular stimuli and maintains normal cytoplasmic pH. Yeast two-hybrid screening was used in order to identify proteins interacting with NHE1 using its cytoplasmic domain as a bait from HeLa cDNA library. One of the interacting cDNA clones was human Lactate dehydrogenase B (LDHB). In vitro translated LDHB was pulled down together with GST-NHE1.cd protein in the GST pull down assay, confirming the interaction in vitro. LDHB antibody immunoprecipitated endogenous LDHB together with NHE1 from H9c2 cells, validating cellular interaction between NHE1 and LDHB. Subsequent analysis revealed that the overexpression of LDHB increased intracellular PH, implying opening of the NHE1 transporter. Moreover, overexpression of LDHB activated caspase 3 and induced cell death, consistent with the expected phenotype of hyper-activation of NHE1. Collectively, our data indicate that LDHB modulates NHE1 activity via physical interaction.

• BMB Reports
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• v.38 no.4
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• pp.381-385
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• 2005

Severe acute respiratory syndrome (SARS) is an emerging infectious disease associated with a novel coronavirus (CoV) that was identified and molecularly characterized in 2003. Previous studies on various coronaviruses indicate that protein-protein interactions amongst various coronavirus proteins are critical for viral assembly and morphogenesis. It is necessary to elucidate the molecular mechanism of SARS-CoV replication and rationalize the anti-SARS therapeutic intervention. In this study, we employed an in vitro GST pull-down assay to investigate the interaction between the membrane (M) and the nucleocapsid (N) proteins. Our results show that the interaction between the M and N proteins does take place in vitro. Moreover, we provide an evidence that 12 amino acids domain (194-205) in the M protein is responsible for binding to N protein. Our work will help shed light on the molecular mechanism of the virus assembly and provide valuable information pertaining to rationalization of future anti-viral strategies.

• BMB Reports
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• v.34 no.1
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• pp.80-84
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• 2001

The human papillomavirus E7 protein can form a specific complex with a retinoblastoma tumor suppressor gene product (p105-Rb) that results in the release of the E2F transcription factor, which is critical for the growth-deregulation and transforming properties of the viral E7 oncoprotein. In an attempt to apply interaction between the E7 oncoprotein and a target cellular protein Rb for an in vitro screening system for drugs against human papillomavirus infection, we primarily investigated the E7Rb binding through a pull down assay and enzyme-linked immunosorbent assay. The pull down assay showed that both glutathione S-transferase-tagged E7 and His-tagged E7 immobilized on resins specifically produced complexes with bacterially expressed Rb in a dose-dependent manner, as determined by immunoblot analyses. This result coincided with that of an enzyme-linked immunosorbent assay, which is a useful system for the mass screening of potential drugs. Taken together, this screening system (based on the interaction between E7 and Rb) can be a promising system in the development of drugs against cervical cancers caused by human papillomavirus infection.

• Journal of Life Science
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• v.15 no.6 s.73
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• pp.928-934
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• 2005

ATF2 is a cellular transcription factor which belongs to the CREB/ATF class and it is leucine zipper protein which generally binds to DNA as dimers. This paper presents the procedure for subcloning the ATF2 gene and the results of experiment used the expressed ATF2. The pET expression vector was used since it produced 6xHis fusion protein for easy purification using affinity column. The Nickel chelating chromatography was used for Purifying the expressed ATE2 from E- codi BL2l. Subsequen시y In vitro binding pull-down assay showed the binding specificity of ATF2 with AP-1 family factors such as BATF, c-Fos, c-Jun and ATF2 itselgf. ATF2 forms homodimer as well as strong heterodimer with BATF. It also forms stable dimer with c-Jun but barely binds with c-Fos.

• Biomedical Science Letters
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• v.9 no.4
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• pp.231-240
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• 2003

Deciphering the molecular interactions of proteins forming Z-lines is pivotal for understanding the regulation of myofibril assembly, sarcomeric organization, and mechanical properties of striated muscle. The purpose of this study is to searched for potential novel ligands of the Z-line portion of nebulin. In this study interacting proteins with intra-Z-line region of nebulin were screened using a yeast two-hybrid approach. The interaction was conformed by GST pull-down assay. We identified 269 residues within villin/gelsolin homology domain of supervillin that intreacts with the serine rich region of nebulin. The specific interactions of nebulin and supervillin were confirmed in vitro by GST pull-down experiments. Our data suggest that supervillin attaches directly to the Z-line through its interaction with the serine rich domain of nebulin in skeletal muscles. This interaction may link myofibrillar Z-discs to the membrane-associated complexes, costameres.

• Journal of Microbiology and Biotechnology
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• v.12 no.2
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• pp.286-291
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• 2002

Histone deacetylase I (HDAC1) works as one of the components in a nucleosome remodeling (NuRD) complex that consists of several proteins, including metastasis-associated protein 1 (MTA1). Since the protein-protein interaction of HDAC1 and MTA1 would appear to be important for both the integrity and functionality of the HDAC1 complex, the interruption of the HDAC1 and MTA1 interaction may be an efficient way to regulate the biological function of the HDAC1 complex. Based on this idea, a yeast two-hybrid system was constructed with HDAC1 and MTA1 expressing vectors in the DNA binding and activation domains, respectively. To verify the efficiency of the assay system, 3,500 microbial metabolite libraries were tested using the paper disc method, and KB0699 was found to inhibit the HDAC1 and MTA1 interaction without any toxicity to the wild-type yeast. Furthermore, KB0699 blocked the interaction of HDAC1 and MTA1 in an in vitro GST pull down assay and induced morphological changes in B16/BL6 melanoma cells, indicating the interruption of the HDAC1 complex function. Accordingly, these results demonstrated that the yeast assay strain developed in this study could be a valuable tool for the isolation of a HDAC1 complex disruptor.

• Journal of Life Science
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• v.20 no.10
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• pp.1451-1457
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• 2010

Serotonin (5-hydroxytryptamine, 5-HT) is an important mediator of cell-cell signaling in neuronal systems. The serotonin transporter (SERT) on the plasma membrane controls the extracellular 5-HT level by reuptake of released 5-HT from the synaptic cleft, but the underlying regulation mechanism is unclear. Here, we used the yeast two-hybrid system to identify the specific binding protein(s) that interacts with the carboxyl (C)-terminal region of SERT and found a specific interaction with protein kinase C-$\varepsilon$ (PKC-$\varepsilon$), a PKC isotype that is characterized as a calcium-independent and phorbol ester/diacylglycerol-sensitive serine/threonine kinase. PKC-$\varepsilon$ bound to the tail region of SERT but not to other members of the $Na^+/Cl^-$ dependent SLC6 gene family in the yeast two-hybrid assay. The C-terminal region of PKC-$\varepsilon$ is essential for interaction with SERT. In addition, these proteins showed specific interactions in the glutathione S-transferase (GST) pull-down assay. PKC-$\varepsilon$ phosphorylated the peptide of the SERT amino (N)-terminus in vitro. These results suggest that the phosphorylation of SERT by PKC-$\varepsilon$ may regulate SERT activity in plasma membrane.