• Journal of Applied Biological Chemistry
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• v.50 no.2
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• pp.58-62
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• 2007

Identification of Dual-specificity protein phosphatase (DSP) substrates is essential in revealing physiological roles of DSPs. We isolated VHZ-interacting proteins from extracts of 293T cells overexpressing a VHZ (C95S, D65A) mutant known to be substrate- trapping mutant. Analysis of specific proteins bound to VHZ by 2D gel electrophoresis and mass spectroscopy revealed that these proteins contained Chaperonin containing TCP1, Type II phosphatidylinositol phosphate kinase ${\gamma}$, Intraflagellar transport 80 homolog, and Kinesin superfamily protein 1B. VHZ-interacting proteins showed that VHZ is involved in many important cellular signal pathways such as protein folding, molecular transportation, and tumor suppression.

• Parasites, Hosts and Diseases
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• v.45 no.3
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• pp.229-232
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• 2007

The WD40-repeat proteins serve as a platform coordinating partner proteins and are involved in a range of regulatory cellular functions. A WD40-repeat protein (CsWD1) of Clonorchis sinensis previously cloned is expressed stage-specifically in the tegumental syncytium of C. sinensis metacercariae. In the present study, interact-ing proteins with the CsWD1 protein was purified by immunoprecipitation and 2 dimension gel electrophoresis from the C. sinensis metacercaria soluble extract, and tryptic peptides were analyzed by LC/ESI-MS. Putative partner proteins were annotated to be actin-2, glyceraldehyde-3-phosphate dehydrogenase, and hypothetical and unmanned proteins. The CsWD1 protein was predicted to contain 3 conserved actin-interacting residues on its functional surface. With these results, the CsWD1 protein is suggested to be an actin-interacting protein of C. sinensis.

• BMB Reports
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• v.47 no.11
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• pp.643-648
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• 2014

Stanniocalcin (STC), a glycoprotein hormone originally discovered in fish, has been implicated in calcium and phosphate homeostasis. While fishes and mammals possess two STC homologs (STC1 and STC2), the physiological roles of STC2 are largely unknown compared with those of STC1. In this study, we identified Ran-binding protein M (RanBPM) as a novel binding partner of STC2 using yeast two-hybrid screening. The interaction between STC2 and RanBPM was confirmed in mammalian cells by immunoprecipitation. STC2 enhanced the RanBPM-mediated transactivation of liganded androgen receptor (AR), but not thyroid receptor ${\beta}$, glucocorticoid receptor, or estrogen receptor ${\beta}$. We also found that AR interacted with RanBPM in both the absence and presence of testosterone (T). Furthermore, we discovered that STC2 recruits RanBPM/AR complex in T-dependent manner. Taken together, our findings suggest that STC2 is a novel RanBPM-interacting protein that promotes AR transactivation.

• Korean Journal of Microbiology
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• v.55 no.3
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• pp.199-205
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• 2019

Yeast two-hybrid (Y2H) technique has been used to study protein-protein interactions, but its application particularly to a large-scale analysis of protein interaction networks, is limited by the fact that the technique is labor-intensive, based on scoring colonies on plate. Here, we develop a new reporter for the measurement of the protein-protein interactions by flow cytometry. The yeast harboring interacting proteins can also be enriched by fluorescence-activated cell sorting (FACS) or magnetic-activated cell sorting (MACS). When two interacting proteins are present in the same yeast cell, a reporter protein containing 10 tandem repeats of c-myc epitope becomes localized on the surface of the cell wall, without affecting cell growth. We successful measured the surface display of c-myc epitope upon interacting p53 with SV40 T antigen by flow cytometry. Thus, the newly developed Y2H assay based on the display of c-myc repeat on yeast cell wall could be used to the simultaneous analysis of multiple protein-protein interactions without laborious counting colonies on plate.

• BMB Reports
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• v.45 no.6
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• pp.331-336
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• 2012

The retinoid-related orphan nuclear receptor gamma ($ROR{\gamma}$) plays critical roles in regulation of development, immunity and metabolism. As transcription factor usually forms a protein complex to function, thus capturing and dissecting of the $ROR{\gamma}$ protein complex will be helpful for exploring the mechanisms underlying those functions. After construction of the recombinant tandem affinity purification (TAP) plasmid, pMSCVpuro $ROR{\gamma}$-CTAP(SG), the nuclear localization of $ROR{\gamma}$-CTAP(SG) fusion protein was verified. Following isolation of $ROR{\gamma}$ protein complex by TAP strategy, seven candidate interacting proteins were identified. Finally, the heat shock protein 90 (HSP90) and receptor-interacting protein 140 (RIP140) were confirmed to interplay with $ROR{\gamma}$ by co-immunoprecipitation. Interference of HSP90 or/and RIP140 genes resulted in dramatically decreased expression of CYP2C8 gene, the $ROR{\gamma}$ target gene. Data from this study demonstrate that HSP90 and RIP140 proteins interact with $ROR{\gamma}$ protein in a complex format and function as co-activators in the $ROR{\gamma}$-mediated regulatory processes of HepG2 cells.

• Bulletin of the Korean Chemical Society
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• v.33 no.9
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• pp.3071-3075
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• 2012

Human ${\alpha}$-synuclein is the major component of the protein aggregates known as Lewy bodies or Lewy neurites, which define the intracellular lesions of Parkinson's disease. Despite extensive efforts, the physiological function of ${\alpha}$-synuclein has not yet been elucidated in detail. As an approach to defining its function, proteins that interacted with ${\alpha}$-synuclein were screened in phage display assays. The SNARE protein vesicle t-SNARE-interacting protein homologous 1B (VTI1B) was identified as an interacting partner. A selective interaction between ${\alpha}$-synuclein and VTI1B was confirmed by coimmunoprecipitation and GST pull-down assays. VTI1B and ${\alpha}$-synuclein were colocalized in N2a neuronal cells, and overexpression of ${\alpha}$-synuclein changed the subcellular localization of VTI1B to be more dispersed throughout the cytosol. Considering the role played by VTI1B, ${\alpha}$-synuclein is likely to modulate vesicle trafficking by interacting with a SNARE complex.

• BMB Reports
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• v.52 no.5
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• pp.304-311
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• 2019

The cytoplasmic FMR1-interacting protein family (CYFIP1 and CYFIP2) are evolutionarily conserved proteins originally identified as binding partners of the fragile X mental retardation protein (FMRP), a messenger RNA (mRNA)-binding protein whose loss causes the fragile X syndrome. Moreover, CYFIP is a key component of the heteropentameric WAVE regulatory complex (WRC), a critical regulator of neuronal actin dynamics. Therefore, CYFIP may play key roles in regulating both mRNA translation and actin polymerization, which are critically involved in proper neuronal development and function. Nevertheless, compared to CYFIP1, neuronal function and dysfunction of CYFIP2 remain largely unknown, possibly due to the relatively less well established association between CYFIP2 and brain disorders. Despite high amino acid sequence homology between CYFIP1 and CYFIP2, several in vitro and animal model studies have suggested that CYFIP2 has some unique neuronal functions distinct from those of CYFIP1. Furthermore, recent whole-exome sequencing studies identified de novo hot spot variants of CYFIP2 in patients with early infantile epileptic encephalopathy (EIEE), clearly implicating CYFIP2 dysfunction in neurological disorders. In this review, we highlight these recent investigations into the neuronal function and dysfunction of CYFIP2, and also discuss several key questions remaining about this intriguing neuronal protein.

• The Korean Journal of Physiology and Pharmacology
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• v.8 no.1
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• pp.27-31
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• 2004

${\Delta}^{12}-Prostaglandin$ $J_2\;({\Delta}^{12}-PGJ_2)$ is one of cyclopentenone prostaglandins. The ${\Delta}^{12}-PGJ_2$ is known to induce apoptosis of tumor cells, however, it's action mechanism is not clear. It has recently been reported that STAT3 is involved in tumorigenesis. In the present study, we investigated the role of STAT3-interacting protein (STIP1) in the cytotoxicity of ${\Delta}^{12}-PGJ_2$, since STIP1 was recently reported as a modulator of STAT3 activation by specifically binding to inactive (unphosphorylated) STAT3. The effect of ${\Delta}^{12}-PGJ_2$ was observed in stably overexpressing Neuro-2A cells transfected with full cDNA of STIP1, and cytotoxicity of ${\Delta}^{12}-PGJ_2$ in the transfected cells was increased, compared with the vector control cells. The cytotoxicity of ${\Delta}^{12}-PGJ_2$ treatment was significantly accentuated by pretreatment of the STIP1-transfected cells with protein kinase inhibitor, genistein, and less activation of STAT3 in STIP1-transfected cells was shown, compared with the vector control cells. Expression of bax was also increased in the STIP1-transfected cells. These data suggest that STIP1 inhibits cell growth via inhibition of STAT3 activation in ${\Delta}^{12}-PGJ_2$ treatment.

• Journal of Microbiology
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• v.38 no.2
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• pp.113-116
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• 2000

Escherichia coli F plasmid partition apparatus is composed of two trans-acting proteins (SopA and SopB) and one cis-acting DNA sequence (sopC). The SopB-sopC complex has been suggested to serve a centromere-like function through its interaction with chromosomally encoded proteins which remain to be identified. In this paper, we are introducing a new yeast 1.5-hybrid system which assembles the two-hybrid and one-hybrid system as a mean to find and additional component of the F plasmid partition system, interacting with DNA (sopC)-bound SopB protein. The results indicates that this system is a promising one, capable of selecting an interacting component.

• BMB Reports
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• v.41 no.6
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• pp.455-460
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• 2008

Calcineurin (Cn) is a serine/threonine phosphatase implicated in a wide variety of biological responses. To identify proteins that mediate Cn signaling pathway effects, we used yeast two-hybrid assays to screen for Cn interacting proteins, discovering a protein encoded by the gene, cnp-2 (Y46G5A.10). Utilizing serially deleted forms of Cn as baits, we demonstrated that the catalytic domain of Cn (TAX-6) binds with CNP-2, and this physical interaction was able to be reconstituted in vitro, supporting our yeast two-hybrid results. cnp-2 is a nematode-specific novel gene found in C. elegans as well as its closest relative, C. briggsae. CNP-2 was strongly expressed in the intestine of C. elegans. To study the function of cnp-2, we performed cnp-2 RNAi knock-down and characterized phenotypes associated with Cn mutants. However, no gross defects were revealed in these RNAi experiments. CNP-2 was proven to be a Cn binding protein; however, its role remains to be elucidated.