• The Journal of Natural Sciences
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• v.17 no.1
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• pp.13-29
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• 2006

Most redox-active proteins have thiol-bearing cysteine residues that are sensitive to oxidation. Cysteine thiols oxidized to sulfenic acid are generally unstable, either forming a disulfide with a nearby thiol or being further oxidized to a stable sulfinic acid, which have been viewed as an irreversible protein modification. However, recent studies showed that cysteine residues of certain thiol peroxidases (Prxs) undergo reversible oxidation to sulfinic acid and the reduction reaction is catalyzed by sulfiredoxin (Srx1). Specific Cys residues of various other proteins are also oxidized to sulfinic acid ($Cys-So_2H$). Srxl is considered one of the oxidant proteins with a role in signaling through catalytic reduction of oxidative modification like in the reduction of glutathionylation, a post-translational, oxidative modification that occurs on numerous proteins. In this study, the role of sulfiredoxin in cellular processes, was investigated by studying its interaction with other proteins. Through the yeast two-hybrid system (Y2HS) technique, we have found that Ams1 is a potential and novel interacting protein partner of Srxl. $\alpha$-mannosidase (Ams1) is a resident vacuolar hydrolase which aids in recycling macromolecular components of the cell through hydrolysis of terminal, non-reducing $\alpha$-D-mannose residues. It forms an oligomer in the cytoplasm and under nutrient rich condition and is delivered to the vacuole by the Cytoplasm to Vacuole (Cvt) pathway. Aside from the role of Srxl as a catalyst in the reduction of cysteine sulfenic acid groups, it may play a completely new function in the cellular process as indicated by its interaction with Ams1 of the yeast Saccharomyces cerevisiae.

• The Korean Journal of Physiology and Pharmacology
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• v.8 no.3
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• pp.167-172
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• 2004

The kinesin proteins (KIFs) make up a large superfamily of molecular motors that transport cargo such as vesicles, protein complexes, and organelles. KIF5 is a heterotetrameric motor that conveys vesicles and plays an important role in neuronal function. Here, we used the yeast two-hybrid system to identify the neuronal protein(s) that interacts with the tail region of KIF5 and found a specific interaction with ${\beta}III$ spectrin. The amino acid residues between 1394 and 1774 of ${\beta}III$ spectrin were required for the interaction with KIF5C. ${\beta}III$ spectrin also bound to the tail region of neuronal KIF5A and ubiquitous KIF5B but not to other kinesin family members in the yeast two-hybrid assay. In addition, these proteins showed specific interactions, confirmed by GST pull-down assay and co-immunoprecipitation. ${\beta}III$ spectrin interacted with GST-KIF5 fusion proteins, but not with GST alone. An antibody to ${\beta}III$ spectrin specifically co-immunoprecipitated KIF5s associated with ${\beta}III$ spectrin from mouse brain extracts. These results suggest that KTF5 motor proteins transport vesicles or organelles that are coated with ${\beta}III$ spectrin.

• Development and Reproduction
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• v.16 no.2
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• pp.129-135
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• 2012

Previously, we identified that the overexpression of IEX-1 induces apoptosis in ovarian cancer cells. Herein we report a new binding partner of IEX-1, CATHEPSIN B, as a lysosomal enzyme which contributes to the various apoptotic signaling in tumor cells. To investigate how IEX-1 regulates cellular survival and death event, we performed yeast two-hybrid screening of rat ovarian cDNA library using IEX-1 as the bait and found CATHEPSIN B. In the present study, CATHEPSIN B and IEX-1 proteins were overexpressed in 293T cells and their specific association was determined by immunoprecipitation and immunoblot analysis. In addition, the endogenous interaction between CATHEPSIN B and IEX-1 was confirmed in HeLa cells. The current finding of lysosomal CATHEPSIN B as the IEX-1-binding partner implies that IEX-1 may involve in lysosome-mediated apoptotic signaling pathways.

• Biomedical Science Letters
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• v.9 no.3
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• pp.159-165
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• 2003

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. Recently, the studies with C-terminus of ankyrins have identified that ankyrin-B is capable of interacting with Hsp40 and sAnkl is capable of interacting with obscurin and titin, but the function of C-terminal domain of ankyrin-G remains unknown. To identify proteins interacting C-terminus of ankyrin-G, we used the C-terminus of ankyrin-G as a bait for a yeast two-hybrid screen of brain cDNA library. Approximately 1.33$\times$l0$^6$ transformants were screened, of which 13 positive clones were obtained as determined by activation of HIS3, ADE2 and MELl reporter genes. Sequence analyses of these 13 plasmids revealed that cDNA inserts of 13 colonies showed highly homologous to 11 genes, including 5 known (i.e., Na$^+$/K$^+$ ATPase $\beta$1, SERBPl, UTF2, cytochrome C oxidase and collagen IV $\alpha$2) and 6 unknown genes. The evaluation of the proteins that emerge from these experiments provides a rational approach to investigate the those proteins significant in interaction with ankyrin-G.

• Biomolecules & Therapeutics
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• v.30 no.4
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• pp.380-388
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• 2022

Snail is implicated in tumour growth and metastasis and is up-regulated in various human tumours. Although the role of Snails in epithelial-mesenchymal transition, which is particularly important in cancer metastasis, is well known, how they regulate tumour growth is poorly described. In this study, the possible molecular mechanisms of Snail in tumour growth were explored. Baculoviral inhibitor of apoptosis protein (IAP) repeat-containing protein 3 (BIRC3), a co-activator of cell proliferation during tumourigenesis, was identified as a Snail-binding protein via a yeast two-hybrid system. Since BIRC3 is important for cell survival, the effect of BIRC3 binding partner Snail on cell survival was investigated in ovarian cancer cell lines. Results revealed that Bax expression was activated, while the expression levels of anti-apoptotic proteins were markedly decreased by small interfering RNA (siRNA) specific for Snail (siSnail). siSnail, the binding partner of siBIRC3, activated the tumour suppressor function of p53 by promoting p53 protein stability. Conversely, BIRC3 could interact with Snail, for this reason, the possibility of BIRC3 involvement in EMT was investigated. BIRC3 overexpression resulted in a decreased expression of the epithelial marker and an increased expression of the mesenchymal markers. siSnail or siBIRC3 reduced the mRNA levels of matrix metalloproteinase (MMP)-2 and MMP-9. These results provide evidence that Snail promotes cell proliferation by interacting with BIRC3 and that BIRC3 might be involved in EMT via binding to Snail in ovarian cancer cells. Therefore, our results suggested the novel relevance of BIRC3, the binding partner of Snail, in ovarian cancer development.

• Development and Reproduction
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• v.12 no.3
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• pp.289-296
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• 2008

BPES (Blepharophimosis/Ptosis/Epicanthus inversus Syndrome) is an autosomal dominant disorder caused by mutations in FOXL2. Affected individuals have premature ovarian failure (POF) in addition to small palpebral fissures, drooping eyelids, and broad nasal bridge. FOXL2 is a member of the forkhead family transcription factors. In FOXL2-deficient ovaries, granulosa cell differentiation dose not progress, leading to arrest of folliculogenesis and oocytes atresia. Using yeast two-hybrid screening of rat ovarian cDNA library with FOXL2 as bait, we found that small ubiquitin-related modifier (SUMO)-conjugating E2 enzyme UBE2I protein interacted with FOXL2 protein. UBE2I also known as UBC9 is an essential protein for processing SUMO modification. Sumoylation is a form of post-translational modification involved in diverse signaling pathways including the regulation of transcriptional activities of many transcriptional factors. In the present study, we confirmed the protein-protein interaction between FOXL2 and UBE2I in human cells, 293T, by in vivo immunoprecipitation. In addition, we generated truncated FOXL2 mutants and identified the region of FOXL2 required for its association with UBE2I using yeast-two hybrid system. Therefore, the identification of UBE2I as an interacting protein of FOXL2 further suggests a presence of novel regulatory mechanism of FOXL2 by sumoylation.

• Journal of Life Science
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• v.24 no.1
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• pp.14-19
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• 2014

Kinesin-1 consists of two heavy chains (KHCs), also called KIF5s, and two light chains (KLCs) that form a heterotetrameric complex. Here, we demonstrate the binding of a neuronal KHC, KIF5A, to the carboxyl (C)-terminal tail region of Fig4 (also known as Sac3), a phosphatase that removes the 5-phosphate from phosphatidylinositol-3,5-bisphosphate ($PtdIns(3,5)P_2$). Fig4 bound to the C-terminal region of KIF5A but not to other KHCs (KIF5B and KIF5C) and KLC1 in yeast two-hybrid assays. The interaction was further confirmed in a glutathione S-transferase pull-down assay and by co-immunoprecipitation. Anti-KIF5A antibody co-immunoprecipitated Fig4 with KIF5A from mouse brain extracts. These results suggest that kinesin-1 could transport the Fig4-associated protein complex or cargo in cells.

• Journal of Sericultural and Entomological Science
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• v.40 no.1
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• pp.52-59
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• 1998

For the construction of plasmid and bmNPV sarrying the FRT recognition site for the FLP recombinases, we synthesized the wild type FRT dligonucleotides. The target FRT sequences consist of three 13bp repeated DNA sequences; two repeats in a direct orientation and one inverted relative to the other two. In addition, there is an 8bp spacer region between the repeats which determune the orientation of the FRT recombination site. In order to place the FRT site both in target BmNPV genome and the transfer vector, we constructed a plasmid, FRT site both in the target BmNPv genome and the transfer vector, we constructed a plasmid, pFRT$\beta$-gal, carrying the FRT sites within the cloning sites of pSV vector and a recombinant BmNPV, vFRTPH, carrying the FRT sites at a downstream of polyhedrin promotor, respectively. In order to test the functionality of the FLP/FRT site-specific recombination system, vFRTPH, pFRT$\beta$-gal and pHsFLP DNA were co-transfected into BmN-4 cells. The resulting recombinant virus was designated a vFRT$\beta$2-gal. From construction analysis of the vFRT$\beta$2-gal with PCR technique it was concluded that the entire pFRT$\beta$-gal plasmid with $\beta$-galactosidase gene and origines of replication flanked by two functional hybrid FRT sequences. The efficiency of recombination was 8.7%, which was higher than that(2.2%) of recombination between a conventional transfer vector and the wild type BmNPV.

• Journal of Life Science
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• v.18 no.7
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• pp.940-946
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• 2008

Neurotransmitter transporters, which remove neurotransmittesr from the synaptic cleft, are regulated by second messenger such as protein kinases and binding proteins. Neuronal ${\gamma}-aminobutyric$ acid transporters (GATs) are responsible for removing the inhibitory neurotransmitter ${\gamma}-aminobutyric$ acid (GABA) from the synaptic cleft. ${\gamma}-aminobutyric$ acid transporters 2 (GAT2/BGT1) is involved in regulating neurotransmitter recycling, but the mechanism how they are stabilized and regulated by the specific binding protein has not yet been elucidated. Here, we used the yeast two-hybrid system to identify the specific binding protein(s) that interacts with the C-terminal region of GAT2 and found a specific interaction with the mammalian LIN-7b (MALS-2). MALS-2 protein bound to the tail region of GAT2 but not to other GAT members in the yeast two-hybrid assay. The "T-X-L" motif at the C-terminal end of GAT2 is essential for interaction with MALS-2. In addition, this protein showed specific interactions in the glutathione S-transferase (GST) pull-down assay. An antibody to GAT2 specifically co-immunoprecipitated MALS associated with GAT2 from mouse brain extracts. These results suggest that MALS may stabilize GAT2 in brain.

• Journal of Life Science
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• v.22 no.9
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• pp.1159-1165
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• 2012

The action of neuronally released ${\gamma}$-aminobutyric acid (GABA) is terminated by uptake into the neurons by GABA transporters (GATs). The mechanism underlying the stabilization and regulation of GAT2 has not yet been elucidated. We used the yeast two-hybrid system to identify proteins that interact with and, thereby, regulate betaine-${\gamma}$-aminobutyric acid transporter 1 (BGT-1/mGAT2). We found an interaction between BGT-1/mGAT2 and Munc-18-interacting proteins (Mints). The "T-H-L" motif at the C-terminal end of BGT-1/mGAT2 was essential for the interaction with Mint2 in the yeast two-hybrid assay. Mint2 bound to the tail region of BGT-1/mGAT2, but not to other GAT members. When co-expressed in HEK-293T cells, Mint2 was co-immunoprecipitated with BGT-1/mGAT2. In addition, we demonstrated the cellular co-localization of BGT-1/mGAT2 and Mint2 in the cells. These results suggest that Mint2 contributes to the regulation of BGT-1/mGAT2.