• Parasites, Hosts and Diseases
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• v.45 no.3
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• pp.165-174
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• 2007

Toxoplasma gondii GRA10 expressed as a GFP-GRA10 fusion protein in HeLa cells moved to the nucleoli within the nucleus rapidly and entirely. GRA10 was concentrated specifically in the dense fibrillar component of the nucleolus morphologically by the overlap of GFP-GRA10 transfection image with IFA images by monoclonal antibodies against GRA10 (Tg378), B23 (nucleophosmin) and C23 (nucleolin). The nucleolar translocalization of GRA10 was caused by a putative nucleolar localizing sequence (NoLS) of GRA10. Interaction of GRA10 with TATA-binding protein associated factor 1B (TAF1B) in the yeast two-hybrid technique was confirmed by GST pull-down assay and immunoprecipitation assay. GRA10 and TAF1B were also co-localized in the nucleolus after co-transfection. The nucleolar condensation of GRA10 was affected by actinomycin D. Expressed GFP-GRA10 was evenly distributed over the nucleoplasm and the nucleolar locations remained as hollows in the nucleoplasm under a low dose of actinomycin D. Nucleolar localizing and interacting of GRA10 with TAF1B suggested the participation of GRA10 in rRNA synthesis of host cells to favor the parasitism of T. gondii.

• Molecules and Cells
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• v.20 no.3
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• pp.446-451
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• 2005

Diap1 is an essential Drosophila cell death regulator that binds to caspases and inhibits their activity. Reaper, Grim and Hid each antagonize Diap1 by binding to its BIR domain, activating the caspases and eventually causing cell death. Reaper and Hid induce cell death in a Ring-dependent manner by stimulating Diap1 auto-ubiquitination and degradation. It was not clear that how Grim causes the ubiquitination and degradation of Diap1 in Grim-dependent cell death. We found that Grim stimulates poly-ubiquitination of Diap1 in the presence of UbcD1 and that it binds to UbcD1 in a GST pull-down assay, so presumably promoting Diap1 degradation. The possibility that dBruce is another E2 interacting with Diap1 was examined. The UBC domain of dBruce slightly stimulated poly-ubiquitination of Diap1 in Drosophila extracts but not in the reconstitution assay. However Grim did not stimulate Diap1 poly-ubiquitination in the presence of the UBC domain of dBruce. Taken together, these results suggest that Grim stimulates the poly-ubiquitination and presumably degradation of Diap1 in a novel way by binding to UbcD1 but not to the UBC domain of dBruce as an E2.

• Biomedical Science Letters
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• v.13 no.4
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• pp.263-272
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• 2007

Nebulin is a giant actin binding protein (600-900 kDa) which is specific to skeletal muscle. This protein is known to regulate thin filaments length in sarcomere as a molecular template. The C-terminus of nebulin is located in the Z-disc of muscle sarcomere and is bound to other proteins such like myopalladin, titin, archvillin, and desmin. The N-terminus of nebulin binds to tropomodulin at the pointed ends of the thin filaments. In recent research, nebulin not only found in brain but also expressed in heart, stomach, and liver. So, the roles of nebulin in non-muscle tissue have been studied. However, lack of information or studies on nebulin binding proteins and nebulin function in brain are available so far. Therefore, the current study have investigated a novel binding partner of Nebulin C-terminus by using yeast two-hybrid screening with human brain cDNA library. Nebulin C-terminus, containing simple repeats, serine rich and SH3 domain, interacts with osteonectin C-terminal region. The specific interaction of nebulin and osteonectin were confirmed in vitro by using GST pull-down assay and reconfirmed in vivo by using transfected COS-7 cells with EGFP-tagged nebulin and DsRed-tagged osteonectin. Consequently, this study identified SH3 domain in nebulin C-terminus specifically binds to extracellular Ca-binding (EeC domain in osteonectin. Also, nebulin C-terminus fusion protein colocalized with osteonectin EC domain fusion protein in transfected COS-7 cells. The current study found the interaction between nebulin and osteonectin in human brain for the first time and suggested the nebulin in brain may be associated with osteonectin, as a regulator of cell cycle progression and mitosis.

• Journal of Life Science
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• v.28 no.7
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• pp.765-771
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• 2018

Peroxisome proliferator-activated receptor gamma ($PPAR{\gamma}$) is a key transcription factor that regulates adipogenesis, and epigenetic control of $PPAR{\gamma}$ is of great interest in obesity-inhibition research. Our previous study showed that CACUL1 (CDK2-associated cullin domain 1) acts as a corepressor that inhibits $PPAR{\gamma}$ transcriptional activity and adipocyte differentiation. Here, we investigated the roles of protein arginine methyltransferase 5 (PRMT5), a novel binding partner of CACUL1, in regulating $PPAR{\gamma}$. The interaction between PRMT5 and CACUL1 was shown by immunoprecipitation assay in vivo and GST pulldown assay in vitro. As shown by luciferase reporter assay, PRMT5 and CACUL1 cooperated to inhibit the transcriptional activity of $PPAR{\gamma}$. The suppressive role of PRMT5 in adipogenesis was examined by Oil Red O staining using 3T3-L1 cells, which stably overexpress or deplete PRMT5. Overexpression of PRMT5 suppresses $PPAR{\gamma}$-mediated adipogenesis, whereas PRMT5 knockdown increases lipid accumulation in 3T3-L1 cells. Consistently, PRMT5 attenuates the expression of Lpl and aP2, the target genes of $PPAR{\gamma}$, as demonstrated by RT-qPCR analysis. Overall, these results suggest that PRMT5 interacts with CACUL1 to impair the transcriptional activity of $PPAR{\gamma}$, leading to the inhibition of adipocyte differentiation. Therefore, the regulation of PRMT5 enzymatic activity may provide a clue to develop an anti-obesity drug.

• Journal of Life Science
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• v.27 no.6
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• pp.673-679
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• 2017

Microtubules are long rods in the cytoplasm of cells that plays a role in cell motility and intracellular transport. Microtubule-based transport by motor proteins is essential in intracellular transport. Kinesin 1 is a molecular motor protein that mediates the intracellular transport of various membranous vesicles, mRNAs, and proteins along microtubules. It is comprised of two heavy chains (KHCs, also called KIF5s) and two light chains (KLCs). KIF5s bear a motor domain in their amino (N)-terminal regions and interact with various cargoes through the cargo-binding domain in their carboxyl (C)-terminal regions. To identify proteins interacting with KIF5B, yeast two-hybrid screening was performed, and a specific interaction with the cytoplasmic linker protein 170 (CLIP-170), a plus end microtubule-binding protein, was found. The coiled-coil domain of CLIP-170 is essential for interactions with KIF5B in the yeast two-hybrid assay. CLIP-170 bound to the cargo-binding domain of KIF5B. Also, other KIF5s, KIF5A and KIF5C, interacted with CLIP-170 in the yeast two-hybrid assay. In addition, glutathione S-transferase (GST) pull-downs showed that KIF5s specifically interacted with CLIP-170. An antibody to KIF5B specifically co-immunoprecipitated CLIP-170 associated with KIF5B from mouse brain extracts. These results suggest that kinesin 1 motor protein may transport CLIP-170 in cells.

• Journal of Life Science
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• v.21 no.9
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• pp.1226-1233
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• 2011

In neurons, kinesin is the molecular motor that transport cargos along microtubules. KIF5s (alias kinesin-I), are heterotetrameric motor conveying cargos, but the mechanism as to how they recognize and bind to a specific cargos has not yet been completely elucidated. To identify the interaction proteins for KIF5C, yeast two-hybrid screening was performed, and specific interaction with the $\underline{S}$am68-$\underline{l}$ike $\underline{m}$ammalian protein $\underline{2}$ (SLM-2), a member of the $\underline{s}$ignal $\underline{t}$ransducers and $\underline{a}$ctivators of $\underline{R}$NA (STAR) family of RNA processing proteins, was found. SLM-2 bound to the carboxyl (C)-terminal region of KIF5C and to other KIF5 members. The C-terminal domain of Sam68, SLM-1, SLM-2 was essential for interaction with KIF5C in the yeast two-hybrid assay. In addition, glutathione S-transferase (GST) pull-downs showed that SAM68, SLM-1, and SLM-2 specifically interacted to Kinesin-I complex. An antibody to SAM68 specifically co-immunoprecipitated SAM68 associated with KIF5s and coprecipitated with a specific set of mRNA. These results suggest that Kinesin-I motor protein transports RNA-associated protein complex in cells.

• Journal of Life Science
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• v.28 no.2
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• pp.170-175
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• 2018

Parkinson's disease (PD) is the most common movement disorder and the second most common neurodegenerative disease after Alzheimer's disease. Approximately 5~10% of PD patients are familial PD cases. Leucine-rich repeat kinase 2 (LRRK2) has been known to be a causal gene of PD when it is mutated. LRRK2 contains the functional kinase and GTPase domains as well as leucine-rich repeat (LRR) and WD40 domains that are known to play critical roles for protein-protein interaction, suggesting that LRRK2-interacting proteins are important regulators for PD pathogenesis. In an effort to identify proteins interacting with LRRK2, we carried out co-immunoprecipitation of LRRK2 antibody using extracts of NIH3T3 cells that express LRRK2 at a relatively high level. The mass spectrometry analysis of a precipitated band revealed that the co-precipitated band was methionyl-tRNA synthetase (MRS), an ancient enzyme that transfers methionin to its cognate tRNA. The interaction of MRS with LRRK2 was confirmed again by co-immunoprecipitation of endogenous proteins and GST pull-down assays. However, LRRK2 did not phosphorylate recombinant MRS protein in in vitro kinase assays, and over-expression of LRRK2 or MRS did not affect the stability of its partner protein. Our data indicate that LRRK2 interacts with but does not phosphorylate MRS, and the stability of each partner is not affected by the other.

• Molecules and Cells
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• v.37 no.3
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• pp.264-269
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• 2014

The molecular interaction between tumor suppressor p53 and the anti-apoptotic Bcl-2 family proteins plays an essential role in the transcription-independent apoptotic pathway of p53. In this study, we investigated the binding of p53 DNA-binding domain (p53DBD) with the anti-apoptotic Bcl-2 family proteins, Bcl-w, Mcl-1, and Bcl-2, using GST pull-down assay and NMR spectroscopy. The GST pull-down assays and NMR experiments demonstrated the direct binding of the p53DBD with Bcl-w, Mcl-1, and Bcl-2. Further, NMR chemical shift perturbation data showed that Bcl-w and Mcl-1 bind to the positively charged DNA-binding surface of p53DBD. Noticeably, the refined structural models of the complexes between p53DBD and Bcl-w, Mcl-1, and Bcl-2 showed that the binding mode of p53DBD is highly conserved among the anti-apoptotic Bcl-2 family proteins. Furthermore, the chemical shift perturbations on Bcl-w, Mcl-1, and Bcl-2 induced by p53DBD binding occurred not only at the p53DBD-binding acidic region but also at the BH3 peptide-binding pocket, which suggests an allosteric conformational change similar to that observed in Bcl-$X_L$. Taken altogether, our results revealed a structural basis for a conserved binding mechanism between p53DBD and the anti-apoptotic Bcl-2 family proteins, which shed light on to the molecular understanding of the transcription-independent apoptosis pathway of p53.

• Animal cells and systems
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• v.6 no.3
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• pp.277-282
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• 2002

Topoisomearse II is an essential enzyme in all organisms with several independent roles in DNA metabolism. Recently, it has been demonstrated that the C-terminal region of topoisomerases II is associated with hetero-logous protein-protein interactions in human and yeast. In this study, we identified that RTP1, a rat homologue of EIA binding protein BS69, is another topoisomerae II interacting protein by yeast two-hybrid screening. RTP1 has an E1A-binding domain and a MYND motif, which are known to be required for transcriptional regulation by binding to other proteins and interaction with the leucine zipper motif of topoisomerase II. The physical interaction between RTP1 and topoisomerase ll$\alpha$ was examined by GST pull-down assay in vitro. The expression level of RTP1 peaks in S phase as that of topoisomerase ll$\alpha$. These results suggest that the interaction between topoisomerase ll$\alpha$ and RTP1 might play an important role in regulating the transcription of genes involved in DNA metabolism in higher eukaryotes.

• Molecules and Cells
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• v.24 no.3
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• pp.372-377
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• 2007

The orphan nuclear receptor, SF-1, plays a pivotal role in the development and differentiation of the endocrine and reproductive systems, and also regulates the transcription of a host of genes, including those encoding several steroidogenic enzymes and gonadotropins. We found that a previously unidentified repressor, EID-1, is an SF-1-interacting protein that inhibits the transactivation of SF-1. A transient transfection assay revealed that EID-1 inhibits SF-1, but not LRH-1, $ERR{\gamma}$, or mCAR. Using the yeast two hybrid and GST pull-down assays, we determined that EID-1 interacted strongly with SF-1. In addition, it colocalized with SF-1 in mammalian cells and interacted specifically with the AF-2 domain of SF-1, competing with SRC-1 to inhibit SF-1 transactivation. EID-1 is expressed in the mouse testis, and its expression decreases during testis development. The results of the present study suggest that EID-1 can act as a repressor, regulating the function of SF-1.