• BMB Reports
• /
• 제34권1호
• /
• pp.15-20
• /
• 2001

One of the innate immune reactions in invertebrates is the pro-phenoloxidase (pro-PO) activation system that is involved in the generation of superoxide, melanin synthesis, and the subsequent sequestration of foreign matter entering the hemocoel of the invertebrates. However, the molecular mechanism of this biological reaction is still obscure. To expand our understanding of the biological roles of the pro-PO activation system in invertebrates, we performed a yeast two-hybrid screening by using three regions of pro-PO as bait and a yeast two-hybrid cDNA library from Tenebrio molitor larvae as prey We isolated a novel partial cDNA clone that encodes a glycine-rich protein that interacted with the active phenoloxidase (termed phenoloxidase interacting protein, POIP). POIP consists of two domains: One is an N-terminal unique domain and the other is a C-terminal glycine-rich domain. The C-terminal glycine-rich domain showed sequential homology with those of insect antifungal proteins. Also, the yeast two-hybrid screen in a reverse orientation (using POIP as bait) yielded PO, suggesting that the PO-POIP interaction is specific. By using a 315 bP PCR fragment of the N-terminal unique region of POIP, we cloned the full-length cDNA of POIP from the Tenebruo cDNA library constructed by using E. coli injected larvae. The interaction analysis between PO, and a truncated fragment lacking the N-terminal unique region of POIP, indicated that the N-terminal unique region is necessary for interaction between PO and POIP. The expression level of the POIP mRNA is increased by bacterial injection into T. molitor larvae. This suggests that POIP might be engaged in the humoral defense reaction.

• 대한의생명과학회지
• /
• 제13권4호
• /
• pp.305-311
• /
• 2007

cAMP-dependent protein kinase A (PKA) is the best-characterized protein kinases and has served as a model of the structure and regulation of cAMP-binding protein as well as of protein kinases. To determine the function of PKA in development, we employed the yeast two-hybrid system to screen for catalytic subunit of PKA $(C\alpha)$ interacting partners in a cDNA library from mouse embryo. A Testis-brain RNA-binding protein (TB-RBP), specifically bound to $C\alpha$. This interaction was verified by several biochemical analysis. Our findings indicate that $C\alpha$ can modulate nucleic acid binding proteins of TB-RBP and provide insights into the diverse role of PKA.

• 한국균학회지
• /
• 제40권4호
• /
• pp.224-228
• /
• 2012

A. nidulans ${\alpha}$-COP과 상호작용하는 단백질을 동정하기 위하여 ${\alpha}$-COP을 암호하는 유전자를 bait로 yeast two-hybrid 스크리닝용 A. nidulans cDNA 라이브러리를 탐색한 결과, COPI 소낭의 구성요소 중 하나인 ${\varepsilon}$-COP을 암호화하고 있는 유전자를 동정하고 $aneA^+$($\underline{A}$spergillus $\underline{n}$idulans $\underline{e}$psilon-COP, $AN{\varepsilon}$-COP)으로 명명하였다. $aneA^+$ 유전자는 총 296개의 아미노산을 암호화하고 있으며, 다른 균류의 ${\varepsilon}$-COP과 높은 상동성을 보였다. Yeast two hybrid 시스템으로 두 단백질 간의 상호작용 부위를 분석한 결과, ${\alpha}$-COP의 COOH 도메인과 ${\varepsilon}$-COP의 C-말단부가 필수 부위였으며, ${\alpha}$-COP N-말단의 WD 도메인과 ${\varepsilon}$-COP의 TPR 부위는 두 단백질 간의 결합을 촉진하는 조절부위로 밝혀졌다. 또한 사상균인 A. nidulans와 효모류인 S. cerevisiae에서 ${\alpha}$-COP과 ${\varepsilon}$-COP 간 작용양상이 유사한 것으로 보아, COPI 소낭의 구성요소인 ${\alpha}$-COP과 ${\varepsilon}$-COP 간의 상호작용 기전은 진핵세포 내에서 진화적으로 잘 보존되어 있는 것으로 추정되었다.

• 농약과학회지
• /
• 제6권3호
• /
• pp.135-156
• /
• 2002

내분비계 장애물질이 환경호르몬이란 용어로 우리들의 실생활에 사용되는 용기 등을 통해 여러 가지 영향을 미치고 있다. 수많은 화학물질들 중에서 환경호르몬은 어떠한 종류의 화학물질이며, 언제 알려지게 되었고, 어떻게 분류되었으며, 이러한 환경호르몬성을 갖는 물질들의 환경호르몬성은 어떠한 연구기법에 의해 검색되는지와 현재까지의 사용실태와 문제점 및 국내외 관련 동향 등을 고찰하여 보았다.

• 대한의생명과학회지
• /
• 제12권4호
• /
• pp.405-411
• /
• 2006

Heterotrimeric GTP binding proteins (G proteins) mediate signal transduction generated by neurotransmitter and hormones. Among G-proteins, Go is classified as a member of the Go/Gi family and the most abundant heterotrimeric G protein in brain. Most of the mechanistic analyses on the activation of Go indicated its action to be mediated by the $G{\beta}{\gamma}$ dimer because downstream effectors for its ${\alpha}$ subunit have not been clearly defined. To determine the downstream effectors of alpha subunits of Go ($Go{\alpha}$), we used yeast two-hybrid system to screen $Go{\alpha}$ interacting partners in cDNA library from the human brain. A brain specific high mobility group box containing protein (BHX), A possible transcription factor, was identified as a $Go{\alpha}$ interacting protein. We confirmed interaction between $Go{\alpha}$ and BHX employing in vitro affinity binding assay. Moreover, active form of $Go{\alpha}$ preferentially interacts with BHX than inactive farm. Our findings indicate that $Go{\alpha}$ could modulate gene expression via interaction with BHX during neuronal or brain development.

• BMB Reports
• /
• 제34권6호
• /
• pp.526-530
• /
• 2001

The translationally controlled tumor protein (TCTP), also known as the IgE-dependent histamine releasing factor (HRF), was used in the yeast two-hybrid system to screen the interacting molecules. We obtained the N-terminus truncated rat fast myosin alkai light chain from the rat skeletal muscle cDNA library in the screening. Since either TCTP/HRF or the myosin light chain is known to be associated with histamine secretion from RBL-2H3 cells, we investigated the possible interaction between rat TCTP/HRF and nonmuscle myosin light chain in these cells. We used affinity chromatography and coimmunoprecipitation. Our data suggests that HRF and the myosin light chain interact, which may play an important role in histamine release in RBL-2H3 cells.

• BMB Reports
• /
• 제33권1호
• /
• pp.59-62
• /
• 2000

Hepatitis C virus (HCV) nonstructural 5B (NS5B) protein is an RNA-dependent RNA polymerase (RdRp). To determine whether it can contribute to viral replication by interaction with cellular proteins, the yeast two-hybrid screening system was employed to screen a human liver cDNA library. Using the HCV NS5B as a bait, we have isolated positive clones encoding a cellular protein. The NS5B interacting protein, 5BIP, is a novel cellular protein of 170 amino acids. Interaction of the HCV NS5B protein with 5BIP was confirmed by a protein-protein blotting assay. Recently, we have demonstrated that NS5B possesses an RdRp activity and thus it is possible that 5BIP, in association with NS5B, plays a role in HCV replication.

• BMB Reports
• /
• 제41권6호
• /
• pp.455-460
• /
• 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.

• BMB Reports
• /
• 제39권6호
• /
• pp.709-716
• /
• 2006

Mammalian polo-like kinase 1 (Plk1) acts at various stages in early and late mitosis. Plk1 localizes at the centrosome and maintains this position through mitosis. Thereafter Plk1 moves to the kinetochore and midbody region, important sites during chromosome separation and cytokinesis. The catalytic domain of Plk1 is in the N-terminus region, whereas the non-catalytic region in the C-terminus of Plk1 has a conserved motif, named the Polobox. This motif is critical for Plk localization. EGFP proteins fused with the N-terminus and C-terminus of Plk1 localize in the nucleus and centrosomes, respectively. The core sequences of the polo-box (50 amino acids) also localize in Plk1 target organelles. To screen for domain-specific target proteins of Plk1, we constructed an N-terminal domain and a tandem repeat polo-box motif, and used them as templates in a yeast two-hybrid screen. The HeLa cell cDNA library indicated several proteins including the centrosome/kinetochore components or regulators, to be characterized as positive clones. Through in vitro protein binding analyses, we confirmed an interaction between these proteins and Plk1. The data reported from this study indicate that the N- and C- termini of Plk1 may function through recruitment and/or activation of domain-specific target proteins in dividing cells. Additionally, tandem repeats of the conserved core motif of the polo-box are sufficient for targeting and may be useful as a centrosome/kinetochore-specific targeting peptide.

• BMB Reports
• /
• 제37권4호
• /
• pp.402-407
• /
• 2004

A human gene T-complex protein 10 like (TCP10L) was cloned in our lab. A previous study showed that it expressed specifically in the liver and testis. A transcription experiment revealed that TCP10L was a transcription factor with transcription inhibition activity. In this study, the human MAD4 was identified to interact with TCP10L by a yeast two-hybrid screen. This finding was confirmed by immunoprecipitation and subcellular localization experiments. As MAD4 is a member of the MAD family, which antagonizes the functions of MYC and promotes cell differentiation, the biological function of the interaction between TCP10L and MAD4 may be to maintain the differentiation state in liver cells. Also, we propose that the up-regulation of Myc is caused by the down-regulation of TCP10L in human hepatocarcinomas.