Yeast Two-Hybrid System을 이용한 단백질-단백질 결합의 검색

석대현 인제대학교 백병원 2002 仁濟醫學 Vol.23 No.2

There is a great need for general methods to characterize the proteins that contemporary biology makes available. The list of such proteins needing further characterization is glowing and includes proteins already known to be important for specific cellular functions, mutant proteins identified in vivo or made in vitro, and very large numbers of protein being identified by genome projects. The recent success of two-hybrid systems is due to the fact that many cellular functions are carried out by proteins that touch one another. A full understanding of the function of any new protein will require knowledge of the interactions it makes with previously identified proteins. Currently, most new proteins are being identified by large scale sequencing projects. For many of these new proteins the sequence alone sheds little or no light on their function. Two-hybrid technology exploits the fact that transcriptional activators are modular in nature. Two physically distinct functional domains are necessary to get transcription: (1) a DNA binding domain (DBD) that binds to the DNA of the promoter and (2) an activation domain (AD) that binds to the basal transcription apparatus and activates transcription. In the yeast two-hybrid system, the known gene encoding X. is cloned into the "bait" vector. Separately, a second gene (or a library of cDNAs encoding potential interactors). Y. is cloned in frame adjacent to an activation domain of a different transcription factor. Thus, in one strain of yeast, a known protein X is fused to the DNA binding domain of a transcription factor: and in another strain, unknown proteins are fused to the activation domain of another transcription factor. If the one of the unknown proteins combines with X, it will bring the AD over to the DBD, and transcription will be activated.

비혈연간 동종골수이식에 의해 치료된 중증 재생불량성빈혈 1예

김현중,이홍기,이방훈,이창근,이종태,정현식,김원석,윤성수,강원기,박근칠,김대식,고영혜,박찬형 대한조혈모세포이식학회 1997 대한조혈모세포이식학회지 Vol.2 No.1

비혈연간 동종골수이식은 혈연간 동종골수이식이 불가능한 환자에서 기존의 면역역제요법으로 효과가 없었던 경우나 재발한 경우에 적응증이 될수 있다. 이식 거부와 이식편대숙주병의 발생이 골수 이식의 성공률을 감소시키므로, 보다 강력한 conditioning regimen의 사용과 T cell이 제거된 골수를 사용하는 것이 필요하다. 기존의 high dose corticosteroid나 cyclosporine에 대하여 불응성인 이식편대숙주병의 치료를 위해서 ATG와 Mycophenolate mofetil (Cellcept®) 의 사용이나 Tacrolimus, Methotrexate와 같은 다른 면역억제제치료를 고려해 볼 수 있다. It is known that allogenic bone marrow transplantation is the most effective treatment for aplastic anemia. However, this treatment is only applicable to a minority of patients because the proportion of patients who have HLA-matched siblings about 25% to 30% of all the aplastic anemia patients. In the case of the absence of HLA-matched donor, unrelated allogenic bone marrow transplantation is considered. However, (because of severe acute and chronic GVHD and graft failure) the success rate of this approach is only 20~30%. Recently, it was reported that increased immunosuppressive condition combined with T cell depletion of the marrow graft would result in greater success. There was no case of successfully treated aplastic anemia patient by unrelated allogenic bone marrow transplantation in Korea. Here, we report a case of severe aplastic anemia successfully treated with unrelated allogenic bone marrow transplantation.

방선균 분리주가 생산하는 Phospholipase C 저해물질인 MT2617-2B의 분리 및 특성

고학룡,이현선,오원근,안순철,김보연,강대욱,민태익,안종석 한국미생물생명공학회 ( 구 한국산업미생물학회 ) 1996 한국미생물·생명공학회지 Vol.24 No.1

방선균 분리주 MT2617-2의 배양액으로 부터 phospholipase C (PLC) 저해물질인 MT2617-2B를 n-butanol 추출 및 column chromatography 법을 이용하여 분리하였다. MT2617-2B는 IR ^13C- 및 ^1H-NMR 그리고 ESI-MS에 의한 구조분석 결과, 한 개의 hemiketal ring, polyhydroxyl 및polymethyl groups으로 구성되었으며 side chain으로 한 개씩의 malonate 및 guanidine group을 가지는 분자량 1057의 macrolide 화합물이었다. 따라서, MT2617-2B를 기존의 macrolide 항생제인 copiamycin 및 niphithricin A로 동정하였다. 한편, MT2617-2B는 methanol 용액에서 실온에서 방치하였을 때 도일한 분자량을 가진 두 개의 이성질체를 생성하였다. PLC γ1과 -β1에 대해 각각 25 및 50㎍/㎖의 IC_50 값을 가지며, Staphylococcus aureus 와 Candida albicans에 대해서는 항균활성을 나타내지만 Escherichia coli에는 나타내지 않았다. A phospholipase C (PLC) inhibitor (MT2617-2B) was isolated from the culture broth of actionmycetes isolate MT2617-2 by the extraction with n-butanol and column chromatographic techniques. The molecular weight of the inhibitor was 1057, by the spectroscopic analyses of IR ^13C- and ^1H-NMR and ESI-MS. The chemical structure of MT2617-2B was found to be a macrolide compound consisted of a hemiketal ring, polyhydroxyl and polymethyl groups, which had a malonate and guanidine group as its side chain. MT2617-2B produced its two isomers having the same molecular weight by standing in methanol solution at room temperature. Therefore, MT2617-2B was identified as copiamycin and niphithricin A, macrolide antibiotics. The values of IC_50 against PLC γ1 and PLC-β1 were 25 and 50㎍/㎖, respectively. MT2617-2B had antimicrobial activities against Staphylococcus aureus and Candida albicans, but not against Escherichia coli.

Interaction of a Kinesin Superfamily Protein 1A (KIF1A) with Calmodulin

Seog, Dae-Hyun Korean Society of Life Science 2002 Journal of Life Science Vol.12 No.2

Kinesin Superfamily Protein 1A (KIF1A) is an anterograde monomeric motor transporting a subset of synaptic vesicle precursors and plays an important role in neuronal function and survival. Here, f have used the yeast two-hybrid system to identify the proteins that interacts with the tail region of KIF1A. Calmodulin was found to interact specifically with the tail region of KIF1A. Calmodulin regulates many diverse cellular functions by modulating the activity of the proteins that interact with it. KIF1A interacts with calmodulin in the yeast two-hybrid assay, which is proved by immunoprecipitation with calmodulin in brain fraction. These results indicate that KIF1A is associated with calmodulin, suggesting that calmodulin may be a key role in the regulation of anterograde transport of synaptic 1 vesicle precursors.

The STAR RNA Binding Proteins SAM68, SLM-1 and SLM-2 Interact with Kinesin-I

Dae-Hyun Seog(석대현) 한국생명과학회 2011 생명과학회지 Vol.21 No.9

키네신은 신경세포에서 미세소관 위를 따라 소포들을 운반하는 분자 motor 단백질로 4개의 단백질로 구성되어있다. 신경세포내에서 발현하는 KIF5C가 세포 내에서 어떤 특정소포를 이동시키는가를 신경세포성장에서 중요문제이다. 이에 본연구는 KIF5C와 결합하는 단백질을 동정하기 위하여 효모 two-hybrid 방법을 사용하여 KIF5C와 특이적으로 rufgkqgksms Sam68-like mammalian protein 2 (SLM-2)을 확인하였다. Signal Transducers and Activators of RNA (STAR) family의 한종류이며 RNA processing에 관여하는 RNA 결합단백질인 SLM-2는 KIF5s의 C-말단과 결합하며, 또한 SLM-2의 C-말단은 KIF5s와 결합하는데 필수영역이였다. 이러한 단백질간의 결합은 Glutathione S-transferase (GST) pull-down assay를 통하여 SAM68, SLM-1, SLM-2은 특이적으로 Kinesin-I과 결합함을 확인하였으며, SAM68의 항체로 면역침강한 결과 KIF5s와 mRNA는 같이 침강하였다. 신경세포의 말단에는 돌기형성에 필요한 단백질들의 주형인 mRNA가 다수 존재하며, 이러한 mRNA는 세포의 중앙에서 세포의 말단쪽으로 이동하여야 하는데, 이번 연구 결과는 Kinesin-I이 특이적으로 mRNA을 운반할 것으로 예상된다. 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 Sam68-like mammalian protein 2 (SLM-2), a member of the signal transducers and activators of RNA (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.

The β Subunit of Heterotrimeric G Protein Interacts Directly with Kinesin Heavy Chains, Kinesin-I

Dae-Hyun Seog(석대현) 한국생명과학회 2010 생명과학회지 Vol.20 No.8

Kinesin-I은 4분자의 단백질로 구성되어 있으며, N-말단의 motor 영역과 C-말단영역을 가지는 장쇄(KHC, 또한 KIF5s로도 통용) 2분자와 KIF5s (KIF5A, KIF5B와 KIF5C)의 줄기영역과 결합하는 단쇄(KLC) 2분자로 구성되어 있다. KIF5A의 결합 단백질을 동정하기 위하여 효모 two-hybrid system을 사용하여 특이적으로 결합하는 heterotrimeric G 단백질의 β 단위체 단백질(Gβ)을 분리하였다. Gβ은 KIF5A의 808에서 935아미노산 부위와 결합하며, 다른 KIF5들과도 결합함을 효모 two-hybrid assay로 확인하였다. 또한 Gβ의 WD40 반복 서열은 KIF5A와의 결합에 필수영역임을 확인하였으며, 이러한 단백질간의 결합은 Glutathione S-transferase (GST) pull-down assay를 통하여 확인하였다. 생쥐의 뇌 파쇄액에 KIF5들의 항체로 면역침강을 행하여 heterotrimeric G 단백질을 확인한 결과, KIF5들은 heterotrimeric G 단백질과 특이적으로 같이 침강하였다. 이러한 결과들은 kinesin-I는 heterotrimeric G 단백질이 포함된 소포를 미세소관을 따라 이동시킴을 시사한다. Kinesin-I exists as a tetramer of two heavy chains (KHCs, also called KIF5s), which contain the amino (N)-terminal motor domain and carboxyl (C)-terminal domain, as well as two light chains (KLCs), which bind to the KIF5s (KIF5A, KIF5B and KIF5C) stalk region. To identify the interaction proteins for KIF5A, yeast two-hybrid screening was performed and a specific interaction with the β subunit of heterotrimeric G proteins (Gβ) was found. G bound to the amino acid residues between 808 and 935 of KIF5A and to other KIF5 members in the yeast two-hybrid assay. The WD40 repeat motif of Gβwas essential for interaction with KIF5A. In addition, these proteins showed specific interactions in the glutathione S-transferase (GST) pull-down assay. An antibody to KIF5s specifically co-immunoprecipitated KIF5s associated with heterotrimeric G proteins from mouse brain extracts. These results suggest that kinesin-I motor protein transports heteroterimeric G protein attachment vesicles along microtubules in the cell.

Sorting Nexin 17 Interacts Directly with Kinesin Superfamily KIF1B<FONT FACE= HCI Tulip >Ղ Protein

Dae-Hyun Seog,Jin Han 대한생리학회-대한약리학회 2008 The Korean Journal of Physiology & Pharmacology Vol.12 No.4

KIF1BՂ is a member of the Kinesin superfamily proteins (KIFs), which are microtubule-dependent molecular motors that are involved in various intracellular organellar transport processes. KIF1BՂ is not restricted to neuronal systems, however, is widely expressed in other tissues, even though the function of KIF1BՂ is still unclear. To elucidate the KIF1BՂ-binding proteins in non-neuronal cells, we used the yeast two-hybrid system, and found a specific interaction of KIF1BՂ and the sorting nexin (SNX) 17. The C-terminal region of SNX17 is required for the binding with KIF1BՂ. SNX17 protein bound to the specific region of KIF1BՂ (813-916. aa), but not to other kinesin family members. In addition, this specific interaction was also observed in the Glutathione S-transferase pull-down assay. An antibody to SNX17 specifically co-immunoprecipitated KIF1BՂ associated with SNX17 from mouse brain extracts. These results suggest that SNX17 might be involved in the KIF1BՂ-mediated transport as a KIF1BՂ adaptor protein.

Sorting Nexin 17 Interacts Directly with Kinesin Superfamily KIF1B${\beta}$ Protein

Seog, Dae-Hyun,Han, Jin The Korean Society of Pharmacology 2008 The Korean Journal of Physiology & Pharmacology Vol.12 No.4

KIF1B${\beta}$ is a member of the Kinesin superfamily proteins (KIFs), which are microtubule-dependent molecular motors that are involved in various intracellular organellar transport processes. KIF1B${\beta}$ is not restricted to neuronal systems, however, is widely expressed in other tissues, even though the function of KIF1B${\beta}$ is still unclear. To elucidate the KIF1B${\beta}$-binding proteins in non-neuronal cells, we used the yeast two-hybrid system, and found a specific interaction of KIF1B${\beta}$ and the sorting nexin (SNX) 17. The C-terminal region of SNX17 is required for the binding with KIF1B${\beta}$. SNX17 protein bound to the specific region of KIF1Bf3 (813-916. aa), but not to other kinesin family members. In addition, this specific interaction was also observed in the Glutathione S-transferase pull-down assay. An antibody to SNX17 specifically co-immunoprecipitated KIF1B${\beta}$ associated with SNX17 from mouse brain extracts. These results suggest that SNX17 might be involved in the KIF1B${\beta}$-mediated transport as a KIF1B${\beta}$ adaptor protein.

Symposium 10 : Neurobiology ; The kinesin superfamily proteins (KIFs) and organelle transport in neuron

( Dae Hyun Seog ) 한국생화학분자생물학회 (구 한국생화학회) 2003 생화학분자생물학회 춘계학술발표논문집 Vol.2003 No.-

γ-Aminobutyric Acid Transporter 2 Binds to the PDZ Domain of Mammalian Lin-7

Dae-Hyun Seog(석대현),Il Soo Moon(문일수) 한국생명과학회 2008 생명과학회지 Vol.18 No.7

신경전달물질을 수송하는 신경전달물질 수송체는 연접전막에서 신경전달물질의 농도를 조절한다. 신경세포에 발현하는 GATs들은 연접에서 억제성 신경전달물질인 GABA의 재흡수에 관여한다. GAT2/BGT1가 어떻게 연접전막에 안정적으로 존재하는지, 어떤 결합단백질과 결합하여 조절을 받는지는 알려져 있지 않다. 본 연구에서 효모 two-hybrid system을 사용하여 GAT2의 C-말단과 특이적으로 결합하는 mammalian Lin-7 (MALS)-2을 분리하였다. GAT2의 C-말단에 존재하는 “T-X-L”아미노산 배열이 MALS-2와의 결합에 필수적으로 관여하였다. 또한 이 단백질간의 결합을 pull-down assay로 확인한 결과 MALS는 glutathione S-transferase (GST)와는 결합하지 않으나 GST-GAT2와는 결합하였다. 또한 생쥐의 뇌 균질액에서 GAT2는 MALS와 함께 침강함을 면역침강으로 확인하였다. 이러한 결과들은 MALS가 GAT2와 결합하여 GAT2를 연접전막에서 안정화시키는 역할을 함을 시사한다. Neurotransmitter transporters, which remove neurotransmittesr from the synaptic cleft, are regulated by second messenger such as protein kinases and binding proteins. Neuronal γ-aminobutyric acid transporters (GATs) are responsible for removing the inhibitory neurotransmitter γ-aminobutyric acid (GABA) from the synaptic cleft. γ-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.