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.

Microtubules and Kinesin Superfamily Proteins and Organelle Transport

Seog, Dae-Hyun 東國大學校醫學硏究所 2001 東國醫學 Vol.8 No.-

The neuron sort proteins and lipids, after their synthesis, to various destinations at appropriate velocities in membranous organelles and protein complexes. Intracellular transport is thus fundamental to cellular morphogenesis and functioning. Microtubules serve as a rail on which motor proteins, such as kinesin and dynein superfamily proteins, convey their cargoes. This review focuses on the kinesin superfamily proteins and the molecular mechanism of organelle transport in neuronal cells.

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

Dae-Hyun Seog 한국생명과학회 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, I 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 vesicle precursors.

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

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

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.

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.

Direct Interaction of KIF5s and Actin-Based Transport Motor, Myo9s

Dae-Hyun Seog(석대현) 한국생명과학회 2011 생명과학회지 Vol.21 No.8

미세소관(microtubule) 위를 이동하는 키네신은 분비소포를 이동시키는 운동단백질이다. KIF5s (KIF5A, KIF5B and KIF5C)는 세포막으로 싸인 각종 세포 내 소기관과 결합하여 미세소관을 따라 목적지까지 이동시킨다는 결과는 알려져 있지만, 어떻게 상대의 cargo를 인식하는지는 밝혀지지 않았다. 본 연구는 KIF5B의 결합 단백질을 동정하기 위하여 효모 two-hybrid system을 사용하여 KIF5B와 특이적으로 결합하는 Myo9b을 확인하였다. Myo9b는 액틴위를 이동하는 운동단백질로 다른 KIF5s들과도 결합함을 효모 two-hybrid assay로 확인하였다. 또한 Myo9s의 GTPase 활성화 단백질(GAP) 영역은 KIF5B와 결합하는데 필수영역임을 확인하였고, 이러한 단백질간의 결합은 Glutathione S-transferase (GST) pull-down assay를 통하여서도 확인하였다. 생쥐의 뇌 파쇄액에 KIF5B들의 항체로 면역침강을 행하여 Myo9s 단백질을 확인한 결과, KIF5s는 Myo9s 단백질과 특이적으로 함께 침강하였다. 이러한 결과들은 kinesin-I는 액틴 결합 운동단백질과 직접 결합함을 보여준다. Microtubule-based kinesin motor proteins are used for long-range vesicular transport. KIF5s (KIF5A, KIF5B and KIF5C) mediate the transport of various membranous vesicles along microtubules, but the mechanism behind how they recognize and bind to a specific cargo has not yet been completely elucidated. To identify the interaction protein for KIF5B, yeast two-hybrid screening was performed and a specific interaction with the unconventional myosin Myo9b, an actin-based vesicle transport motor, was found. The GTPase-activating protein (GAP) domain of Myo9s was essential for interaction with KIF5B in the yeast two-hybrid assay. Myo9b bound to the carboxyl-terminal region of KIF5B and to other KIF5 members. In addition, glutathione S-transferase (GST) pull-downs showed that Myo9s specifically interact to the complete Kinesin-I complex. An antibody to KIF5B specifically co-immunoprecipitated KIF5B associated with Myo9s from mouse brain extracts. These results suggest that kinesin-I motor protein interacts directly with actin-based motor proteins in the cell.