View : 760 Download: 0

Full metadata record

DC Field Value Language
dc.contributor.advisor이공주-
dc.contributor.author김윤경-
dc.creator김윤경-
dc.date.accessioned2016-08-26T12:08:10Z-
dc.date.available2016-08-26T12:08:10Z-
dc.date.issued2005-
dc.identifier.otherOAK-000000011801-
dc.identifier.urihttps://dspace.ewha.ac.kr/handle/2015.oak/191435-
dc.identifier.urihttp://dcollection.ewha.ac.kr/jsp/common/DcLoOrgPer.jsp?sItemId=000000011801-
dc.description.abstract핵인내 인산화 단백질 p140 (hNopp140)는 핵인의 생합성과정에 관여하며 항암약제인 독소루비신에 결합 할 수 있다. 대장균에서 생성한 제 조합형 hNopp140단백질은 3.3 x10-7 M의 분리상수를 가지며 특이적으로 독소루비신에 결합한다. 그리고 이들간의 친화력은 ATP의 존재 하에 매우 증가한다. 반면에 카세인 인산화 효소II에 의해 광범위하게 인산화된 hNopp140는 독소루비신에 결합하지 못한다. 더욱이 독소루비신은 세포 추출물에 있는 hNopp140와 생체 외 반응으로 제 조합하여 만든 hNopp140모두의 인사화 정도를 감소시킨다. 이는 독소루비신이 생체 내 hNopp140의 인산화 과정을 조절함으로써 hNopp140의 세포 내 기능을 방해 할 것임을 가리킨다. 흥미롭게도 hNopp140는 카세인 인산화 효소 II의 작용을 조절한다. 카세인 인산화 효소II에 의한 알파 카세인의 인사화는 원형의 hNopp140와 인산화된 형태의 hNopp140 모두에 의해서 인산화가 현격하게 줄어든다. 생체내의 또 다른 카세인 인산화 효소II의 기질인 geminin의 인산화 역시 hNopp140에 의해 감소한다. 원형의 hNopp140는 카세인 인산화 효소의 베타 기본단위와 결합하고 인사화 형태의 hNopp140는 알파 기본단위와 결합한다. 그러나 이러한 결합은 카세인 인산화 효소II의 알파 베타 기본단위간의 거대 효소 형성을 위한 조합을 방해하진 않는다. 그러므로 우리는 hNopp140 단백질이 카세인 인산화 효소II에 결합하여 거대효소 형성 방해가 아닌 다른 방법으로 인산화 효소II의 기능을 조절할 것이라고 추정한다. 게다가 이미 hNopp140에 결합한 독소루비신은 세포추출물 내에 존재하는 내재 hNopp140와 마찬가지로, 세포 밖에서 생산된 hNopp140단백질의 인산화 정도를 감소시킨다. 이러한 결과는 독소루비신이 hNopp140의 인산화 정도를 조절함으로써 세포 내 기능을 변화시킬 수 있음을 제안한다. hNopp140는 비록 열변성에 강한 저항성을 가지고 있지만 이차구조가 결여되어있고 단백질 분해 반응에 매우 민감한 특성을 가지고 있다. 이러한 특성은 hNopp140가 원래 풀린 구조 단백질 중 하나임을 시사하고 이는 핵 형성 과정에 관여하는 다양한 단백질과의 결합이나 독소루비신과 같은 약제와의 결합에 대한 설명도 가능하게 해준다.; Human nucleolar phosphoprotein p140, hNopp140, is involved in the biogenesis of nucleoli and can bind to an anti-cancer agent, doxorubicin. Recombinant hNopp140 prepared from E. coli specifically bound to doxorubicin with KD value of 3.3 × 10-7 M, and the affinity was significantly enhanced in the presence of ATP. On the contrary, extensively phosphorylated hNopp140 by protein kinase CKII failed to bind doxorubicin. Furthermore doxorubicin lowered the degree of phosphorylation of the recombinant hNopp140 in vitro as well as the endogenous hNopp140 in cell extract, indicating that doxorubicin may perturb the cellular function of hNopp140 by regulating the level of phosphorylation. Interestingly, hNopp140 regulated phosphorylation activity of CKII. Phosphorylation of α-casein by CKII was dramatically reduced in presence of both native and phosphorylated hNopp140. Phosphorylation of geminin, CKII substrate in vitro and in vivo, also was decreased by hNopp140. Native form of hNopp140 bound to CKII β-subunit and phosphorylated form interacted with CKII α-subunit. But these interactions did not perturb the assembly of CKII α- and β- subunits for formation of holoenzyme. So we assumed that hNopp140 regulated CKII activity via interaction with CKII subunits but not disturbing the formation of CKII complex. Furthermore, previously-bound doxorubicin lowered the degree of phosphorylation of recombinant hNopp140 in vitro, as well as of endogenous hNopp140 in a cell extract. These results suggest that doxorubicin may perturb the cellular function of hNopp140 by regulating its level of phosphorylation. Although hNopp140 exhibited a strong resistance to thermal denaturation, it had little secondary structure and was highly sensitive to proteolysis. These characteristics imply that hNopp140 is a member of natively unfolded proteins, which can interact with a variety of proteins involved in nucleogenesis, as well as therapeutic molecules such as doxorubicin.-
dc.description.tableofcontentsAbstract = xi I. Introduction = 1 I-1. Identification of drug receptor = 1 I-2. Identification of doxorubicin binding protein = 7 I-2-1. Doxorubicin, an anti tumor agent = 7 I-2-1. Isolation of doxorubicin binding protein = 9 I-3. Human nucleolar phosphoprotein p140, hNopp140 = 9 II. Materials and Methods = 13 II-1.Materials = 13 II-1-1. Chemicals and media = 13 II-1-2. Bacterial strains and plasmid = 14 II-1-3. Mammalian cell line and expression = 14 II-2. Methods = 15 II-2-1. Expression and purification of proteins = 15 II-2-2. Biotinylation of doxorubicin = 18 II-2-3. Phosphorylation of proteins = 19 II-2-4. Cell culture and transfection = 20 II-2-5. Size exclusion chromatography = 20 II-2-6. Circular dichroism spectroscopy = 21 II-2-7. Ion dependent aggregation = 21 II-2-8. ATPase / GTPase activity = 21 II-2-9. Heat stability test and limited proteolysis = 22 II-2-10. Surface plasmon resonance = 22 II-2-11. Pull down assay using biotinylated doxorubicin = 23 II-2-12. GST-pull down assay = 24 III. Results = 25 III-1. Production of hNopp140 proteins = 25 III-1-1. Cloning of the expression vector = 25 III-1-2. Expression and purification of hNopp140 proteins = 28 III-1-3. Phosphorylation of hNopp140 proteins = 32 III-2. Interaction between hNopp140 and doxorubicin = 32 III-2-1. Binding of native hNopp140 to doxorubicin = 32 III-2-2. Enhancement of interaction by ATP = 36 III-2-3. Diminishing of interaction by phosphorylation of hNopp140 = 38 III-3. Inhibitory effect of doxorubicin on hNopp140 phosphorylation = 41 III-3-1. Doxorubicin inhibited the phosphorylation of recombinant hNopp140 by protein kinase CKII = 41 III-3-2. Doxorubicin inhibited the phosphorylation of hNopp140 expressed from 293T cell. = 46 III-3-3. Protein kinases, cdc2 kinase and PKA phosphorylate hNopp140 in presence of doxorubicin. = 46 III-4. Characterization of hNopp140 = 47 III-4-1. Oligomeric state of hNopp140 =47 III-4-2. Ion dependent complex formation of phosphorylated hNopp140 = 51 III-4-3. Weak ATPase/GTPase activity of recombinant hNopp140 = 52 III-4-4. Heat stability and protease sensitivity of hNopp140 = 52 III-4-5. Secondary structure prediction and circular dichroism spectrometry = 59 III-5. Regulation of protein kinase CKII by hNopp140 = 60 III-5-1. Expression and purification of protein kinase CKII α-/β-subunit = 60 III-5-2. Preparation of Geminin protein as CKII substrate = 69 III-5-3. hNopp140 regulation of protein kinase CKII activity = 69 III-5-4. Interaction of hNopp140 and protein kinase CKII subunits = 77 III-5-5. Phosphorylation of hNopp140 by GST-CKII α-subunit = 77 III-5-6. hNopp140 effect on CKII holoenzyme assembly = 79 IV. Discussion = 83 IV-1. Interaction between hNopp140 and doxorubicin = 83 IV-2. Regulation of protein kinase CKII activity by hNopp140 = 85 IV-3. hNopp140, novel ‘disordered protein’ = 89 V. References = 92 국문요약 = 98-
dc.formatapplication/pdf-
dc.format.extent5153068 bytes-
dc.languageeng-
dc.publisher이화여자대학교 대학원-
dc.titleCharacterization of the interaction of human nucleolar phosphoprotein p140 (hNopp140) with doxorubicin and protein kinase CKII-
dc.typeDoctoral Thesis-
dc.title.translated핵인내 인산화 단백질 p140 (hNopp140)의 독소루비신과 단백질 인산화 효소 CKII와의 결합특성 연구-
dc.format.pagexii, 99p.-
dc.identifier.thesisdegreeDoctor-
dc.identifier.major대학원 분자생명과학부-
dc.date.awarded2006. 2-
Appears in Collections:
일반대학원 > 생명·약학부 > Theses_Ph.D
Files in This Item:
There are no files associated with this item.
Export
RIS (EndNote)
XLS (Excel)
XML


qrcode

BROWSE