Stress granule 형성에 있어 PDCD4의 기능적 연구

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Programmed cell death 4 (PDCD4)는 새로운 종양 억제자이고 핵과 세포질에서 전사와 번역을 조절하는 기능을 한다. Stress granules (SGs)는 세포가 환경적 스트레스에 노출이 되면 비번역 mRNA가 축적된 세포질 군집을 일컫는다. 한때 PDCD4는 진핵세포의 번역개시인자 4A (eIF4A) 억제를 통하여 번역 억제와 연관이 있는 것으로 알려졌다. 여기서 우리는 PDCD4가 산화적 스트레스에서 SG 형성의 과정에 기능적인 역할을 하는지 연구했다. 우리는 면역형광염색법을 이용하여 산화적 스트레스에서 PDCD4가 SGs에 국한되는 것을 발견했다. 다음에 우리는 PDCD4 특이적 siRNA를 사용하여 감소된 PDCD4가 SG 형성에 영향을 주는지 평가했다. 흥미롭게도 SG 형성이 eIF2α의 인산화와 eIF4E 결합 단백질 (4E-BP) 의 탈인산화의 결과로 촉진되어지는 것을 확인했다. 우리는 PDCD4의 감소는 polysome disassembly을 촉진 시키고 과산화수소 스트레스에서 eIF4F 복합체를 파괴시키고 eIF4E:4EBP 상호작용을 강요한다. 게다가, PDCD4의 과발현은 스트레스 상태에서 SG 형성을 감소시킨다.
이러한 결과는 PDCD4가 SG에 역학적인 역할 형성에 관여하고 스트레스 상태에서 cap 의존성 번역 억제에 관여 한다는 것을 밝혔다.|Programmed cell death 4 (PDCD4) is a novel tumor suppressor that function in the nucleus and the cytoplasm and appears to be involved in the regulation of transcription and translation. Stress granules (SGs) are cytoplasmic foci at which untranslated mRNAs accumulate when cells exposed to environmental stresses. Since PDCD4 has implicated in translation repression through direct inhibition of eukaryotic translation initiation factor 4A (eIF4A), we here investigated if PDCD4 has a functional role in the process of SG assembly under oxidative stresses. Using immunofluorescence microscopy, we found that PDCD4 is relocalized to SGs under oxidative stresses. Next, we tested if knockdown of PDCD4 has an effect on the assembly of SG using PDCD4-specific siRNA.
Interestingly, SG assembly was accelerated and this effect was caused by sensitization of phosphorylation of eIF2a and dephosphorylation of eIF4E binding protein (4E-BP). We demonstrate that knockdown of PDCD4 accelerated polysome disassembly and causes eIF4F complex disruption and ehances eIF4E:4EBP1 interaction under H2O2 stress. Moreover, Overexpression of PDCD4 decrease stress granule assembly under stress conditions.
These results suggest that PDCD4 has an effect on SG dynamics and possibly involved in cap-dependent translation repression under stress conditions.
Alternative Title
Functional analysis of PDCD4 on the assembly of stress granule
Alternative Author(s)
Park, Ra Young
일반대학원 생물신소재학과
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Table Of Contents
List of Figures i
Abstract 1
국문초록 3
l. Introduction 4
ll. Materials and Methods 8
ll-1. Cell culture and transfection 8
ll-2. Reagents and antibodies. 8
ll-3. Construction of plasmids 9
ll-4. Preparation of cell lysate and western blot analysis 9
ll-5. Immunofluorescence microscopic analysis 10
ll-6. Polysome profiling analysis 11
ll-7. RNA interference 11
ll-8. Methyl Cap Pull-Down Assay. 12
ll-9. Cytosolic and Nuclear Protein Fractionation. 13
lll. Results 14
lll-1. Knockdown of PDCD4 accelerates stress granule assembly 14
lll-2. PDCD4 is component of SGs under oxidative stresses 17
lll-3. PDCD4 is colocalized to SGs under oxidative stresses 20
lll-4. Relocalization of PDCD4 from nucleus to cytoplasm under oxidative
stress condition. 23
lll-5. SG assembly was accelerated by sensitization of phosphorylation of eIF2α under sodium arsenite stress 26
lll-6. SG assembly was accelerated by sensitization of dephospho-rylation eIF4E binding protein under H2O2 stress 29
lll-7. Overexpression of PDCD4 decrease stress granule assembly under SA stress 32
lll-8. Knockdown of PDCD4 accelerated polysome disassembly under SA stress 35
lll-9. knockdown of PDCD4 causes eIF4F complex disruption and enhance eIF4E:4E-BP1 interaction under H2O2 stress 38
lV. Discussion 43
V. References 46
VI. Acknowledgements 50
조선대학교 대학원
박라영. (2014). Stress granule 형성에 있어 PDCD4의 기능적 연구.
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General Graduate School > 4. Theses(Ph.D)
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