Targeting PSMD14 induces paraptosis by disrupting proteostasis and Ca2+ homeostasis
- 주제(키워드) PSMD14 , Paraptosis , Proteasome , Ca2+ homeostasis , Protein trafficking
- 주제(DDC) 570
- 발행기관 아주대학교
- 지도교수 최경숙
- 발행년도 2022
- 학위수여년월 2022. 2
- 학위명 석사
- 학과 및 전공 일반대학원 의생명과학과
- 실제URI http://www.dcollection.net/handler/ajou/000000031654
- 본문언어 영어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
PSMD14 is a subunit of the 19S regulatory particle of the proteasome with deubiquitinating enzyme activity and its high expression was reported to be associated with the poor prognosis of many cancers. In the present study, I showed that the silencing of PSMD14, as well as pharmacological inhibition of PSMD14 with capzimin (CZM), induces paraptosis, a cell death mode accompanied by dilation of the endoplasmic reticulum (ER) and mitochondria in breast cancer cells. I found that CZM markedly induced ER stress and cycloheximide, a protein synthesis inhibitor, almost completely blocked CZM-induced paraptosis. I also found that CZM induced the depletion and ER Ca2+ and subsequent increases in cytosolic and mitochondrial Ca2+ in a proteasome-independent manner. Interestingly, pretreatment with BAPTA-AM, an intracellular Ca2+ chelator, effectively attenuated CZM-induced increase in cytosolic Ca2+ levels and paraptotic cell death. Furthermore, CZM induced disruption of the Golgi apparatus and possibly protein transport system, whereas bortezomib, a proteasome inhibitor, did not. Unexpectedly, inhibition of IRE1α or JNK enhanced CZM-induced increase in cytosolic Ca2+ levels and accelerated vacuolization. Taken together, these results demonstrate that disruption of both proteostasis and Ca2+ homeostasis may contribute to the paraptosis induced by PSMD14 inhibition.
more목차
I. INTRODUCTION 1
II. MATERIALS AND METHODS 11
A. Chemicals and antibodies. 11
B. Cell culture 11
C. Cell viability assay 12
D. Live-cell imaging 12
E. Morphological examination of the ER and mitochondria 13
F. Immunoblot analysis 13
G. Immunofluorescence microscopy 14
H. Measurement of cytosolic and mitochondrial Ca2+ levels 14
I. Small interfering RNA-mediated knockdown 15
J. Transmission electron microscopy 15
K. Assessment of proteasome activity using UbG76V-GFP accumulation 16
L. Statistical analysis 16
III. RESULTS 17
1. PSMD14 inhibition induces cell death accompanied by cytoplasmic vacuolation in MDA-MB 435S cells 17
2. Proteasome inhibition is not enough to explain the paraptosis induced by PSMD14 inhibition 30
3. CZM induces disruption of intracellular Ca2+ homeostasis 36
4. Both proteasome inhibition and Ca2+ imbalance critically contribute to CZM-induced paraptosis in MDA-MB 435S cells 44
5. Dysregulation of ER-to-Golgi protein trafficking may be associated with the paraptosis induced by PSMD14 inhibition 55
IV. DISCUSSION 62
V. REFERENCES 71
VI. 국문요약 87
