배양근원세포의 산화스트레스에 대한 신트로핀의 기능 연구
Role of α-syntrophin under oxidative stress in cultured muscle cells
- 주제(키워드) α-Syntrophin , oxidative stress , myoblast differentiation , ER stress
- 발행기관 아주대학교
- 지도교수 김혜선
- 발행년도 2019
- 학위수여년월 2019. 2
- 학위명 박사
- 학과 및 전공 일반대학원 생명과학과
- 실제URI http://www.dcollection.net/handler/ajou/000000028542
- 본문언어 영어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
α-Syntrophin is a component of the dystrophin-glycoprotein complex that interacts with various intracellular signaling proteins in muscle cells. The α-syntrophin knock-down C2 cell line (SNKD), established by infecting lentivirus particles with α-syntrophin shRNA, is characterized by a defect in terminal differentiation and increase in cell death. Since myoblast differentiation is accompanied by intensive mitochondrial biogenesis, the generation of intracellular reactive oxygen species (ROS) is also increased during myogenesis. Two-photon microscopy imaging showed that excessive intracellular ROS accumulated during the differentiation of SNKD cells as compared to control cells. The formation of 4-hydroxynonenal adduct, a byproduct of lipid peroxidation during oxidative stress, significantly increased in differentiated SNKD myotubes and was dramatically reduced by epigallocatechin-3-gallate, a well-known ROS scavenger. Among antioxidant enzymes, catalase was significantly decreased during differentiation of SNKD cells without changes at the mRNA level. Of interest was the finding that the degradation of catalase was rescued by MG132, a proteasome inhibitor, in the SNKD cells. These results suggest that α-syntrophin plays an important role in the regulation of oxidative stress from endogenously generated ROS during myoblast differentiation by modulating the protein stability of catalase. In addition, ER stress was increased and the rate of protein synthesis was significantly decreased during differentiation of SNKD cells. α-Syntrophin overexpression suppressed ER stress induced by endogenously and exogenously. Protein synthesis during differentiation was also increased by overexpression of α-syntrophin. Moreover, as the result of immunoprecipitation, proteins interacting with α-syntrophin under menadione-induced oxidative stress were increased. These results demonstrate that α-syntrophin has a protective role in response to oxidative stress and ER stress by interacting and stabilizing other proteins. This study suggests a novel function of α-syntrophin in the maintenance of proteostasis during myoblast differentiation.
more초록/요약
α-Syntrophin은 dystrophin-glycoprotein 복합체의 구성요소로서, 근육세포 내의 다양한 신호전달물질과 상호작용한다. C2 근육세포에 α-Syntrophin 특이적인 shRNA를 주입하여 α-syntrophin knock-down (SNKD) 세포주를 제작하여 특성을 분석한 결과 분화 후기의 결함이 나타났으며, 세포 사멸이 증가했다. 근원세포의 분화 과정은 광범위한 미토콘드리아 생합성을 동반하기 때문에 세포 내 활성산소종 (ROS)의 생성 또한 분화 과정 동안에 증가하게 된다. Two-photon microscopy를 이용하여 미토콘드리아의 ROS를 측정한 결과, 대조군에 비하여 SNKD 세포에서 과도한 양의 ROS가 분화 과정 동안 축적되어 있었으며, 산화스트레스에 의한 지질 과산화의 부산물인 4-hydroxynonenal adduct가 분화한 SNKD 세포에서 증가했다. 항산화물질인 epigallocatechin-3-gallate (EGCG)를 이용하여 SNKD 세포를 분화 시킨 결과 4-hydroxynonenal adduct 생성이 크게 감소했다. SNKD 세포의 분화 과정에서 catalase의 단백질 발현이 유의하게 감소하였으나 mRNA 수준의 변화는 없었다. Catalase의 분해와 관련된 단백질 분해효소를 스크리닝한 결과 proteasome inhibitor인 MG132에 의해 SNKD 세포에서의 catalase 분해가 억제되었다. 이러한 결과는 α-syntrophin이 catalase의 stability에 관여함으로써 분화 과정 동안에 생성되는 ROS를 조절하는 과정에 중요한 역할을 한다는 것을 의미한다. 또한 SNKD 세포의 분화 과정 동안에 ER stress가 증가했으며 단백질 합성이 크게 감소하였다. α-Syntrophin을 과발현시킴으로써 내재적으로 유도된 ER stress 및 menadione에 의한 산화스트레스로 유도된 ER stress가 억제되었다. 분화 과정 동안의 단백질 합성 또한 α-syntrophin 과발현 세포에서 증가하였다. Menadione을 처리하여 산화스트레스를 유도하면 α-syntrophin과 결합하는 단백질이 증가하는 것을 immunoprecipitation으로 확인하였다. 이러한 결과는 α-syntrophin이 다른 단백질과 결합하고 안정화시킴으로써 산화스트레스 및 ER stress에 대한 세포의 반응 과정에 보호적 역할을 한다는 것을 의미한다. 이 연구는 분화 과정 동안의 단백질 항상성 유지에 관련한 α-syntrophin의 새로운 역할을 규명하였다.
more목차
I. Introduction 1
A. Differentiation of muscle cells 1
B. α-Syntrophin 5
C. Oxidative stress 9
1. Reactive oxygen species (ROS) 9
2. Antioxidant system 10
D. Endoplasmic reticulum (ER) stress and ubiquitin-proteasome system (UPS) 13
E. Protein homeostasis in muscle cells 16
F. The aims of study 18
II. Materials and Methods 19
A. Reagents and antibodies 19
B. Cell culture 20
C. Transfection of shRNA 20
D. Reverse transcriptase-PCR and conventional PCR 20
E. Western blot 21
F. Hematoxylin staining and fusion index 22
G. Trypan blue exclusion assay 22
H. DCFDA staining 23
I. Two-photon microscopy 23
J. Real-time PCR 24
K. Cycloheximide pulse chase assay 25
L. Plasmid DNA and siRNA transfection 25
M. Immunoprecipitation 25
N. Calculation of protein/DNA ratio 26
O. Statistical analysis 26
III. Results 28
A. Establishment of α-syntrophin knock-down cell line 28
B. Deficiency of α-syntrophin causes cell death during myogenic differentiation 31
C. Accumulation of endogenous ROS induces oxidative stress in SNKD cells 36
D. EGCG alleviated oxidative stress generated by endogenous ROS in SNKD cells 40
E. Protein stability of catalase is significantly decreased in differentiated SNKD cells 45
F. Catalase in SNKD cells is subject to UPS regulation 53
G. α-Syntrophin is involved in the ER stress during myoblast differentiation 58
H. α-Syntrophin modulates the protein synthesis during myoblast differentiation 63
I. α-Syntrophin has a protective role in the menadione-induced ER stress 68
IV. Discussion 73
A. Role of α-syntrophin under oxidative stress during myoblast differentiation 73
B. Oxidative stress and protein homeostasis 78
V. References 82
VI. 국문요약 96
