氏 名 林下
ハヤシシタ瑞
ミ ズ希
キ所 属 理工学研究科 生命科学専攻 学 位 の 種 類 博士（理学）
学 位 記 番 号 理工博 第
303号 学位授与の日付 令和元年
9月
30日
課程・論文の別 学位規則第４条第１項該当
学 位 論 文 題 名
BAG6 deficiency induces mis-distribution of mitochondrial clusters under inhibition of oxidative phosphorylation.脱分極ミトコンドリアの移動プロセスにおける
BAG6の新規機能
（英文）
論 文 審 査 委 員 主査 教授 川原 裕之 委員 教授 岡本 龍史 委員 准教授 安藤 香奈絵
【論文の内容の要旨】
Mitochondria are dynamic organelles that continue fission, fusion, and redistribution in cells. Mitochondria play pivotal roles in cells such as energy production, calcium storage, and apoptosis. Because the quality control of mitochondria is essential for the maintenance of cellular homeostasis, dysfunction of mitochondria was implicated in many neurodegenerative diseases.
PTEN induced kinase 1 (PINK1) and Parkin that have been identified from patients of Parkinson’s disease regulate mitophagy in cells. In stressed conditions, PINK1 accumulates on the surface of the outer mitochondrial membrane and recruits Parkin from the cytosol to damaged mitochondria where relocated parkin subsequently ubiquitinates mitochondrial outer membrane proteins. The polyubiquitinated proteins recruit an autophagic adapter p62/SQSTM1 to damaged mitochondria. p62/SQSTM1 mediates the aggregation of the damaged mitochondria in the perinuclear region.
However, detailed molecular mechanism how mitochondrial translocation is controlled under oxidative stressed conditions had not been elucidated adequately.
BAG6 contains a ubiquitin-like domain at its N-terminus and a nuclear localization signal and a BAGS domain at its C-terminus. It was reported that the N-terminal region is responsible for interaction with E3 ubiquitin ligases such as RNF126, and is assumed to play roles in the defective protein recognition and degradation. While the
C-terminus was found to form a complex with UBL4A, TRC35 and TRC40 to sort tail-anchored (TA) membrane protein to the endoplasmic reticulum. Thus, BAG6 plays distinct roles depend on its interacting proteins. Here, I tried to identify novel BAG6-interacting proteins by mass spectrometry and identified 471 proteins as candidates for BAG6-interacting proteins. I analyzed the localization of the identified proteins and found that 56%, 31%, and 6% of the proteins as cytosolic, nuclear, and mitochondrial proteins, respectively. To examine whether BAG6 is a genuine mitochondrion localizing protein, I fractionated HeLa cells lysate into cytosolic, microsome, MAM (mitochondria-associated membranes), and pure mitochondrial fractions. I found that BAG6 was in the pure-mitochondrial fraction and its signal disappeared after sequential wash with high salt buffer, indicating that BAG6 is bound to the mitochondrial surface by interaction with other mitochondrial proteins.
To examine the roles of BAG6 in mitochondria, I knocked down the BAG6 gene with specific small interfering RNA (siRNA) in HeLa cells. Any significant differences of mitochondrial distribution were observed in the BAG6 knockdown cells. The result suggests that BAG6 does not have essential roles in the maintenance of mitochondrial dynamics in basal conditions. To examine the roles of BAG6 in the Parkin-dependent pathway, I examined the distribution of mitochondria under stressed conditions induced by CCCP (carbonyl cyanide m-chlorophenyl hydrazine) in HeLa cells transiently expressing Parkin. In oxidative stressed conditions, mitochondria showed Parkin-dependent relocalization of mitochondrial clusters to the perinuclear cytoplasmic region, while most of the mitochondria in BAG6 knockdown cells formed clusters after CCCP treatment, but failed to translocate to the perinuclear region and were scattered in the cytoplasm. Since Parkin and p62/SQSTSM1 relocalize to the mitochondrial surface in the oxidative stressed conditions, I examined whether BAG6 knockdown affects the relocalization of these proteins. The result shows that BAG6 knockdown did not impair the relocalization of Parkin and p62/SQSTSM1 to the mitochondrial surface, suggesting that BAG6 acts downstream of these proteins in the pathway. Collectively, these results demonstrate that BAG6 might possess an essential function in the perinuclear translocation of damaged mitochondria that were mediated by the Parkin-dependent pathway.
