S-Space College of Natural Sciences (자연과학대학) Dept. of Biological Sciences (생명과학부) Theses (Ph.D. / Sc.D._생명과학부)
Molecular functions of PINK1 in regulating Parkin and VDAC
- 자연과학대학 생명과학부
- Issue Date
- 서울대학교 대학원
- 학위논문 (박사)-- 서울대학교 대학원 : 생명과학부, 2016. 2. 정종경.
- Parkinsons disease (PD) is the second most common neurodegenerative disease caused by degeneration of dopaminergic neurons in substantia nigra pars compacta. Although majority of PD cases are sporadic types, recent studies have consistently suggested that genetic backgrounds also play a critical role in PD pathogenesis. Indeed, more than twenty PD-associated genes have been discovered so far, and their physiological roles have been extensively studied to decipher the molecular mechanism of the disease. Among the PD-associated genes, PINK1 and Parkin have been most thoroughly studied, and their functions in regulating mitochondrial homeostasis, such as mitochondrial dynamics, trafficking, and mitophagy, are highly implicated in PD pathogenesis.
Firstly, to further investigate the physiological functions and patho-mechanism of PINK1, I studied the effects of 17 representative PINK1 mutations identified in PD patients in both mammalian cells and Drosophila model system. Interestingly, these mutations did not affect the typical cleavage patterns and subcellular localization of PINK1 under both normal and damaged mitochondria conditions in mammalian cells. However, PINK1 mutants with mutations in the kinase domain failed to translocate Parkin to mitochondria and to induce mitochondrial aggregation. Consistent with the mammalian data, Drosophila PINK1 mutants with mutations in the kinase domain (G426D and L464P) did not genetically interact with Parkin during eye development. Furthermore, PINK1-deficient flies expressing the transgenic PINK1 G426D mutant displayed defective phenotypes with increasing age, whereas PINK1 L464P mutant-expressing flies showed defects at an earlier age. These results consistently supported the hypothesis that the kinase activity of PINK1 is essential for its function and for Parkin regulation in mitochondria.
Secondly, I found a novel post-translational modification on VDAC/Porin by Parkin. When Parkin was activated by dissipating mitochondrial membrane potential or PINK1 co-expression, Parkin mono-ubiquitinated VDAC1. Although the VDAC/Porin which could not be mono-ubiquitinated did not affect PINK1/Parkin-mediated mitophagy, it selectively induced apoptosis in both mammalian cells and Drosophila in a Bax-dependent manner. Furthermore, downregulation of VDAC1/Porin suppressed defective phenotypes of PINK1-deficient fly, including downturned wing posture and crushed thorax morphology. These data strongly implicated that regulation of VDAC/Porin mono-ubiquitination by the PINK1/Parkin pathway is important for PD pathogenesis.
Thirdly, using an RNAi-based genetic screen, I screened 369 Drosophila genes encoding mitochondrial inner membrane proteins to discover novel components of the PINK1/Parkin pathway. Fortunately, I identified a novel component of the PINK1/Parkin pathway (NCP), a small G-protein, as a positive regulator of the PINK1/Parkin pathway. Expression of RNAi for NCP strongly suppressed the defects induced by PINK1 overexpression during Drosophila eye development. Conversely, NCP overexpression suppressed the downturned wing and crushed thorax phenotypes of PINK1-deficient fly, which correlated well with improved ATP contents and climbing ability. These results strongly suggested that NCP is a critical downstream target of PINK1 with novel characteristics in mitochondria.
In summary, I found that the kinase activity of PINK1 is indispensable in regulating its functions and downstream targets, and defective kinase activities of PINK1 tightly correlate with PD pathogenesis. I also demonstrated that PINK1 regulates apoptosis through Parkin-dependent VDAC/Porin mono-ubiquitination, which provides a novel molecular mechanism of how the PINK1/Parkin pathway controls apoptosis in PD pathogenesis. With understanding of these new functions of the PINK1/Parkin pathway and identification a novel component of the pathway, my thesis studies would provide us a new insight into the molecular mechanisms of PD pathogenesis.