BRCA1 (乳癌相關蛋白1)為一種乳癌及卵巢癌專一性的抑制因子，且基因上的突變會大幅提升女性得到乳癌及卵巢癌的機率。研究發現BRCA1及BARD1 藉由RING 結構域之間的交互作用形成異源二聚體，進而參與脫氧核醣核酸的損傷修復、中心體及轉錄的調控。中心體為動物細胞內主要的微管組織中心，當調控異常時會導致中心體不正常增生及分裂。研究發現BRCA1/BARD1 的RING 結構域具有E3 泛素連接酶的活性，能促使中心體內 -微管蛋白進行泛素化修飾，抑制微管生成及調控中心體數目。在細胞週期中的任一時期BRCA1/BARD1皆在中心體中，且在試管實驗中發現BRCA1/BARD1進行自我泛素化修飾，可能導致蛋白質降解。目前對於如何調控E3 泛素連接酶仍然還是未知。有趣的是，在中心體中BRCA1/BARD1被發現能與蛋白OLA1結合，同時也發現 OLA1基因表現減量會導致中心體不正常增生及過度活化微管生成。因此，我的論文主要探討BRCA1/BARD1是否調控OLA1酵素活性及OLA1是否調控BRCA1/BARD1 E3泛素連接酶的活性。實驗數據顯示BRCA1/BARD1刺激OLA1的酵素活性但不調控BRCA1/BARD1的自我泛素化，此外我們發現BRCA1/BARD1能泛素化OLA1。因此，藉由酵素活性實驗及泛素化實驗，我們提供了一個新的見解來解釋BRCA1/BARD1及OLA1的功能。

BRCA1 (breast cancer associated gene 1) is a breast- and ovary-specific tumor suppressor. Germline mutations of this protein increase woman's risk of breast and ovarian cancers. BRCA1 is involved in DNA repair, centrosome regulation and transcription. Centrosomes are the major microtubule-organizing center (MTOC) in animal cells and misregulation of centrosomes leads to centrosome amplification and abnormal cell division. The RING domains of BRCA1 and BARD1 (BRCA1-associated RING domain protein 1) form a heterodimer and function as an E3 ligase. The BRCA1/BARD1 complex ubiquitinates K48 of -tubulin, a major microtubule-nucleating component of centrosomes, to inhibit microtubule aster formation and regulate centrosome number during cell division. However, BRCA1/BARD1 localizes at the centrosome throughout the cell cycle. In addition, BRCA1/BARD has been observed to ubiquitinate itself in vitro and in cells and may promote its proteasomal degradation. The regulatory mechanism of BRCA1/BARD1 E3 ligase is still unknown. The previous study showed that Obg-like ATPase 1 (OLA1) interacts with the RING domain of BRCA1 and BARD1 BRCT domain in the centrosome. Knockdown of OLA1 causes centrosome amplification and hyperactive microtubule nucleation. To understand the biochemical and functional interplay between BRCA1/BARD1 with OLA1, we asked whether BRCA1/BARD1 can regulate the ATPase activity of OLA1 and whether OLA1 can regulate the E3 ligase activity of BRCA1/BARD1. Our data revealed that BRCA1/BARD1 stimulates the ATPase activity of OLA1, but OLA1 has no specific effect on the auto-ubiquitination of BRCA1/BARD1. Interestingly, OLA1 is ubiquitinated by BRCA1/BARD1 in vitro. These data provides a new insight to the regulation of OLA1 by BRCA1/BARD1.
 

中文摘要 II
Abstract III
Contents VI
Abbreviation 1
Chapter 1. Introduction 3
Chapter 2. Materials and methods 9
Chapter 3. Result 20
Chapter 4. Conclusion & Discussion 27
Reference 31
Figure 4
Supplementary data 52

 

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