中心體是哺乳動物細胞中組織微管的中心，它在細胞分裂及紡錘絲的生成過程中同時扮演著重要的角色。BRCA1 是乳癌及卵巢癌的腫瘤抑制基因，它能夠與BARD1形成一個具有E3泛素連接酶活性的異二聚體，並且參與DNA的修復、轉譯、泛素化以及中心體的調控。重要的是，當BRCA1表達異常或是產生突變，會導致中心體不正常的增生。OLA1會結合BRCA1及BARD1，此複合體對於維持正確的中心體數量非常重要，但結構上的結合機制尚未被揭曉。在這篇研究中，我們透過一系列生物物理與生物化學的分析，探討BARD1 BRCT與OLA1之間的分子交互作用關係。
根據NMR及水解酶活性的測定，當環境中存在ATP時，BRCT會結合OLA1，並幫助提升OLA1的活性。我們合成了一個融合重組蛋白，藉此加強蛋白質之間的結合，以及促進OLA1/BRCT複合體的形成。我們也透過酵素活性的測試，發現當BARD1 BRCT存在時，OLA1的酵素轉換率被提升了。我們還另外利用蛋白質交聯法以及序列比對法，探討蛋白質交互作用在生物演化上的重要性。結構上分析了多個突變位點組合，均顯示BRCT和OLA1產生結合的表面具有高度保守性。
雖然我們沒有得到OLA1/BRCT複合體的蛋白質晶體結構，序列保守性的分析也給予了一些線索，暗示著高度保留的胺基酸具有維持蛋白質功能的意義。蛋白質沉降法也利用突變後的重組蛋白證明結合面被破壞後，會降低蛋白質之間的結合，此結果也與我們預測會參與結合面的特定胺基酸提供了強而有力的證據。綜合以上實驗，我們得知BARD1 BRCT會透特定胺基酸與OLA1結合，並且主導OLA1的水解活性，進而影響於中心體數目的調控。在未來，我們希望此份基礎研究，可以幫助其他研究學者們更深入地探討由中心體數量所導致的癌症相關疾病以及藥物的治療。

Centrosomes, the major microtubule organizing centers in mammalian cells, play a critical role in cell division and spindle formation. The breast cancer-associated gene 1 (BRCA1) is a tumor suppressor of breast and ovarian cancers. It forms a heterodimer with BRCA1-associated RING domain protein (BARD1) and acts as an E3 ubiquitin ligase in DNA repair, transcription, ubiquitination, and centrosome regulation. Importantly, BRCA1 silencing and mutations result in abnormal centrosome amplification. An Obg-like ATPase (OLA1) interacts with BRCA1 and BARD1 which is critical for the correct centrosome number, but the structural mechanism is unknown. Here, we determined the molecular interplay between the BARD1 BRCT domain and OLA1 by a series of biophysical and biochemical analyses. Based on the NMR and ATPase assay, the BRCT domain binds to ATP-OLA1 and enhances the OLA’s activity. We made a fusion construct to improve the weak interaction and enhance OLA1/BRCT complexes in solution. We also did cross-linking and multiple sequence alignment to explore the function of protein-protein interaction in biological evolution. Structural analyses in combination with the mutagenesis study indicated that a highly conserved region on the BRCT domain contributes to the binding of OLA1. Even though we did not obtain a crystal structure of the OLA1/BRCT complex, conservation analyses gave us some clues. Mutational studies and pull-down assays provide strong evidence to support that the binding interface is composed of these conserved residues. Altogether, these data identify a functional surface on the BARD1 BRCT domain that contributes to centrosome regulation by modulating the activity of OLA1. In the future, we expect our basic research will facilitate other cancer studies and drug discoveries that are related to centrosomes.

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