2007-01-012024-05-18https://scholars.lib.ntu.edu.tw/handle/123456789/713960摘要：在細胞中，蛋白解體（proteasome）負責對一些病毒蛋白或損壞及短期性的蛋白質進行分解處置的工作。而其部分產物會被進一步轉送至內質網，由內質網氨&#32957;&#37238; （endoplasmic reticulum aminopeptidase I, ERAP1）將過長的胜&#32957;片段處理為結合在主要組織相容性複合體I （major histocompatibility complex class I, MHC class I）上的抗原胜&#32957;，最後呈現於細胞表面。接著透過CD8+ T細胞的辨識，進而引發一連串的免疫反應。目前對於內質網氨&#32957;&#37238;的基質選擇性差異並無相關報告，因此本研究擬建立內質網氨&#32957;&#37238;的蛋白質表現系統與高效能的胜&#32957;分析系統，用以研究蛋白解體及內質網氨&#32957;&#37238;對抗原胜&#32957;前軀物的基質選擇性。此外亦將建立另兩個新發現的內質網氨&#32957;&#37238;（ERAP2與P-LAP）之蛋白質表現系統，以探討其生化性質及生理角色。並希望透過立體結構的解析，以探討內質網氨&#32957;&#37238;之作用機轉。此外，對於缺乏KDEL內質網回收訊號的ERAP1/ERAP2，為何可以停留於內質網中，我們亦將以免疫共沈澱等方法來探究原因。另一方面，將探討proteasome activating nucleotidase (PAN) 如何以其C-端之保留區域與蛋白解體產生交互作用。本研究將以archaea之20S蛋白解體為研究系統，以定位點突變技術將次單元體上的特定胺基酸突變後，探討其與PAN結合時之重要胺基酸序列，並透過立體結構的解析，設計有別於抑制蛋白解體活性中心之新型態專一性抑制劑。值得一提的是，以蛋白解體抑制劑類似物所研發的藥物VELCADE，已證實可治療多發性骨髓癌，並已獲美國食品暨藥物管理局（FDA），歐盟及世界四十幾個<br> Abstract: It is now firmly established that the proteasome pathway is responsible for the generation of the great majority of antigenic peptides from viral proteins or intracellular proteins. Proteasomes generally degrade proteins to peptide fragments ranging from 2-25 residues long, but most are too short (<8 residues) for antigen presentation. Only the 9-16 amino acids antigenic precursors are then transported efficiently into the ER by the TAP transporter, and further processed by ERAP1 to proper size (8-9 residues) for MHC binding. MHC class I molecules bind tightly and present antigenic peptides on the cell surface for recognition by cytotoxic T lymphocytes. Many results including RNAi gene silencing have revealed that ERAP1 plays a key role in trimming antigenic precursors in the antigen presentation pathway. To understand ERAP1’s novel peptide trimming mechanism and immunological importance, we shall characterize its selectivity for peptide substrates. We will also study the biological properties of the homologous new aminopeptidases, ERAP2 and P-LAP, and clarify further their importance in antigen presentation. To explore and elucidate the trimming mechanism of ERAP, we shall solve their structures and explore the substrate diversity among ERAP and MHC class I molecules. Also, we shall study the ER localization of ERAP1/ERAP2 without containing the C-terminal KDEL sequence by using the co-immunoprecipitation to discover the unknown ERAP-binding protein. Another major aim is to learn more about 19S/20S assembly and substrate entry and product release from 26S proteasome. By using site-directed mutagenesis to replace the potential amino acid residues in the alpha-subunit, we shall identify the important residues of 20S which interact with proteasome activating nucleotidase, PAN. Further, we shall mutate the corresponding C-terminal residues of PAN and analyze the PAN/20S structures. This study would provide the important information for designing new proteasome inhibitors by interrupting the binding of regulatory particles, 19S and PAN. It is also noted that VELCADE, a new type of cancer drug called proteasome inhibitor, is the first medicine to be approved by The Food and Drug Administration (FDA) , European Union and more than 40 countries for the treatment of patients with multiple myeloma cancer.蛋白解體內質網氨&#32957&#37238主要組織相容性複合體抗原胜&#32957抗原處理抗原呈現proteasome activating nucleotidaseproteasomeantigenic peptideantigen presentationtransporter associated with antigen processingTAPendoplasmic reticulum aminopeptidaseERAPmajor histocompatibility complexMHCantigen processingproteasome activating nucleotidasePAN