Abstract
摘要：關鍵字：蛋白解體；內質網氨肽酶；主要組織相容性複合體；抗原胜肽；抗原處理；抗原呈現；proteasome activating nucleotidase
將內生性或病毒蛋白質所衍生而來的抗原胜肽結合於主要組織相容性複合體I （major histocompatibility complex class I），並於細胞表面呈現給CD8+ T細胞進行辨識，為免疫監控系統極為重要的步驟與作用機制，用以移除不正常或被病毒所感染的細胞。在細胞中，泛素及蛋白解體降解系統（ubiquitin-proteasome system）與內質網氨肽酶（endoplasmic reticulum aminopeptidase I, ERAP1）即分別於細胞質與內質網中，負責對一些病毒蛋白質或損壞及短期性的蛋白質進行分解與處理成抗原胜肽的工作。目前對於內質網氨肽酶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肽酶的蛋白質表現系統與高效能的胜肽分析系統，用以研究蛋白解體及內質網氨肽酶對抗原胜肽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肽酶（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，來深入瞭解內質網氨肽酶之分子作用機轉。此外，我們亦將分析抗原胜肽結合於ERAP1之異位調節區時，如何增進ERAP1的活性，及此現象與細胞中抗原呈現系統之關聯性。而對於缺乏KDEL內質網回收訊號的ERAP1/ERAP2，為何可以停留於內質網中，我們預計進行免疫共沈澱等方法來探究原因，並鑑定未知之結合蛋白質。另一方面，將探討proteasome activating nuc
Abstract: Key words: antigenic peptide; major histocompatibility complex, MHC; proteasome; endoplasmic reticulum aminopeptidase, ERAP; proteasome activating nucleotidase, PAN
The display of antigenic peptides representing endogenous and viral proteins by major histocompatibility complex (MHC) class I molecules is important and essential for immune surveillance. It is now firmly established that these peptides are mainly generated by the ubiquitin-proteaosme system in the cytoplasm and then in the endoplasmic reticulum (ER) by the ER aminopeptidase I (ERAP1). However, the substrate preference of ERAP1 is still largely unknown. To understand ERAP1’s novel peptide trimming mechanism and immunological importance, we shall characterize its selectivity for peptide substrates. Further, we will characterize the allosteric activation by binding a suitable peptide in a different site from the catalytic site of ERAP1 to elucidate the possible regulatory mechanism. We will also study the biological properties of the homologous new aminopeptidases, ERAP2 and P-LAP, and clarify further their importance in antigen presentation by RNAi gene silencing techniques. 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. Importantly, all the experiments described above are based on the reliable baculovirus protein expression system which we would like to generate at the beginning. Also, we shall study the ER localization of ERAP1/ERAP2 without containing the C-terminal KDEL sequence by using the co-immunoprecipitation to identify 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-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.
Keyword(s)
內質網氨肽
酶
主要組織相容性複合體
抗原胜肽
抗原處理
抗原呈現
antigenic peptide
major histocompatibility complex
MHC
proteasome
endoplasmic reticulum aminopeptidase
ERAP
proteasome activating nucleotidase
PAN
