ELR-CXC趨化素是導致嗜中性球性發炎的關鍵角色。過度嗜中性球浸潤將損害組織及產生若干種發炎性疾病。由上皮細胞或巨噬細胞分泌的一種趨化素，介白素8，可與ELR-CXC受器，CXCR1和CXCR2，緊密結合並在人類嗜中性球中觸發二級訊號傳導，其中包括了細胞趨化性、成粒作用以及產生過氧化物(ROS)。在我們先前研究中已設計介白素8類似物，RP4，並可有效抑制IL-8與CXCR1/2所導致的嗜中性球趨化作用。本研究中，培養出RP4晶體並且利用晶體X-光繞射得到解析度1.8Å的蛋白質結構。根據結構顯示，在介白素8的 N-loop 上，T12S以及K11R兩個點突變使蛋白質分子靜電力以及胺基酸主鏈發生變化，進而促使RP4與CXCR1/2有更強的親合力，而RP4與CXCR1/2的生物活性則是透過改變ELR結構而被抑制。ELISA蛋白親合力實驗則指出RP4與CXCR1/2有比介白素8與CXCR1/2低於3至4倍的解離常數。
介白素8已被證實與多種慢性疾病有關，包括類風濕關節炎、數種癌症及炎症等，本實驗結果中清楚呈現RP4何以作為CXCR1/2的強力拮抗劑， RP4可望藉抑制上游GPCR訊號而發展成為新一代蛋白質藥物的潛力。

ELR-CXC chemokines play an important role in many neutrophilic inflammations. Excessive neutrophil infiltration mediated by the ELR-CXC chemokine usually leads to tissue damage and several inflammatory diseases. Interleukin 8 (IL-8, CXCL8), one of chemokine is secreted by epithelial cells or macrophages has been reported can binds to ELR-CXC receptor, CXCR1 and CXCR2. Then, it triggers signal transduction in human neutrophils, including chemotaxis, granulation, and generation of reactive oxygen species (ROS). Our previous studies have designed an IL-8 analogue, RP4, significantly inhibits CXCR1/2 triggered neutrophil chemotaxis. In this study, we cultivated crystals of RP4 and determined the molecular structure of RP4 at 1.8Å resolution by crystallography. According to structure, T12S and K11R site-direct mutagenesis on N-loops of IL-8 enhances the binding affinity between IL-8 and CXCR1 with higher charge–charge interaction and backbone slight displacement. Although RP4 has a high binding affinity of CXCR1/2, the RP4-CXCR1/2 complexes cannot pass signal transduction. ELISA binding experiments showed dissociation constant of RP4 and CXCR2 was lower 3-4 folds than IL-8-CXCR2 binding.
It has shown IL-8 related to a number of chronic diseases, including rheumatoid arthritis (RA), some cancers and some inflammatory diseases. From our results, we could explain easily why RP4 acted as an aggressive CXCR1/2 antagonist. RP4 showed potential as new chances in the development of anti-inflammatory drugs.

Abstract------------------------------------------------------------- 3
Table of content----------------------------------------------------- 4
Chapter 1 Introduction----------------------------------------------- 5
Chapter 2 Material and Methods--------------------------------------- 11
2.1 Chemical and Reagents-------------------------------------------- 11
2.2 Tricine–SDS-PAGE------------------------------------------------ 11
2.3 Construct and transform the vector of IL-8 and pRP4-------------- 12
2.4 Expression and purification of recombinant IL-8 and RP4---------- 13
2.5 RP4 Crystallization---------------------------------------------- 14
2.6 RP4 crystals X-ray diffraction and structure modeling------------ 15
2.7 Determination of dissociation constants by ELISA----------------- 16
Chapter 3 Results---------------------------------------------------- 20
3.1 Structure overview----------------------------------------------- 20
3.2 Structure Comparison with IL-8----------------------------------- 20
3.3 RP4 inhibits activities of CXCR1/2 signal transduction by RP4 F33 adjusted ELR motif modification-------------------------------------- 21
3.4 K11R and T12S site mutagenesis enhance RP4 and CXCR1 binding affinity --------------------------------------------------------------------- 22
3.5 The binding affinity of RP4 are 3-4 folds stronger than IL-8 to both CXCR1 and CXCR2------------------------------------------------------ 23
Chapter 4 Discussion and Conclusion---------------------------------- 25
Abbreviation Index--------------------------------------------------- 47
Reference------------------------------------------------------------ 48

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