以核磁共振研究S100蛋白質、RAGE V領域、Lysozyme與Tranilast之間的蛋白質相互作用

NMR Study of Protein-Protein Interactions Amongst S100 Proteins, RAGE V Domain, Lysozyme and Tranilast

伊姆蘭(Khan, Md Imran)

指導教授 ： 余靖

國立清華大學/理學院/化學系所/博士(2019年)

摘要

本篇論文研究，我們整合了功能及分子階層的資訊，研究溶菌酶、S100蛋白質以及曲尼司特(Tranilast) 其蛋白質-蛋白質/蛋白質-配體之間的交互作用形成的巨分子複合物，其改變與下游訊號傳遞及細胞增殖相關，影響多種人類疾病。S100蛋白質與其目標蛋白質的交互作用以及結合後的特性依然不清楚。S100蛋白質-蛋白質複合物在弱的交互作用情況下，在藥理研究上對於鑑定與選擇潛力的標靶將擁有直接的影響。我們選擇了S100A1、S100A6及S100B蛋白質(其屬於S100蛋白質) 、 溶菌酶、以及Tranilast來進行研究。
第二章我們簡短的報告與鈣離子結合的人類S100A1蛋白質及S100B蛋白質的生物功能，其皆屬於S100蛋白家族的成員。當鈣離子與S100A1及S100B的EF-hand motifs結合後成為發炎過程中的中間調節者。最終糖化蛋白受體 (Receptors for advanced glycation end products, RAGE) 已知道有五個不同的結構域(domains)：變異(V)區，兩個不變(C1、C2) 區，跨膜區以及膜內區。大部分的S100蛋白，特別是S100A1會以RAGE V domain為標靶結合。目前已知S100A1會結合在特定的疏水區域表面，並且會增進細胞增殖、訊息傳遞、細胞生長以及腫瘤生成。為了瞭解RAGE V domain與S100A1的交互作用，我們透過NMR(核磁共振)方法，利用2D NMR 1H-15N HSQC，滴定S100A1至S100B (以及滴定S100B至S100A1) ，結果顯示S100A可以結合S100B。
根據NMR滴定實驗的結果，我們利用HADDOCK (High Ambiguity Driven biomolecular DOCKing) 程式模擬兩個二元複合物：S100A1-RAGE V domain 以及 S100A1-S100B。比較這兩個複合物，我們清楚觀察到S100A1對於RAGE V domain以及對於S100B擁有相似的結合表面。利用PyMOL軟體將此兩個複合物結構重疊，發現S100B擋住了S100A1-RAGE V domain的結合面。我們進一步利用生物細胞增殖測定實驗(WST-1) 確認此擋住的區域，實驗結果支持我們的假設。此結果可能在神經退化性疾病或癌症治療中對新蛋白的改進帶來益處。
在第三章，我們利用NMR與分子模型(HADDOCK) 展現了複合物的結構，我們探討了溶菌酶- S100A6複合物的結構。結果顯示溶菌酶是一個抗增生作用劑，可以阻斷S100A6 與 RAGE V domain的交互作用面，抑制了細胞訊息傳遞及增生。我們也發現溶菌酶與Tranilast [N-(3, 4-dimethoxy cinnamoyl) anthranilic acid] 的交互作用，Tranilast是一種抗過敏藥物，其顯著的影響溶菌酶- S100A6複合物與S100A6-RAGE V-domain複合物的形成，過程中Tranilast阻斷了溶菌酶- S100A6複合物的交互作用面，而溶菌酶- S100A6會增進細胞訊息傳遞與增生。最後，我們利用WST-1細胞增殖測定實驗去觀察活性的抑制與促進，結果顯示，溶菌酶抑制細胞增殖 (阻斷S100A6-RAGE V-domain) 但可能透過Tranilast與溶菌酶結合而恢復活性。
結論，這些研究揭露了蛋白質-蛋白質以及蛋白質-藥物間的交互作用力，對於設計新療法於細胞增殖相關疾病（如癌症）是至關重要的。

關鍵字

RAGE V Domain ； Lysozyme ； S100 protein ； NMR ； HADDOCK

並列摘要

In this thesis, we have integrated the functional and molecular information of lysozyme, S100 protein and tranilast, protein-protein/ protein-ligand interactions, these information are used for understanding the macro-molecular complex formation, and resultant, changes associated in the downstream signaling cascade, cell proliferation leading to the various disease for human health. Interaction of the S100 protein and its behavior after binding to its target protein remains unclear. In this case the weak interaction between S100 protein-protein complexes will have direct implications in pharmaceutical research to identify and select the potential targets. We have selected S100A1, S100A6 and S100B proteins belong to S100 protein, and lysozyme as well as tranilast molecule to investigate the biological functions.
The Ca2+ binding human S100A1 and S100B protein belonging to a member of the S100 protein family, are substantial intermediators for inflammation when Ca2+ attaches to its EF-hand motifs. Receptors for advanced glycation end products (RAGE) are known to have five different domains: variable (V) domains, two constant (C1, C2) domain, transmembrane and the cytoplasmic domain. Most of the S100 protein, specially S100A1 targets the RAGE V domain to interact. S100A1 is known to bind to hydrophobic region of RAGE V domain and therefore enhance the cell proliferation, signaling transduction cascades and cell growth and tumorigenesis. To find out the interaction between RAGE V domain and S100A1, we utilized NMR (nuclear magnetic resonance) spectroscopy method. Our investigation is found that the S100A1 protein could interact and bind to the S100B protein via 2D NMR 1H-15N HSQC titrations of S100A1 against S100B and vice versa. On the basis of the NMR titration consequences, we utilized the HADDOCK (High ambiguity ariven biomolecular DOCKing) program to generate the two binary complex: S100A1-RAGE V domain and S100A1-S100B. From these two complexes, we clearly observed that S100A1 have a similar binding interface with RAGE V domain and S100B protein. Using PyMOL program we overlapped these two complex where S100B protein aligns between S100A1 and RAGE V domain which blocks the binding interface of S100A1-RAGE V domain complex. We also confirmed this blocking by cell proliferation assay WST-1. This information could possibly be beneficial for new protein improvement for neurodegenerative disease and cancer treatment.
In chapter III, we educidted the structure of a molecular complex using NMR and molecular modelling (HADDOCK), we have studied the structure of the lysozyme-S100A6 complex. We found lysozyme as an anti-proliferative agent to block the interface of the S100A6 and RAGE V domain, which results in the inhibition of signal transduction and cell proliferation. We also found the interaction of lysozyme with the tranilast [N-(3, 4-dimethoxy cinnamoyl) anthranilic acid], an antiallergic drug which has the dramatic influence on lysozyme-S100A6 and S100A6-RAGE V domain complex formation, where tranilast blocks the interaction between lysozyme-S100A6 complex which enhances the signal transduction and cell proliferation. Finally, we utilized the WST-1 cell proliferation assay to observed the inhibition and enhancement activities of these protein. From this data, we have found out that lysozyme, most believably by blocking the interaction between S100A6 and RAGE V domain, inhibits cell proliferation while tranilast may reverse this effect by binding to lysozyme.
In conclusion, we demonstrated that the protein-protein and protein-drug interactions, are of most importance for understanding the unclear interactions designing new therapies to treat diseases and possibly such as cancers.

並列關鍵字

RAGE V Domain ； Lysozyme ； S100 protein ； NMR ； HADDOCK

參考文獻

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