Reactive Peptides for Protein-Binding Domains

過去數十年裏，多肽的藥物研究取得了長足的進展。越來越多的研究組和生物製藥公司從小分子轉向多肽。受此激勵，我們也致力於具有細胞活性的多肽的開發，並且成功设计得到了一類新型“反應活性多肽”。这些多肽可以通過发生親和力引导的共价结合反应而實現對目標蛋白的定點反應。比如半胱氨酸的巰基於反應活性多肽的α氯乙酸官能團可以發生共價交聯反應。利用此親和力誘導反應的位點特異性，我們預計反應活性多肽可以用作研究蛋白-多肽相互作用，從而揭示通常的生物化學實驗所無法揭示的新的細節。以Src同源結構域2（SH2）蛋白為例，本論文開展了應用反應活性多肽來研究磷酸化酪氨酸多肽與SH2蛋白域的結合研究。首先，根據SH2蛋白二級結構中具有反應活性的半胱氨酸的不同位置，我們可以將人類基因組120個SH2結構域劃分為在多肽結合位點有或者沒有半胱氨酸的兩類，進而把含有半胱氨酸的SH2蛋白劃分為五組。從這些蛋白類別中，我們選取了10個有代表性的蛋白進行基因重組和表達純化，以它們為模型設計了反應活性多肽，並且研究了這些多肽與蛋白之間的共價交聯反應。以此證明了反應活性多肽的位點特異性。在此過程中我們發現了一個非常有趣的現象：部分SH2蛋白在與磷酸化酪氨酸多肽結合時，採取不同於已知結合模式（成為經典模式）的反向結合模式。我們進一步通過設計合成新的反應活性多肽來驗證並且解釋這種反向結合模式的特性。除此之外，我們與計算物理組合作，獲得了證據證明LCK SH2蛋白與磷酸化酪氨酸多肽結合中確實存在經典模式和反向模式。因此我們發展出了一類新型的分子探針：反應活性多肽，用這類分子首次揭示出了SH2蛋白的新的不為人所致的多肽結合特性。除了作為研究蛋白-蛋白相互作用的探針之外，我們還利用反應活性多肽作為蛋白組學的研究工具發展得到了定向共價固化的方法。新方法在不影響目標蛋白的結合能力的情況下，優化了甲基化蛋白組學中的樣品富集工作。同時我們開發了特異性與Nck1的SH2蛋白域共價反應的多肽，有望能夠開發出針對Nck1這一重要的信號分子的多肽探針。總之，我們的實驗證明活性反應多肽作為活性多肽的一個新成員，有望在多個領域成為強有力的化學生物學探針，甚至對多肽藥物的發展起到推動作用。Past several decades have witnessed a rapid development of peptide drugs. More and more medicinal groups and pharmaceutical companies are turning to peptides for drug leads. Inspired by this surge, we devoted ourselves to the development of active peptides that have intracellular activities. In particular, we pioneered the development of a new type of active peptides, which are call “reactive peptides”. Reactive peptides react with protein targets through proximity-induced covalent reactions guided by the binding affinity between the peptide and the protein, thereby realizing site specific covalent reaction at the selected protein, selected residue site. For example, using the mild reaction between cysteine thiol group and chloroacetyl group, we developed reactive peptides that only target a particular protein at the cysteine site. The site-specificity of the proximity-induced reaction inspired us that we may utilize this type of peptides to unravel the details of protein-peptide binding interactions that are unattainable otherwise.This thesis showcases that reactive peptides can be used as a new type of probes for SH2 domains to reveal a new binding mode between SH2 domain and phosphotyrosine peptides. The reverse binding mode of SH2 domains is rarely documented and has long been ignored in the literature of SH2 proteins. In fact, there is only one example of reversely bound SH2-peptide complex in the literature, which is considered an uncommon exception. Our study defines the norm as we reveal that the reverse binding mode exists in more than one SH2-peptide binding interactions, and is likely a general mechanism during the process that phosphotyrosine peptide seeks for the optimal binding pose on the surface of SH2 proteins. This work in turn supports our postulation that reactive peptides could be superior probes for binding interactions.In a second project, we developed Nck1-specific reactive peptide probes which are likely used in unravel the function of the adaptor protein Nck1. Besides acting as probes, reactive peptides are also developed into a site-specific covalent conjugation strategy that found use in proteomics. Taken together, our work illustrates that reactive peptides, as a new type of active peptides, will find wide uses in the area of chemical biology, or even medicinal chemistry.Wang, Rui.Thesis Ph.D. Chinese University of Hong Kong 2016.Includes bibliographical references (leaves ).Abstracts also in Chinese.Title from PDF title page (viewed on …).Detailed summary in vernacular field only.Detailed summary in vernacular field only