設計新穎含金屬酵素催化胰島素轉譯後修飾

Abstract

隨著現代飲食精緻化，糖尿病的盛行率逐年提升。世界衛生組織統計全世界約超過4.2億患有糖尿病，臺灣在擁有200多萬糖尿病患者成因下，此疾病也成為2018年國人第五大死因。自1922年首次將胰島素作為藥物使用以來，胰島素一直是糖尿病患者最主要的治療方式，而為了因應不同的血糖調控需求，各式胺基酸序列改變及化學修飾的胰島素類似物也相繼研究開發。然而，由於胰島素的修飾受到了自然胺基酸側鏈官能基的蛋白質化學限制，此研究藉由設計新穎的金屬酶以增加胰島素類似物多樣性，並放眼未來用於活體內進行蛋白質轉譯後修飾的催化。 文獻研究已指出銅離子可作為路易斯酸用以催化傅-克烷基化，為了催化α,β-不飽和羰基化合物與胰島素的芳香族胺基酸之間的專一反應，此研究改造人類重鏈攜鐵蛋白設計了新穎的含銅酵素。攜鐵蛋白是一種普遍存在於生物體內用於儲存和釋放鐵離子的蛋白質，屬於24蛋白單體自組裝的籠蛋白群組。在這此研究中，我們將胰島素受體羧基末端α螺旋的αCT片段連接於攜鐵蛋白末端，並利用吡咯-轉核糖核酸合成酶•tRNAPyl突變株配對將非典型胺基酸嵌入籠狀攜鐵蛋白的C2界面，建構新的金屬螯合位。經由蛋白質譜分析，新型攜鐵蛋白球在螯合銅離子下，專一地對胰島素的B鏈的HisB5位點進行位置特異的烷基化修飾。總結本研究，新型含銅攜鐵蛋白球酵素具有胰島素特定位置修飾功能，可以催化胰島素B鏈上HisB5的傅-克烷基化反應，球體外露αCT胜肽鏈和胰島素的親合鍵結設計也能增進傅-克烷基化的效率。

According to WHO, more than 422 million people are diagnosed with diabetes around the world, and in Taiwan, 2 million people are suffering from diabetes. Since insulin is the major treatment to those patients, development of efficient insulin drugs is of great significance. However, modifications of insulin analogues are limited by the deficiency of protein chemistry, therefore our goal is to construct a post-translational modification (PTM) enzyme aimed to explore new protein chemistry and enrich the chemical diversity of insulin. Previous literature has performed Friedel-Crafts alkylation using copper ion, however, without specificity. To catalyze reactions between small molecules with α,β-unsaturated carbonyl and aromatic amino acids on insulin, a metalloprotein based biocatalysis platform using human heavy chain ferritin was designed. Ferritin is a family of self-assembled 24-subunits protein cages who act as antioxidants by storing and releasing irons. In this study, a ferritin-αCT fusion protein containing the insulin receptor carboxy-terminal α-chain (αCT) segment on its C-terminus was constructed. Non-canonical amino acids (ncAAs), L-2-(5-Bromothienyl)alanine, H-N-3-Methyl-L-histidine, and 3-(4-Thiazolyl)-L-alanine, are then incorporated at the C2 interface by evoluted pyrrolysyl-tRNA synthetase (PylRS)•tRNAPyl pair, followed by the introduction of Cu(II) after iron ion removal. With evidences of protein mass spectrometry analysis, engineered ferritin-αCT variants have demonstrated specificity toward insulin modification by catalyzing Friedel-Crafts alkylation between the first histidine on B chain and diethyl ethylidenemalonate (DEEM).