K27、K29與K33鍵結專一性抗體應用於非典型泛素化蛋白質體之探索

Abstract

泛素為一種人體當中相當重要的小分子蛋白質。這些蛋白質可藉由轉譯後修飾(泛素化)作用，將泛素以單一的、或是以泛素鏈的形式，共價鍵結至特定蛋白質標的上。泛素的胺基酸序列中有7個賴氨酸(Lysine, K)，分別在第6、11、27、29、33、48與63的位置 (K6, K11, K27, K29, K33, K48, and K63)。一般認為，藉由其自身在此7個位置不同的泛素化鍵結形式與修飾程度，生物體可調控不同的生理機制。然而較為廣泛研究的泛素化鍵結大多以K48 與K63為主，其他的泛素鍵結的功能則不明確。主因是由於目前仍缺乏適當的分子辨識工具可用以分析以及純化這些非典型的泛素鏈與被這些泛素鏈修飾的目標蛋白質。在本研究中，我們針對三種可分別辨識K27、K29與K33泛素鏈鍵結的抗體進行評估測試，成功的利用西方墨點法與流式微陣列技術，確認抗體的專一性。在實際應用上，則藉由這些抗體純化其個別泛素鏈修飾蛋白質分子，結合質譜進行蛋白質鑑定。此外，我們也將這些抗體應用在組織微陣列晶片的免疫螢光染色，藉此評估正常與癌症組織之間不同病理狀態下，這些非典型泛素化的差異。所建構之泛素化辨識抗體應可加速這類非典型泛素化蛋白質體的研究。

Ubiquitin (Ub) is an important small protein in human body. The mono Ub or Ub linkages can be covalently linked to target protein substrates via posttranslational modifications (ubiquitination). There are seven lysines (K6, K11, K27, K29, K33, K48 and K63) in the amino acid sequence of ubiquitin. It is generally believed that various physiological mechanisms in organisms can be regulated by different types and levels of polyubiquitin linkages. The K48 and K63 were the most extensively studied Ub linkages. The other Ub linkages, such as K27-, K29- or K33, on the other hand have been relatively unknown in their functions due to the lack of suitable recognition binders to enable the investigation and purification of these atypical Ub linkages and the associated ubiquitinated proteins. In this study, we have employed three antibodies which can specifically recognize three atypical Ub linkages (the K27, K29 and K33-specific antibodies). We confirmed their respective specificity by western blot and the bead-based suspension microarray. The practical applications of these antibodies have been successfully demonstrated in the enrichment of ubiquitinated proteome, followed by the protein identification using mass spectrometry. Besides, we also applied these antibodies to the tissue microarrays to enable fluorescent immunohistochemistry staining (FIHC), evaluating the atypical ubiquitination levels between malignant and normal biosies. We expect these antibodies shall facilitate the atypical linkage-specific ubiquitome discovery.