建構一新穎胜肽辨識分子以應用於特定泛素化修飾蛋白之偵測

Abstract

泛素(Ubiquitin, Ub)是一種普遍存在於真核細胞中的調控蛋白，它可以調控細胞中各種生理反應，而調控的機制是藉由修飾在蛋白質上不同的共價鍵結來決定，在泛素的胺基酸序列中有7個不同位置的離胺酸(lysine)，泛素藉由這7個不同的離胺酸(lysine)在蛋白質上形成共價鍵修飾，7種修飾分別為K6、K11、K27、K29、K33、K48、K63，大致上K48的修飾會與細胞內蛋白質的降解有關；K63的修飾則與細胞內訊息傳遞、DNA修復、Kinase活化有關，但是目前由於缺乏對於其他泛素修飾有關的鍵結蛋白辨識分子，所以受這些特殊泛素修飾調控的生理作用還不是很清楚，近年來，許多研究已在不同的蛋白質上發現各種泛素結合區(ubiquitin binding domains, UBDs)，如NEMO、MUD1、TAB2_NZF等，這些UBDs對個別泛素修飾具專一性親和力，本研究期望利用此一特性評估UBDs應用於各種不同泛素修飾蛋白純化與偵測的可行性。在實驗過程中利用石英晶體微天平(Quartz crystal microbalance, QCM)、HaloTag Resin based assays等技術來確認UBDs與各個泛素修飾之間的專一性親和力。此外，在本研究中，建構了一新型“類抗體”之胜肽辨識分子“A Novel Ubiquitin peptide Binder Opted by tailored Tandem Iteration” (ANUBOTI)，實驗結果顯示，其對高度泛素化的大分子蛋白質具極佳的專一親和力。所建構之新型泛素鍵結辨識分子未來應能作為研究泛素化蛋白質體的新工具並加速泛素化蛋白分子的發現。

Ubiquitin (Ub) regulates various cell signaling pathways by covalently conjugation to targets. There are seven lysine residues in the Ub sequence (Lysine (K)6, K11, K27, K29, K33, K48, and K63). K48-linked ubiquitin chains send substrates to the proteasome, whereas mono-ubiquitination or K63-linked chains usually trigger signaling events that are not related to proteolysis, however, due to the lack of corresponding binders, most of properties of these topological variants remain uncharacterized. As there are increasing ubiquitin binding domains (UBDs) identified from diverse protein sources, we proposed that these small peptide molecules may serve as useful affinity binders suitable for varied ubiquitome detection and characterization. A proof-of-principle study is performed, in which selective UBDs including NEMO, MUD1 and TAB2_NZF have been constructed as specific linkages binders. Both QCM (Quartz crystal microbalance) and the HaloTag Resin based assays have been applied to validate the specificity between the constructed binders and their corresponding linkages. Furthermore, “A Novel Ubiquitin peptide Binder Opted by tailored Tandem Iteration” (ANUBOTI) has been charaterized as an antibody-like molecular tool with specificity to higly ubiquitinated large molecules. The construction of these novel peptide binders shall facilitate the ubiquitinylated proteome discovery, providing a tool readily for specific ubiquitin linkages profiling.