Rapid Multilabel Detection of Geranylgeranylated Proteins by Using Bioorthogonal Ligation Chemistry
Rapid Multilabel Detection of Geranylgeranylated Proteins by Using Bioorthogonal Ligation Chemistry
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DOI：
10.1002/cbic.201000087
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发表时间：
2010-04-12
期刊：
影响因子：
3.2
通讯作者：
Tate, Edward W.
中科院分区：
文献类型：
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作者：
Berry, Alexandra F. H.;Heal, William P.;Tate, Edward W.
蛋白质翻译后异戊二烯化是真核细胞中的一个重要过程，在信号转导和囊泡运输中发挥着关键作用。 [1]在蛋白质香叶基香叶基化的情况下，香叶基香叶基焦磷酸 (GGpp) 充当异戊烯基供体，该过程由香叶基香叶基转移酶-1 (GGT-1) 或 Rab 香叶基香叶基转移酶 (RGGT) 催化，后者需要 Rab 护送蛋白（REP-1 或 REP-2）。 GGT-1 作用于 Rho 家族蛋白，而 RGGT 作用于 Rab 小 GTP 酶；两组蛋白质均在 C 端异戊二烯化基序处被异戊二烯化。 [2] Rab 的错误异戊二烯化会导致多种严重疾病，[3] 为了设计有效的疗法，更深入地了解这一过程至关重要。然而，有效检测和鉴定异戊二烯化蛋白质仍然具有挑战性，需要使用大量 3H-GGpp，检测时间通常从几天延长到几周。[4]生物素化或含有荧光团的异戊二烯类似物可增强检测能力，[5, 6]，但其灵活性受到异戊二烯转移酶底物特异性的限制，并且与天然 RGGT 的亲和力显着降低有关。[6]我们和其他人最近证明，叠氮化物或炔烃标签与生物正交连接化学相结合，可用于检测无细胞和活细胞系统中翻译后蛋白质脂化（酰化或异戊二烯化）。 [4, 7–10] 这些高度生物相容性和仿生标签的破坏性极小，并且能够使用各种潜在报告基因进行后续标记。我们在此描述了一种多功能叠氮标签/生物正交连接系统（图 1），该系统能够使用多个标记快速检测和亲和纯化 RGGT 或 GGT-1 香叶基香叶基化的蛋白质，适用于重组蛋白和来自细胞系或哺乳动物组织的蛋白质。
Post-translational prenylation of proteins is an essential process in eukaryotic cells, and plays a key role in signal transduction and vesicular trafficking.[1] In the case of protein geranylgeranylation, geranylgeranyl pyrophosphate (GGpp) acts as the prenyl donor, and the process is catalyzed either by geranylgeranyl transferase-1 (GGT-1), or by Rab geranylgeranyl transferase (RGGT), the latter requiring a Rab escort protein (REP-1 or REP-2). GGT-1 targets Rho family proteins, whilst RGGT acts on the Rab small GTPases; both sets of proteins are prenylated at C-terminal prenylation motifs.[2] Mis-prenylation of Rabs leads to several severe diseases,[3] and a deeper understanding of this process is essential in order to design effective therapies.However, efficient detection and identification of prenylated proteins remains challenging, making use of quantities of 3H-GGpp with detection times typically extending from days to weeks.[4] Biotinylated or fluorophore-containing prenyl analogues offer enhanced detection,[5, 6] but their flexibility is limited by the substrate specificity of the prenyl transferase and they are associated with a significant reduction in affinity for native RGGT.[6] We and others have recently demonstrated that azide or alkyne tagging combined with bioorthogonal ligation chemistry can be used to detect post-translational protein lipidation (acylation or prenylation) in cell-free and live-cell systems.[4, 7–10] These highly biocompatible and biomimetic tags are minimally disruptive and enable subsequent labeling with a wide range of potential reporters. We describe here a versatile azidetag/bioorthogonal ligation system (Figure 1) that enables the rapid detection and affinity purification of proteins geranylgeranylated by either RGGT or GGT-1 by using multiple labels, applicable both to recombinant proteins and proteins from cell lines or mammalian tissues.