Wollack James W, Zeliadt Nicholette A, Mullen Daniel G, Amundson Gregg, Geier Suzanne, Falkum Stacy, Wattenberg Elizabeth V, Barany George, Distefano Mark D
Department of Chemistry, University of Minnesota, Minneapolis, Minnesota 55455, USA.
J Am Chem Soc. 2009 Jun 3;131(21):7293-303. doi: 10.1021/ja805174z.
Protein prenylation is a common post-translational modification present in eukaryotic cells. Many key proteins involved in signal transduction pathways are prenylated, and inhibition of prenylation can be useful as a therapeutic intervention. While significant progress has been made in understanding protein prenylation in vitro, we have been interested in studying this process in living cells, including the question of where prenylated molecules localize. Here, we describe the synthesis and live cell analysis of a series of fluorescently labeled multifunctional peptides, based on the C-terminus of the naturally prenylated protein CDC42. A synthetic route was developed that features a key Acm to Scm protecting group conversion. This strategy was compatible with acid-sensitive isoprenoid moieties and allowed incorporation of an appropriate fluorophore as well as a cell-penetrating sequence (penetratin). These peptides are able to enter cells through different mechanisms, depending on the presence or absence of the penetratin vehicle and the nature of the prenyl group attached. Interestingly, prenylated peptides lacking penetratin are able to enter cells freely through an energy-independent process and localize in a perinuclear fashion. This effect extends to a prenylated peptide that includes a full "CAAX box" sequence (specifically, CVLL). Hence, these peptides open the door for studies of protein prenylation in living cells, including enzymatic processing and intracellular peptide trafficking. Moreover, the synthetic strategy developed here should be useful for the assembly of other types of peptides that contain acid-sensitive functionalities.
蛋白质异戊二烯化是真核细胞中常见的一种翻译后修饰。许多参与信号转导途径的关键蛋白都会发生异戊二烯化，抑制异戊二烯化可作为一种治疗干预手段。虽然在体外理解蛋白质异戊二烯化方面已经取得了显著进展，但我们一直对在活细胞中研究这一过程感兴趣，包括异戊二烯化分子的定位问题。在这里，我们描述了基于天然异戊二烯化蛋白CDC42的C末端合成一系列荧光标记的多功能肽并进行活细胞分析。开发了一种合成路线，其特点是关键的Acm到Scm保护基转化。该策略与酸敏感的类异戊二烯部分兼容，并允许掺入合适的荧光团以及细胞穿透序列（穿膜肽）。这些肽能够通过不同机制进入细胞，这取决于穿膜肽载体的存在与否以及所连接异戊二烯基团的性质。有趣的是，缺乏穿膜肽的异戊二烯化肽能够通过一个不依赖能量的过程自由进入细胞，并以核周方式定位。这种效应扩展到一种包含完整“CAAX框”序列（具体为CVLL）的异戊二烯化肽。因此，这些肽为活细胞中蛋白质异戊二烯化的研究打开了大门，包括酶促加工和细胞内肽运输。此外，这里开发的合成策略应该对组装其他类型含有酸敏感功能的肽有用。
