Department of Medicinal Chemistry, University of Minnesota, Minneapolis, MN 55455, USA.
Department of Chemistry, University of Minnesota, Minneapolis, MN 55455, USA.
Int J Mol Sci. 2021 Nov 7;22(21):12042. doi: 10.3390/ijms222112042.
Protein farnesylation is a post-translational modification where a 15-carbon farnesyl isoprenoid is appended to the C-terminal end of a protein by farnesyltransferase (FTase). This modification typically causes proteins to associate with the membrane and allows them to participate in signaling pathways. In the canonical understanding of FTase, the isoprenoids are attached to the cysteine residue of a four-amino-acid CaaX box sequence. However, recent work has shown that five-amino-acid sequences can be recognized, including the pentapeptide CMIIM. This paper describes a new systematic approach to discover novel peptide substrates for FTase by combining the combinatorial power of solid-phase peptide synthesis (SPPS) with the ease of matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS). The workflow consists of synthesizing focused libraries containing 10-20 sequences obtained by randomizing a synthetic peptide at a single position. Incubation of the library with FTase and farnesyl pyrophosphate (FPP) followed by mass spectrometric analysis allows the enzymatic products to be clearly resolved from starting peptides due to the increase in mass that occurs upon farnesylation. Using this method, 30 hits were obtained from a series of libraries containing a total of 80 members. Eight of the above peptides were selected for further evaluation, reflecting a mixture that represented a sampling of diverse substrate space. Six of these sequences were found to be bona fide substrates for FTase, with several meeting or surpassing the in vitro efficiency of the benchmark sequence CMIIM. Experiments in yeast demonstrated that proteins bearing these sequences can be efficiently farnesylated within live cells. Additionally, a bioinformatics search showed that a variety of pentapeptide CaaaX sequences can be found in the mammalian genome, and several of these sequences display excellent farnesylation in vitro and in yeast cells, suggesting that the number of farnesylated proteins within mammalian cells may be larger than previously thought.
蛋白质法尼基化是一种翻译后修饰，其中一个 15 碳法尼基异戊二烯通过法尼基转移酶（FTase）连接到蛋白质的 C 末端。这种修饰通常导致蛋白质与膜结合，并允许它们参与信号通路。在 FTase 的规范理解中，异戊二烯连接到四氨基酸 CaaX 框序列的半胱氨酸残基上。然而，最近的工作表明，五氨基酸序列也可以被识别，包括五肽 CMIIM。本文描述了一种新的系统方法，通过将固相肽合成（SPPS）的组合能力与基质辅助激光解吸电离质谱（MALDI-MS）的易用性相结合，来发现新型 FTase 肽底物。该工作流程包括合成含有 10-20 个序列的聚焦文库，这些序列是通过在单个位置随机化合成肽获得的。文库与 FTase 和法尼基焦磷酸（FPP）孵育，然后进行质谱分析，由于法尼基化导致的质量增加，酶促产物可以与起始肽明显区分开来。使用这种方法，从包含总共 80 个成员的一系列文库中获得了 30 个命中。选择了上述 8 个肽进行进一步评估，反映了代表不同底物空间的混合体。其中 6 个序列被发现是 FTase 的真正底物，其中几个序列的体外效率达到或超过了基准序列 CMIIM。酵母实验表明，携带这些序列的蛋白质可以在活细胞中有效地法尼基化。此外，生物信息学搜索表明，哺乳动物基因组中存在多种五肽 CaaaX 序列，其中一些序列在体外和酵母细胞中显示出良好的法尼基化，这表明哺乳动物细胞中法尼基化蛋白的数量可能比以前认为的要多。
