Highly sensitive and adaptable fluorescence-quenched pair discloses the substrate specificity profiles in diverse protease families.
Highly sensitive and adaptable fluorescence-quenched pair discloses the substrate specificity profiles in diverse protease families.
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DOI：
10.1038/srep43135
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发表时间：
2017-02-23
影响因子：
4.6
通讯作者：
Drag M
中科院分区：
文献类型：
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作者：
Poreba M;Szalek A;Rut W;Kasperkiewicz P;Rutkowska-Wlodarczyk I;Snipas SJ;Itoh Y;Turk D;Turk B;Overall CM;Kaczmarek L;Salvesen GS;Drag M
源于FRET(Förster Resonance Energy Transfer，Förster Resonance Energy Transfer，Förster Resonance Energy Transfer)的内猝灭荧光(IQF)多肽底物是检测蛋白酶活性和特异性的有力工具，各种供体/受体对被广泛用于设计单个底物和组合文库。我们开发了一种高度敏感和适应性强的供体/受体对，可以用来研究半胱氨酸蛋白酶、丝氨酸蛋白酶和金属蛋白酶的底物特异性。该对化合物由7-氨基-4-氨甲酰甲基香豆素(ACC)和2，4-二硝基苯基赖氨酸(Lys(DNP))组成。使用caspase-3、caspase-7、caspase-8、中性粒细胞弹性蛋白酶、豆蛋白和两种基质金属蛋白酶(MMP2和MMP9)，我们证明了含有ACC/Lys(DNP)的底物比传统的7-甲氧基-香豆素-4-基乙酸(MCA)/Lys(DNP)底物的灵敏度高7-10倍；因此，每次检测所使用的底物和酶的量都大大减少。因此，我们建议ACC/Lys(DNP)对可以被认为是设计任何一组内肽酶底物的一种新颖和敏感的支架。我们进一步证明，含有非天然氨基酸的IQF底物可用于研究含有翻译后修饰氨基酸的多肽的蛋白酶活性/特异性。最后，我们使用IQF底物重新研究了caspase的P1-Asp特性，从而证明了一些人caspase也可以在谷氨酸之后对底物进行水解。
Internally quenched fluorescent (IQF) peptide substrates originating from FRET (Förster Resonance Energy Transfer) are powerful tool for examining the activity and specificity of proteases, and a variety of donor/acceptor pairs are extensively used to design individual substrates and combinatorial libraries. We developed a highly sensitive and adaptable donor/acceptor pair that can be used to investigate the substrate specificity of cysteine proteases, serine proteases and metalloproteinases. This novel pair comprises 7-amino-4-carbamoylmethylcoumarin (ACC) as the fluorophore and 2,4-dinitrophenyl-lysine (Lys(DNP)) as the quencher. Using caspase-3, caspase-7, caspase-8, neutrophil elastase, legumain, and two matrix metalloproteinases (MMP2 and MMP9), we demonstrated that substrates containing ACC/Lys(DNP) exhibit 7 to 10 times higher sensitivity than conventional 7-methoxy-coumarin-4-yl acetic acid (MCA)/Lys(DNP) substrates; thus, substantially lower amounts of substrate and enzyme can be used for each assay. We therefore propose that the ACC/Lys(DNP) pair can be considered a novel and sensitive scaffold for designing substrates for any group of endopeptidases. We further demonstrate that IQF substrates containing unnatural amino acids can be used to investigate protease activities/specificities for peptides containing post-translationally modified amino acids. Finally, we used IQF substrates to re-investigate the P1-Asp characteristic of caspases, thus demonstrating that some human caspases can also hydrolyze substrates after glutamic acid.