A Quantitative FRET Assay for the Upstream Cleavage Activity of the Integral Membrane Proteases Human ZMPSTE24 and Yeast Ste24
A Quantitative FRET Assay for the Upstream Cleavage Activity of the Integral Membrane Proteases Human ZMPSTE24 and Yeast Ste24
复制标题
人 ZMPSTE24 和酵母 Ste24 整体膜蛋白酶上游裂解活性的定量 FRET 测定
DOI：
10.1007/978-1-4939-9532-5_21
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发表时间：
2019
影响因子：
--
通讯作者：
Hrycyna, Christine A.
中科院分区：
文献类型：
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作者：
Hsu, Erh-Ting;Vervacke, Jeffrey S;Distefano, Mark D;Hrycyna, Christine A.
整体膜蛋白酶ZMPSTE24在lamin A成熟途径中起重要作用。ZMPSTE24是唯一已知的能够切割前纤层蛋白A c端最后15个残基的酶，包括法酰化和羧甲基化半胱氨酸。ZMPSTE24基因突变可导致核内异常前纤层蛋白A积累的类早衰疾病。Ste24是ZMPSTE24的酵母功能同源物，在其翻译后成熟过程中类似地切割茶因子信息素前体。为了补充现有的定性技术，用于检测上游酶裂解ZMPSTE24和Ste24，包括凝胶移法和质谱分析，我们开发了一种酶体外基于fret的方法来定量测量这两种酶的上游裂解活性。该试验使用纯化酶或粗膜制剂中的酶，以及作为Ste24和ZMPSTE24底物的33个氨基酸因子类似肽。该肽在其n端含有一个荧光基团（2-氨基苯甲酸- abz）和位于裂解位点下游四个残基的猝灭基团（二硝基苯酚- dnp）。裂解后，在420 nm处实时产生荧光信号，该荧光信号与肽的裂解成正比，这些动力学数据用于量化活性。该方法可为ZMPSTE24和Ste24的动力学分析和催化机理的研究提供有用的工具。
The integral membrane protease ZMPSTE24 plays an important role in the lamin A maturation pathway. ZMPSTE24 is the only known enzyme to cleave the last 15 residues from the C-terminus of prelamin A, including a farnesylated and carboxyl methylated cysteine. Mutations in ZMPSTE24 lead to progeroid diseases with abnormal prelamin A accumulation in the nucleus. Ste24 is the yeast functional homolog of ZMPSTE24 and similarly cleaves thea-factor pheromone precursor during its posttranslational maturation. To complement established qualitative techniques used to detect the upstream enzymatic cleavage by ZMPSTE24 and Ste24, including gel-shift assays and mass spectrometry analyses, we developed an enzymatic in vitro FRET-based assay to quantitatively measure the upstream cleavage activities of these two enzymes. This assay uses either purified enzyme or enzyme in crude membrane preparations and a 33-amino acida-factor analog peptide that is a substrate for both Ste24 and ZMPSTE24. This peptide contains a fluorophore (2-aminobenzoic acid—Abz) at its N-terminus and a quencher moiety (dinitrophenol—DNP) positioned four residues downstream from the cleavage site. Upon cleavage, a fluorescent signal is generated in real time at 420 nm that is proportional to cleavage of the peptide and these kinetic data are used to quantify activity. This assay should provide a useful tool for kinetic analysis and for studying the catalytic mechanism of both ZMPSTE24 and Ste24.