Количественные FRET (Ферстер резонансный перенос энергии) анализ для определения протеазы SENP1 кинетики
Summary
Новый метод с участием количественный анализ FRET (Ферстер резонансный перенос энергии) сигналов описано для изучения кинетики ферментов.<em> K<sub> M</sub</em> И<em> К<sub> Кот</sub</em> Были получены для гидролиза каталитический домен SENP1 (SUMO / Sentrin специфической протеазы 1) предварительно SUMO1 (Small Убиквитин-как модификатор). Общие принципы количественного лада на основе исследования кинетической протеазы могут быть применены в других протеаз.
Abstract
Reversible posttranslational modifications of proteins with ubiquitin or ubiquitin-like proteins (Ubls) are widely used to dynamically regulate protein activity and have diverse roles in many biological processes. For example, SUMO covalently modifies a large number or proteins with important roles in many cellular processes, including cell-cycle regulation, cell survival and death, DNA damage response, and stress response 1-5. SENP, as SUMO-specific protease, functions as an endopeptidase in the maturation of SUMO precursors or as an isopeptidase to remove SUMO from its target proteins and refresh the SUMOylation cycle 1,3,6,7.
The catalytic efficiency or specificity of an enzyme is best characterized by the ratio of the kinetic constants, kcat/KM. In several studies, the kinetic parameters of SUMO-SENP pairs have been determined by various methods, including polyacrylamide gel-based western-blot, radioactive-labeled substrate, fluorescent compound or protein labeled substrate 8-13. However, the polyacrylamide-gel-based techniques, which used the “native” proteins but are laborious and technically demanding, that do not readily lend themselves to detailed quantitative analysis. The obtained kcat/KM from studies using tetrapeptides or proteins with an ACC (7-amino-4-carbamoylmetylcoumarin) or AMC (7-amino-4-methylcoumarin) fluorophore were either up to two orders of magnitude lower than the natural substrates or cannot clearly differentiate the iso- and endopeptidase activities of SENPs.
Recently, FRET-based protease assays were used to study the deubiquitinating enzymes (DUBs) or SENPs with the FRET pair of cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) 9,10,14,15. The ratio of acceptor emission to donor emission was used as the quantitative parameter for FRET signal monitor for protease activity determination. However, this method ignored signal cross-contaminations at the acceptor and donor emission wavelengths by acceptor and donor self-fluorescence and thus was not accurate.
We developed a novel highly sensitive and quantitative FRET-based protease assay for determining the kinetic parameters of pre-SUMO1 maturation by SENP1. An engineered FRET pair CyPet and YPet with significantly improved FRET efficiency and fluorescence quantum yield, were used to generate the CyPet-(pre-SUMO1)-YPet substrate 16. We differentiated and quantified absolute fluorescence signals contributed by the donor and acceptor and FRET at the acceptor and emission wavelengths, respectively. The value of kcat/KM was obtained as (3.2 ± 0.55) x107 M-1s-1 of SENP1 toward pre-SUMO1, which is in agreement with general enzymatic kinetic parameters. Therefore, this methodology is valid and can be used as a general approach to characterize other proteases as well.
Protocol
Discussion
FRET технология была использована для изучения созревания предварительно SUMO1 путем SENP1 9. CFP-YFP был использован в качестве FRET пар и радиометрический анализ, который представляет собой отношение акцепторных доноров выбросов, был использован для характеристики кинетические свойства….
Disclosures
The authors have nothing to disclose.
Acknowledgements
Мы очень благодарны Виктору GJ Rodgers за ценные советы. Мы благодарим всех членов группы Ляо очень близко совместную работу и помощь в этом исследовании. Это исследование было поддержано Национальным институтом здравоохранения (грант AI076504 для JL).
Materials
| Name of the reagent | Company | Catalogue number |
| PCR II TOPO Kit | Invitrogen | K466040 |
| 2xYT | Research Products Internationa.l Corp. | X15600 |
| Ni-NTA Agrose | Thermo Scientific | 88222 |
| Coomassie plus (Bradford) Assay Regent | Thermo Scientific | 23238 |
| 384-well plate (glass bottom) | Greiner | 781892 |
| FlexStation II 384 plate reader | Molecular Device |
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