SENP1 Protease कैनेटीक्स निर्धारण के लिए मात्रात्मक झल्लाहट विश्लेषण (Forster प्रतिध्वनि ऊर्जा हस्तांतरण)
Summary
एक उपन्यास विधि झल्लाहट (Forster प्रतिध्वनि ऊर्जा हस्तांतरण) संकेतों के मात्रात्मक विश्लेषण शामिल एंजाइम कैनेटीक्स का अध्ययन करने के लिए वर्णित है.<em> कश्मीर<sub> पुरूष</sub</em> और<em> कश्मीर<sub> बिल्ली</sub</em> पूर्व SUMO1 (लघु आपरिवर्तक Ubiquitin तरह) के लिए उत्प्रेरक SENP1 (सूमो Sentrin / विशिष्ट protease 1) डोमेन की hydrolysis के लिए प्राप्त किया गया. इस protease मात्रात्मक झल्लाहट आधारित गतिज अध्ययन के सामान्य सिद्धांतों अन्य proteases के लिए लागू किया जा सकता है.
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
झल्लाहट प्रौद्योगिकी 9 SENP1 पूर्व SUMO1 परिपक्वता का अध्ययन करने के लिए इस्तेमाल किया गया है. CFP YFP जोड़ी झल्लाहट और ratiometric विश्लेषण, जो स्वीकर्ता के दाता उत्सर्जन के लिए अनुपात के रूप में इस्तेमाल किया गया थ…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
हम बहुत मूल्यवान सलाह के लिए विक्टर GJ Rodgers आभारी हैं. हम लियाओ समूह के सदस्यों के बहुत करीबी सहयोगी और इस अध्ययन के साथ मदद के लिए काम करने के लिए सभी को धन्यवाद. इस अध्ययन स्वास्थ्य के राष्ट्रीय संस्थान (जीएल AI076504 अनुदान) द्वारा समर्थित किया गया था.
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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