Ubiquitin Proteasome Activity Measurement in Total Plant Extracts
植物总提取物中泛素蛋白酶体活性测定
发布: 2017年09月05日第7卷第17期 DOI: 10.21769/BioProtoc.2532 浏览次数: 8829
评审: Marisa RosaJinping ZhaoNoelia Foresi
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参见作者原研究论文
The authors used this protocol in:
Nov 2016
Abstract
The fine-tuned balance of protein level, conformation and location within the cell is vital for the dynamic changes required for a cell to respond to a given stimulus. This requires the regulated turnover of damaged or short-lived proteins through the ubiquitin proteasome system (UPS). Thus, the protease activity of the proteasome is adjusted to meet the current demands of protein degradation via the UPS within the cell. We describe the adaptation of an intramolecular quenched fluorescence assay utilizing substrate-mimic peptides for the measurement of proteasome activity in total plant extracts. The peptide substrates contain donor-quencher pairs that flank the scissile bond. Following cleavage, the increase in dequenched donor emission of the product is subsequently measured over time and used to calculate the relative proteasome activity.
Keywords: Proteasome (蛋白酶体)
Immunity (免疫力)
Plant defense (植物防御)
Targeted proteolysis (靶向蛋白水解)
Fluorogenic substrate (荧光底物)
Background
The ubiquitin proteasome system (UPS) is the major protein degradative machinery in eukaryotic cells and as such the UPS is essential for the regulation of many cellular processes, including signaling, cell cycle, vesicle trafficking, and immunity. Proteins destined for turnover are marked by the covalent attachment of ubiquitin and then degraded by the 26S proteasome. The 26S proteasome is composed of two subparticles, the 20S core protease (CP) that compartmentalizes the protease active sites and the 19S regulatory particle that recognizes and translocates appropriate substrates into the CP lumen for breakdown. Proteasome activity is modulated in order to maintain proteostasis in response to fluctuating internal and external conditions. We have recently shown that the UPS is involved in several aspects of plant immunity and a range of plant and animal pathogens subvert the UPS to enhance their virulence (Üstün et al., 2013; Üstün et al., 2014; Üstün and Börnke, 2015; Üstün et al., 2016). In plants, proteasome activity is strongly induced during basal defense and adapted bacterial pathogens can interfere with this induction using specific virulence factors. This protocol describes an assay to assess the relative chymotrypsin-like proteolytic activity of the proteasome in total plant extracts using a fluorogenic substrate. This assay is carried out in a 96-well format using a plate reader and thus is amendable to medium to high throughput and can easily be modified to measure additional proteolytic activities of the proteasome by exchanging the substrate accordingly.
Materials and Reagents
- Pipette tips
- 1.5 ml microcentrifuge tubes
- Black walled 96-well plates for proteasome activity measurements (Corning, catalog number: 3603 )
- Clear 96-well plates for protein measurements (Greiner Bio One International, catalog number: 655101 )
- Plant material (the protocol is optimized for leaf samples from Nicotiana benthamiana, Capsicum annuum and Arabidopsis thaliana but can also be applied to other plant species or tissues)
- Murashige and Skoog (MS) agar (Sigma-Aldrich)
- Sucrose (MP Biomedicals, catalog number: 04821713 )
- Liquid nitrogen (N2)
- Bio-Rad Protein Assay Kit II (Bio-Rad Laboratories, catalog number: 5000002 )
- Proteasome substrate
Suc-LLVY-AMC (chymotrypsin-like activity substrate) (Sigma-Aldrich, catalog number: S6510 )
Note: Other proteolytic activities of the proteasome can be measured using the same protocol by using appropriate substrates (Z-ARR-AMC for measuring the trypsin-like activity [Bachem, catalog number: I-1125.0050 ] and Z-LLE-AMC for the determination of the peptidylglutamyl-peptide-hydrolyzing [PGPH] activity of the 20S proteasome [Bachem, catalog number: I-1945.0005 ]). - Dimethyl sulfoxide (DMSO) (Sigma-Aldrich, catalog number: D8418 )
- Adenosine 5’-triphosphate disodium salt hydrate (ATP) (Sigma-Aldrich, catalog number: A3377 )
- 4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid, N-(2-Hydroxyethyl)piperazine-N’-(2-ethanesulfonic acid) (HEPES) (Sigma-Aldrich, catalog number: H3375 )
- Potassium hydroxide (KOH) (Sigma-Aldrich, catalog number: 60377 )
- 1,4-Dithiothreitol (DTT) (Thermo Fisher Scientific, InvitrogenTM, catalog number: 15508013 )
- Magnesium chloride hexahydrate (MgCl2·6H2O) (Sigma-Aldrich, catalog number: M2670 )
- Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P9541 )
- Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A2153 )
- Proteasome substrate solution (see Recipes)
- Extraction buffer (see Recipes)
- Proteasome lysis/assay buffer (see Recipes)
Equipment
- Pipettes
- pH-meter
- Cork borer 0.7 cm diameter (Sigma-Aldrich, catalog number: Z165220 )
- Overhead stirrer (Heidolph Instruments, model: RZR 1 )
- Cooled microcentrifuge (4 °C)
- Adjustable thermoblock for 96-well plates (Eppendorf, model: ThermoMixer® C )
- Plate reader spectrophotometer (Absorbance, Fluorescence) (e.g., BioTek Instruments, model: Synergy HT )
Software
- Gen5 software (Biotek Instruments)
Procedure
文章信息
版权信息
© 2017 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
- Üstün, S. and Börnke, F. (2017). Ubiquitin Proteasome Activity Measurement in Total Plant Extracts. Bio-protocol 7(17): e2532. DOI: 10.21769/BioProtoc.2532.
- Üstün, S., Sheikh, A., Gimenez-Ibanez, S., Jones, A., Ntoukakis, V. and Börnke, F. (2016). The proteasome acts as a hub for plant immunity and is targeted by Pseudomonas type III effectors. Plant Physiol 172(3): 1941-1958.
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分类
植物科学 > 植物生物化学 > 蛋白质
生物化学 > 蛋白质 > 降解
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