Published: Vol 7, Iss 17, Sep 5, 2017 DOI: 10.21769/BioProtoc.2532 Views: 8829
Reviewed by: Marisa RosaJinping ZhaoNoelia Foresi
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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: ProteasomeBackground
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
Equipment
Software
Procedure
Data analysis
The representation of the results can be done in relative fluorescent units (RFUs) by subtracting the RFU levels of the blank wells (containing all solutions except protein extract) from all values. Calculate proteasome activity from the mean linear slope of the emission curve (Figure 1) and express it as fluorescence units per minute (RFU min-1) or as percentage relative to controls, respectively. The mean slope (Mean V in Gen5) is calculated by a linear regression on points in the calculation zone (marked by a green bar in Figure 1) by the software. In Gen5 (Biotek Instruments) go to Protocols > Data Reduction > Well Analysis and tic the Mean V box. For Mean V, the calculation zone is typically adjusted automatically to ignore misleading data points generated at the beginning of a kinetic assay due to noise. The Mean V values can then be exported to Excel by pressing the ‘Edit Table’ button within the ‘Statistics’ view for further analysis (calculation of mean values and statistics). Refer to the user manual of your plate reader for specific details of your instrument.
A typical result of data analysis is shown in Figure 2. Here, pepper plants were infected with different bacterial strains alongside with MgCl2, as a control. For the experiments it is important to use such controls and leaf material from different plants (at least n = 3 to be able to perform statistics) that are of the same age.
Figure 1. Screenshot of a typical output of a proteasome activity measurement in a leaf sample from Arabidopsis thaliana using the above described procedure. The sample was measured on a Biotek Synergy HT plate reader controlled by the Gen5 software (Biotek Instruments). The green bar indicates the part of the linear slope that has been used to calculate the relative fluorescence units per minute (RFU min-1).
Figure 2. XopJ reduces proteasome activity during Xanthomonas-pepper interaction. Pepper leaves were infiltrated with Xanthomonas campestris pv vesicatoria (Xcv) strains indicated in the figure (WT, wild type; ∆xopJ, strain lacking the type-III effector protein XopJ; ∆xopD strain lacking the type-III effector protein XopD). At 3 dpi proteasome activity in total leaf extracts was determined by monitoring the breakdown of the fluorogenic peptide suc-LLVY-NH-AMC in a fluorescence spectrophotometer. Data represent the mean SD (n = 5). Significant differences were calculated using Student’s t-test and are indicated by: *P < 0.05; **P < 0.01.
Recipes
Acknowledgments
This work was supported by the Federation of the European Biochemical Societies (long-term fellowship to S.Ü.), and by the Deutsche Forschungsgemeinschaft (grant Nos. CRC973 and BO1916/5-2 to F.B.). This protocol is modified from previous work described in Reinheckel et al. (2000).
References
Article Information
Copyright
© 2017 The Authors; exclusive licensee Bio-protocol LLC.
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Category
Plant Science > Plant biochemistry > Protein
Biochemistry > Protein > Degradation
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