Recombinant α- β- and γ-Synucleins Stimulate Protein Phosphatase 2A Catalytic Subunit Activity in Cell Free Assays
August 13th, 2017
This publication presents a protocol showing how to measure the activity of recombinant protein phosphatase 2A catalytic subunit (PP2Ac) in response to recombinant α-synuclein, β-synuclein, or γ-synuclein proteins using a simple colorimetric assay. We show findings regarding α-synuclein specificity toward PP2Ac in mouse brain.
Recombinant alpha, beta, and gamma synucleins stimulate protein phosphatase 2A catalytic subunit activity in cell free assays. Hello, my name is Sovanarak Lek. I am a medical student at Texas Tech University Health Sciences Center, El Paso, Paul L.Foster School of Medicine.
In this video, I will be demonstrating a protein phosphatase two way color metric assay using recombinant synuclein proteins. Preparation of buffers and radiants. Preparing p-Nitrophenol Phosphate Buffer.
To prepare 50 mils of of pNPP buffer dissolve Trizma base in 25 mils ultrapure deionized water in a beaker. Add a clean stire bar and place on magnetic stirrer. Once Trizma dissolves, add calcium chloride with continued stirring.
Adjust pH to 7.0. Transfer the solution to a graduated cylinder. Bring the final total volume to 50 mils with ultrapure deionized water.
Preparing malachite green solution. Malachite green solution requires three solutions, parts A, B, and C.Solution A is four molar hydrochloric acid. Solution is a combination of ammonium molybdate plus solution A.Solution C is 0.045%malachite green oxalate salt.
To prepare solution D, mix one part B to three parts C, one to three ratio. Stir one hour at room temperature. Finally, filter solution D through a 22 micrums membrane and store at four degrees Celsius.
Protect from light. Preparation of activator. Activator is one percent Tween 20 solution.
Pippette 10 microliters of Tween 20 in 990 microliters of ultrapure deionized water. Vortex until Tween 20 dissolves completely. Preparing malachite green plus Tween 20 working solution.
Add activator to solution D in a ration of one to a 100. Preparing threonine phosphopeptide. To prepare a two mM pT stock solution, add 550 microliters of ultrapure deionized water to one milligram pT.
Vortex until phosphopeptide is completely dissolved. Place on ice to use immediately. Store extra pT aliquots at minus 20 degrees Celsius.
Synuclein preparation. Resuspend one milligram of synuclein in mil ultrapure deionized water at a final concentration of one milligram per mil. Vortex gently to dissolve.
Use immediately and store unused aliquots at minus 80 degrees Celsius. Preparing phosphate standards. Phosphate standards step one.
To prepare 1 mM phosphate stock solution dissolve potassium phosphate in ultrapure deionized water in a beaker with a stir bar. Transfer to graduation cylinder and bring the final volume to 100 mils. This is a 10 mM solution.
Filter the entire solution through a 22 micros filter using a large syringe. Dilute this to 1 to 100 to make 1 mM phosphate solution to be used for assay standards. Phosphate standards step two.
Add the appropriate amount of phosphate plus ultrapure deionized water to 1.5 mils tubes as per table. This will yield tubes of 150, 300, 600, 1, 200, and 2, 400 pmol phosphate. And store aliquots at minus 20 degrees Celsius.
PP2A activity assay. Prepare PP2A assay in 1.5 mil tubes on ice. Add 43 microliters pNPP buffer.
Add one microliter of human recombinant PP2Ac. Add synuclein and incubate PP2A plus synuclein 30 minutes on a Nutator at four degrees Celsius. Next, add 16 microliters of 2mM pT.
Incubate 10 minutes at 30 degrees Celsius in water bath. On a flat bottom 96-well plate, add 25 microliters of each phosphate standards in duplicate from low to high. Pipet 24 microliters of each PP2A assay in duplicate onto the same 96 well plate with the standards.
Pipet 75 microliters of solution D containing Tween 20 to each sample and standard. Upon addition of MGT, you will see a color change in wells when free phosphates are present. Photometric analysis.
Analyze on a spectrophotometer set to 630 nanometers. Representative results. Here we have a representative PP2A assay and the graph demonstrating the standard curve.
On the left are the phosphate standards. The more phosphate that the wells contain, the darker the shade of green. One the right we have PP2A that was incubated with alpha, beta, or gamma synucleins at different molar concentrations.
Higher concentrations were more effective at stimulating PP2A catalytic subunit activity. Here we see the graphical representation of PP2A activity in response to varying concentrations of alpha synuclein, beta synuclein, and gamma synuclein. Alpha synuclein, beta synuclein, and gamma synuclein were all significant stimulators of PP2A activity at concentration of one to two micromolar.
Only alpha synuclein and beta synuclein were significant stimulators of PP2A activity at 0.5 micromolar concentration. Figure three shows the interaction of PP2Ac with endogenous synucleins in mouse brain. In A, recombinant alpha, beta, and gamma synucleins are shown as positive controls.
In B, co-immunoprecipitation was done using the PP2Ac-specific antibody 1D6, which brought down PP2Ac and also brought down considerable interacting alpha synuclein but almost no beta synuclein. No gamma synuclein came down with PP2Ac in the co-immunoprecipitation. This co-immunoprecipitation data suggests that alpha synuclein is the endogenous modulator of PP2Ac in vivo.
We want to thank everyone who helped us in making this video. So, in conclusion this is Ruth Perez. Sonavarak Lek.
Javier Vargas. Wishing you great success. Goodbye and good luck.
Overview
This publication presents a protocol for measuring the activity of recombinant protein phosphatase 2A catalytic subunit (PP2Ac) in response to recombinant α-synuclein, β-synuclein, or γ-synuclein proteins using a colorimetric assay. The findings indicate α-synuclein's specificity toward PP2Ac in mouse brain.
Key Study Components
Area of Science
- Neuroscience
- Biochemistry
- Cell Biology
Background
- Protein phosphatase 2A (PP2A) is a critical enzyme involved in various cellular processes.
- Synucleins are a family of proteins implicated in neurodegenerative diseases.
- Understanding the interaction between synucleins and PP2A can provide insights into their roles in neuronal function.
- Colorimetric assays are effective for measuring enzyme activity in vitro.
Purpose of Study
- To develop a protocol for assessing PP2Ac activity in the presence of synucleins.
- To evaluate the specificity of α-synuclein towards PP2Ac.
- To provide a detailed methodology for researchers in the field.
Methods Used
- Preparation of buffers and reagents for the colorimetric assay.
- Incubation of PP2Ac with varying concentrations of synucleins.
- Measurement of enzyme activity using a spectrophotometer.
- Co-immunoprecipitation to assess interactions in mouse brain samples.
Main Results
- All three synucleins (α, β, γ) stimulated PP2Ac activity at specific concentrations.
- α-synuclein showed the highest specificity and interaction with PP2Ac.
- Co-immunoprecipitation indicated α-synuclein as the primary endogenous modulator of PP2Ac.
- Colorimetric changes correlated with phosphate levels in the assay.
Conclusions
- The developed protocol is effective for studying PP2Ac activity.
- α-synuclein is a significant modulator of PP2Ac in vivo.
- This study enhances understanding of synuclein interactions in neurobiology.
Frequently Asked Questions
Executive Industry Relevance
This study establishes a cell-free assay to evaluate synuclein-mediated modulation of PP2Ac activity, providing a mechanistic tool for target validation in neurodegenerative disease research. By demonstrating that all three synuclein isoforms can stimulate PP2Ac in vitro, the method supports hypothesis testing around conserved functional homology and its implications for pathway de-risking. The co-immunoprecipitation data further refine target specificity, highlighting α-synuclein as the endogenous modulator in vivo, which aids in prioritizing therapeutic hypotheses and reducing mechanistic ambiguity in early discovery.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Scientific Value: Enables interrogation of therapeutic hypotheses regarding synuclein-PP2Ac interactions in neurodegeneration.
- Scientific Value: Supports biological de-risking by clarifying whether observed effects stem from direct enzyme modulation or indirect pathways.
- Scientific Value: Facilitates target confidence assessment by comparing isoform-specific activity in a controlled, reproducible system.
Screening & Assay Development
- Scientific Value: Provides a standardized, quantitative readout (phosphate release via malachite green assay) for evaluating compound effects on PP2Ac activity.
- Operational Value: Uses recombinant proteins and defined buffers to ensure assay reproducibility across laboratories and screening campaigns.
- Operational Value: Enables preparation of validated biological tools (e.g., dose-response curves) for downstream compound evaluation and hit confirmation.
Translational & Preclinical Research
- Scientific Value: Links in vitro findings to endogenous interactions observed in mouse brain, supporting translational continuity from discovery to preclinical validation.
- Scientific Value: Helps de-risk target engagement strategies by distinguishing between promiscuous in vitro activity and selective in vivo modulation.
- Operational Value: Informs preclinical model selection by indicating which synuclein isoforms are likely to engage PP2Ac in physiological contexts.
Pipeline & Workflow Integration
The assay fits within the early discovery continuum, supporting target validation and lead identification by providing quantitative, mechanism-based readouts that inform go/no-go decisions before significant investment in compound optimization.
- Discovery Biology: Supports hypothesis testing and pathway clarification by enabling dose-dependent measurement of PP2Ac activation by synuclein isoforms.
- Screening: Delivers assay readiness through standardized reagent preparation and a colorimetric output compatible with plate-based formats.
- Analytics: Generates quantitative phosphate measurements that allow comparison of modulator potency and efficacy across experimental conditions.
- Translational Research: Connects to preclinical work by validating that observed in vitro effects align with endogenous interactions in brain tissue.
- Enterprise Reuse: Establishes a reusable platform for assessing modulator effects on PP2Ac, applicable beyond synucleins to other regulatory proteins or small molecules.
Operational & Enterprise Impact
- Scientific Value: Increases predictive confidence in target modulation claims by providing direct, quantitative enzyme activity data.
- Operational Value: Promotes standardization and reproducibility through detailed reagent preparation and defined incubation conditions.
- Strategic Value: Improves go/no-go decision efficiency by reducing reliance on indirect or correlative biomarkers.
- Portfolio Impact: Enables risk-adjusted prioritization of targets based on mechanistic evidence of pathway engagement.
Implementation Considerations
- Requires expertise in protein biochemistry and enzyme assay techniques, including handling of recombinant proteins and phosphorylation detection.
- Dependent on access to spectrophotometric equipment capable of 630 nm absorbance readings and temperature-controlled incubation systems.
- Necessitates cross-team standardization of reagent preparation (e.g., malachite green solution, pNPP buffer) to ensure assay consistency.
- Involves adaptation considerations when extending the assay to non-neuronal model systems or alternative PP2Ac sources.
- Includes practical limitations such as the need for fresh phosphopeptide substrates and strict temperature control during incubation steps.
Biopharma FAQs
Chapters in this video
0:00
Title
0:33
Preparation of Buffers and Reagents
0:38
Preparing p-Nitrophenol Phosphate (pNPP) Buffer
1:21
Preparing Malachite Green Solution
2:21
Preparation of Activator
2:42
Preparing Malachite Green + Tween 20 (MGT) Working Solution
2:58
Preparing Threonine Phosphopeptide (pT)
3:31
Synuclein Preparation
3:59
Preparing Phosphate Standards
4:45
Phosphate Standards Step 2
5:17
PP2A Activity Assay
6:42
Photometric Analysis
6:53
Representative Results
9:01
Conclusion
