Isolating Potentiated Hsp104 Variants Using Yeast Proteinopathy Models
November 11th, 2014
Yeast proteinopathy models are valuable tools to assess the toxicity and aggregation of proteins implicated in disease. Here, we present methods for screening Hsp104 variant libraries for toxicity suppressors. This protocol could be adapted to screen any protein library for toxicity suppressors of any protein that is toxic in yeast.
The aim of the following experiment is to screen HS P 1 0 4 libraries for the selection of variants that suppress the toxicity of the proteins associated with protein misfolding disorders. This is achieved by co transforming HSP 1 0 4 variant libraries with a disease associated substrate in yeast and coex expressing the two proteins as a second step. The selected yeast are treated with five Fluor utic acid or five FOA to counter select for the HSP 1 0 4 plasmid.
Next, the hits are sequenced by amplifying the mutagen region by colony PCR or identification of the mutations in the HSP 1 0 4 gene. Ultimately, the suppression of toxicity of the disease associated substrate can be evaluated to identify the HS P 1 0 4 variants that are most promising for further characterization. The main advantage of this technique over existing methods like binding based selections, is that this technique allows for the selection of variants that suppress the toxicity of the disease associated substrate.
This method can help answer key questions in the protein folding field by identifying the proteins that can reverse protein misfolding. Though this method can provide insight into HS P 1 0 4 potentiation, it can also be applied to screening libraries of any protein for the toxicity suppression of any substrate that is toxic in yeast. Generally, individuals new to this method will struggle because many colonies may need to be screened to find valid hits To screen for protea toxicity suppression begin by washing HSP 1 0 4 colonies off the plates with raffinose supplemented dropout media, a serological pipette and sterile wood and applicators transfer the liquid washes to a 50 milliliter conical tube and vortex the colonies thoroughly to separate any cell clumps.
Next, dilute the cell suspension to a slightly cloudy culture and grow the cells overnight in Raffinose dropout media with shaking at 30 degrees Celsius. Grow the HSP 1 0 4 wild type and vector controls in parallel at this time as well. The following morning plate, a range of cell concentrations onto individual galactose plates to generate at least one plate per group with single colonies.
Normalize the vector and HSP 1 0 4 world type cultures to the OD 600 of the library and plate. Equal volumes of these cells to the experimental strain concentrations to compare the growth of the library relative to the controls to assess the selection. Stringency also plate the library on glucose repressing media and then incubate all of the plates for two to three days at 30 degrees Celsius until colonies appear to use five FOA for selection of the cells, which no longer maintain HSP 1 0 4 expressing plasmids next streak out the single colonies from each strain in duplicate onto double and single dropout media plates per overnight incubation at 30 degrees Celsius.
These plates will be used for sequencing later to prevent the plates from drying out. Wrap the SD hiss Euro plates in para film and install them at four degrees Celsius while performing the five FOA screen. Then streak the colonies from the SD hiss plates to single colonies on five FOA plates for a one to two day incubation at 30 degrees Celsius until single colonies appear.Next.
From each five FOA plate, select and streak. Three surviving colonies onto one SD Euro and one SD his plate per surviving colony after another one to two days of incubation at 30 degrees Celsius. The colonies that have lost the HSP 1 0 4 plasmid will grow on SD hiss plates, but not SD Euro plates.
Grow these strains to saturation in raffinose dropout media in 96. Deep well two milliliter plates overnight at 30 degrees Celsius with shaking. Then to eliminate any false positives, perform a spotting assay.
Near the end of the incubation is a multi-channel pipette to aliquot 200 microliters of raffinose dropout media per well to a new 96 well plate reserving columns one and seven for the neat cultures. Next aliquot 250 microliters of each of the 16 saturated cultures into columns one and seven of the plate and serially dilute the cultures five fold into each column of the plate, mixing thoroughly with the multichannel pipette. Then to ensure even spotting, remove 50 microliters of the diluted culture from column six and 12 to ensure the final volume of each well is 200 microliters.
Then use a 96 volt replicator tool to spot the cultures in duplicate onto SD hiss and escal hiss plates. After a two to three day incubation at 30 degrees Celsius, select for the sequencing strains that exhibit toxicity on the escal hiss plates. Similar to that of the controls false positive strains, which are not as toxic as the controls can be discarded.
Also discard strains that exhibit a growth defect on the sd. His plates or that exhibit substantially greater toxicity than the disease substrate alone. To sequence the HSP 1 0 4 variants by colony PCR first scrape approximately 10 microliters of yeast from the SD hiss Euro plates into three microliters of 20 milli sodium hydroxide in PCR stripped tubes to ly the cells by freeze thaw.
First place the strips in a minus 80 degrees Celsius freezer. After 10 minutes in the freezer, incubate the tubes at 99 degrees Celsius in the thermocycler for 10 minutes, and then dilute the strains to 100 microliters with PCR grade water using a high primer concentration. Next, run a standard PCR program on the samples, and then analyze the samples by agel electrophoresis to confirm the amplification of A PCR product of the appropriate size.
Finally, sequence the PCR products taking care to thoroughly analyze the sequencing chromatograms for mutations. Mutations may be observed as a mixture of two nucleotide to given site as yeast often harbor multiple plasmids. In this representative experiment, after the construction of a library of HSP 1 0 4 variants randomized in the middle domain, the library was transformed into yeast harboring a plasmid for galactose inducible TDP 43 expression.
The transformants were then recovered and plated onto glucose and galactose plates to assess the stringency of the screen Hits were selected from the galactose plate. Following five FOA treatment spotting assays of a subset of the variants selected in the initial screen. Indicated that of the four colonies selected, two displayed HSP 1 0 4, mediated TDP 43 toxicity suppression.
One was a false positive and one displayed enhanced toxicity. Following five FOA treatment, the two true hits were then sequenced by colony PCR to identify the middle domain mutations. These HS P 1 0 4 mutations can then be made in the parental HSP 1 0 4 plasmid by site directed mutagenesis to confirm their toxicity suppression Once mastered, this technique can be used to screen up to 200 hits at a time in the five FO, a step over the course of one to two weeks if it is performed properly.
While attempting this procedure, it's important to carefully analyze the sequencing results as mixtures of plasmids are often obtained Following this procedure. Other methods like fluorescence microscopy can be performed to help answer additional questions like, how do the HSP 1 0 4 variants affect the aggregation of the disease associated substrate? After watching this video, you should have a good understanding of how to screen libraries of HSP 1 0 4 variants for the selection of toxicity suppressors.
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Overview
This article presents a method for screening Hsp104 variant libraries to identify suppressors of protein toxicity associated with misfolding disorders. The approach utilizes yeast models to evaluate the effectiveness of these variants in reducing toxicity.
Key Study Components
Area of Science
- Neuroscience
- Protein Misfolding Disorders
- Yeast Models
Background
- Protein misfolding disorders are linked to toxic protein aggregation.
- Hsp104 is a chaperone protein that can influence protein aggregation.
- Identifying variants that suppress toxicity can provide insights into therapeutic strategies.
- Yeast models are effective for studying protein toxicity and aggregation.
Purpose of Study
- To screen Hsp104 variant libraries for variants that suppress protein toxicity.
- To evaluate the effectiveness of these variants in a yeast model.
- To enhance understanding of protein misfolding disorders.
Methods Used
- Co-transforming Hsp104 variant libraries with disease-associated substrates in yeast.
- Co-expressing the two proteins to assess interactions.
- Treating selected yeast with five Fluor utic acid to counter-select for the Hsp104 plasmid.
- Sequencing hits to identify mutations in the Hsp104 gene.
Main Results
- Identification of Hsp104 variants that effectively suppress toxicity.
- Evaluation of the suppression of toxicity in yeast models.
- Insights into the mechanisms of protein misfolding disorders.
- Comparison of this method to existing binding-based selections.
Conclusions
- The method allows for the selection of variants that suppress toxicity.
- It provides a promising approach for further characterization of Hsp104 variants.
- This research contributes to the understanding of protein misfolding disorders.
Frequently Asked Questions
Executive Industry Relevance
This method enables biopharma R&D teams to identify engineered protein disaggregase variants that suppress toxicity in neurodegenerative disease models, supporting target validation and mechanistic de-risking. By using yeast proteinopathy models to screen Hsp104 libraries, the approach provides a scalable, genetically tractable system for evaluating functional rescue of proteotoxic proteins like TDP-43, FUS, and α-synuclein. The two-step screening process reduces false positives, increasing confidence in hit selection for downstream preclinical evaluation.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Scientific Value: Enables interrogation of Hsp104 variant functionality in suppressing TDP-43, FUS, and α-synuclein toxicity and aggregation in yeast.
- Operational Value: Supports biological de-risking by distinguishing true suppressors from spontaneous suppressors via counter-selection with 5-FOA.
Screening & Assay Development
- Scientific Value: Generates quantitative growth-based readouts on selective media to assess variant-mediated toxicity suppression.
- Operational Value: Standardizes library screening through co-transformation, inducible expression, and plasmid loss selection for reproducible hit identification.
Translational & Preclinical Research
- Scientific Value: Uses yeast models that recapitulate cytoplasmic aggregation of human disease-linked proteins, enabling phenotypic screening relevant to ALS and Parkinson’s disease pathology.
- Operational Value: Facilitates hit-to-lead progression by linking genetic suppression to protein conformational rescue, supporting mechanistic follow-up in mammalian systems.
Pipeline & Workflow Integration
The method fits within early discovery workflows, enabling lead identification through functional screening of Hsp104 variant libraries before mechanistic and preclinical validation.
- Discovery Biology: Supports hypothesis testing by evaluating whether Hsp104 variants can reverse proteotoxicity associated with disease-linked substrates.
- Screening: Delivers assay-ready yeast strains with inducible expression and counter-selection for high-throughput variant evaluation.
- Analytics: Enables colony PCR and sequencing to correlate genetic mutations with functional suppression, guiding structure-function analysis.
- Translational Research: Connects yeast-based suppression data to human-relevant protein misfolding phenotypes, informing target confidence.
- Enterprise Reuse: The protocol is adaptable to any protein backbone and toxic substrate, allowing platform reuse across multiple neurodegeneration targets.
Operational & Enterprise Impact
- Scientific Value: Increases predictive confidence in target modulation by validating functional suppression of aggregation and toxicity.
- Operational Value: Enhances reproducibility through standardized counter-selection and spotting assays to eliminate false positives.
- Strategic Value: Improves go/no-go decisions by prioritizing variants with demonstrated efficacy in disease-relevant models.
- Portfolio Impact: Enables risk-adjusted advancement of Hsp104 variants as potential therapeutic leads for protein misfolding disorders.
Implementation Considerations
- Requires expertise in yeast genetics, plasmid transformation, and selective media handling.
- Dependent on inducible expression systems (e.g., galactose promoters) and counter-selection markers (e.g., 5-FOA sensitivity).
- Necessitates standardized workflows for library transformation, plating, and phenotypic screening across replicates.
- Adaptation to other protein backbones requires validation of expression, folding, and toxicity in yeast.
- Practical limitation: Screening efficiency depends on library size and false positive rate, necessitating the two-step validation to ensure hit quality.
