We thank Sue Lindquist, Aaron Gitler, and Martin Duennwald for kindly sharing reagents. Our studies were supported by: an American Heart Association Post-Doctoral Fellowship (M.E.J); NIH Director's New Innovator Award DP2OD002177, NIH grants R21NS067354, R21HD074510, and R01GM099836, a Muscular Dystrophy Association Research Award (MDA277268), Packard Center for ALS Research at Johns Hopkins University, Target ALS, and an Ellison Medical Foundation New Scholar in Aging Award (J.S.).

1. Library Generation
<ol>
	<li>To construct libraries of Hsp104 using domain-specific error-prone PCR, first amplify the domain of interest with an error prone DNA polymerase20.</li>
	<li>Purify the PCR product by gel extraction.</li>
	<li>Perform a megaprimer extension step using a standard site-directed mutagenesis protocol: combine 50 ng template plasmid, 250 ng megaprimer, 200 &#956;M dNTPs, and high-fidelity DNA polymerase in PCR buffer, and dilute to 50 &#956;l total volume with PCR grade water20. Run a standard PCR program. 
	NOTE: Specific primers used will vary based on the particular region of the gene that is to be amplified.
	<ol>
		<li>Following PCR, digest parental template DNA with 1 &#956;l DpnI restriction enzyme for 2 hr at 37 &#176;C.</li>
	</ol>
	</li>
	<li>Transform the library by electroporation or other means in ultracompetent E. coli and purify it by miniprep. 
	NOTE: Library generation varies substantially based upon the aims of a given experiment. Hsp104 is a very large protein, so it is impractical to randomize the entire gene, which is why we utilize domain-specific error prone PCR. Additionally, the structure of Hsp104 remains poorly understood, making the design of directed libraries challenging21. Libraries can be constructed using directed or random approaches to mutagenesis, with the only restriction being that the template plasmid backbone should contain the URA3 gene to allow for 5-FOA counter selection on dextrose media.</li>
</ol>
2. Transformation of the Hsp104 Library
<ol>
	<li>Integrate the disease-associated substrate into W303a&#916;hsp104 yeast using a standard lithium acetate/PEG transformation protocol22. Select single colonies and screen them for toxicity to isolate a strain with high toxicity of the disease associated substrate23. 
	NOTE: Use an integrated strain and isolate a single colony to ensure equal expression in all cells. Clone the disease substrate into a plasmid allowing integration of the gene under any marker other than uracil (we use histidine) and the Hsp104 library into the pAG416GAL plasmid (uracil marker)24. To allow for 5-FOA counterselection, ensure that the library is expressed from a plasmid with the uracil marker. Other yeast strains can also be employed. We have noted a similar toxicity suppression phenotype using W303a and BY4741 yeast strains in both WT and &#916;hsp104 backgrounds (M.E.J. and J.S., unpublished observations).</li>
	<li>Transform the Hsp104 library into this strain using the same lithium acetate/PEG transformation protocol22. Scale up the transformation appropriately to maintain the sequence space of the library and to preserve the predicted library size.</li>
	<li>Plate the transformation mixture onto noninducing, selective plates (e.g. SD-His-Ura) using enough plates to ensure growth of a large number of colonies. Use large petri dishes (150 mm) to minimize the number of plates needed. Recovering transformants using plates allows transformation efficiency to be assessed.</li>
	<li>Transform Hsp104WT and vector negative controls in parallel.</li>
</ol>
3. Screening for Suppression of Proteotoxicity
<ol>
	<li>Wash the colonies off the plates using raffinose supplemented dropout media (e.g. SRaff-His-Ura). Use a serological pipette and sterile wooden applicators to loosen the colonies from the plates. Transfer the liquid washes to a 50 ml conical tube and vortex thoroughly to separate any clumps of cells. Dilute to a slightly cloudy culture, OD600 ~0.025. 
	NOTE: Here raffinose serves to relieve the cells of glucose-mediated repression of induction, thus priming the cells for galactose-mediated induction.</li>
	<li>Grow the diluted culture overnight in raffinose dropout media with shaking at 30 &#186;C. Grow the Hsp104WT and vector controls in parallel.</li>
	<li>The following morning, plate a range of concentrations onto individual galactose (e.g. SGal-His-Ura) plates (1 &#956;l to 2 ml concentrated culture in 400 &#956;l total volume). Plating a broad range of concentrations will ensure that a plate will have single colonies. The actual concentration required depends upon the toxicity of the disease substrate of interest.</li>
	<li>Normalize the vector and Hsp104WT cultures to the OD600 of the library and plate equal volumes to compare growth of the library relative to the controls.</li>
	<li>To assess selection stringency, plate the library on glucose (repressing) media. Incubate plates for 2-3 days at 30 &#186;C, until colonies appear (Figure 2). Assess the resulting colonies and compare to the controls. 
	NOTE: Often both large and small colonies are obtained, but no correlation between colony size and activity is observed. Screen these potential hits using a 5-FOA counter selection technique (Step 4) to minimize false positives carried over to sequencing.</li>
</ol>
4. 5-FOA Counterselection and Spotting to Eliminate False Positives
<ol>
	<li>Streak out single colonies in duplicate onto double and single dropout media (e.g. SD-His-Ura and SD-His plates) and grow overnight at 30 &#186;C. Repeat with vector and Hsp104WT controls. Plating on SD-His prior to 5-FOA increases the efficiency of the 5-FOA step by increasing the likelihood that cells will have lost the Hsp104 plasmid when they are plated on 5-FOA.</li>
	<li>To prevent plates from drying out, wrap the SD-His-Ura plates in parafilm and store at 4 &#186;C while performing the 5-FOA screen. These plates will eventually be used for sequencing.</li>
	<li>Streak the colonies from the SD-His plates to single colonies on 5-FOA plates (YPD media + 1g/L 5-FOA) and incubate for 1-2 days at 30 &#186;C until single colonies appear. Here, 5-FOA is converted into a toxic product (5-fluorodeoxyuridine) in cells that contain the URA3 gene. Thus, any cells still harboring the Hsp104 plasmid will die.</li>
	<li>Streak the single colonies from the 5-FOA plates in duplicate onto SD-Ura and SD-His plates. Test 3 colonies for each hit to increase the likelihood that at least one colony for each hit will have lost the Hsp104 plasmid. Incubate for 1-2 days at 30 &#176;C. Colonies that have lost the Hsp104 plasmid will grow on SD-His plates but not SD-Ura plates.</li>
	<li>Grow the strains that have lost the Hsp104 plasmids for spotting assays. Grow the strains to saturation in raffinose dropout media (e.g. SRaff-His) in 96 DeepWell 2 ml plates overnight at 30 &#186;C with shaking. Be sure to include the 5-FOA treated control strains.</li>
	<li>Prepare plates for spotting assay. Using a multichannel pipette, aliquot 200 &#181;l raffinose dropout media (e.g. SRaff-His) per well of a 96 well plate, reserving columns 1 and 7 for the neat cultures. Aliquot 250 &#181;l of each of the 16 saturated cultures into columns 1 and 7 of the plate.</li>
	<li>Serially dilute the cultures 5 fold into each column of the plate, mixing thoroughly with a multichannel pipette. Remove 50 &#181;l of the diluted culture from columns 6 and 12 to ensure the final volume of each well is 200 &#181;l. This is essential to ensure even spotting.</li>
	<li>Using a 96-bolt replicator tool (frogger), spot the cultures in duplicate onto SD-His and SGal-His plates. Incubate the plates at 30 &#186;C for 2-3 days.</li>
	<li>Select for sequencing strains that exhibit toxicity on the SGal-His plates similar to that of the controls. Discard as false positives strains that are not as toxic as the controls. 5-FOA is also often toxic on its own, so discard strains that exhibit a growth defect on SD-His plates or that exhibit substantially greater toxicity than the disease substrate alone (Figure 3).</li>
</ol>
5. Sequencing the Hsp104 Variants by Colony PCR
<ol>
	<li>Scrape ~10 &#956;l yeast from the SD-His-Ura plates in 3 &#956;l 20 mM NaOH in PCR strip tubes and lyse the cells by freeze-thaw. Place the strip tubes in a -80 &#186;C freezer for 10 min or in liquid nitrogen, then incubate at 99 &#186;C in a thermocycler for 10 min. Dilute the strains to 100 &#181;l with PCR grade water.</li>
	<li>Prepare PCR reactions: 5 &#956;l PCR template, 0.5 &#956;l 100 &#956;M each primer, 0.5 &#956;l 10mM dNTPs, 0.25 &#956;l DNA polymerase in PCR buffer, and make up to 25 &#956;l total volume with PCR grade water. Design primers to amplify the randomized region plus approximately 100 bp on either end. Minimize the size of the amplified region to increase the likelihood of successful amplification. Run a standard PCR program.</li>
	<li>Analyze samples by agarose gel electrophoresis to confirm amplification of a PCR product of the appropriate size. Repeat PCR if no product is present. 
	NOTE: PCR failure is typically due to too much yeast being used in step 5.1.</li>
	<li>Prepare samples for DNA sequencing. Remove PCR primers either by PCR purification or using a PCR product cleanup reagent such as Exonuclease I and Shrimp Alkaline Phosphatase enzyme (ExoSAP-IT) to degrade single stranded DNA. This reagent enzymatically degrades unincorporated primers and dNTPs, allowing for higher throughput.</li>
	<li>Sequence the PCR products. Be sure to thoroughly analyze sequencing chromatograms for mutations. Mutations may be observed as a mixture of two nucleotides at a given site (Figure 4), as yeast often harbor multiple plasmids.</li>
</ol>

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The authors have nothing to disclose.

Here we present our approach to isolating potentiated Hsp104 variants that suppress the toxicity of disease-associated substrates using yeast proteinopathy models. Using this approach, large libraries of variants can be screened in high throughput, with the only limitation being the number of variants that pass the 5-FOA secondary screen. By performing these steps in 96 well format, we routinely screen up to 200 hits at a time in the 5-FOA step over the course of 1-2 weeks. The number of hits obtained during the initial screening step will vary largely depending upon substrate toxicity and the makeup of each particular library. When sequencing hits, it is essential to carefully analyze all sequencing chromatograms since mixtures of plasmids are typically present in each cell.
We have sometimes noted a large percentage of Hsp104WT being isolated as &#8216;hits&#8217;. This is likely due to the presence of spontaneous genetic suppressors or very weak activity. To circumvent this problem, we have found that screening at elevated temperatures (e.g. 34 &#176;C) can decrease the percentage of &#8216;hits&#8217; that are Hsp104WT. It is also essential to re-clone any sequenced hits to independently assess the activity of any novel variants and to ensure plasmid purity. Once new hits are identified, they can be assessed for suppression of aggregation in yeast and characterized biochemically.
We have used these methods to isolate potentiated Hsp104 variants against TDP-43, FUS, and &#945;-synuclein and verified their activity using pure protein biochemistry assays17. Ultimately, these methods could be used to screen protein libraries against any substrate of interest that is toxic in yeast.

Yeast proteinopathy models have been developed for protein-misfolding disorders including amyotrophic lateral sclerosis (ALS) and Parkinson&#8217;s disease (PD)1-3. Expression of the proteins TDP-43 and FUS, which misfold in ALS patients, are toxic and mislocalize to form cytoplasmic aggregates in yeast1,2. Similarly, expression of &#945;-synuclein (&#945;-syn), which is implicated in PD, is toxic and mislocalizes to form cytoplasmic aggregates in yeast3. These features recapitulate phenotypes in patients with these disorders4,5. Thus, yeast models provide a useful platform for screening for proteins or small molecules that prevent or reverse these phenotypes2,6-13. We are interested in the development of proteins that are capable of reversing aggregation and toxicity due to TDP-43, FUS, and &#945;-syn. We focus on Hsp104, an AAA+ protein from yeast that is uniquely capable of disaggregating proteins both from amorphous aggregates and amyloid in yeast, yet it has no human homologue14,15. Hsp104 is finely tuned to disaggregate endogenous yeast prions and has only limited ability to disaggregate substrates implicated in human neurodegenerative diseases, which it never ordinarily encounters16,17. Thus, we aim to engineer enhanced versions of Hsp104 that are able to efficaciously disaggregate these human substrates. To do so, we construct large libraries of Hsp104 variants using error-prone PCR; these libraries can be screened using the yeast proteinopathy models17. We have adopted a domain-targeted approach to constructing and screening libraries, as Hsp104 is very large17. We initially focused on the middle domain (MD) of Hsp10417, though similar approaches can be employed to screen other domains. These models enable screening for disaggregase activity directly, as opposed to alternative techniques such as surface display, which is restricted to use for monitoring binding18.
Our protocol is based on two screening steps (Figure 1). First, Hsp104 variants that suppress the toxicity of the disease substrate in yeast are selected. To do so, the Hsp104 variants and disease-associated substrate are cotransformed into &#8710;hsp104 yeast. We employ &#8710;hsp104 yeast to explore Hsp104 sequence space in the absence of wild-type (WT) Hsp10417. Importantly, deletion of Hsp104 does not affect &#945;-syn, FUS, or TDP-43 toxicity in yeast, and expression of Hsp104WT provides minimal rescue1,13,17. The yeast is then plated on inducing media to induce expression of both proteins. Yeast harboring Hsp104 variants that suppress toxicity of the disease-associated substrate confer growth of the colony. These variants are selected for further analysis, while colonies maintaining variants that do not suppress toxicity die. However, false positives are a substantial problem in this screen. Expression of TDP-43, FUS, and &#945;-syn are highly toxic, which creates a strong selective pressure for the appearance of spontaneous genetic suppressors of toxicity unrelated to the Hsp104 variant being expressed. Thus, we have used a secondary screen that is also relatively high throughput to eliminate these nonspecific toxicity suppressors17. In this secondary screen, selected yeast are treated with 5-Fluorootic Acid (5-FOA) to counter select for the Hsp104 plasmid19. The strains are then assessed for substrate (TDP-43, FUS, or &#945;-syn) toxicity via spotting assay to ensure that the toxicity of the substrate is restored after loss of the Hsp104 plasmid. Thus, yeast in which toxicity is restored in this secondary screen presumably originally displayed toxicity suppression due to the presence of the Hsp104 variant. These yeast are designated as &#8216;hits&#8217; and the Hsp104 plasmid should then be recovered and sequenced to identify the mutations in the Hsp104 gene17 (Figure 1). Any hits should then be reconfirmed by constructing the mutation independently using site-directed mutagenesis and then retesting for toxicity suppression. The potential applications for this protocol are broad. Using these methods, libraries of any type of protein could be screened for variants that suppress toxicity of any substrate protein that is toxic in yeast.

<table><tbody><tr><td>GeneMorphII EZClone Domain Mutagenesis Kit</td><td>Agilent</td><td>200552</td><td></td></tr><tr><td>150 mm Petri dishes</td><td>Falcon</td><td>351058</td><td></td></tr><tr><td>5-Fluorootic Acid</td><td>Research Products International</td><td>f10501-5.0</td><td></td></tr><tr><td>96-DeepWell 2 ml Plates</td><td>Eppendorf</td><td>0030 502.302</td><td></td></tr><tr><td>96 bold replicator tool</td><td>V&amp;amp;P Scientific</td><td>vp-404</td><td></td></tr><tr><td>ExoSAP-IT</td><td>Affymetrix</td><td>78200</td><td></td></tr></tbody></table>

isolating potentiated hsp104 variants using yeast proteinopathy models

Many protein-misfolding disorders can be modeled in the budding yeast Saccharomyces cerevisiae. Proteins such as TDP-43 and FUS, implicated in amyotrophic lateral sclerosis, and &#945;-synuclein, implicated in Parkinson&#8217;s disease, are toxic and form cytoplasmic aggregates in yeast. These features recapitulate protein pathologies observed in patients with these disorders. Thus, yeast are an ideal platform for isolating toxicity suppressors from libraries of protein variants. We are interested in applying protein disaggregases to eliminate misfolded toxic protein conformers. Specifically, we are engineering Hsp104, a hexameric AAA+ protein from yeast that is uniquely capable of solubilizing both disordered aggregates and amyloid and returning the proteins to their native conformations. While Hsp104 is highly conserved in eukaryotes and eubacteria, it has no known metazoan homologue. Hsp104 has only limited ability to eliminate disordered aggregates and amyloid fibers implicated in human disease. Thus, we aim to engineer Hsp104 variants to reverse the protein misfolding implicated in neurodegenerative disorders. We have developed methods to screen large libraries of Hsp104 variants for suppression of proteotoxicity in yeast. As yeast are prone to spontaneous nonspecific suppression of toxicity, a two-step screening process has been developed to eliminate false positives. Using these methods, we have identified a series of potentiated Hsp104 variants that potently suppress the toxicity and aggregation of TDP-43, FUS, and &#945;-synuclein. Here, we describe this optimized protocol, which could be adapted to screen libraries constructed using any protein backbone for suppression of toxicity of any protein that is toxic in yeast.

We have constructed a library of Hsp104 variants randomized in the middle domain and screened it for suppression of TDP-43 toxicity. The library was transformed and plated onto glucose and galactose plates (Figure 2) to assess the stringency of the screen. Single colonies were selected and the strains were counter selected using 5-FOA to eliminate the Hsp104 plasmid. These strains were then assessed to confirm that toxicity was due to TDP-43 alone, without the Hsp104 variants. Spotting assays of a subset of the variants selected in the initial screen showed that of the 4 colonies selected, 2 displayed Hsp104-mediated TDP-43 toxicity suppression, 1 was a false positive, and 1 displayed enhanced toxicity following 5-FOA treatment (Figure 3). The 2 true hits were then sequenced by colony PCR to identify the middle domain mutations (Figure 4). Once these mutations were identified, the Hsp104 mutation was constructed afresh in the parental Hsp104 plasmid by site-directed mutagenesis to confirm toxicity suppression. Variants selected using these methods were later confirmed to suppress aggregation in yeast, clear preformed aggregates in biochemical assays, and suppress dopaminergic neurodegeneration in a C. elegans model of PD17.
<img alt="Figure 1" fo:content-width="7in" src="/files/ftp_upload/52089/52089fig1highres.jpg" width="700" /> 
Figure 1: Flow-chart for isolating potentiated Hsp104 variants. Hsp104 libraries (URA3 marker, GAL1 promoter) are transformed in yeast containing the disease substrates (HIS3 marker, GAL1 promoter) and screened for toxicity suppressors by plating on inducing media. Potential hits are then screened again using a 5-FOA counterselection step to eliminate nonspecific toxicity suppressors. Variants selected in this second step are then sequenced by colony PCR. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52089/52089fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/52089/52089fig2highres.jpg" /> 
Figure 2: Screening for toxicity suppressors. Yeast cotransformed with pAG303GAL-TDP-43 and the pAG416GAL-Hsp104 library were plated onto glucose (repressing) or galactose (inducing) media. TDP-43 is highly toxic, so very few Hsp104 variants are capable of suppressing this toxicity, and this screen is very stringent. Single colonies are selected as hits from the galactose plate for the 5-FOA secondary screen. Both large and small colonies are typically obtained, but we have not observed any trends that dictate how colony size correlates with activity.
<img alt="Figure 3" src="/files/ftp_upload/52089/52089fig3highres.jpg" /> 
Figure 3: 5-FOA secondary screen. Yeast treated with 5-FOA to counter select for the Hsp104 plasmid were assessed by spotting assay. Strains were grown in SRaff-His media, serially diluted 5 fold, and spotted in duplicate onto SD-His and SGal-His plates. Hsp104A503V is a true hit that we have previously verified and show here as a control along with vector alone17. Rows 3 and 4 are library hits that retain TDP-43 toxicity following loss of the Hsp104 suppressor. Row 5 shows a false positive, where TDP-43 is no longer toxic, so a nonspecific toxicity suppressor is present. Row 6 shows a strain that is more toxic than TDP-43 typically is, possibly due to 5-FOA toxicity. This strain could still be sequenced but may not be a valid hit.
<img alt="Figure 4" fo:content-width="3in" src="/files/ftp_upload/52089/52089fig4highres.jpg" width="300" /> 
Figure 4. Analyzing sequencing results. Selected yeast often contains multiple plasmids. Therefore, following sequencing of the colony PCR products, care must be taken to ensure any mutations are not overlooked in the chromatograms. In this chromatogram, two potential mutations (denoted by *) could be overlooked. In the first instance, there is a mixture of A and T and in the second, a mixture of T and C.

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Isolating Potentiated Hsp104 Variants Using Yeast Proteinopathy Models

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.

Many protein-misfolding disorders can be modeled in the budding yeast Saccharomyces cerevisiae. Proteins such as TDP-43 and FUS, implicated in amyotrophic ...

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Nanyang Technological University

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8.1K Views.  Perelman School of Medicine at the University of Pennsylvania. 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.

Video: Isolating Potentiated Hsp104 Variants Using Yeast Proteinopathy Models

Mating-based Overexpression Library Screening in Yeast

Budding yeast has been widely used as a model in studying proteins associated with human diseases. Genome-wide genetic screening is a powerful tool commonly used in yeast studies. The expression of a number of neurodegenerative disease-associated proteins in yeast causes cytotoxicity and aggregate formation, recapitulating findings seen in patients with these disorders. Here, we describe a method for screening a yeast model of the Amyotrophic Lateral Sclerosis-associated protein FUS for modifiers of its toxicity. Instead of using transformation, this new screening platform relies on the mating of yeast to introduce an arrayed library of plasmids into the yeast model. The mating method has two clear advantages: first, it is highly efficient; second, the pre-transformed arrayed library of plasmids can be stored for long-term as a glycerol stock, and quickly applied to other screens without the labor-intensive step of transformation into the yeast model each time. We demonstrate how this method can successfully be used to screen for genes that modify the toxicity of FUS.

This article presents a mating-based method to facilitate overexpression screening in budding yeast using an arrayed plasmid library.

Budding yeast has been widely used as a model in studying proteins associated with human diseases. Genome-wide genetic screening is a powerful tool ...

Genetics

Yeast As a Chassis for Developing Functional Assays to Study Human P53

The finding that the well-known mammalian P53 protein can act as a transcription factor (TF) in the yeast S. cerevisiae has allowed for the development of different functional assays to study the impacts of 1) binding site [i.e., response element (RE)] sequence variants on P53 transactivation specificity or 2) TP53 mutations, co-expressed cofactors, or small molecules on P53 transactivation activity. Different basic and translational research applications have been developed. Experimentally, these approaches exploit two major advantages of the yeast model. On one hand, the ease of genome editing enables quick construction of qualitative or quantitative reporter systems by exploiting isogenic strains that differ only at the level of a specific P53-RE to investigate sequence-specificity of P53-dependent transactivation. On the other hand, the availability of regulated systems for ectopic P53 expression allows the evaluation of transactivation in a wide range of protein expression. Reviewed in this report are extensively used systems that are based on color reporter genes, luciferase, and the growth of yeast to illustrate their main methodological steps and to critically assess their predictive power. Moreover, the extreme versatility of these approaches can be easily exploited to study different TFs including P63 and P73, which are other members of TP53 gene family.

Presented here are four protocols to construct and exploit yeast Saccharomyces cerevisiae reporter strains to study human P53 transactivation potential, impacts of its various cancer-associated mutations, co-expressed interacting proteins, and the effects of specific small molecules.

The finding that the well-known mammalian P53 protein can act as a transcription factor (TF) in the yeast S. cerevisiae has allowed for the development of ...

Cancer Research

Characterizing Histone Post-translational Modification Alterations in Yeast Neurodegenerative Proteinopathy Models

Neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS) and Parkinson&#39;s disease (PD), cause the loss of hundreds of thousands of lives each year. Effective treatment options able to halt disease progression are lacking. Despite the extensive sequencing efforts in large patient populations, the majority of ALS and PD cases remain unexplained by genetic mutations alone. Epigenetics mechanisms, such as the post-translational modification of histone proteins, may be involved in neurodegenerative disease etiology and progression and lead to new targets for pharmaceutical intervention. Mammalian in vivo and in vitro models of ALS and PD are costly and often require prolonged and laborious experimental protocols. Here, we outline a practical, fast, and cost-effective approach to determining genome-wide alterations in histone modification levels using Saccharomyces cerevisiae as a model system. This protocol allows for comprehensive investigations into epigenetic changes connected to neurodegenerative proteinopathies that corroborate previous findings in different model systems while significantly expanding our knowledge of the neurodegenerative disease epigenome.

This protocol outlines experimental procedures to characterize genome-wide changes in the levels of histone post-translational modifications (PTM) occurring in connection with the overexpression of proteins associated with ALS and Parkinson&#39;s disease in Saccharomyces cerevisiae models. After SDS-PAGE separation, individual histone PTM levels are detected with modification-specific antibodies via Western blotting.

Neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS) and Parkinson&#39;s disease (PD), cause the loss of hundreds of thousands of lives ...

Genetic Studies of Human DNA Repair Proteins Using Yeast as a Model System 

Understanding the roles of human DNA repair proteins in genetic pathways is a formidable challenge to many researchers. Genetic studies in mammalian systems have been limited due to the lack of readily available tools including defined mutant genetic cell lines, regulatory expression systems, and appropriate selectable markers. To circumvent these difficulties, model genetic systems in lower eukaryotes have become an attractive choice for the study of functionally conserved DNA repair proteins and pathways. We have developed a model yeast system to study the poorly defined genetic functions of the Werner syndrome helicase-nuclease (WRN) in nucleic acid metabolism. Cellular phenotypes associated with defined genetic mutant backgrounds can be investigated to clarify the cellular and molecular functions of WRN through its catalytic activities and protein interactions. The human WRN gene and associated variants, cloned into DNA plasmids for expression in yeast, can be placed under the control of a regulatory plasmid element. The expression construct can then be transformed into the appropriate yeast mutant background, and genetic function assayed by a variety of methodologies. Using this approach, we determined that WRN, like its related RecQ family members BLM and Sgs1, operates in a Top3-dependent pathway that is likely to be important for genomic stability. This is described in our recent publication [1] at <a href="http://www.impactaging.com">www.impactaging.com</a>. Detailed methods of specific assays for genetic complementation studies in yeast are provided in this paper.

Genetic Studies of Human DNA Repair Proteins Using Yeast as a Model System

Genetic studies in yeast can be employed to investigate the molecular and cellular functions of human genes in cellular DNA metabolism. Methods are described for the genetic characterization of the human WRN gene product defective in the premature aging disorder Werner syndrome in functionally conserved pathways using yeast as a tractable model system.

Understanding the roles of human DNA repair proteins in genetic pathways is a formidable challenge to many researchers.  Genetic studies in mammalian ...

Purification of Hsp104, a Protein Disaggregase

Hsp104 is a hexameric AAA+ protein1 from yeast, which couples ATP hydrolysis to protein disaggregation2-10 (Fig. 1). This activity imparts two key selective advantages. First, renaturation of disordered aggregates by Hsp104 empowers yeast survival after various protein-misfolding stresses, including heat shock3,5,11,12. Second, remodeling of cross-beta amyloid fibrils by Hsp104 enables yeast to exploit myriad prions (infectious amyloids) as a reservoir of beneficial and heritable phenotypic variation13-22. Remarkably, Hsp104 directly remodels preamyloid oligomers and amyloid fibrils, including those comprised of the yeast prion proteins Sup35 and Ure223-30. This amyloid-remodeling functionality is a specialized facet of yeast Hsp104. The E. coli orthologue, ClpB, fails to remodel preamyloid oligomers or amyloid fibrils26,31,32.
Hsp104 orthologues are found in all kingdoms of life except, perplexingly, animals. Indeed, whether animal cells possess any enzymatic system that couples protein disaggregation to renaturation (rather than degradation) remains unknown33-35. Thus, we and others have proposed that Hsp104 might be developed as a therapeutic agent for various neurodegenerative diseases connected with the misfolding of specific proteins into toxic preamyloid oligomers and amyloid fibrils4,7,23,36-38. There are no treatments that directly target the aggregated species associated with these diseases. Yet, Hsp104 dissolves toxic oligomers and amyloid fibrils composed of alpha-synuclein, which are connected with Parkinson's Disease23 as well as amyloid forms of PrP39. Importantly, Hsp104 reduces protein aggregation and ameliorates neurodegeneration in rodent models of Parkinson's Disease23 and Huntington's disease38. Ideally, to optimize therapy and minimize side effects, Hsp104 would be engineered and potentiated to selectively remodel specific aggregates central to the disease in question4,7. However, the limited structural and mechanistic understanding of how Hsp104 disaggregates such a diverse repertoire of aggregated structures and unrelated proteins frustrates these endeavors30,40-42.
To understand the structure and mechanism of Hsp104, it is essential to study the pure protein and reconstitute its disaggregase activity with minimal components. Hsp104 is a 102kDa protein with a pI of ~5.3, which hexamerizes in the presence of ADP or ATP, or at high protein concentrations in the absence of nucleotide43-46. Here, we describe an optimized protocol for the purification of highly active, stable Hsp104 from E. coli. The use of E. coli allows simplified large-scale production and our method can be performed quickly and reliably for numerous Hsp104 variants. Our protocol increases Hsp104 purity and simplifies His6-tag removal compared to a previous purification method from E. coli47. Moreover, our protocol is more facile and convenient than two more recent protocols26,48.

Here, we describe a protocol for the purification of highly active Hsp104, a hexameric AAA+ protein from yeast, which couples ATP hydrolysis to protein disaggregation. This scheme exploits a His6-tagged construct for affinity purification from E. coli followed by anion-exchange chromatography, His6-tag removal with TEV protease, and size-exclusion chromatography.

Hsp104 is a hexameric AAA+ protein1 from yeast, which couples ATP hydrolysis to protein disaggregation2-10 (Fig. 1). This activity imparts two key ...

Growth Assays to Assess Polyglutamine Toxicity in Yeast

Protein misfolding is associated with many human diseases, particularly neurodegenerative diseases, such as Alzheimer&#x2019;s disease, Parkinson's disease, and Huntington's disease 1. Huntington's disease (HD) is caused by the abnormal expansion of a polyglutamine (polyQ) region within the protein huntingtin. The polyQ-expanded huntingtin protein attains an aberrant conformation (i.e. it misfolds) and causes cellular toxicity 2. At least eight further neurodegenerative diseases are caused by polyQ-expansions, including the Spinocerebellar Ataxias and Kennedy&#x2019;s disease 3.
The model organism yeast has facilitated significant insights into the cellular and molecular basis of polyQ-toxicity, including the impact of intra- and inter-molecular factors of polyQ-toxicity, and the identification of cellular pathways that are impaired in cells expressing polyQ-expansion proteins 3-8. Importantly, many aspects of polyQ-toxicity that were found in yeast were reproduced in other experimental systems and to some extent in samples from HD patients, thus demonstrating the significance of the yeast model for the discovery of basic mechanisms underpinning polyQ-toxicity. 
A direct and relatively simple way to determine polyQ-toxicity in yeast is to measure growth defects of yeast cells expressing polyQ-expansion proteins. This manuscript describes three complementary experimental approaches to determine polyQ-toxicity in yeast by measuring the growth of yeast cells expressing polyQ-expansion proteins. The first two experimental approaches monitor yeast growth on plates, the third approach monitors the growth of liquid yeast cultures using the BioscreenC instrument. 
Furthermore, this manuscript describes experimental difficulties that can occur when handling yeast polyQ models and outlines strategies that will help to avoid or minimize these difficulties. The protocols described here can be used to identify and to characterize genetic pathways and small molecules that modulate polyQ-toxicity. Moreover, the described assays may serve as templates for accurate analyses of the toxicity caused by other disease-associated misfolded proteins in yeast models.

This manuscript describes three complementary protocols for assessing the toxicity of polyglutamine (polyQ)-expansion proteins in the yeast Saccharomyces cerevisiae. These protocols can easily be modified to monitor the toxicity of other misfolded proteins in yeast.

Protein misfolding is associated with many human diseases, particularly neurodegenerative diseases, such as Alzheimer&#x2019;s disease, Parkinson's ...

Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

Proteostasis, defined as the combined processes of protein folding/biogenesis, refolding/repair, and degradation, is a delicate cellular balance that must be maintained to avoid deleterious consequences 1. External or internal factors that disrupt this balance can lead to protein aggregation, toxicity and cell death. In humans this is a major contributing factor to the symptoms associated with neurodegenerative disorders such as Huntington's, Parkinson's, and Alzheimer's diseases 10. It is therefore essential that the proteins involved in maintenance of proteostasis be identified in order to develop treatments for these debilitating diseases. This article describes techniques for monitoring in vivo protein folding at near-real time resolution using the model protein firefly luciferase fused to green fluorescent protein (FFL-GFP). FFL-GFP is a unique model chimeric protein as the FFL moiety is extremely sensitive to stress-induced misfolding and aggregation, which inactivates the enzyme 12. Luciferase activity is monitored using an enzymatic assay, and the GFP moiety provides a method of visualizing soluble or aggregated FFL using automated microscopy. These coupled methods incorporate two parallel and technically independent approaches to analyze both refolding and functional reactivation of an enzyme after stress. Activity recovery can be directly correlated with kinetics of disaggregation and re-solubilization to better understand how protein quality control factors such as protein chaperones collaborate to perform these functions. In addition, gene deletions or mutations can be used to test contributions of specific proteins or protein subunits to this process. In this article we examine the contributions of the protein disaggregase Hsp104 13, known to partner with the Hsp40/70/nucleotide exchange factor (NEF) refolding system 5, to protein refolding to validate this approach.

Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

This article describes the use of a firefly luciferase-GFP fusion protein to investigate in vivo protein folding in Saccharomyces cerevisiae. Using this reagent, refolding of a model heat-denatured protein can be monitored simultaneously by fluorescence microscopy and an enzymatic assay to probe the roles of proteostasis network components in protein quality control.

Proteostasis, defined as the combined processes of protein folding/biogenesis, refolding/repair, and degradation, is a delicate cellular balance that must ...

High-throughput Screening for Protein-based Inheritance in S. cerevisiae

The encoding of biological information that is accessible to future generations is generally achieved via changes to the DNA sequence. Long-lived inheritance encoded in protein conformation (rather than sequence) has long been viewed as paradigm-shifting but rare. The best characterized examples of such epigenetic elements are prions, which possess a self-assembling behavior that can drive the heritable manifestation of new phenotypes. Many archetypal prions display a striking N/Q-rich sequence bias and assemble into an amyloid fold. These unusual features have informed most screening efforts to identify new prion proteins. However, at least three known prions (including the founding prion, PrPSc) do not harbor these biochemical characteristics. We therefore developed an alternative method to probe the scope of protein-based inheritance based on a property of mass action: the transient overexpression of prion proteins increases the frequency at which they acquire a self-templating conformation. This paper describes a method for analyzing the capacity of the yeast ORFeome to elicit protein-based inheritance. Using this strategy, we previously found that &gt;1% of yeast proteins could fuel the emergence of biological traits that were long-lived, stable, and arose more frequently than genetic mutation. This approach can be employed in high throughput across entire ORFeomes or as a targeted screening paradigm for specific genetic networks or environmental stimuli. Just as forward genetic screens define numerous developmental and signaling pathways, these techniques provide a methodology to investigate the influence of protein-based inheritance in biological processes.

High-throughput Screening for Protein-based Inheritance in S. cerevisiae

This protocol describes a high-throughput methodology to functionally screen for protein-based inheritance in S. cerevisiae.

The encoding of biological information that is accessible to future generations is generally achieved via changes to the DNA sequence. Long-lived ...

Effect of Fluorescent Proteins on Fusion Partners Using Polyglutamine Toxicity Assays in Yeast

For the investigation of protein localization and trafficking using live cell imaging, researchers often rely on fusing their protein of interest to a fluorescent reporter. The constantly evolving list of genetically encoded fluorescent proteins (FPs) presents users with several alternatives when it comes to fluorescent fusion design. Each FP has specific optical and biophysical properties that can affect the biochemical, cellular, and functional properties of the resulting fluorescent fusions. For instance, several FPs tend to form nonspecific oligomers that are susceptible to impede on the function of the fusion partner. Unfortunately, only a few methods exist to test the impact of FPs on the behavior of the fluorescent reporter. Here, we describe a simple method that enables the rapid assessment of the impact of FPs using polyglutamine (polyQ) toxicity assays in the budding yeast Saccharomyces cerevisiae. PolyQ-expanded huntingtin proteins are associated with the onset of Huntington&#39;s disease (HD), where the expanded huntingtin aggregates into toxic oligomers and inclusion bodies. The aggregation and toxicity of polyQ expansions in yeast are highly dependent on the sequences flanking the polyQ region, including the presence of fluorescent tags, thus providing an ideal experimental platform to study the impact of FPs on the behavior of their fusion partner.

This article describes protocols to assess the effect of fluorescent proteins on the aggregation and toxicity of misfolded polyglutamine expansion for the rapid evaluation of a newly uncharacterized fluorescent protein in the context of fluorescent reporters.

For the investigation of protein localization and trafficking using live cell imaging, researchers often rely on fusing their protein of interest to a ...

A Method to Study &#945;-Synuclein Toxicity and Aggregation Using a Humanized Yeast Model

Parkinson's disease is the second most common neurodegenerative disorder and is characterized by progressive cell death caused by the formation of Lewy bodies containing misfolded and aggregated &#945;-synuclein. &#945;-synuclein is an abundant presynaptic protein that regulates synaptic vesicle trafficking, but the accumulation of its proteinaceous inclusions results in neurotoxicity. Recent studies have revealed that various genetic factors, including bacterial chaperones, could reduce the formation of &#945;-synuclein aggregates in vitro. However, it is also important to monitor the anti-aggregation effect in the cell to apply this as a potential treatment for the patients. It would be ideal to use neuronal cells, but these cells are difficult to handle and take a long time to exhibit the anti-aggregation phenotype. Therefore, a quick and effective in vivo tool is required for the further evaluation of in vivo anti-aggregation activity. The method described here was used to monitor and analyze the anti-aggregation phenotype in the humanized yeast Saccharomyces cerevisiae, which expressed human &#945;-synuclein. This protocol demonstrates in vivo tools that could be used for monitoring &#945;-synuclein-induced cellular toxicity, as well as the formation of &#945;-synuclein aggregates in cells.

An in vivo physiological model of &#945;-synuclein is required to study and understand the pathogenesis of Parkinson's disease. We describe a method to monitor the cytotoxicity and aggregate formation of &#945;-synuclein using a humanized yeast model.

Parkinson's disease is the second most common neurodegenerative disorder and is characterized by progressive cell death caused by the formation of Lewy ...

Isolating Potentiated Hsp104 Variants Using Yeast Proteinopathy Models

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