Name: assays for the degradation of misfolded proteins in cells
Uploaded: 2016-08-28T15:00:00
Description: Watch this Scientific Journal Video about Assays for the Degradation of Misfolded Proteins in Cells at JoVE.com

We thank S. Raychaudhuri for providing the destabilized firefly luciferase mutant plasmid, and A. Glavis-bloom and N. Charan for technical assistance. This work was supported, in part, by grants from NIH (CA088868, GM060911, and CA182675).

1. Preparation of Reagent
<ol>
	<li>Prepare cell lysis buffer (50 mM Tris, pH 8.8, 100 mM NaCl, 5 mM MgCl2, 0.5% NP-40). Supplement 2 mM DTT, 1x complete protease cocktail, and 250 IU/ml benzonase before use.</li>
	<li>Prepare pellet buffer (20 mM Tris, pH 8.0, 15 mM MgCl2). Supplement 2 mM DTT, 1x complete protease cocktail and 250 IU/ml benzonase before use.</li>
	<li>Prepare 3x boiling buffer (6% SDS, 20 mM Tris, pH 8.0). Supplement 150 mM DTT before use.</li>
	<li>Prepare low-fluorescence DMEM...

<ol>
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Mechanisms that regulate the degradation of misfolded proteins are essential for maintaining the homeostasis of cellular proteins, and they likely represent valuable drug targets for treating neurodegenerative disorders and other protein-misfolding diseases. Here, assays that examine the degradation of misfolded proteins are described, using a pathogenic Atxn1 protein (Atxn1 82Q) and a nuclear localized luciferase mutant (NLS-LucDM) as examples.
To examine the degradation Atxn1 82Q, which has ...

Proteins are the most abundant macromolecules in cells, and they play an essential role in virtually all biological processes. The biological activity of most proteins requires their folding into, and maintaining, the native three-dimensional structures. Proteins with aberrant conformations not only lose their normal functions, but also frequently form soluble oligomeric species or aggregates that impair the functions of other proteins and are toxic to cells1,2. To counteract protein misfolding, cells employ both molecular chaperones, which assist unfolded or partially folded polypeptides to reach their native conformation, and degradation pathways, which eliminate misfolded proteins3. Given the complexity and stochastic nature of the folding process, protein misfolding is inevitable, and it may not be reversed in the case of mutations, biosynthetic errors, and post-translational damages1. Hence, cells ultimately rely on degradation pathways to maintain their protein quality.
The importance of cellular protein quality control (PQC) systems is underscored by the prevalence of protein-misfolding diseases, including cancer, diabetes, and many neurodegenerative disorders such as Alzheimer&#39;s disease, Parkinson&#39;s disease, amyotrophic lateral sclerosis, Huntington&#39;s disease (HD), and spinocerebellar ataxias (SCA)4,5. For example, mutations in the tumor suppressor p53 is the single most frequently genetic lesion in tumors, associated with ~50-70% of all cases6. A substantial fraction of p53 mutations are missense mutations that alter the conformation of p53, leading to the formation of aggregates7. Furthermore, proteins with expanded polyQ stretches are genetically and pathologically associated with HD and SCA. These progressive and often fatal diseases manifest when the length of the polyQ stretch in the affected proteins exceeds a certain threshold, and becomes increasingly severe as the length of the polyQ stretch prolongs8.
An attractive approach for treating these diseases is to therapeutically bolster cellular PQC systems, especially the degradation pathways. However, the pathways involved in the degradation of defective proteins, particularly those in mammalian cells, remain poorly understood. Although it has been recognized that the proteasome is critically important for the degradation of misfolded proteins, a vital issue remains undefined: how misfolded proteins are specifically recognized and targeted for degradation. Moreover, although PQC systems have been identified in cellular compartments including the cytoplasm, the endoplasmic reticulum, and the mitochondrion, the PQC systems in the nucleus remain unclear2.
Recent studies by our lab have identified a system that recognizes and degrades a variety of misfolded proteins in the nucleus of mammalian cells9. This system is comprised of the promyelocytic protein (PML), a nuclear protein and a member of the tripartite motif-containing (TRIM) protein family, and RNF4, a RING-domain containing protein. PML, and several other TRIM proteins, possess SUMO (small ubiquitin-like modifier) E3 ligase activity, which facilitates the specificity and efficiency of protein SUMOylation10. RNF4 belongs to a small group of SUMO-targeted ubiquitin ligases (STUbL), which contain one or more sumo-interacting motifs (SIMs) in addition to the RING domain that affords them the ubiquitin ligase activity11. We found that PML specifically recognizes misfolded proteins through discrete substrate recognition sites that can discern distinct features on misfolded proteins. Upon binding, PML tags misfolded proteins with poly-chains of SUMO2/3, two nearly identical mammalian SUMO proteins that can form poly-chains due to the existence of an internal SUMOylation site. SUMOylated misfolded proteins are then recognized by RNF4, which leads to their ubiquitination and proteasomal degradation. We further demonstrated that the PML-RNF4 system is important for protection against neurodegeneration, as deficiency in PML exacerbates the behavioral and neuropathological defects of a mouse model of SCA type 1 (SCA1)9.
To distinguish protein degradation from other cellular mechanisms that may regulate protein levels, the rates of protein turnover was measured9. Among the most frequently used methods to determine protein turnover are pulse chase and cycloheximide (CHX) chase. These two methods examine over time, respectively, the radioisotope-labeled proteins of interest in translation-proficient cells and total preexisting proteins of interest in translation-inhibited cells. However, a major challenge for studying pathogenic and misfolding-prone proteins is that the half-life of these proteins can be extremely long. For example, Ataxin-1, Ataxin-7, Huntingtin, &#945;-synuclein, and TDP-43 all have half-lives of more than 12 to 24 hr9,12-16. The slow turnover rates of these proteins preclude the use of the CHX chase analysis because the cells harboring these proteins may not survive prolonged translation inhibition, especially because the misfolded proteins themselves can be highly toxic to cells. The pulse-chase analysis with isotopic labeling may also be challenging for proteins that are highly aggregation-prone. Most pulse-chase assays rely on immunoprecipitation to separate the protein of interest from all the other proteins that are also radioactively labeled. This procedure normally includes lengthy immunoprecipitation and wash procedures, during which SDS-insoluble aggregates can form, making the analysis with SDS-PAGE electrophoresis inaccurate.
Here a protocol to analyze nuclear misfolded proteins with a slow turnover rate is described9. A pathogenic form of Ataxin-1 (Atxn1) that contains a stretch of 82 glutamines (Atxn1 82Q) is used for this purpose8. When expressed in cells, an enhanced green fluorescent protein (GFP) fusion of Atxn1 82Q forms microscopically visible inclusions in the nucleus (Figure 1A). Pulse chase analysis reveals that the half-life of Ataxn1 82Q is over 18 hr9. Atxn1 82Q is composed of species with misfolded conformations of different characteristics, as well as species with native conformation. It is likely that these species are degraded at different rates, and thus should be analyzed separately. Lysates from Atxn1 82Q-GFP-expressing cells are fractionated into NP-40-soluble (soluble or NS, likely representing native proteins or misfolded monomeric/oligomeric proteins) and NP-40-insoluble (NI, aggregated/misfolded) portions. The latter can be further divided into SDS-soluble (SS; likely disordered aggregates) or SDS-resistant (SR; likely amyloid fibrils) fractions (Figure 1B). NS and SS fractions can be analyzed by SDS-PAGE followed by western blotting, whereas the SR fraction can be detected by filter retardation assays followed by immunoblotting. CHX chase is combined with the detergent fractionation method, and discovered that the half-life of SS Atxn1 82Q is much shorter than that of NS Atxn1 82Q and total Atxn1 82Q (Figure 2A), indicating that the SS fraction can be readily recognized and degraded in cells9. Thus, this method provides a powerful tool to study the dynamics of misfolded proteins and to compare their degradation pattern.
We also describe a method that is suitable for high throughput screening for identifying macromolecules or small compounds that can modulate the degradation of misfolded proteins. This method is based on a conformationally destabilized mutant of firefly luciferase (LucDM)17, a model chaperone substrate. We have fused LucDM to a nuclear localization signal (NLS) to probe the PQC systems in this cellular compartment, and GFP (NLS-LucDM-GFP) for convenience of detection. NLS-LucDM-GFP forms microscopically visible nuclear aggregates in a small percentage of cells (Figure 3A). Similar to Atxn1 82Q, NLS-LucDM-GFP-but not its wild-type counterpart NLS-LucWT-GFP-is modified by SUMO2/3 and regulated by the PML-RNF4 pathway9. NLS-LucDM-GFP also forms SS and SR species, although the relative amounts of SS and SR species are minimal compared to NS, using the conditions described in protocol 3 below. To simplify the assay, we only analyze SDS soluble LucDM (including both the NS and SS fractions) by SDS-PAGE and western blot. Importantly, CHX chase assay showed that the half-life of SDS-soluble NLS-LucDM-GFP is much shorter than that of its wild-type counterpart (Figure 3B), suggesting that LucDM is a specific substrate for the system that recognizes and degrades misfolded proteins.
The degradation of LucDM-GFP causes a significant drop in overall fluorescence signal. Therefore, we have also developed a protocol for real-time detection of cellular LucDM-GFP using microplate fluorescence-based assay. Many high-throughput screen (HTS) systems are developed for drugs or genes modifying cellular aggregates and cellular viability caused by aberrant proteins18-20. However, very few HTSs are specifically designed for targeting degradation in mammalian cells. This protocol serves as a robust system for rapid and large-scale analysis of the effects of protein expression, knockdown, and drug treatment on cellular misfolded protein degradation. Using HeLa cells as example, below we describe the protocols for the analysis of these two misfolded proteins. The assays can also be applied to other cell lines, although transfection conditions and time course may need to be optimized for individual cell lines.

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		<tr height="21">
			<td height="21" style="height:21px;width:308px;">Dulbecco&#39;s Modified Eagle Medium</td>
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			<td style="width:119px;">11995-092</td>
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			<td height="21" style="height:21px;width:308px;">Fetal Bovine Serum</td>
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			<td style="width:119px;">10082147</td>
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			<td height="21" style="height:21px;width:308px;">Lipofectamine 2000</td>
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			<td style="width:119px;">11668019</td>
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			<td height="21" style="height:21px;width:308px;">NP-40</td>
			<td style="width:175px;">Sigma-Aldrich</td>
			<td style="width:119px;">NP40S-500ML</td>
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			<td height="21" style="height:21px;width:308px;">SDS</td>
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			<td height="21" style="height:21px;width:308px;">MG132</td>
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			<td height="21" style="height:21px;width:308px;">Cycloheximide</td>
			<td style="width:175px;">Sigma-Aldrich</td>
			<td style="width:119px;">C7698</td>
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			<td height="21" style="height:21px;width:308px;">Dithiothreitol (DTT)</td>
			<td style="width:175px;">Fisher Scientific</td>
			<td style="width:119px;">45000232</td>
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		</tr>
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			<td height="42" style="height:42px;width:308px;">Complete Protease Inhibitor Cocktail Tablets</td>
			<td style="width:175px;">Roche Boehringer Mannheim</td>
			<td style="width:119px;">4693159001</td>
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		</tr>
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			<td height="21" style="height:21px;width:308px;">Bio-Dot Apparatus&#160;</td>
			<td style="width:175px;">Bio-Rad</td>
			<td style="width:119px;">1706545</td>
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			<td height="21" style="height:21px;width:308px;">Living Colors GFP Monoclonal Antibody</td>
			<td style="width:175px;">Clonetech</td>
			<td style="width:119px;">632375</td>
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			<td height="21" style="height:21px;width:308px;">Anti-Actin mAb Rabbit, IgG</td>
			<td style="width:175px;">Sigma-Aldrich</td>
			<td style="width:119px;">A45060-200UL</td>
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			<td height="21" style="height:21px;width:308px;">Amino acids</td>
			<td style="width:175px;">Sigma-Aldrich</td>
			<td style="width:119px;"></td>
			<td>Amino acids are used for making low fluorecence culturing medium</td>
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			<td height="42" style="height:42px;width:308px;">Olympus IX-81&#160; Inverted Fluorescence Microscope</td>
			<td style="width:175px;">Olympus</td>
			<td style="width:119px;">IX71/IX81</td>
			<td></td>
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		<tr height="42">
			<td height="42" style="height:42px;width:308px;">96 Well Black TC Plate w/ Transluscent Clear Bottom</td>
			<td style="width:175px;">Sigma-Greiner</td>
			<td style="width:119px;">89135-048</td>
			<td></td>
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		<tr height="42">
			<td height="42" style="height:42px;width:308px;">Fluorescence Bottom Plate Reader Infinite 200&#174; PRO</td>
			<td style="width:175px;">TECAN</td>
			<td style="width:119px;">Infinite 200&#174; PRO</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:308px;">Cellulose acetate membrane 0.2 &#181;m</td>
			<td style="width:175px;">Sterlitech</td>
			<td style="width:119px;">CA023001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">Prism 5</td>
			<td style="width:175px;">GraphPad</td>
			<td style="width:119px;"></td>
			<td>Statistical analysis software</td>
		</tr>
	</tbody>
</table>

assays for the degradation of misfolded proteins in cells

Protein misfolding and aggregation are associated with various neurodegenerative diseases. Cellular mechanisms that recognize and degrade misfolded proteins may serve as potential therapeutic targets. To distinguish degradation of misfolding-prone proteins from other mechanisms that regulate their levels, one important method is to measure protein half-life in cells. However, this can be challenging because misfolding-prone proteins may exist in different forms, including the native form and misfolded forms of distinct characteristics. Here we describe assays to examine the half-life of misfolded proteins in mammalian cells using a highly aggregation-prone protein, Ataxin-1 with an extended polyglutamine (polyQ) stretch, and a conformationally unstable luciferase mutant as models. Cycloheximide chase is combined with cell fractionation to examine the turnover rate of misfolding-prone proteins in various cellular fractions. We further depict a fluorescence-based assay using an enhanced green fluorescence protein (EGFP)-fusion of the luciferase mutant, which can be adapted for high throughput screening on a microplate-reader.

In a steady state analysis, microscopically visible Atxn1 82Q-GFP nuclear aggregates can be observed in 30 - 50% of HeLa cells 20 hr after transfection (Figure 1A). Western blot analysis of NS and SS fractions using anti-GFP antibody shows a distinct band of Atxn1 82Q-GFP between 100 kDa and 150 kDa markers, corresponding to the protein&#39;s molecular weight (Figure 1B). Atxn1 82Q-GFP in the SR fraction can be detected either by filter retardation assay,...

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Assays for the Degradation of Misfolded Proteins in Cells

This report describes protocols for measuring degradation rates of misfolded proteins by either western blot or fluorescence-based assays. The methods can be applied to analysis of other misfolded proteins and for high throughput screening.

Protein misfolding and aggregation are associated with various neurodegenerative diseases. Cellular mechanisms that recognize and degrade misfolded ...

The overall goal of this assay is to measure the cellular degradation rates of misfolded proteins. Misfolded proteins are associated with many neurodegenerative diseases. This assay helps to investigate the cellular protein quality control systems that are protectors against aberrant proteins.Misfolding prone proteins may exist in different forms in cells. By combining cell fractionation with the cyclohexane method chase, the main advantage of this technique is to specifically measure the half life of the misfolded species. We use two model proteins as examples.A highly aggregation prone polyglutamate protein, Atxn1 82Q and a conformationally unstable nuclear luciferase mutant. The protocol can also be applied to other misfolded protein measurements. To carry out a degradation assay for Atxn1 82Q GFP, plate approximately 3x10^5 HeLa cells in 35 millimeter plates with DMEM medium and 10%FBS.Incubate the cells overnight so that they reach 40 to 60%confluence at the time of transfection. Four to five hours after transfecting the cells with Atxn1 82Q GFP PRK5, under a florescence microscope, use an excitation wavelength of 450 to 490 nanometers to examine the live cells for GFP expression in the nuclei, characterized by the presence of diffused as well as small speckles of GFP signals. Just before cycloheximide treatment, harvest one plate of cells by removing the medium and using three milliliters of ice cold PBS to wash the cells twice.Then snap freeze the plate on dry ice. For the remaining plates of cells, remove the medium by vacuum aspiration and add two milliliters of fresh DMEM containing 50 micrograms per milliliter of cycloheximide. Also add 10 micromolar of the proteasome inhibitor, MG132 to one plate.Incubate treated cells for four, eight, 12 and 16 hours before freezing on dry ice. After harvesting the MG132 treated cells at 16 hours, scrape the frozen cells from all the plates into 150 microliter of ice cold cell lysis buffer and incubate on ice for 30 minutes. Centrifuge the lysates in a benchtop centrifuge at 17, 000x g and four degrees Celsius for 15 minutes.Then transfer the supernatant which contains MP40 soluble proteins to another tube. Rinse the pellets by gently adding approximately 200 microliters of 1x PBS to the tubes without disturbing the pellets. Carefully remove the PBS by aspiration or pipette, resuspend the pellets in 150 microliters of ice cold pellet buffer and then incubate them on ice for 15 to 30 minutes.Next add 75 microliters of 3x boiling buffer into MP40 soluble fractions and MP40 insoluble fractions resuspended from the pellets. Then heat the samples at 95 degrees Celsius on a heat block for five minutes. Add SDS gel loading buffer to an aliquot of boiled MP40 soluble and insoluble fractions.Load equal volumes of samples collected from all time points onto an SDS-PAGE gel. Detect MP40 soluble and SDS soluble Atxn1 82Q GFP by Western blot using anti GFP antibody and enhanced chemiluminescence. To examine SDS resistant Atxn1 82Q from the pellet fraction with a filter retardation assay, set up a dot plot apparatus holding a 0.2 micron cellulose acetate membrane.Then load 80 to 120 microliters of boiled MP40 insoluble samples into each well of the dot blot apparatus. After filtering the samples through the membrane by vacuum, detect Atxn1 82Q GFP aggregates stuck on the membrane by anti GFP immunoblotting. It is critical to use a filter retardation assay to examine the levels of SDS resistant form over time.This is because SDS soluble form may transform into SDS resistant form rather than being degraded. In this example, SDS resistant form is minimal and remains a similar level over the chase experiment. To carry out a degradation assay, after an overnight transfection of HeLa cells with NLS-luciferase-GFP, examine the live cells under an inverted fluorescent microscope for GFP expression with an excitation wavelength of 450 to 490 nanometers.Just before cycloheximide treatment, harvest one plate of cells by removing the medium and use three milliliters of ice cold PBS to wash the cells twice. Then snap freeze the plate on dry ice. For the remaining plates, after removing the medium, add two milliliters of fresh DMEM containing 50 micrograms per milliliter of cycloheximide and add 10 micromolar of the proteasome inhibitor MG132 to one plate.Treat the cells for 1.5, three, 4.5 and six hours before harvesting as just described, harvesting the MG132 treated cells at six hours. After harvesting the last time point of cells, scrape each plate into 150 microliters of ice cold cell lysis buffer and incubate on ice for 30 minutes. Incubate the samples on heat block at 95 degrees Celsius for five minutes, before carrying out SDS-PAGE and Western blotting according to the text protocol.Add SDS gel loading buffer to whole cell lysates for a final concentration of 2%SDS and 50 millimolar DTT. After seeding and transfecting HeLa cells in 96 well plates according to the text protocol, 20 to 24 hours after transfection, examine the cells for GFP expression. Remove the medium, add approximately 200 microliters of 1x PBS to each well and then aspirate it to remove the residual DMEM.Add 60 microliters of low fluorescence DMEM with 5%FBS and 50 micrograms per milliliter of cycloheximide and add 10 micromolar MG132 to one set of samples. With a fluorescence plate reader, measure the GFP signal, reading the plates every hour for up to eight to 10 hours. Export the data and carry out statistical analysis according to the text protocol.In a steady state analysis, microscopically visible Atxn1 82Q GFP nuclear aggregates can be observed in 30 to 50%of HeLa cells 20 hours after transfection. As seen here by Western blot, the half life of Atxn1 82Q GFP in the SDS soluble fraction is around five hours. In contrast to a slight or no decrease in Atxn1 82Q GFP in the MP40 soluble fraction over 16 hours.The degradation of Atxn1 82Q GFP is partially inhibited by treating cells with MG132. These images illustrate that 20 hours after transfection, NLS-luciferase DM mutant GFP aggregates become microscopically visible in five to 15%of HeLa cells. Western blots revealed that the half life of SDS soluble NLS-luciferase DM mutant GFP is two to three hours.In addition, the florescence intensity of transfected cells also decreases over time upon cycloheximide treatment. At approximately six hours after cycloheximide treatment, soluble NLS-luciferase DM mutant GFP is largely degraded suggesting that the remaining fluorescence is generated from aggregated species resistant to degradation. These florescence images confirm that aggregated, but not diffused GFP, remains in cells nine hours after cycloheximide treatment.Well the immunblotting based assay takes a few days to accomplish. The protein degradation rates can be obtained immediately after the cycloheximide chase using a fluorescence based assay. Both immunoblotting and fluorescence based assays can be applied to studies of other misfolded protein.The latter is also suitable for high throughput screening for identifying macromolecules or small compounds that can modulate the degradation of misfolded proteins. It is important to remember that for some misfolded proteins such Atxn1 82Q, the half life can only be measured through cell fractionation and immunoblotting and this is because the misfolded species that degraded quickly are only a small portion of the overall Atxn1 82Q expression. To uncover the mechanism of a protein quality controls, protein degradation assays need to be coupled with analysis of the steady state levels of aggregates as well as that they are partitions in different cell fractions.Other experiments like in vitro protein folding and aggregation assays can also be performed. After watching this video, you should have a good understanding of how to measure the degradation rates of misfolding proteins using different strategies. Thanks for watching and good luck with your experiments.

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