Name: quantifying subcellular ubiquitin-proteasome activity in the rodent brain
Uploaded: 2019-05-21T14:30:00
Description: Watch this Scientific Journal Video about Quantifying Subcellular Ubiquitin-proteasome Activity in the Rodent Brain at JoVE.com

This work was supported by startup funds from the College of Agricultural and Life Sciences and the College of Science at Virginia Tech. T.M. is supported by the George Washington Carver Program at Virginia Tech.

All procedures including animal subjects have been approved by the Virginia Polytechnic Institute and State University Institutional Animal Care and Use Committee (IACUC).
1. Collection and Dissection of Rodent Brain Tissue
NOTE: This protocol can be applied to a variety of brain regions and used with various tissue collection procedures. Below is the procedure used in our lab for subcellular of rat brain tissue, using 8-9 week old male Sprague Dawley...

<ol>
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Here, we demonstrate an efficient method for quantifying changes in ubiquitin-proteasome activity across different subcellular compartments in the same animal. Currently, most attempts at measuring subcellular changes in activity of the ubiquitin-proteasome system have been limited to a single compartment per sample, resulting in the need to repeat experiments. This leads to significant costs and loss of animal life. Our protocol alleviates this problem by splitting hemispheres, allowing different cellular fractions to b...

The ubiquitin-proteasome system (UPS) is a complex network of interconnected protein structures and ligases that controls the degradation of most short-lived proteins in cells1. In this system, proteins are marked for degradation or other cellular processes/fates by the small modifier ubiquitin. A target protein can acquire 1-7 ubiquitin modifications, which can link together at one of seven lysine (K) sites (K6, K11, K27, K29, K33, K48 and K63) or the N-terminal methionine (M1; as known as linear) in the previous ubiquitin2. Some of these polyubiquitin tags are degradation-specific (K48)3, while others are largely independent of the protein degradation process (M1)4,5,6. Thus, the protein ubiquitination process is incredibly complex and the ability to quantify changes in a specific polyubiquitin tag is critical for ultimately understanding the role of that given modification in cellular functioning. Further complicating the study of this system, the proteasome, which is the catalytic structure of the UPS7, both degrades proteins but can also be involved in other non-proteolytic processes8,9. Not surprisingly then, since its initial discovery, normal and aberrant ubiquitin-proteasome activity has been implicated in long-term memory formation and a variety of disease states, including many neurological, neurodegenerative and psychiatric disorders10,11. As a result, methods which can effectively and efficiently quantify UPS activity in the brain are critical for ultimately understanding how this system is dysregulated in disease states and the eventual development of treatment options targeting ubiquitin and/or proteasome functioning.
There are a number of issues in quantifying ubiquitin-proteasome activity in brain tissue from rats and mice, which are the most common model systems used to study UPS function, including 1) the diversity of ubiquitin modifications, and 2) distribution and differential regulation of UPS functioning across subcellular compartments12,13,14. For example, many of the early demonstrations of ubiquitin-proteasome function in the brain during memory formation used whole cell lysates and indicated time-dependent increases in both protein ubiquitination and proteasome activity15,16,17,18,19,20. However, we recently found that ubiquitin-proteasome activity varied widely across subcellular compartments in response to learning, with simultaneous increases in some regions and decreases in others, a pattern that differs significantly from what was previously reported in whole cell lysates21. This is consistent with the limitation of a whole cell approach, as it cannot dissociate the contribution of changes in UPS activity across different subcellular compartments. Though more recent studies have employed synaptic fraction protocols to study the UPS specifically at synapses in response to learning22,23,24, the methods used occlude the ability to measure nuclear and cytoplasmic ubiquitin-proteasome changes in the same animal. This results in an unnecessary need to repeat experiments multiple times, collecting a different subcellular fraction in each. Not only does this result in a greater loss of animal lives, but it eliminates the ability to directly compare UPS activity across different subcellular compartments in response to a given event or during a specific disease state. Considering that protein targets of ubiquitin and the proteasome vary widely throughout the cell, understanding how ubiquitin-proteasome signaling differs in distinct subcellular compartments is critical for identifying the functional role of the UPS in the brain during memory formation and neurological, neurodegenerative and psychiatric disorders.
To address this need, we recently developed a procedure in which nuclear, cytoplasmic and synaptic fractions could be collected for a given brain region from the same animal21. Additionally, to account for the limited amount of protein that can be obtained from collecting multiple subcellular fractions from the same sample, we optimized previously established protocols to assay in vitro&#160;proteasome activity and linkage-specific protein ubiquitination in lysed cells collected from rodent brain tissue. Using this protocol, we were able to collect and directly compare learning-dependent changes in proteasome activity, K48, K63, M1 and overall protein polyubiquitination levels in the nucleus and cytoplasm and at synapses in the lateral amygdala of rats. Here, we describe in detail our procedure (Figure 1), which could significantly improve our understanding of how the UPS is involved in long-term memory formation and various disease states. However, it should be noted that the in vitro proteasome activity discussed in our protocol, while widely used, does not directly measure the activity of complete 26S proteasome complexes. Rather, this assay measures the activity of the 20S core, meaning it can only serve as a proxy to understand the activity of the core itself as opposed to the entire 26S proteasome complex.

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			<td height="40" style="height:40px;width:222px;">0.5M EDTA</td>
			<td style="width:143px;">Fisher</td>
			<td style="width:259px;">15575020</td>
			<td style="width:255px;">Various other vendors</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">20S Proteasome Activity Kit</td>
			<td>Millipore Sigma</td>
			<td>APT280</td>
			<td>Other vendors carry different versions</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:222px;">ATP</td>
			<td style="width:143px;">Fisher</td>
			<td style="width:259px;">FERR1441</td>
			<td style="width:255px;">Various other vendors</td>
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			<td>Cell signaling</td>
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			<td>Various other vendors</td>
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			<td height="40" style="height:40px;">Beta-tubulin antibody</td>
			<td>Cell signaling</td>
			<td>2128T</td>
			<td>Various other vendors</td>
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		<tr height="40">
			<td height="40" style="height:40px;">BioTek Synergy H1 plate reader</td>
			<td>BioTek</td>
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			<td>Other vendors carry different versions</td>
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			<td height="40" style="height:40px;">clasto lactacystin b-lactone</td>
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			<td>L7035</td>
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			<td>Fisher</td>
			<td>033377B</td>
			<td>Various other vendors</td>
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		<tr height="40">
			<td height="40" style="height:40px;">DMSO</td>
			<td>DMSO</td>
			<td>D8418</td>
			<td>Varous other vendors</td>
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			<td height="40" style="height:40px;width:222px;">DTT</td>
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			<td style="width:143px;">Millipore Sigma</td>
			<td style="width:259px;">G5516</td>
			<td style="width:255px;">Various other vendors</td>
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			<td height="40" style="height:40px;">H3 antibody</td>
			<td>Abcam</td>
			<td>ab1791</td>
			<td>Various other vendors</td>
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			<td height="40" style="height:40px;width:222px;">HEPES</td>
			<td style="width:143px;">Millipore Sigma</td>
			<td style="width:259px;">H3375</td>
			<td style="width:255px;">Various other vendors</td>
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			<td height="40" style="height:40px;">Hydrochloric acid</td>
			<td>Fisher</td>
			<td>SA48</td>
			<td>Various other vendors</td>
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		<tr height="40">
			<td height="40" style="height:40px;width:222px;">IGEPAL (NP-40)</td>
			<td style="width:143px;">Millipore Sigma</td>
			<td style="width:259px;">I3021</td>
			<td style="width:255px;">Various other vendors</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">K48 Ubiquitin Antibody</td>
			<td>Abcam</td>
			<td>ab140601</td>
			<td>Various other vendors</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">K63 Ubiquitin Antibody</td>
			<td>Abcam</td>
			<td>ab179434</td>
			<td>Various other vendors</td>
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		<tr height="40">
			<td height="40" style="height:40px;width:222px;">KCl</td>
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			<td>Life Sensors</td>
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			<td>Only M1 antibody</td>
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			<td>Carried by Millipore-Sigma</td>
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			<td height="40" style="height:40px;width:222px;">NaCl</td>
			<td style="width:143px;">Millipore Sigma</td>
			<td style="width:259px;">S3014</td>
			<td style="width:255px;">Various other vendors</td>
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			<td height="40" style="height:40px;width:222px;">Phosphatase Inhibitor</td>
			<td style="width:143px;">Millipore Sigma</td>
			<td style="width:259px;">524625</td>
			<td style="width:255px;">Various other vendors</td>
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			<td>Various other vendors</td>
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		<tr height="40">
			<td height="40" style="height:40px;width:222px;">Protease Inhibitor</td>
			<td style="width:143px;">Millipore Sigma</td>
			<td style="width:259px;">P8340</td>
			<td style="width:255px;">Various other vendors</td>
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		<tr height="40">
			<td height="40" style="height:40px;">PSD95 antibody</td>
			<td>Cell signaling</td>
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			<td>Various other vendors</td>
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			<td height="50" style="height:50px;width:222px;">SDS</td>
			<td style="width:143px;">Millipore Sigma</td>
			<td style="width:259px;">L3771</td>
			<td style="width:255px;">Various other vendors</td>
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			<td height="45" style="height:46px;">Sodium hydroxide</td>
			<td>Fisher</td>
			<td>SS255</td>
			<td>Various other vendors</td>
		</tr>
		<tr height="48">
			<td height="48" style="height:48px;width:222px;">Sucrose</td>
			<td style="width:143px;">Millipore Sigma</td>
			<td style="width:259px;">S0389</td>
			<td style="width:255px;">Various other vendors</td>
		</tr>
		<tr height="58">
			<td height="58" style="height:58px;">TBS</td>
			<td>Alfa Aesar</td>
			<td>J62938</td>
			<td>Varous other vendors</td>
		</tr>
		<tr height="49">
			<td height="49" style="height:50px;width:222px;">Tris</td>
			<td style="width:143px;">Millipore Sigma</td>
			<td style="width:259px;">T1503</td>
			<td style="width:255px;">Various other vendors</td>
		</tr>
		<tr height="36">
			<td height="36" style="height:36px;">Tween-20</td>
			<td>Fisher</td>
			<td>BP337-100</td>
			<td>Various other vendors</td>
		</tr>
		<tr height="48">
			<td height="48" style="height:48px;">Ubiquitin Antibody</td>
			<td>Enzo Life Sciences</td>
			<td>BML-PW8810</td>
			<td>Various other vendors</td>
		</tr>
	</tbody>
</table>

quantifying subcellular ubiquitin-proteasome activity in the rodent brain

The ubiquitin-proteasome system is a key regulator of protein degradation and a variety of other cellular processes in eukaryotes. In the brain, increases in ubiquitin-proteasome activity are critical for synaptic plasticity and memory formation and aberrant changes in this system are associated with a variety of neurological, neurodegenerative and psychiatric disorders. One of the issues in studying ubiquitin-proteasome functioning in the brain is that it is present in all cellular compartments, in which the protein targets, functional role and mechanisms of regulation can vary widely. As a result, the ability to directly compare brain ubiquitin protein targeting and proteasome catalytic activity in different subcellular compartments within the same animal is critical for fully understanding how the UPS contributes to synaptic plasticity, memory and disease. The method described here allows collection of nuclear, cytoplasmic and crude synaptic fractions from the same rodent (rat) brain, followed by simultaneous quantification of proteasome catalytic activity (indirectly, providing activity of the proteasome core only) and linkage-specific ubiquitin protein tagging. Thus, the method can be used to directly compare subcellular changes in ubiquitin-proteasome activity in different brain regions in the same animal during synaptic plasticity, memory formation and different disease states. This method can also be used to assess the subcellular distribution and function of other proteins within the same animal.

Using the procedure described here, nuclear, cytoplasmic and synaptic fractions were collected from the lateral amygdala of the rat brain (Figure 1). Purity of the individual fractions were confirmed via Western blotting, probing with antibodies against proteins that should be enriched or depleted in the lysate. In the first hemisphere where a crude synaptic fraction was collected, postsynaptic density protein 95 (PSD95) was present in the synaptic, but not n...

Watch this Scientific Journal Video about Quantifying Subcellular Ubiquitin-proteasome Activity in the Rodent Brain at JoVE.com

Quantifying Subcellular Ubiquitin-proteasome Activity in the Rodent Brain

This protocol is designed to efficiently quantify ubiquitin-proteasome system (UPS) activity in different cellular compartments of the rodent brain. Users are able to examine UPS functioning in nuclear, cytoplasmic and synaptic fractions in the same animal, reducing the amount of time and number of animals needed to perform these complex analyses.

The ubiquitin-proteasome system is a key regulator of protein degradation and a variety of other cellular processes in eukaryotes. In the brain, increases ...

This protocol measures subcellular protein ubiquitination levels and proteasome activity in the rodent brain allowing within subjects comparisons of how ubiquitin-proteasome activity changes in response to cellular activity, learning, or disease. The protocol allows collection of synaptic, cytoplasmic, and nuclear fractions from the same rodent brain. Minimizing loss, it could be performed with small tissue quantities and basic laboratory equipment.Demonstrating the procedure will be Taylor McFadden, a graduate student from my laboratory. Begin this procedure with collection and dissection of rodent brain tissue as described in the text protocol. Ensure that the hemisphere used is counter balanced across extraction conditions for each experimental group.Remove the 1.5 milliliter centrifuge microtube containing one hemisphere of the region of interest from the minus 80 degree Celsius freezer. Using a scalpel, transfer the frozen brain tissue to a two milliliter glass Teflon homogenizer. Add 500 microliters of lysis buffer to the Teflon tube.Using pestle B, homogenize the same tissue with 15 strokes until no visible amount of solid material is present. Use a turning motion during each stroke. With a 1, 000 microliter pipette, transfer the homogenized sample to a new 1.5 milliliter microcentrifuge tube.Place the tube on wet ice and incubate for 30 minutes. Place the tube in the microcentrifuge and spin for 10 minutes at 845 times g in four degrees Celsius. After completion, carefully remove the supernatant by pipetting and place into a new 1.5 milliliter microcentrifuge tube.This is the cytoplasmic fraction. Add 50 microliters of extraction buffer to the resulting pellet and resuspend by pipetting. Do not vortex the pellet.Place the tube containing the resuspended pellet on ice and incubate for 30 minutes. Then centrifuge the tube for 20 minutes at 21, 456 times g in four degrees Celsius. Following centrifugation, carefully remove the supernatant by pipetting and place into a new 1.5 milliliter microcentrifuge tube.This is the nuclear fraction. Homogenize one hemisphere of the region of interest as before except to use 500 microliters of TEVP buffer instead of lysis buffer. Using a 1, 000 microliter pipette, transfer the homogenized sample to a new 1.5 milliliter microcentrifuge tube.Centrifuge the sample at 1, 000 times g for 10 minutes at four degrees Celsius. Collect the supernatant and transfer it to a new 1.5 milliliter microcentrifuge tube using a 1, 000 microliter pipette. Centrifuge the sample at 10, 000 times g for 10 minutes at four degrees Celsius.Discard the original pellet which contains nuclei and the large debris. Transfer the supernatant to a new 1.5 milliliter microcentrifuge tube. This is the cytosolic fraction.Add 50 microliters of homogenization buffer to the pellet and resuspend by pipetting until no solid material is visible. Centrifuge the sample at 20, 000 times g for 10 minutes at four degrees Celsius. Transfer the supernatant to a new 1.5 milliliter microcentrifuge tube.This is the crude synaptosomal membrane fraction. To set up the assay, prewarm the plate reader to 37 degrees Celsius and hold through the run. Set the excitation to 360 nanometers and emission to 460 nanometers.If the 96 well plate used is clear, set the optics position to bottom. If a dark 96 well plate is used, set optics position to top. Program a kinetic run with a time of two hours scanning every 30 minutes.Reconstitute the 20S 10X assay buffer provided in the kit with 13.5 milliliters of ultrapure water. Add 14 microliters of 100 millimolar ATP to the now 1X buffer. This significantly enhances proteasome activity in the samples and improves assay reliability.Reconstitute the AMC standard provided in the kit with 100 microliters of DMSO. Perform steps using the AMC standard in the dark or under low light conditions as the standard is light sensitive. Create a standard curve of AMC from a series of high to low AMC concentrations.This curve will be used for plate reader calibration and analysis of proteasome activity in the homogenized samples. Reconstitute the proteasome subtrate provided in the kit with 65 microliters of DMSO. Also perform this step in the dark or under low light conditions as the substrate is light sensitive.Then create a one to 20 dilution of the proteasome substrate in a new 1.5 milliliter microcentrifuge tube using 20S assay buffer. Add a normalized amount of the desired samples to a 96 well plate. Run each sample in duplicate.The amount of sample needed varies based on tissue preparation. Generally, 10 to 20 micrograms is sufficient for any subcellular fraction. Bring the sample well volume to 80 microliters with ultrapure water.The amount added depends on the volume of sample added. In two separate wells, add 80 microliters of water alone. These will be the assay blanks.Add 10 microliters of 20S assay buffer to each well including assay blanks. Use a repeater or automated pipette to ensure consistent assay volume across the wells. Turn off the lights or enter a dark room.Add all 100 microliters of diluted AMC standards to a new well using a single well for each standard. In the dark, use a repeater or automated pipette to add 10 microliters of diluted proteasome substrate to wells containing sample and assay blanks but not the AMC standard. Place the plate into the plate reader and start the kinetic run.Perform quantification of diverse polyubiquitin tags in different subcellular fractions collected from rodent brain tissue using a variety of standard Western blotting protocols in combination with unique linkage-specific polyubiquitin antibodies. Some ubiquitin Western blot images will provide columns of distinct bands while others produces smear-like pattern with few or no clear lines. For quantification of imaged ubiquitin Western blots, draw a box around the column that extends the entire molecular standards ladder.Adjust the box if ubiquitin staining does extend through the entire ladder. This is common for lysine 48 modifications and varies widely across subcellular compartments. Finally, subtract out the background which is calculated as the mean optical density of the background immediately surrounding the column on all sides.Shown here is the quantification of proteasome activity in different fractions collected from the lateral amygdala of the same animal. During the in vitro proteasome activity assay, relative fluorescent units detected increased from the beginning to the end of the assay in the synaptic fraction, the cytoplasmic fraction, and the nuclear fraction. The proteasome inhibitor beta-lac prevented RFUs from changing across the session.Subcellular differences were observed in proteasome activity in the lateral amygdala of the same animal. An increase in nuclear proteasome activity was detected in trained animals relative to controls which corresponded to a decrease in activity within the synaptic fraction. Cytoplasmic proteasome activity remained at baseline.Shown here are subcellular differences in linkage-specific protein ubiquitination in the lateral amygdala of the same animal following learning. There was an increase in overall ubiquitination in the nuclear fraction following learning which correlated with a decrease in the cytoplasmic fraction. Following learning, linear ubiquitination increased in the nuclear fraction but not cytoplasmic or synaptic fractions.Interestingly, K63 ubiquitination increased in the nuclear fraction following learning which correlated with a decrease in the synaptic fraction. Whereas K48 uniquitination increased in the nuclear and cytoplasmic fractions following learning but not in the synaptic fraction. This protocol can also be used to understand the subcellular distribution and function of other proteins within the same animal.

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