Which cell process does the nucleus control?

Quantification of subcellular ubiquitin proteasome activity in rodent brain

Summary

This protocol was developed to efficiently quantify the activity of the ubiquitin proteasome system (UPS) in different cellular compartments of the rodent brain. Users are able to study USB function in nuclear, cytoplasmic and synaptic fractions in the same animal, reducing the time and number of animals required to perform these complex analyzes.

Abstract

The ubiquitin-proteasome system is an important regulator of protein breakdown and a variety of other cellular processes in eukaryotes. In the brain, increases in enrubized-proteasome activity are critical to 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 problems with studying ubiquitin proteasome function in the brain is that it is present in all cellular compartments, where protein targets, functional role, and regulatory mechanisms 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 to fully understanding how the UPS contributes to synaptic plasticity. Memory and sickness. The method described here enables the removal of nuclear, cytoplasmic and crude synaptic fractions from the same rodent (rat) brain, followed by a simultaneous quantification of the proteasome catalytic activity (indirect, which provides the activity of the proteasome nucleus). ) 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.

Introduction

Log in or Start trial to access full content. Learn more about your institution’s access to JoVE content here

The Ubiquitin Proteasome System (UPS) is a complex network of interconnected protein structures and ligases that enable the breakdown of most of the short-lived proteins in cells1controls. 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 that occur at one of seven lysine (K) sites (K6, K11, K27, K29, K33, K48, and K63) or the N-terminal methionine (M1; known as linear) in the previous ubiquitin2can be linked to each other. Some of these polyubiquitin tags are degradation-specific (K48)3, while others are largely independent of the protein breakdown process (M1)4,5,6are. Hence, the protein ubiquitination process is incredibly complex and the ability to quantify changes in a given polyubiquitin tag is critical to ultimately understanding the role of that given change in cellular function. To make matters worse, the investigation of this system, the proteasome, the catalytic structure of the UPS 7, both breaks down proteins and can also be involved in other non-proteolytic processes8,9. Not surprisingly, since its first discovery, normal and abnormal ubiquitin-proteasomic 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 that can effectively and efficiently quantify USS activity in the brain are critical to ultimately understanding how this system is dysregulated in disease states and ultimately developing treatment options that work on ubiquitin and / or proteasome.

There are a number of problems in quantifying ubiquitin proteasome activity in rat and mouse brain tissue, which are the most common model systems used to study UPS function, including 1) the variety of ubiquitin modifications and 2) The distribution and differential regulation of the USUs function across subcellular compartments12,13,14. For example, many of the early demonstrations of ubiquitin proteasome function in the brain used whole cell lysates during memory formation and indicated a time-dependent increase in both protein ubiquitination and proteasome activity15,16,17,18,19,20. Recently, however, we found that ubiquitin proteasome activity in the subcellular compartments was very different in response to learning, with some regions increasing at the same time and others decreasing a pattern that was significantly different. from what has previously been reported in whole cell lysates21. This is consistent with the limitation of a whole cell approach as it cannot separate the contribution of changes in USB activity across different subcellular compartments. Although recent studies have used synaptic rupture protocols, the UPS specifically connects to synapses in response to learning22,23,24To study the methods used include the ability to measure nuclear and cytoplasmic ubiquitin proteasome changes in the same animal. This leads to an unnecessary need to repeat experiments several times, each time collecting a different subcellular fraction. Not only does this result in greater loss of animal life, but it also eliminates the ability to directly compare THE UPS activity in different subcellular compartments in response to a particular event or during a particular disease state. Considering that the protein targets of ubiquitin and the proteasome are very different in the cell, understanding how ubiquitin-proteasome signaling differs in different subcellular compartments is crucial to the functional role of USB in memory formation and neurological , neurodegenerative and psychiatric disorders.

To meet this need, we recently developed a method by which nuclear, cytoplasmic and synaptic fractions could be collected for a specific brain region from the same animal21. To take into account the limited amount of protein that can be obtained from the removal of multiple subcellular fractions from the same sample, we optimized previously established protocols to measure in vitro proteasome activity and linkage-specific protein ubiquitination in lysed rodent cells -Brain tissue collected. With this protocol, we were able to collect and directly compare learning-dependent changes in proteasome activity K48, K63, M1 and total protein polyubiquitination in the nucleus and cytoplasm as well as at synapses in the lateral amygdala of rats. Here we describe our procedure in detail (illustration 1), which could greatly improve our understanding of how the UPS is involved in long-term memory formation and various disease states. It should be noted, however, that while the in vitro proteasome activity discussed in our protocol is widespread, it does not directly measure the activity of full 26S proteasome complexes. Rather, this assay measures the activity of the 20S core; H. it can only serve as a proxy to understand the activity of the nucleus itself as opposed to the entire 26S proteasome complex.

Subscription Required. Please recommend JoVE to your librarian.

Protocol

Log in or Start trial to access full content. Learn more about your institution’s access to JoVE content here

All procedures, including animal subjects, have been approved by the Virginia Polytechnic Institute and the 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 wide variety of brain regions and used with various tissue-harvesting procedures. Below is the procedure used in our laboratory for subcellular rat brain tissue, using 8-9 week old male Sprague Dawley rats. To process all cell compartments in the same animal, section 1.3. be followed regardless of the tissue collection procedure used.

  1. Extract the rat brain and place it in a cryogenic cup pre-chilled on dry ice. Freeze the brain on dry ice or use liquid nitrogen if available and transfer to a -80 ° C freezer. Brains can be dissected the same day or at a later time.
  2. Remove the frozen brain from the cryogenic cup and place it in a rat brain matrix cooled with dry ice. Each slot on the matrix corresponds to 0.5mm which can be used to determine the approximate location of the region of interest (ROI).
    1. With a Rat Brain Atlas, insert a razor blade into the matrix just before the predicted ROI start. Next, immediately insert a razor blade at the predicted end (posterior) of the ROI.
    2. Remove the disc between the razor blades with a scalpel and place on a microscope slide cooled on dry ice. Typically the sections are 2-3mm thick, but this will vary based on the ROI.
  3. Scalpel Using a scalpel, dissect the ROI one hemisphere at a time. Place each hemisphere in separate 1.5 ml microcentrifuge tubes pre-chilled on dry ice. Frozen tissue can be used immediately for subcellular fractionation or processed at a later time when stored at -80 ° C.

2. Nuclear and cytoplasmic extraction

NOTE: This protocol uses pre-made storage solutions of common laboratory chemicals, including 0.1 M HEPES, 1 M MgCl2, 1 M dithiothreitol (DTT), 0.5 M ethylenediaminetetraacetic acid (EDTA), 5 M NaCl, 10% NP-40 (IGEPAL) and 50% glycerin. When Endpoint is used in proteasome activity tests, glycerol and ATP can be added to all buffers to prevent disassembly of proteasome complexes during lysis.

  1. Prepare the lysis buffer. Add 8.63 ml of ultrapure (deionized and distilled) water to a sterile 15 ml conical tube.
    1. Add 1,000 liters of 0.1 m HEPES, 15 liters of 1 M MgCl, 100 liters of 1 M DTT and 50 liters of 10% NP-40 to the conical tube.
    2. Add 100 l protease inhibitor cocktail and 100 l phosphatase inhibitor cocktail. Briefly swirl the solution to mix and cool on wet ice. Note that the solution may be yellow in color from the phosphatase inhibitor.
      NOTE: The use of protease inhibitors is important to preserve the protein and to ensure the specificity of the in vitro proteasome activity assay. However, these inhibitors can also lead to a slight reduction in proteasome activity, which means that the activity measured in the in vitro test can be an underestimation of the actual activity level.
  2. Prepare the extraction buffer. Add 5.925 ml of ultrapure (deionized and distilled) water to a sterile 15 ml conical tube.
    1. In the conical tube add 2,000 liters of 0.1 m HEPES, 1250 liters of 50% glycerin, 15 liters of 1 M MgCl2, 5 L of 1 M DTT, 5 L with 0.5 M EDTA and 600 L 5 M NaCl.
    2. Add 100 l protease inhibitor cocktail and 100 l phosphatase inhibitor cocktail. Briefly swirl the solution to mix and cool on wet ice. Note that the solution may be yellow in color from the phosphatase inhibitor.
  3. Remove the 1.5 ml centrifuge microtube containing a hemisphere of the ROI from the -80 ° C freezer. Make sure that the hemisphere used for each experimental group is balanced across the extraction conditions. For example, in a two-group experiment, use the left hemisphere for the first two animals in each group and the right half for the next two samples, and so on.
  4. Transfer the frozen brain tissue to a 2 ml glass-Teflon homogenizer with a scalpel. Add 500 L of Lysis Buffer to the Teflon tube.
    1. Using the plunger B, homogenize the same tissue with 15 strokes until there is no visible amount of solid material. Use a rotary motion (clockwise or counterclockwise) with each stroke.
  5. Transfer the homogenized sample to a new 1.5 ml microcentrifuge tube using a 1,000 L pipette. Place the tube on wet ice and incubate for 30 min.
  6. Put tube in microcentrifuge and 10 min at 845 x G and turn 4 ° C. When the supernatant is complete, carefully remove it with a pipette and place it in a new 1.5 ml microcentrifuge tube; This is the cytoplasmic fraction which can be stored on ice or at 4 ° C up to section 2.9.
  7. Add 50 L extraction buffer to the resulting pellet and reapply by pipetting. Do not swirl the pellet. Place the tube with the resuspended pellet on ice and incubate for 30 min.
  8. Place the tube in the microcentrifuge and 20 min at 21,456 x G, Turn 4 ° C. When the supernatant is complete, carefully remove it with a pipette and place it in a new 1.5 ml microcentrifuge tube; that is the core faction. The pellet can now be disposed of.
  9. Measure the protein concentration of the cytoplasmic and nuclear extractions using the Dc Protein Assay (per manufacturer's instructions) or an equivalent assay for samples collected with nonionic detergents. Proceed immediately with the proteasome activity test (Section 4) or Western blotting (Section 5). Alternatively, samples can be stored at -80 ° C until needed.

3. Synaptic Fraction Collection

NOTE: This protocol uses pre-made storage solutions of common laboratory chemicals, including 1 M Tris (pH 7.5), 0.5 M EDTA, 5 M NaCl, and 10% SDS. When Endpoint is used in proteasome activity tests, glycerol and ATP can be added to all buffers to prevent disassembly of proteasome complexes during lysis

  1. Prepare the TEVP buffer with 320 mM sucrose. Add 60 ml of ultrapure (deionized and distilled) water to a clean 100 ml beaker.
    1. Add 1,300 l of 1 M Tris (pH 7.5), 260 l with 0.5 m EDTA, 100 l protease inhibitor cocktail, 100 l phosphatase inhibitor cocktail and 10.944 g sucrose to the beaker. Mix with a stir stick until sucrose is completely dissolved.
    2. Bring the pH to 7.4 by adding the solution drop by drop with a pipette.
    3. Transfer the solution to a 100 ml volumetric flask. Bring the volume to 100 ml by adding ultrapure water. Chill the final solution on ice until needed.
  2. Prepare the homogenization buffer. Add 60 ml of ultrapure (deionized and distilled) water to a clean 100 ml beaker.
    1. Add to the beaker, add 5,000 l of 1 M Tris (pH 7.5), 3,000 l of 5 M NaCl, 100 l protease inhibitor cocktail, 100 l phosphatase inhibitor cocktail and 2,000 l 10% SDS. Mix with a stirring rod.
      NOTE: Ionic detergents can interfere with proteasome activity tests by causing denaturation of the proteasome complex. The volume of SDS could be decreased to preserve proteasome function if desired.
    2. Bring the pH to 7.4 by adding hCl drop by drop into the solution with a pipette.
    3. Transfer the solution to a 100 ml volumetric flask. Bring the volume to 100 ml by adding ultrapure water. Store at room temperature; do not cool, as SDS will fall out of the solution.
  3. Remove the 1.5 ml centrifuge containing a hemisphere of the ROI from the -80 ° C freezer. Make sure that the hemisphere used for each experimental group is balanced across the extraction conditions. For example, in a two-group experiment, use the left hemisphere for the first two animals in each group and the right half for the next two samples, and so on.
  4. Transfer the frozen brain tissue to a 2 ml glass-Teflon homogenizer with a scalpel. Add 500 L of TEVP buffer to the Teflon tube.
    1. Using the plunger B, homogenize the same tissue with 15 strokes until there are no visible transfers of solid material. Use a rotary motion (clockwise or counter-clockwise) with each stroke.
  5. Transfer the homogenized sample to a new 1.5 ml microcentrifuge tube using a 1,000 L pipette. Centrifuge the sample at 1,000 x G for 10 min, 4 ° C.
  6. Collect the supernatant and transfer to a new 1.5 ml microcentrifuge tube using a 1,000 L pipette. Centrifuge the sample at 10,000 x G for 10 min, 4 ° C. The original pellet (P1) contains seeds and the large debris and can be disposed of.
  7. Transfer the supernatant to a new 1.5 ml microcentrifuge tube. This is a cytosolic fraction.Add 50 L of homogenization buffer to the pellet (P2) and replace the pellet (P2) until no solid material is visible.
  8. Centrifuge the sample at 20,000 x G for 10 min, 4 ° C. Transfer the supernatant to a new 1.5 ml microcentrifuge tube; This is the Crude Synaptosomal Membrane (Synaptic) fraction. The pellet can be disposed of.
  9. Measure the protein concentration of the synaptic fraction using the Dc protein assay (according to the manufacturer's instructions) or an equivalent assay for samples collected with ionic detergents. Proceed immediately with the proteasome activity test (Section 4) or Western blotting (Section 5). Alternatively, samples can be stored at -80 ° C until needed.

4. Proteasome Activity Assay

NOTE: Proteasome activity can be measured in homogenized brain tissue with a slightly modified version of the 20S Proteasome Activity Kit. This test does not directly measure the activity of complete 26S proteasome complexes. Rather, it measures the activity of the 20S core; H. it can only serve as a proxy to understand the activity of the nucleus itself as opposed to the entire 26S proteasome complex. The success of this test decreases with repeated freeze-thaw cycles and / or increasing detergents, especially ionic ones, and requires the use of a plate reader with a filter set 360/460 (excitation / emission) and heating capabilities up to 37 ° C.

  1. Plate reader settings: preheat to 37 ° C and keep it warm.
    1. Set the excitation to 360 and the emission to 460. If the 96-well plate being used is clear, set the optics position up Belowa. If a dark / black 96-well plate is used, set up the optics position Topa.
    2. Place older plate reader models under the fluorescent reading options Auto gain a; newer models are preset for this. Program a kinetic run with a time of 2 h, scanning (reading) every 30 min.
  2. Reconstitute the 10x Assay Buffer in the kit with 13.5 ml ultrapure water. Add 14 l of 100 mM ATP to the now 1x buffer; this significantly improves the proteasome activity in the samples and improves the reliability of the assay. The final 20S Assay Buffer can be stored on ice or at 4 ° C until needed and is stable for several months.
  3. Reconstitute the AMC standard included in the kit with 100 L DMSO. Do this step in the dark or in low light as the standard is sensitive to light.
  4. Create a stand curve of AMC with the reconstituted standard, in the dark or in poor light conditions.
    1. In separate 0.5 ml microcentrifuge tubes, add 16, 8, 6.4, 3.2, 1.6, 0.8, 0.4 and 0 L of the AMC standard, making the AMC concentration 20, 10, 8 , 4, 2, 1, 0.5 and 0 M AMC.
    2. To these tubes, in the same order, add 84, 92, 93.6, 96.8, 98.4, 99.2, 99.6, and 100 μL of 20S Assay Buffer. This creates a range of high to low AMC concentrations that are used to calibrate and analyze proteasome activity in the homogenized samples.
    3. Store all diluted standards on ice in the dark until needed.
  5. Reconstitute the proteamic substrate (Suc-LLVY-AMC) in the kit with 65 L DMSO. Do this step in the dark or in low light conditions as the substrate is sensitive to light. Make a 1:20 dilution of the proteasome substrate in a new 1.5 ml microcentrifuge tube with 20S Assay Buffer. Store the diluted substrate on ice in the dark until needed.
    NOTE: If the plate z. B. has 10 samples and 1 blank, you need enough diluted substrate for 22 wells (with duplicates) at 10 l per well. This corresponds to 220 l requirements + 30 l for pipetting errors that require 12.5 l substrate and 237.5 l 20S assay buffer.
  6. Thaw desired samples (if frozen) and add a normalized amount to a 96-well plate. Run each example in duplicates. The amount of sample required varies depending on the tissue preparation. Generally 10-20 g for each subcellular fraction is sufficient.
  7. Bring the sample volume to 80 l with ultrapure water. The amount added depends on the volume of the sample added. If z. B. Sample 1 was 4.5 liters of protein and Sample 2 was 8.7 liters, the amount of water required is 75.5 liters and 71.3 liters, respectively. In two separate wells, add 80 L of water alone; these will be the assay blanks.
    NOTE: To limit changes in protein volume due to pipetting errors, protein concentrations can be normalized to the least concentrated sample. This enables the same sample volume to be used under all conditions.
  8. Add 10 L of 20S Assay Buffer, including Assay Blanks, to each well. A repeater / automated pipette is recommended here to ensure consistent assay volume across wells.
  9. Optional: At this stage, introduce in vitro manipulations if desired; This requires each sample to have an additional 2 wells per treatment, including the vehicle. If so, add 2 of the sample wells and an appropriate control / vehicle volume to another 2 wells. Place the plate on the prewarmed plate reader or in a 37 ° C incubator for 30 min.
  10. Turn off the lights or go into a dark room. Add every 100 L of diluted AMC standards to a new well; each standard has a single well.
  11. In the dark, add 10 L of diluted proteasome substrate to wells with specimens and assay blanks, but not the AMC standard. A repeater / automated pipette is recommended here to ensure consistent assay volume across wells.
  12. Place the plate in the plate reader and start the kinetic run.
    NOTE: The plate does not have to be constantly stirred during the kinetic run, but the user can, if desired, choose
  13. At the end of the kinetic run, export raw 360/460 fluorescence values ​​to Microsoft Excel.
    1. Average total duplicate wells for each standard, each sample and the assay blank for all 5 scans. Raw fluorescence readings should increase on scans for the samples but remain stable (or decrease slightly) for standards and assay blanks.
    2. Take the highest AMC standard well average and divide by the known concentration (20 m). Divide this value by the sample concentration used in the test to obtain a standardized AMC value. For each sample and assay blank average, divide by the standardized AMC value.
    3. Take this final value and divide by the sample concentration used in the test to obtain the normalized value for each sample and the assay blank. Do this for every 5 scans.
    4. Subtract the normalized assay blank value from each normalized sample value for all 5 scans.

5. Quantification of the linkage-specific protein ubiquitination

  1. Quantitation of various polyubiquitin tags in various subcellular fractions collected from rodent brain tissue should be done using a variety of standard Western blotting protocols in combination with unique, linkage-specific polyubiquitin antibodies.
    1. For denaturation, mix the normalized samples with an equal volume of Laemmli sample buffer, supplemented by the Mitband mercaptoethanol to a volume percentage of 5 volume percent, as specified by the manufacturer.
    2. To quantify all monoubiquitination and polyubiquitinization modifications regardless of the compound, use a pan-ubiquitin antibody.
    3. To detect all polyubiquitinated proteins, use a ubiquitin antibody that does not cross-react with monoubiquitination.
    4. For linkage-specific polyubiquitination, use antibodies that can recognize lysine-27, lysine-48, lysine-63 and linear (M1) polyubiquitination.
  2. To prevent cross-contamination between developments, which could lead to false positive or interfering imaging of another ubiquitin modification, rub membranes between developments with 0.1 M NaOH for 10 min.
    1. Wash the membranes in TBS with 0.1% Tween twice for 10 min and reblock (with any blocking agent is preferred in the Western blot protocol).
    2. Incubate the membrane with the secondary antibody and re-develop to confirm that the primary and secondary antibody membrane has been properly removed.
    3. Recommended: Use fluorescent or near-infrared imaging systems to successfully develop various ubiquitin antibodies without contamination. This can often prevent transmission between antibodies.
  3. Some ubiquitous Western blot images will feature gaps of disparate bands (like M1), while others will produce a smear pattern with few or no clear lines (common with K48). To quantify the depicted ubiquitin western blots, draw a box around the column that extends the entire molecular standards ladder.
    1. Adjust the box up (or down) if the ubiquitin stain extends all the way down the ladder; This is common with lysine 48 modifications and varies widely in subcellular subjects.
    2. Subtract the background, which is calculated as the mean optical density of the background immediately surrounding the column on all sides.

Subscription Required. Please recommend JoVE to your librarian.

Representative Results

Log in or Start trial to access full content. Learn more about your institution’s access to JoVE content here

With the method described here, nuclear, cytoplasmic and synaptic fractions from the lateral amygdala of the rat brain (Figure1) taken. The purity of the individual fractions was confirmed by Western blotting, whereby antibodies against proteins that were supposed to accumulate or exhausted in the lysate were examined. In the first hemisphere where a coarse synaptic fraction was collected, the postsynaptic density protein 95 (PSD95) was present in the synaptic but non-nuclear fraction with lower concentrations in the cytoplasm (Figure 2A). This is consistent with previous work showing that synaptic hernia dissection has both presynaptic and postsynaptic componentsisolated 25. Conversely, the core protein histone H3 was present in the nuclear force, but not in the synaptic fraction, with lower concentrations in the cytoplasm (Figure 2B). The presence of PSD95 and H3 in the cytoplasm is consistent with their cytoplasmic translation. The cytoplasmic protein tubulin was present in our cytoplasmic fraction, but was largely absent from the nuclear lysate (Figure 2C), with lower concentrations in the synaptic region. This suggests that we were able to produce a nuclear fraction that was largely lacking of cytoplasmic proteins. The presence of tubulin in the synaptic region is consistent with previous studies26. All three fractions showed similar concentrations of the housing, the dasminiati actin (Figure 2D) that was used as a charge control. Together, these results confirm that the purity of the nuclear, cytoplasmic and synaptic fractions collected from a single lateral amygdala of the single rat.

Next, all lysates were confirmed for functional proteasome activity using the described modified version of the in vitro 20S proteasome activity assay. In all lysates, the success of the assay was defined as an increase in the raw fluorescence units (RFU) detected from the first scan (0 min) to the fifth / last scan (120 min). For all these analyzes, 10 M AMC was used as the highest standard for normalizing the raw fluorescence units (RFU). In the raw synaptic fraction, RFU on scan 5 reached (Figure 3A) a peak value, which led to a normalized RFU of just under 0.1 (Figure 3B). In the cytoplasmic fraction, rFU increased across scans (Figure 3C) with a final normalized RFU of 1.6 (3D illustration). The core portion also showed time-dependent changes in RFU (Figure 3E), with a final normalized RFU of 0.3 (Figure 3F). The differences in proteasome activity in the compartments likely reflect the availability of proteasomes in the fraction, which are generally most abundant in the cytoplasmic and nuclear areas27, Compartments that have the highest activity in our preparation. The lowest activity in the synaptic region is consistent with it being the only lysate collected with ionic detergents that can reduce proteasome activity due to the harsher denaturation state. It is important that RFU has not increased over time in the assay blanks or in lysates (synaptically) incubated with the highly specific and potent proteasome inhibitor clasto-lactacystin - lactone (Figure 3G, 0.01 or 0.001 (Figure 3H). This suggests that the observed change in RFU was specifically due to the activity of the proteaso and not to other proteases. In addition, when analyzed across experimental conditions, there was an increase in nuclear, but not cytoplasmic, proteasomic activity in the lateral amygdala after learning, which coincided with a decrease in synaptic proteasome activity when compared to control animals (Figure 4). Together, these results confirm that proteasome activity could be accurately measured in all three subcellular fractions collected from a single rat lateral amygdala.

One of the advantages of the proteasome activity assay is that in vitro manipulations can be introduced into the samples immediately prior to the addition of the proteaso substrate, allowing the identification of specific molecules that make the proteasome in that particular subcellular fraction. As an example of this, the role of CaMKII (calcium / calmodulin-dependent protein kinase II) in the synaptic fraction, the hardest denatured lysate collected since CaMKII presumably regulates proteasome function at synapses, was evaluated. A 30 min incubation with the CaMKII inhibitor myr-AIP (myristolayted autocamtide-2 related inhibitory peptide) led to a significantly reduced increase in proteasome activity on the assay, which only reached values ​​that (Figure5A). Conversely, the same manipulation that was applied to the cytoplasm did not result in a change in the proteasome activity of vehicle-treated wells (Figure 5B). These results confirm that proteasome activity can be further manipulated in vitro and that this manipulation can have different effects depending on the subcellular fraction.

In addition to quantifying proteasome activity, the protocol described can be used to measure subcellular differences in various ubiquitin modifications using Western blotting methods. It is important to note that the ubiquitin tags that can be recognized are limited by the availability of linkage-specific antibodies, which currently include K48, K63 and M1 for rats (Note: a K27 antibody is available but has no detectable image in lateral amygdala fraction or whole cell lysed under a variety of conditions). Overall, polyubiquitination, degradation-independent linear / M1 and K63 ubiquitination and degradation-specific K48 ubiquitination were detected in all subcellular fractions. It is important that when analyzing various test-related conditions in total (Figure 6A), linear (Figure 6B), K63 (Figure 6C) and K48 (Figure 6D) Polyubiquitination in the lateral amygdala nuclear fraction after learning compared to controls in animals. At the same time, polyubiquitination in the cytoplasmic region decreased and K48 ubiquitination increased after learning, while synaptic K63 ubiquitination was reduced. Together, these results show that subcellular differences in linkage-specific protein ubiquitination can be accurately detected within the same animal.


illustration 1: Schematic for subcellular fractionation of rat brain tissue. The rodent's brain is extracted, the brain region dissected, and the hemispheres split. With a series of buffers and centrifugation steps, nuclear and cytoplasmic fractions are collected from one hemisphere while a gross synaptic fraction is collected from the other. Both fractions are then used for proteasome activity assays and Western blotting to study protein polyubiquitination levels. Please click here to view a larger version of this image.


Figure 2: Confirmation of raw synaptic, nuclear and cytoplasmic fraction purity. (A.) The synaptic protein postsynaptic density protein 95 (PSD95; 1: 1,000) was present in the synaptic but not nuclear fraction with lower levels in the cytoplasm. (B.) Histone H3 (1: 500) was present in the nuclear but not synaptic fraction with lower expression in the cytoplasm. (C.) -Tubulin (1: 1,000) was present in the cytoplasmic, but largely not in the nuclear power, fraction with lower expression in the synaptic lysate. (D.) The housekeeping protein actin (1: 1,000) was present in all subcellular compartments. The ranges indicate the expected size of the target protein. This figure was obtained from Orsi, S.A. et al.21changed. Please click here to view a larger version of this image.


Figure 3: Quantification of proteasome activity in nuclear, cytoplasmic and synaptic fractions collected from the lateral amygdala of the same animal. During the in vitro proteasome activity test, relative fluorescent units (RFU) were measured from the beginning (scan 1) to the end (scan 5) of the assay in the synaptic (A.), Cytoplasmic (C.) and nuclear (E.) Fractions detected. The quantification of this change from the initial value resulted in a normalized RFU value (relative to 10 M AMC) of 0.1 in the synaptic (B.), 1.6 in the cytoplasmic (D.) and 0.3 in the core fractions (F.). The proteasome inhibitor 'lac prevented the RFUs from changing over the session (G-H). This figure was obtained from Orsi, S.A. et al.21changed. Please click here to view a larger version of this image.


Figure 4: Subcellular differences in proteasome activity in the lateral amygdala of the same animal. In trained (fear-conditioned) animals, an increase in nuclear proteasome activity was found compared to controls, which corresponded to a decrease in activity within the synaptic fraction. The cytoplasmic proteasome activity remained at the starting point. *P. < 0,05="" von="" control.="" diese="" zahl="" wurde="" von="" orsi,="" s.a.="" et="">21changed. Please click here to view a larger version of this image.


Figure 5: In vitro manipulation of proteasome activity in pooled synaptic and cytoplasmic fractions. In vitro manipulation of CaMKII signaling via the inhibitor AIP reduced the proteasome activity in the synaptic (A.), but not the cytoplasmic (B.), Fraction from the rat lateral amygdala. This figure was found by Jarome, T.J. et al.23changed. Please click here to view a larger version of this image.


Figure 6: Subcellular differences in linkage-specific protein ubiquitination in the lateral amygdala of the same animal after learning. (A.After learning, the total ubiquitination in the nuclear fraction increased, which correlated with a decrease in the cytoplasmic fraction. (B.) There was an increase in linear ubiquitination in the nuclear, but not cytoplasmic or synaptic, fraction after learning. (C.After learning, the K63 ubiquitination increased in the nuclear fraction, which correlated with a decrease in the synaptic fraction. (D.) K48 ubiquitination in the nuclear and cytoplasmic, but not synaptic, fraction increased after learning. *P. < 0,05="" von="" control.="" alle="" erhaltenen="" optischen="" ubiquitin-dichten="" wurden="" auf="" die="" a-actin-spiegel="" normalisiert="" (niedrigere="" repräsentative="" westliche="" blot-bilder="" in="" a),="" die="" als="" ladekontrolle="" verwendet="" wurde.="" diese="" zahl="" wurde="" von="" orsi,="" s.a.="" et="">21changed. Please click here to view a larger version of this image.

Subscription Required. Please recommend JoVE to your librarian.

Discussion

Log in or Start trial to access full content. Learn more about your institution’s access to JoVE content here

Here we show an efficient method for quantifying changes in ubiquitin proteasome activity in different subcellular compartments in the same animal. Currently, most attempts to measure subcellular activity changes of the ubiquitin-proteasome system are limited to a single compartment per sample, which means that experiments have to be repeated. This leads to significant costs and loss of animal life. Our protocol alleviates this problem by splitting hemispheres so that different cellular fractions can be collected from each hemisphere of the same animal. With this protocol we were able to show for the first time that in the same animal ubiquitin proteasome activity there are differential changes in nuclear, cytoplasmic and synaptic fractions in response to learning21.

The main limitation of the protocol described here is that it depends on the amount of brain tissue (sample) obtained. For example, as described above, this protocol requires the division of the hemispheres of a specific brain region. However, this may not always be possible, e.g. B. in certain areas that are only present in one hemisphere. In these cases, the protocol could be changed by first homogenizing the entire brain region in the TEVP buffer used in the synaptic rupture step (Section 3.1), as this buffer is free of all denaturants. The sample can then be divided into two equal parts by volume. The first part can be used for the synaptic fraction, following the protocol as described. For the second half of the sample, the non-ionic detergent NP-40 can be added to a final concentration of 0.05%, followed by centrifugation according to section 2.6. This enables the separation of cytoplasmic proteins into the supernatant and nuclear proteins into the pellet, which can be further isolated after the remaining steps in Section 2. Another concern with the amount of tissue is that some areas of the brain are very small, such as the prelimbic cortex. In these cases, however, the above protocol can still be used by reducing the volumes of the buffers used, which would have to be determined empirically from the size of the brain region collected. Thus, this protocol can also be changed in the more difficult brain regions where less tissue is available.

One of the main advantages of the protocol we are outlining here is that it uses common laboratory equipment and reagents found in most facilities, so this method can be changed even on a budget or with limited resources. While we outline this protocol as a way to measure subcellular changes in ubiquitin proteasome signaling, this method can also be applied to any other protein or cell process in which understanding cellular location and function is important. Hence, this protocol could have wide applications to understand the subcellular functions of certain proteins or complexes during learning and memory or various disease states.

Subscription Required. Please recommend JoVE to your librarian.

Disclosures

The authors have nothing to reveal.

Acknowledgments

This work was supported by start-up 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.

Materials

SurnameCompanyCatalog NumberComments
0.5 M EDTAFisher15575020Various other vendors
20S Proteasome Activity KitMillipore SigmaAPT280Other vendors carry different versions
ATPFisherFERR1441Various other vendors
Beta-actin antibodyCell signaling4967SVarious other vendors
Beta-tubulin antibodyCell signaling2128TVarious other vendors
BioTek Synergy H1 plate readerBioTekVATECHH1MT3Other vendors carry different versions
B-mercaptoethanolFisherICN19024280Various other vendors
Clasto lactacystin b-lactoneMillipore SigmaL7035Various other vendors
Cryogenic cupFisher033377BVarious other vendors
DMSODMSOD8418Varous other vendors
DTTMillipore SigmaD0632Various other vendors
GlycerolMillipore SigmaG5516Various other vendors
H3 antibodyAbcamfrom1791Various other vendors
HEPESMillipore SigmaH3375Various other vendors
Hydrochloric acidFisherSA48Various other vendors
IGEPAL (NP-40)Millipore SigmaI3021Various other vendors
K48 Ubiquitin AntibodyAbcamfrom140601Various other vendors
K63 Ubiquitin AntibodyAbcamfrom179434Various other vendors
KClMillipore SigmaP9541Various other vendors
KONTES tissue grinderVWRKT885300-0002Various other vendors
Laemmli sample bufferBio-rad161-0737Various other vendors
Linear Ubiquitin AntibodyLife sensorsAB-0130-0100Only M1 antibody
MgClMillipore Sigma442611Various other vendors
MicrocentrifugeEppendorf2231000213Various other manufacturers / models
myr-AIPEnzo Life SciencesBML-P212-0500Carried by Millipore-Sigma
NaClMillipore SigmaS3014Various other vendors
Odyssey Fc Imaging SystemLiCor2800-02Other vendors carry different versions
Phosphatase inhibitorMillipore Sigma524625Various other vendors
Precision Plus Protein StandardBio-rad161-0373Various other vendors
Protease inhibitorMillipore SigmaP8340Various other vendors
PSD95 antibodyCell signaling3450TVarious other vendors
SDSMillipore SigmaL3771Various other vendors
Sodium hydroxideFisherSS255Various other vendors
SucroseMillipore SigmaS0389Various other vendors
TBSAlfa AesarJ62938Varous other vendors
TrisMillipore SigmaT1503Various other vendors
Tween-20FisherBP337-100Various other vendors
Ubiquitin AntibodyEnzo Life SciencesBML-PW8810Various other vendors

DOWNLOAD MATERIALS LIST

References

  1. Hershko, A., Ciechanover, A. The ubiquitin system.Annu Rev Biochem. 67, 425-479 (1998).
  2. Akutsu, M., Dikic, I., Bremm, A. Ubiquitin chain diversity at a glance.Journal of Cell Science. 129, (5), 875-880 (2016).
  3. Ravid, T., Hochstrasser, M. Diversity of degradation signals in the ubiquitin-proteasome system.Nature Reviews Molecular Cell Biology. 9, (9), 679-690 (2008).
  4. Erpapazoglou, Z., Walker, O., Haguenauer-Tsapis, R. Versatile roles of k63-linked ubiquitin chains in trafficking.Cells. 3, (4), 1027-1088 (2014).
  5. Iwai, K., Fujita, H., Sasaki, Y. Linear ubiquitin chains: NF-kappaB signaling, cell death and beyond.Nature Review Molecular Cell Biology. 15, (8), 503-508 (2014).
  6. Rieser, E., Cordier, S. M., Walczak, H. Linear ubiquitination: a newly discovered regulator of cell signaling.Trends in Biochemical Sciences. 38, (2), 94-102 (2013).
  7. Collins, G.A., Goldberg, A.L. The Logic of the 26S Proteasome.Cell. 169, (5), 792-806 (2017).
  8. Bhat, K. P., et al.The 19S proteasome ATPase Sug1 plays a critical role in regulating MHC class II transcription.Molecular Immunology. 45, (8), 2214-2224 (2008).
  9. Ezhkova, E., Tansey, W. P. Proteasomal ATPases link ubiquitylation of histone H2B to methylation of histone H3.Molecular Cell. 13, (3), 435-442 (2004).
  10. Jarome, T. J., Helmstetter, F. J. The ubiquitin-proteasome system as a critical regulator of synaptic plasticity and long-term memory formation.Neurobiology of Learning and Memory. 105, 107-116 (2013).
  11. Hegde, A.N., Upadhya, S.C. The ubiquitin-proteasome pathway in health and disease of the nervous system.Trends in Neuroscience. 30, (11), 587-595 (2007).
  12. Adori, C., et al.Subcellular distribution of components of the ubiquitin-proteasome system in non-diseased human and rat brain.Journal of Histochemistry and Cytochemistry. 54, (2), 263-267 (2006).
  13. Upadhya, S. C., Ding, L., Smith, T. K., Hegde, A. N. Differential regulation of proteasome activity in the nucleus and the synaptic terminals.Neurochemistry International. 48, (4), 296-305 (2006).
  14. Enenkel, C., Lehmann, A., Kloetzel, P. M. Subcellular distribution of proteasomes implicates a major location of protein degradation in the nuclear envelope-ER network in yeast.EMBO Journal. 17, (21), 6144-6154 (1998).
  15. Jarome, T. J., Kwapis, J. L., Ruenzel, W. L., Helmstetter, F. J. CaMKII, but not protein kinase A, regulates Rpt6 phosphorylation and proteasome activity during the formation of long-term memories.Frontiers in Behavioral Neuroscience. 7, 115 (2013).
  16. Jarome, T. J., Werner, C. T., Kwapis, J. L., Helmstetter, F. J. Activity dependent protein degradation is critical for the formation and stability of fear memory in the amygdala.PLoS One. 6, (9), e24349 (2011).
  17. Lopez Salon, M., et al.The ubiquitin-proteasome cascade is required for mammalian long-term memory formation.European Journal of Neuroscience. 14, (11), 1820-1826 (2001).
  18. Reis, D. S., Jarome, T. J., Helmstetter, F. J. Memory formation for trace fear conditioning requires ubiquitin-proteasome mediated protein degradation in the prefrontal cortex.Frontiers in Behavior Neuroscience. 7, 150 (2013).
  19. Rosenberg, T., Elkobi, A., Rosenblum, K. mAChR-dependent decrease in proteasome activity in the gustatory cortex is necessary for novel taste learning.Neurobiology of Learning and Memory. 135, 115-124 (2016).
  20. Rosenberg, T., Elkobi, A., Dieterich, D. C., Rosenblum, K. NMDAR-dependent proteasome activity in the gustatory cortex is necessary for conditioned taste aversion.Neurobiology of Learning and Memory. 130, 7-16 (2016).
  21. Orsi, S. A., et al.Distinct subcellular changes in proteasome activity and linkage-specific protein polyubiquitination in the amygdala during the consolidation and reconsolidation of a fear memory.Neurobiology of Learning and Memory. 157, 1-11 (2019).
  22. Cullen, P. K., Ferrara, N. C., Pullins, S. E., Helmstetter, F. J. Context memory formation requires activity-dependent protein degradation in the hippocampus.Learning and memory. 24, (11), 589-596 (2017).
  23. Jarome, T. J., Ferrara, N. C., Kwapis, J. L., Helmstetter, F. J. CaMKII regulates proteasome phosphorylation and activity and promotes memory destabilization following retrieval.Neurobiology of Learning and Memory. 128, 103-109 (2016).
  24. Lee, S. H., et al.Synaptic protein degradation underlies destabilization of retrieved fear memory.Science. 319, (5867), 1253-1256 (2008).
  25. Dunah, A. W., Standaert, D. G. Dopamine D1 receptor-dependent trafficking of striatal NMDA glutamate receptors to the postsynaptic membrane.Journal of Neuroscience. 21, (15), 5546-5558 (2001).
  26. Kelly, P.T., Cotman, C.W. Synaptic proteins. Characterization of tubulin and actin and identification of a distinct postsynaptic density polypeptide.Journal of Cell Biology. 79, (1), 173-183 (1978).
  27. Mengual, E., Arizti, P., Rodrigo, J., Gimenez-Amaya, J.M., Castano, J.G. Immunohistochemical distribution and electron microscopic subcellular localization of the proteasome in the rat CNS.Journal of Neuroscience. 16, (20), 6331-6341 (1996).