Quantifying Subcellular Ubiquitin-proteasome Activity in the Rodent Brain
Summary
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.
Abstract
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.
Introduction
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 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.
Protocol
Representative Results
Discussion
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…
Declarações
The authors have nothing to disclose.
Acknowledgements
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.
Materials
| 0.5M EDTA | Fisher | 15575020 | Various other vendors |
| 20S Proteasome Activity Kit | Millipore Sigma | APT280 | Other vendors carry different versions |
| ATP | Fisher | FERR1441 | Various other vendors |
| Beta-actin antibody | Cell signaling | 4967S | Various other vendors |
| Beta-tubulin antibody | Cell signaling | 2128T | Various other vendors |
| BioTek Synergy H1 plate reader | BioTek | VATECHH1MT3 | Other vendors carry different versions |
| B-mercaptoethanol | Fisher | ICN19024280 | Various other vendors |
| clasto lactacystin b-lactone | Millipore Sigma | L7035 | Various other vendors |
| Cryogenic cup | Fisher | 033377B | Various other vendors |
| DMSO | DMSO | D8418 | Varous other vendors |
| DTT | Millipore Sigma | D0632 | Various other vendors |
| Glycerol | Millipore Sigma | G5516 | Various other vendors |
| H3 antibody | Abcam | ab1791 | Various other vendors |
| HEPES | Millipore Sigma | H3375 | Various other vendors |
| Hydrochloric acid | Fisher | SA48 | Various other vendors |
| IGEPAL (NP-40) | Millipore Sigma | I3021 | Various other vendors |
| K48 Ubiquitin Antibody | Abcam | ab140601 | Various other vendors |
| K63 Ubiquitin Antibody | Abcam | ab179434 | Various other vendors |
| KCl | Millipore Sigma | P9541 | Various other vendors |
| KONTES tissue grinder | VWR | KT885300-0002 | Various other vendors |
| Laemmli sample buffer | Bio-rad | 161-0737 | Various other vendors |
| Linear Ubiquitin Antibody | Life Sensors | AB-0130-0100 | Only M1 antibody |
| MgCl | Millipore Sigma | 442611 | Various other vendors |
| Microcentrifuge | Eppendorf | 2231000213 | Various other manufacturers/models |
| myr-AIP | Enzo Life Sciences | BML-P212-0500 | Carried by Millipore-Sigma |
| NaCl | Millipore Sigma | S3014 | Various other vendors |
| Odyssey Fc Imaging System | LiCor | 2800-02 | Other vendors carry different versions |
| Phosphatase Inhibitor | Millipore Sigma | 524625 | Various other vendors |
| Precision Plus Protein Standard | Bio-rad | 161-0373 | Various other vendors |
| Protease Inhibitor | Millipore Sigma | P8340 | Various other vendors |
| PSD95 antibody | Cell signaling | 3450T | Various other vendors |
| SDS | Millipore Sigma | L3771 | Various other vendors |
| Sodium hydroxide | Fisher | SS255 | Various other vendors |
| Sucrose | Millipore Sigma | S0389 | Various other vendors |
| TBS | Alfa Aesar | J62938 | Varous other vendors |
| Tris | Millipore Sigma | T1503 | Various other vendors |
| Tween-20 | Fisher | BP337-100 | Various other vendors |
| Ubiquitin Antibody | Enzo Life Sciences | BML-PW8810 | Various other vendors |
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