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 ....

Protocol

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.......

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.......

Materials

Name	Company	Catalog Number	Comments
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