Examining Ubiquitinated Protein Aggregates in Tissue Sections - Protein Misfolding and Cellular Stress in Disease and Aging - page 167

Chapter 11
Examining Ubiquitinated Protein Aggregates in Tissue
Sections
Natalia A. Kaniuk and John H. Brumell
Abstract
In the cell, the binding of ubiquitin to abnormal or misfolded proteins marks them for degradation by
the proteasome or lysosome via autophagy. Ubiquitinated-protein aggregates form when an increase
in protein misfolding exceeds the degradation capacity of the cell. Many cellular stresses can cause an
increase in the amount of ubiquitinated misfolded protein and failure to eliminate these proteins can
disrupt cellular homeostasis and cause cellular toxicity. Ubiquitinated-protein aggregates accumulate
in the cytosol and can be detected in tissues of patients with a variety of diseases, including Alzheimer's,
Parkinson's, and Huntington's. Using a diabetic rat model, we have shown that ubiquitinated-protein
aggregates form in pancreatic beta cells during diabetes-induced oxidative stress. Aggregates were also
evident in the hippocampus, kidney, and liver of these animals. Our detailed protocol is provided here.
Mounted tissue sections were first deparaffinized, then boiled in sodium citrate buffer to expose the
antigen, followed by a specific staining procedure that allows for detection of ubiquitinated-protein
aggregates. The ability to visualize ubiquitinated-protein aggregates in tissue sections can provide further
understanding of the pathobiology of diseases associated with misfolded protein.
Key words: Ubiquitination, Immunofluorescence, Cellular stress, Aggregates, In vivo, Confocal
microscopy, Tissue
1. Introduction
In disease situations such as diabetes and bacterial infection,
cellular stress is particularly prevalent. As a result, the production
of misfolded protein is greatly increased and cellular mechanisms
that normally protect against this form of stress cannot keep up
( 1, 2 ). When the production of misfolded proteins exceeds deg-
radation, ubiquitinated-protein aggregates form and accumulate
in the cytosol of pancreatic cells during diabetes ( 3 ). Large cytosolic