Biomedical Engineering Reference
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ubiquitinated-protein aggregates termed DALIS (dendritic cell
aggresome-like inducible structures) have also been observed in
mature dendritic cells from murine lymphatic tissue (
4
). Several
examples of pathological aggregation of ubiquitinated-proteins
characteristic of amyloid diseases have been reported including
Huntington's and Alzheimer's (
5, 6
). A model of ubiquitinated-
protein aggregate formation and clearance can be seen in Fig.
1
.
Paraffin-embedded tissues require deparaffinization and rehy-
dration treatment to remove the wax, and this is done chemically
with a series of xylene and ethanol washes. Following this, tissue
sections may undergo an antigen retrieval treatment in order to
visualize ubiquitinated-protein aggregates (
7
). Finally, a staining
procedure has been suggested that allows fluorescent visualiza-
tion of the aggregates using confocal microscopy. The micro-
scopic visualization of individual proteins localizing within the
cell is an important technique demonstrated in cell biology. Here,
we describe a protocol to process diabetic pancreatic tissue in
order to image ubiquitinated-protein aggregates within the cyto-
sol of individual cells. Observing these aggregates within tissue is
a significant step in characterizing this disease phenotype.
Mutation
ROS
Translation
ERAD
Ub-protein
Ub-protein
Ub-protein
Ub-protein
p62
Autophagy
Proteasome
Fig. 1. Model of ubiquitinated-protein aggregate formation during cellular stress. Tissues
are susceptible to many stresses during disease. Endoplasmic reticulum-association
degradation (ERAD) targets damaged and misfolded proteins for proteasomal degrada-
tion by tagging them with ubiquitin (
8
). Mutated proteins can also accumulate into ubiq-
uitinated aggregates (
6
). Reactive oxygen species (ROS) in the form of oxidative stress
causes the formation of ubiquitinated-protein aggregates in the cytosol of
b
-cells during
diabetes and plays a part in the accumulation of misfolded protein aggregates in neuro-
degenerative diseases (
3, 9
). It has also been documented that an increase in protein
transcription and translation in dendritic cells can cause newly synthesized protein to be
sequestered into aggregates where ubiquitination occurs (
4, 10
). Clearance may occur
by proteasomal degradation or by the autophagy pathway, which degrades these ubiq-
uitinated-protein aggregates by mediating their delivery to lysosomes (
3, 11-14
). Nbr1
and p62 adaptor proteins are thought to recruit autophagic machinery (
15, 16
).
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