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Table 1 Assays used to study UPS function in polyglutamine expansion disorders. See text for further details.

From: The ubiquitin proteasome system in Huntington's disease and the spinocerebellar ataxias

Method Measures UPS component assayed Advantages Disadvantage Reference
Fluorogenic substrate peptides 20S proteasome activity Direct measure of chymotrypsin-like, trypsin-like or peptidyl-glutamyl activity of the 20S proteasome Quantitative analysis of proteolytic activity in cell and tissue lysates Does not measure ubiquitylation, substrate interaction, unfolding or effects on other components of the UPS other than proteasome activity [14, 15, 25, 30, 32, 33]
Degron-tagged fluorescent proteins Levels of fluorescent reporter protein tagged with a signal targeting it for proteasome degradation Proteasome activity and targeting to proteasome Functional analysis of UPS system in vivo Does not measure all aspects of UPS function [13, 14, 29, 37, 38]
Levels of endogenous UPS substrates Degradation of well characterised, endogenous UPS substrates e.g. p53 Ubiquitylation, proteasome activity, chaperones Functional analysis of entire UPS system. Substrate expressed at endogenous levels Levels of endogenous substrate may be altered due to effects of the polyglutamine expansion independent of the UPS [15]
Yeast two-hybrid assay Interaction of polyglutamine-containing proteins with UPS components Proteasome subunits and components that interact with polyglutamine proteins Shows direct interactions Does not give functional data [26-28]
In vitro assay of proteasome activity Effect of synthetic peptides, purifed aggregates and fibrillar species on activity of purified proteasomes Activity of purified proteasomes Shows direct effects of poly-glutamine containing proteins on proteasome activity Does not measure other components of the UPS [14, 23-25]