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

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]