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