Short-lived green fluorescent proteins for quantifying ubiquitin/proteasome-dependent proteolysis in living cells

Abstract

The ubiquitin/proteasome-dependent proteolytic pathway is an attractive target for therapeutics because of its critical involvement in cell cycle progression and antigen presentation. However, dissection of the pathway and development of modulators are hampered by the complexity of the system and the lack of easily detectable authentic substrates. We have developed a convenient reporter system by producing N-end rule and ubiquitin fusion degradation (UFD)-targeted green fluorescent proteins that allow quantification of ubiquitin/proteasome-dependent proteolysis in living cells. Accumulation of these reporters serves as an early predictor of G2/M arrest and apoptosis in cells treated with proteasome inhibitors. Comparison of reporter accumulation and cleavage of fluorogenic substrates demonstrates that the rate-limiting chymotrypsin-like activity of the proteasome can be substantially curtailed without significant effect on ubiquitin-dependent proteolysis. These reporters provide a new powerful tool for elucidation of the ubiquitin/proteasome pathway and for high throughput screening of compounds that selectively modify proteolysis in vivo.

The arginylation branch of the N-end rule pathway positively regulates cellular autophagic flux and clearance of proteotoxic proteins

“…S9). Consistent with the inhibitory role of PCA on the autophagic flux, ectopically expressed Arg-HSPA5-GFP, generated from Ub-Arg-HSPA5-GFP, 46 was also significantly stabilized after PCA treatment, along with endogenous Arg-HSPA5, when examined with antiArg-HSPA5-specific antibodies 34 (Fig. 3C).…”

“…For example, Arg-GFP-which is generated from Ub-Arg-GFP fusion proteins 46 after translational cleavage of the Ub-Arg junction by deubiquitinating enzymes and therefore not a substrate of ATE1-was short-lived in WT and ate1 ¡/¡ MEFs but stable in ubr1 ¡/¡ ubr2 ¡/¡ MEFs, under normal conditions ( Fig. 2E to 2G).…”

Proteasomal degradation of human release factor eRF3a regulates translation termination complex formation

“…2B, lane GFP-3a-FRAA). As it has been shown that GFP is not a substrate for ubiquitin-proteasome-dependent proteolysis (Dantuma et al 2000), the target for polyubiquitination was likely the eRF3a moiety of GFP3a-FRAA. Of course, we cannot rule out the possibility that eRF3a forms containing mutations in the eRF1-binding site were misfolded and targeted to ubiquitin-proteasomedependent proteolysis.…”

Roles of heat shock factor 1 and 2 in response to proteasome inhibition: consequence on p53 stability

“…A Ubi-GFP was previously used to quantify ubiquitinproteasome-dependent proteolysis in living cells (Dantuma et al, 2000). For this purpose, the DNA fragment from the pDG268 plasmid (kindly provided by Dr D Gray, University of Ottawa, Ottawa, Ontario, Canada), encoding an ubiquitin-EGFP fusion protein (Tsirigotis et al, 2001), was transferred into the pCDNA3 expression vector (Invitrogen Life Science, Carlsbad, CA, USA).…”