Combined chemical and genetic approach to inhibit proteolysis by the proteasome

Abstract

Regulated protein destruction by the proteasome is crucial for the maintenance of normal cellular homeostasis. Much of our understanding of proteasome function stems from the use of drugs that inhibit its activity. Curiously, despite the importance of proteasomal proteolysis, previous studies have found that proliferation of the yeast Saccharomyces cerevisiae is relatively resistant to the effects of proteasome inhibitors such as MG132, even in the presence of mutations that increase inhibitor levels in cells. We reasoned that part of the resistance of S. cerevisiae to proteasome inhibitors stems from the fact that most proteasome inhibitors preferentially target the chymotryptic activity of the proteasome, and that the caspase-like and tryptic sites within the 20S core could compensate for proteasome function under these conditions. To test this hypothesis, we generated a strain of yeast in which the gene encoding the drug efflux pump Pdr5 is deleted, and the tryptic and caspase-like proteasome activities are inactivated by mutation. We find that this strain has dramatically increased sensitivity to the proteasome inhibitor MG132. Under these conditions, treatment of yeast with MG132 blocks progression through the cell cycle, increases the accumulation of polyubiquitylated proteins and decreases the ability to induce transcription of certain genes. These results highlight the contribution of the caspase-like and tryptic activities of the proteasome to its function, and provide a strategy to potently block proteasomal proteolysis in yeast that has practical applications.

Similar temporal and spatial recruitment of native 19S and 20S proteasome subunits to transcriptionally active chromatin

“…Support for APIS comes from genetic and biochemical evidence tying 19S base components to transcriptional activation domains (1,2), and from results of chromatin immunoprecipitation (ChIP) experiments showing that 19S base proteins are recruited to activated genes separately from 20S core components (1, 3, 4). These observations, however, are countered by reports that the proteolytic activity of the proteasome is important for transcriptional activation in yeast (5,6) and for repression of cryptic transcription in mammalian cells (7), and by the finding that 20S proteins also associate with active chromatin by ChIP, albeit with different temporal and spatial distributions than 19S proteins (1,8). Disparate observations on the involvement of 19S versus 26S proteasomes in transcription make it difficult to arrive at a unified view of how proteasome components interact with chromatin.…”

“…1A), but whether 20S function is required for this process is controversial. Recently, we learned that the proteasome inhibitor MG132 is only partially effective in yeast because the tryptic-and caspase-like sites of the proteasome compensate for chemical inhibition of the chymotryptic site by MG132 (6). We therefore used a chemical-genetic strategy in which MG132 is combined with inactivating point mutations in the tryptic-(PUP1) and caspase-(PRE3) like sites to effect rapid and comprehensive inhibition of proteasome function (6, 13) (Fig.…”

Proteolytic Degradation of the Yap1 Transcription Factor Is Regulated by Subcellular Localization and the E3 Ubiquitin Ligase Not4

“…To determine whether the proteasome was similarly required for Yap1 degradation, a cycloheximide chase experiment was carried out in the presence or absence of the proteasome inhibitor MG132. These experiments were performed in a strain lacking the Pdr5 ABC transporter protein as this genetic background has been found to facilitate use of MG132, likely through increasing the uptake of this drug (29). MG132 was added to one culture prior to beginning the cycloheximide chase assay.…”

Non-repair Pathways for Minimizing Protein Isoaspartyl Damage in the Yeast Saccharomyces cerevisiae

“…Cell pellets were stored at Ϫ80°C until lysis. To analyze the effect of combining proteasome mutations with chemical inhibition, 5-ml overnight cultures of PUP1PRE3pdr5⌬ and pup1pre3pdr5⌬ cells (obtained as a gift from the William Tansey laboratory at Vanderbilt University) (48) were prepared in YPAD (YPD with 0.002% adenine hemisulfate (Sigma, catalog number A9126)) and used to inoculate 30 ml of YPAD with or without 50 M MG132. The cells were cultured for 8 (to 0.6 -0.8 A 600 ) or 24 h (to Ͼ5 A 600 ) before centrifugation at 2,000 ϫ g for 5 min at 4°C.…”

“…6A). Recent studies have shown that although the inhibition of proteasome activity by MG132 targets mainly the chymotryptic activity of the proteasome the tryptic and caspase-like activities imparted by the Pup1 and Pre3 proteases are still present (48). Therefore, we treated pup1pre3pdr5⌬ S. cerevisiae with MG132 to see whether there was an increase in isoaspartyl damage.…”