Proteasome Inhibitors Cause Induction of Heat Shock Proteins and Trehalose, Which Together Confer Thermotolerance in Saccharomyces cerevisiae

Abstract

An accumulation in cells of unfolded proteins is believed to be the common signal triggering the induction of heat shock proteins (hsps). Accordingly, in Saccharomyces cerevisiae, inhibition of protein breakdown at 30°C with the proteasome inhibitor MG132 caused a coordinate induction of many heat shock proteins within 1 to 2 h. Concomitantly, MG132, at concentrations that had little or no effect on growth rate, caused a dramatic increase in the cells' resistance to very high temperature. The magnitude of this effect depended on the extent and duration of the inhibition of proteolysis. A similar induction of hsps and thermotolerance was seen with another proteasome inhibitor, clasto-lactacystin ␤-lactone, but not with an inhibitor of vacuolar proteases. Surprisingly, when the reversible inhibitor MG132 was removed, thermotolerance decreased rapidly, while synthesis of hsps continued to increase. In addition, exposure to MG132 and 37°C together had synergistic effects in promoting thermotolerance but did not increase hsp expression beyond that seen with either stimulus alone. Although thermotolerance did not correlate with hsp content, another thermoprotectant trehalose accumulated upon exposure of cells to MG132, and the cellular content of this disaccharide, unlike that of hsps, quickly decreased upon removal of MG132. Also, MG132 and 37°C had additive effects in causing trehalose accumulation. Thus, the resistance to heat induced by proteasome inhibitors is not just due to induction of hsps but also requires a short-lived metabolite, probably trehalose, which accumulates when proteolysis is reduced.

z-Leucinyl-Leucinyl-Norleucinal Induces Apoptosis of Human Glioblastoma Tumor–Initiating Cells by Proteasome Inhibition and Mitotic Arrest Response

“…The central role of proteasome inhibition in the activity of LLNle on GBM TICs is further supported by the observation that LLNle induces accumulation of poly-ubiquitinylated proteins that cannot be processed by the inhibited proteasome and by the induction of genes related to the unfolded protein response and endoplasmic reticulum stress (including HSPA1B). Similar results have been previously described in different cell types with proteasome inhibitors (35,36,(52)(53)(54). Furthermore, the close similarity of the chemical structure of LLNle with those of GM132 and N-acetyl-leucyl-leucyl-norleucinal, well-known proteasome inhibitors, should be underlined (55).…”

“…To investigate in more detail the effects of LLNle on the proteasome pathway in GBM TICs, we studied the expression of the Hsp70, a known marker of proteasome inhibition, by Western blot analysis (35,36). Our results show that Hsp70 expression is higher in PT1 cells treated with LLNle for 24 hours than Equal amount of total proteins were loaded in each lane, and after blotting, the membrane was stained using Ponceau Red to show integrity of total protein pattern and loading before challenging it with specific antibodies.…”

Complementary whole-genome technologies reveal the cellular response to proteasome inhibition by PS-341

“…Yeast total RNA was isolated by using Trizol (Invitrogen) according to the manufacturer's directions. Fifteen micrograms of total RNA and 1.5 g of oligo(dT) [12][13][14][15][16][17][18] were incubated with SuperScript II (Invitrogen) at 42°C for 1 h in the presence of 160 M each dATP, dGTP, and dTTP, 1.6 M dCTP, and 50 Ci ␣ 33 P-dCTP (2,000-4,000 Ci͞mmol). Labeled cDNA was purified (Micro Bio-Spin 6 column, Bio-Rad), treated with 50 mM NaOH at 68°C for 15 min, and split into separate tubes containing duplicate nylon arrays.…”

Proteasome Activity Is Required for Androgen Receptor Transcriptional Activity via Regulation of Androgen Receptor Nuclear Translocation and Interaction with Coregulators in Prostate Cancer Cells

“…The proteasome inhibitors, such as MG132, suppress the inflammatory response by blocking NF-B activation or induction of heat shock protein expression, which may allow cells to resist higher temperatures and other toxic agents as well as prevent leukemia cell apoptosis (13)(14)(15). In contrast, proteasome inhibitors can also induce cancer cell apoptosis, accompanied by activation of several caspases, such as caspase-3 or caspase-7 (16,17).…”