The effect of acetaminophen on ubiquitin homeostasis in Saccharomyces cerevisiae

Abstract

Acetaminophen (APAP), although considered a safe drug, is one of the major causes of acute liver failure by overdose, and therapeutic chronic use can cause serious health problems. Although the reactive APAP metabolite N-acetyl-p-benzoquinoneimine (NAPQI) is clearly linked to liver toxicity, toxicity of APAP is also found without drug metabolism of APAP to NAPQI. To get more insight into mechanisms of APAP toxicity, a genome-wide screen in Saccharomyces cerevisiae for APAP-resistant deletion strains was performed. In this screen we identified genes related to the DNA damage response. Next, we investigated the link between genotype and APAP-induced toxicity or resistance by performing a more detailed screen with a library containing mutants of 1522 genes related to nuclear processes, like DNA repair and chromatin remodelling. We identified 233 strains that had an altered growth rate relative to wild type, of which 107 showed increased resistance to APAP and 126 showed increased sensitivity. Gene Ontology analysis identified ubiquitin homeostasis, regulation of transcription of RNA polymerase II genes, and the mitochondria-to-nucleus signalling pathway to be associated with APAP resistance, while histone exchange and modification, and vesicular transport were connected to APAP sensitivity. Indeed, we observed a link between ubiquitin levels and APAP resistance, whereby ubiquitin deficiency conferred resistance to APAP toxicity while ubiquitin overexpression resulted in sensitivity. The toxicity profile of various chemicals, APAP, and its positional isomer AMAP on a series of deletion strains with ubiquitin deficiency showed a unique resistance pattern for APAP. Furthermore, exposure to APAP increased the level of free ubiquitin and influenced the ubiquitination of proteins. Together, these results uncover a role for ubiquitin homeostasis in APAP-induced toxicity.

Fig 1. Deletion of DDR genes MMS2, UBC13, and RFX1 confers resistance to APAP.

Fivefold serial dilutions of a cell suspension of the mutant strains with optical density OD₆₀₀ = 0.05 were plated on YPD plates containing 0, 50 and 75 mM APAP and incubated for one (control) and three days at 37°C. After three days, the 75 mM plate was transferred to room temperature (RT) for 6 days. The strains used were WT (BY4741), Δmms2, Δubc13, Δrad5 and Δrfx1.

Fig 2. APAP resistance of ubiquitin deficient strains.

Five-fold dilution of WT (BY4741), Δubi4, Δdoa1, Δubp6 and Δdoa4 cells were spotted on YPD plates with or without 70 mM APAP and grown at 37°C for three and four days, respectively.

Fig 3. Ubiquitin overexpression confers APAP sensitivity.

Strains WT, Δmms2, Δubc13, Δubi4, Δubp6 and Δdoa1 were transformed with a multicopy plasmid expressing a ubiquitin gene from the UBI4 promoter and an empty plasmid as a control. A spotting assay was performed on YPD plates containing 70, 80 and 90 mM APAP (A). A spotting assay performed with WT cells on YPD plates containing 50 and 60 mM APAP (B). YPD plates without APAP were the control. The plates were incubated at 37°C for 3 days for APAP containing plates and 2 days for the control plate. All strains exhibited higher sensitivity to APAP upon ubiquitin overexpression.

Fig 4. APAP affects levels of free ubiquitin in yeast.

The WT, Δubi4, Δdoa1 and Δubp6 cells were grown exponentially and treated with 0, 25 or 50 mM APAP for 2 hrs at 37°C before harvesting. 4A) Western blot with antibodies against ubiquitin. 4B) Western blot with anti-actin antibodies was used as an internal control for loading.

Fig 5. Ubiquitin deficient strains are uniquely APAP resistant and sensitive for a variety of chemicals.

The individual dots represent five-fold dilution of the cells: they were spotted on YPD plates with and without the chemicals. The overall conditions were: 70 mM APAP, 90 mM AMAP, 4 mM quinine and 400 μg/μl rapamycin all grown for 5 days; 1 μg/ml cadmium, 0.1 mM arsenic (III)-oxide, 50 mM H₂O₂, 0.25 μM cycloheximide (CHX), 30μg/ml benomyl, 0.01% MMS and 15 μM FTY20, all grown for three days; and 2.5 mM ibuprofen grown for 6 days. The plates were incubated at 37°C.

Fig 6. Model to illustrate the effect of APAP on various cellular processes through ubiquitination.

In WT cells, the APAP-induced changes in ubiquitination lead to a growth arrest, while in ubiquitin-depleted mutants growth arrest is prevented and cells become resistant.