Gi-Wook Hwang

A ubiquitin-proteasome system is responsible for the protection of yeast and human cells against methylmercury

The mechanism responsible for the toxic effects of methylmercury (MeHg), an important environmental pollutant, is poorly understood. We have identified a gene, CDC34, that confers resistance to MeHg in Saccharomyces cerevisiae by screening a yeast genomic DNA library. CDC34 encodes a ubiquitin‐conjugating enzyme, Cdc34, which is involved in ubiquitin‐dependent proteolysis. Overexpression of Cdc34 results in significant resistance to MeHg both in yeast and human cells, and it increases the cellular level of ubiquitinated proteins. The ubiquitinconjugating activity of Cdc34 is essential for the Cdc34‐mediated resistance to MeHg, and the protective effect of the overexpression of Cdc34 is depressed by inhibition of proteasome activity. Our results support the hypothesis that MeHg induces the cellular accumulation of a certain protein(s) that causes cell damage and that this protein(s) is degraded after its ubiquitination in proteasomes.

The ubiquitin-conjugating enzymes, Ubc4 and Cdc34, mediate cadmium resistance in budding yeast through different mechanisms

The expression of the genes encoding the ubiquitin-conjugating enzymes, Ubc4, Ubc5, and Ubc7, has been reported to be induced by cadmium in budding yeast. In contrast, we have reported that the overexpression of Cdc34, another ubiquitin-conjugating enzyme, confers resistance to cadmium. In the present study, we examined the effects of overexpression of Ubc4, Ubc5, or Ubc7 on the sensitivity of budding yeast to cadmium. We found that yeast cells that overexpressed Ubc4, but not Ubc5 or Ubc7, showed similar cadmium resistance as yeast cells that overexpressed Cdc34. The ubiquitination levels of cellular proteins were significantly increased by overexpression of Ubc4 as well as by Cdc34. As previously reported, yeast cells overexpressing Cdc34 were resistant to cadmium even in the presence of the proteasome inhibitor MG132. However, the acquired resistance to cadmium by overexpression of Ubc4 was not observed in the presence of MG132. Cdc34 overexpression has been shown to inactivate the transcriptional activity of Met4 by accelerating its ubiquitination and to reduce expression of the MET25 gene, a target gene of Met4. Unlike Cdc34, overexpression of Ubc4 did not affect the expression of the MET25 gene. These findings suggest that the mechanism of acquired resistance to cadmium by overexpression of Ubc4 is different from that of Cdc34 and that Ubc4 confers resistance to cadmium by ubiquitination of proteins other than Met4 and accelerates the degradation of these proteins in the proteasomes.

Chronotoxicity of bromobenzene-induced hepatic injury in mice

The aim of the present study is to investigate whether or not bromobenzene (BB) toxicity varies with circadian periodicity. Seven-week-old male ICR mice were injected with 900 mg/kg (5.73 mmol/kg) BB intraperitoneally at 4 different time points of a day (zeitgeber time [ZT]: ZT0, ZT6, ZT12, and ZT18). Mortality was then monitored for 7 days after injection. Interestingly, mice were sensitive to BB acute toxicity at ZT6 while tolerant at ZT18. Moreover, in mice that were given a non-lethal dose of BB (540 mg (3.44 mmol)/kg), levels of alanine aminotransferase and aspartate aminotransferase, used as markers of hepatic injury, markedly increased in response to injection at ZT6, but did not increase significantly in response to injection at ZT18. In contrast, the markers of renal injury (creatinine and blood urea nitrogen), showed no significant difference in response to the two injection times. To further investigate this extreme circadian variation, we examined hepatic and renal lipid peroxidation levels, and conducted histopathological studies. Similar to our observation with alanine aminotransferase and creatinine, hepatic lipid peroxidation and histopathological changes were more pronounced than renal changes, and showed circadian variation. Our present investigation demonstrated that BB-induced mortality had clear circadian variation, and suggested that hepatic injury was one of the important factors for determination of this variation.

Methylmercury induces the expression of TNF-α selectively in the brain of mice.

Methylmercury selectively damages the central nervous system (CNS). The tumor necrosis factor (TNF) superfamily includes representative cytokines that participate in the inflammatory response as well as cell survival, and apoptosis. In this study, we found that administration of methylmercury selectively induced TNF-α expression in the brain of mice. Although the accumulated mercury concentration in the liver and kidneys was greater than in the brain, TNF-α expression was induced to a greater extent in brain. Thus, it is possible that there may exist a selective mechanism by which methylmercury induces TNF-α expression in the brain. We also found that TNF-α expression was induced by methylmercury in C17.2 cells (mouse neural stem cells) and NF-κB may participate as a transcription factor in that induction. Further, we showed that the addition of TNF-α antagonist (WP9QY) reduced the toxicity of methylmercury to C17.2 cells. In contrast, the addition of recombinant TNF-α to the culture medium decreased the cell viability. We suggest that TNF-α may play a part in the selective damage of the CNS by methylmercury. Furthermore, our results indicate that the higher TNF-α expression induced by methylmercury maybe the cause of cell death, as TNF-α binds to its receptor after being released extracellularly.

The protein transportation pathway from Golgi to vacuoles via endosomes plays a role in enhancement of methylmercury toxicity

Methylmercury causes serious damage to the central nervous system, but the molecular mechanisms of methylmercury toxicity are only marginally understood. In this study, we used a gene-deletion mutant library of budding yeast to conduct genome-wide screening for gene knockouts affecting the sensitivity of methylmercury toxicity. We successfully identified 31 genes whose deletions confer resistance to methylmercury in yeast, and 18 genes whose deletions confer hypersensitivity to methylmercury. Yeast genes whose deletions conferred resistance to methylmercury included many gene encoding factors involved in protein transport to vacuoles. Detailed examination of the relationship between the factors involved in this transport system and methylmercury toxicity revealed that mutants with loss of the factors involved in the transportation pathway from the trans-Golgi network (TGN) to the endosome, protein uptake into the endosome, and endosome-vacuole fusion showed higher methylmercury resistance than did wild-type yeast. The results of our genetic engineering study suggest that this vesicle transport system (proteins moving from the TGN to vacuole via endosome) is responsible for enhancing methylmercury toxicity due to the interrelationship between the pathways. There is a possibility that there may be proteins in the cell that enhance methylmercury toxicity through the protein transport system.

Identification of substrates of F-box protein involved in methylmercury toxicity in yeast cells.

We previously reported that some of the substrate proteins recognized by Hrt3 or Ucc1, a component of Skp1/Cdc53/F‐box protein ubiquitin ligase, may include proteins that are involved in the methylmercury toxicity and degraded by the proteasome. In this study, we found that Dld3 and Grs1 bound to Hrt3 and that Eno2 bound to Ucc1 using a yeast two‐hybrid screening. We demonstrated that Dld3 and Grs1 are substrates that are ubiquitinated by Hrt3, and Eno2 is a substrate that is ubiquitinated by Ucc1. Moreover, any yeast showing overexpression of Dld3, Grs1, and Eno2 demonstrated higher methylmercury sensitivity. This indicates that Hrt3 and Ucc1 are involved in alleviating the methylmercury toxicity by promoting proteasomal degradation through the ubiquitination of Dld3, Grs1, and Eno2.

Transport of pyruvate into mitochondria is involved in methylmercury toxicity

We have previously demonstrated that the overexpression of enzymes involved in the production of pyruvate, enolase 2 (Eno2) and D-lactate dehydrogenase (Dld3) renders yeast highly sensitive to methylmercury and that the promotion of intracellular pyruvate synthesis may be involved in intensifying the toxicity of methylmercury. In the present study, we showed that the addition of pyruvate to culture media in non-toxic concentrations significantly enhanced the sensitivity of yeast and human neuroblastoma cells to methylmercury. The results also suggested that methylmercury promoted the transport of pyruvate into mitochondria and that the increased pyruvate concentrations in mitochondria were involved in intensifying the toxicity of methylmercury without pyruvate being converted to acetyl-CoA. Furthermore, in human neuroblastoma cells, methylmercury treatment alone decreased the mitochondrial membrane potential, and the addition of pyruvate led to a further significant decrease. In addition, treatment with N-acetylcysteine (an antioxidant) significantly alleviated the toxicity of methylmercury and significantly inhibited the intensification of methylmercury toxicity by pyruvate. Based on these data, we hypothesize that methylmercury exerts its toxicity by raising the level of pyruvate in mitochondria and that mitochondrial dysfunction and increased levels of reactive oxygen species are involved in the action of pyruvate.

Whi2 enhances methylmercury toxicity in yeast via inhibition of Akr1 palmitoyltransferase activity

BACKGROUND We have previously reported that Whi2 enhances the toxicity of methylmercury in yeast. In the present study we examined the proteins known to interact with Whi2 to find those that influence the toxicity of methylmercury. METHODS Gene disruption and site-directed mutagenesis were employed to examine the relationship of mercury toxicity and palmitoylation. Protein palmitoylation was examined using the acyl-biotinyl exchange method. Protein-protein interactions were detected by immunoprecipitation and immunoblotting. RESULTS We found that deletion of Akr1, a palmitoyltransferase, rendered yeast cells highly sensitive to methylmercury, and Akr1 is necessary for the methylmercury resistance of Whi2-deleted yeast. Palmitoyltransferase activity of Akr1 has an important role in the alleviation of methylmercury toxicity. Whi2 deletion or methylmercury treatment enhanced the palmitoyltransferase activity of Akr1, and methylmercury treatment reduced the binding between Akr1 and Whi2. CONCLUSIONS Whi2 bonds to Akr1 (a protein that is able to alleviate methylmercury toxicity) and thus inhibits Akr1s palmitoyltransferase activity, which leads to enhanced methylmercury toxicity. In contrast, methylmercury might break the bond between Whi2 and Akr1, which enhances the palmitoyltransferase activity of Akr1 to alleviate methylmercury toxicity. GENERAL SIGNIFICANCE This studys findings propose that the Whi2/Akr1 system can be regarded as a defense mechanism that detects methylmercury incorporation of yeast cells and alleviates its toxicity.

The Phospholipid:Diacylglycerol Acyltransferase Lro1 Is Responsible for Hepatitis C Virus Core-Induced Lipid Droplet Formation in a Yeast Model System.

Chronic infection with the hepatitis C virus frequently induces steatosis, which is a significant risk factor for liver pathogenesis. Steatosis is characterized by the accumulation of lipid droplets in hepatocytes. The structural protein core of the virus induces lipid droplet formation and localizes on the surface of the lipid droplets. However, the precise molecular mechanisms for the core-induced formation of lipid droplets remain elusive. Recently, we showed that the expression of the core protein in yeast as a model system could induce lipid droplet formation. In this study, we probed the cellular factors responsible for the formation of core-induced lipid-droplets in yeast cells. We demonstrated that one of the enzymes responsible for triglyceride synthesis, a phospholipid:diacylglycerol acyltransferase (Lro1), is required for the core-induced lipid droplet formation. While core proteins inhibit Lro1 degradation and alter Lro1 localization, the characteristic localization of Lro1 adjacent to the lipid droplets appeared to be responsible for the core-induced lipid droplet formation. RNA virus genomes have evolved using high mutation rates to maintain their ability to replicate. Our observations suggest a functional relationship between the core protein with hepatocytes and yeast cells. The possible interactions between core proteins and the endoplasmic reticulum membrane affect the mobilization of specific proteins.

Effects of long-term cadmium exposure on urinary metabolite profiles in mice

Cadmium (Cd) is a common environmental pollutant with known toxic effects on the kidney. Urinary metabolomics is a promising approach to study mechanism by which Cd-induced nephrotoxicity. The aim of this study was to elucidate the mechanism of Cd toxicity and to develop specific biomarkers by identifying urinary metabolic changes after a long-term of Cd exposure and with the critical concentration of Cd in the kidney. Urine samples were collected from wild-type 129/Sv mice after 67 weeks of 300 ppm Cd exposure and analyzed by ultra performance liquid chromatography connected with quadrupole time of flight mass spectrometer (UPLC-QTOF-MS) based metabolomics approach. A total of 40 most differentiated metabolites (9 down-regulated and 31 up-regulated) between the control and Cd-exposed group were identified. The majority of the regulated metabolites are amino acids (glutamine, L-aspartic acid, phenylalanine, tryptophan, and D-proline) indicating that amino acid metabolism pathways are affected by long-term exposure of Cd. However, there are also some nucleotides (guanosine, guanosine monophosphate, cyclic AMP, uridine), amino acid derivatives (homoserine, N-acetyl-L-aspartate, N-acetylglutamine, acetyl-phenylalanine, carboxymethyllysine), and peptides. Results of pathway analysis showed that the arginine and proline metabolism, purine metabolism, alanine, aspartate and glutamate metabolism, and aminoacyl-tRNA biosynthesis were affected compared to the control. This study demonstrates that metabolomics is useful to elucidate the metabolic responses and biological effects induced by Cd-exposure.