S-Mercuration of rat sorbitol dehydrogenase by methylmercury causes its aggregation and the release of the zinc ion from the active site

Abstract

We previously developed a screening method to identify proteins that undergo aggregation through S-mercuration by methylmercury (MeHg) and found that rat arginase I is a target protein for MeHg (Kanda et al. in Arch Toxicol 82:803-808, 2008). In the present study, we characterized another S-mercurated protein from a rat hepatic preparation that has a subunit mass of 42 kDa, thereby facilitating its aggregation. Two-dimensional SDS-polyacrylamide gel electrophoresis and subsequent peptide mass fingerprinting using matrix-assisted laser desorption and ionization time-of-flight mass spectrometry revealed that the 42 kDa protein was NAD-dependent sorbitol dehydrogenase (SDH). With recombinant rat SDH, we found that MeHg is covalently bound to SDH through Cys44, Cys119, Cys129 and Cys164, resulting in the inhibition of its catalytic activity, release of zinc ions and facilitates protein aggregation. Mutation analysis indicated that Cys44, which ligates the active site zinc atom, and Cys129 play a crucial role in the MeHg-mediated aggregation of SDH. Pretreatment with the cofactor NAD, but not NADP or FAD, markedly prevented aggregation of SDH. Such a protective effect of NAD on the aggregation of SDH caused by MeHg is discussed.

<i>S</i>-Mercuration of cellular proteins by methylmercury and its toxicological implications

“…In the experiments that we investigated the effect of MeHg on hepatic arginase I described above, we coincidentally detected other proteins in the same hepatic preparations that also readily underwent covalent modifi cation by MeHg and subsequently became aggregated (Kanda et al, 2012). The rat hepatic preparation was incubated with different concentrations of MeHg (1-200 μM) at 37°C for 1 hr, and then each mixture was centrifuged at 12,000 × g for 10 min.…”

“…The lane numbers 1, 2, 3, 4, and 5 correspond to MeHg concentrations of 0, 1, 10, 100, and 200 μM, respectively. Reprinted from Kanda et al, 2012. The assay was used to detect BPM-modifi ed thiols in proteins that had been exposed to MeHg.…”

The Role of the Keap1/Nrf2 Pathway in the Cellular Response to Methylmercury

“…Two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis was used to separate the protein, and it was identified by peptide mass fingerprinting using matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF/MS). This 42 kDa protein was identified as sorbitol dehydrogenase (SDH) [19]. Using recombinant rat SDH possessing 10 cysteine residues in a subunit [20], we found that MeHg was covalently bound to SDH through Cys44, Cys119, Cys129, and Cys164, resulting in the inhibition of its catalytic activity and the release of zinc ions, facilitating protein aggregation [19].…”

<i>S</i>-Mercuration of ubiquitin carboxyl-terminal hydrolase L1 through Cys152 by methylmercury causes inhibition of its catalytic activity and reduction of monoubiquitin levels in SH-SY5Y cells

“…High levels of MeHg target proteins such as tubulin and a disintegrin and metalloproteinases (Imura et al, 1980;Wasteneys et al, 1988;Tamm et al, 2008). We also found that MeHg inactivates neuronal nitric oxide synthase, sorbitol dehydrogenase and arginase-I through S-mercuration of these proteins (Shinyashiki et al, 1998;Kanda et al, 2012Kanda et al, , 2008. We therefore hypothesize that an approach to detect electrophilic modification of proteins by MeHg is required for understanding the toxicity or health effects of MeHg.…”