alpha-chymotrypsin and sodium-arsenite

Sodium arsenite (NA) and cadmium chloride (CdCl(2)) are relatively abundant environmental toxicants that have multiple toxic effects including carcinogenesis, dysfunction of gene regulation and DNA and protein damage. In the present study, treatment of Xenopus laevis A6 kidney epithelial cells with concentrations of NA (20-30 μM) or CdCl(2) (100-200 μM) that induced HSP30 and HSP70 accumulation also produced an increase in the relative levels of ubiquitinated protein. Actin protein levels were unchanged in these experiments. In time course experiments, the levels of ubiquitinated protein and HSPs increased over a 24h exposure to NA or CdCl(2). Furthermore, treatment of cells with NA or CdCl(2) reduced the relative levels of proteasome chymotrypsin (CT)-like activity compared to control. Interestingly, pretreatment of cells with the HSP accumulation inhibitor, KNK437, prior to NA or CdCl(2) exposure decreased the relative levels of ubiquitinated protein as well as HSP30 and HSP70. A similar finding was made with ubiquitinated protein induced by proteasomal inhibitors, MG132 and celastrol, known to induce HSP accumulation in A6 cells. However, the NA- or CdCl(2)-induced decrease in proteasome CT-like activity was not altered by KNK437 pretreatment. This study has shown for the first time in poikilothermic vertebrates that NA and CdCl(2) can inhibit proteasomal activity and that there is a possible association between HSP accumulation and the mechanism of protein ubiquitination.

Topics: Animals; Arsenites; Benzhydryl Compounds; Cadmium Chloride; Cell Line; Chymotrypsin; Cysteine Proteinase Inhibitors; Dose-Response Relationship, Drug; Environmental Pollutants; Epithelial Cells; Heat-Shock Proteins; HSP30 Heat-Shock Proteins; HSP70 Heat-Shock Proteins; Immunoblotting; Kidney; Leupeptins; Pentacyclic Triterpenes; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Pyrrolidinones; Sodium Compounds; Time Factors; Triterpenes; Ubiquitinated Proteins; Ubiquitination; Xenopus laevis; Xenopus Proteins

Our previous studies with the thiol-specific reagent methyl methanethiolsulfonate (MMTS) and the vicinal dithiol-specific reagent sodium arsenite have established that 2 spatially close thiols (i.e. vicinal dithiols) are involved in steroid binding to the intact 98 K rat glucocorticoid receptor. These 2 thiols form an intramolecular disulfide after treatment with low concentrations of MMTS. One of these thiols was proposed to by Cys-656. In an effort to identify both thiols, we have examined the effects of MMTS and arsenite on proteolytic fragments of the receptor, which contain progressively fewer cysteines. MMTS and arsenite are now found to cause the same dithiothreitol-reversible inhibition of steroid binding and affinity labeling of both the 42 K chymotrypsin fragment and the 16 K steroid-binding core fragment of the receptor as was seen for the intact receptor. Characteristic responses include a bimodal inhibition curve for steroid binding after preincubation with MMTS and an inhibition of binding by very low concentrations of arsenite. Low concentrations of MMTS could block steroid binding by forming a disulfide bond between the receptor and a tightly associated, nonreceptor protein. However, no evidence for such cross-linking was observed when intact 98 K receptors, 42 K chymotrypsin fragments, or 16 K trypsin fragments were treated with various concentrations of MMTS, separated on nonreducing sodium dodecyl sulfate-polyacrylamide gels, and visualized by Western blotting with antiheat shock protein 90 or antireceptor antibodies. One of the antireceptor antibodies (aP1) that had been raised against the rat receptor sequence 440-795 was now found to recognize at least 1 epitope in the 16 K core fragment. We conclude that the vicinal dithiols involved in steroid binding are 2 of the 3 cysteines in the sequence of Thr537-Arg673.

Topics: Animals; Antibodies; Arsenic; Arsenites; Chemical Phenomena; Chemistry; Chymotrypsin; Disulfides; Methyl Methanesulfonate; Peptide Fragments; Rats; Receptors, Glucocorticoid; Sodium Compounds; Steroids; Sulfhydryl Compounds