Sporidesmin Induced Inactivation of Thioltransferase and Modification of Cellular Protein Thiols

Abstract

The mycotoxin sporidesmin contains a disulfide-bridged ring that is responsible for cytopathic activity. Sporidesmin toxicity may be mediated by a cyclic conversion of the disulfide and dithiol that produces superoxide anion and other cytotoxic oxygen species or by covalent modification of intracellular protein thiols resulting in altered protein activity. TTase is a cytosolic enzyme that belongs to the TDOR family of enzymes. TTase catalyses reversible thiol-disulfide exchange reactions in the presence of GSH and is involved in the maintenance of the cellular redox status. The effect of sporidesmin on purified recombinant human TTase, HepG2 thiols and HepG2 protein thiol reactivity was investigated. Sporidesmin (m/z 474) covalently modified TTase (m/z 11,645) in vitro to form a TTase-sporidesmin adduct (m/z 12,054) that resulted in loss of 2 sulfurs (-64), one each from TTase and sporidesmin. Adduct formation involved C22 and at least one of C25, C78, and C82 residues. The single sulfur loss from TTase and sporidesmin during adduct formation may result from the interaction of sporidesmin with TTase but might also be affected by sulfur elimination during MALDI. Sporidesmin exposure resulted in a general decrease in TCA precipitated HepG2 protein thiols possibly due to mixed disulfide formation. Sporidesmin caused an increase in TTase activity in HepG2 cells since sporidesmin is a substrate of TTase in the presence of GSH. A decrease in TTase activity occurred in BSO treated cells after sporidesmin exposure. GSH (m/z 308) convalently modified TTase to form an adduct (11,952) that did not involve sulfur loss. GSH protected TTase from sporidesmin induced modification. HepG2 proteins that showed altered thiol accessibility to IAA after sporidesmin exposure included chaperones (PDI, TCP, GRP78), TDOR enzymes (PDI) and cytoskeletal proteins (ezrin, merlin and moesin). An increase in thiol accessibility (eg PDI, GST, TCP, moesin) may indicate reduction of the disulfide forms of the proteins while a decrease (ezrin and merlin) may indicate formation of protein-S-S-sporidesmin mixed disulfides. The mechanism of sporidesmin induced cytotoxicity and the specific intracellular targets of sporidesmin are not known. Sporidesmin covalently modified TTase in vitro, inactivated TTase in the absence of GSH in vivo and altered the thiol accessibility of certain HepG2 cellular proteins. These observations suggest that sporidesmin targets TDOR enzymes (PDI, TTase) as well as other cellular proteins and may exert its effects both by disruption of the cellular redox status and as well as through mixed disulfide formation that may result in aberrant protein function.