Identification of small GTPases as potential target proteins of the mycotoxin and renal carcinogen ochratoxin A

Ochratoxin A (OTA), a mycotoxin commonly found as a contaminant in a variety of foods, is known for its ability to cause kidney damage and tumors in rodents. Recent research indicates that replicative stress leading to aberrant mitoses and subsequent genetic instability may play a key role in OTA carcinogenicity. However, the specific molecular targets of OTA and early key events leading to replicative stress and mitotic disruption remain to be determined. In this study, a chemoproteomic workflow was employed to identify proteins that directly interact with OTA and its non-chlorinated analog ochratoxin B (OTB). To this end, OTA and OTB were immobilized on a stationary phase through covalent coupling to amine-functionalized agarose beads via their carboxy group. OTA-and OTB-functionalized beads were then incubated with kidney epithelial cell lysates to capture binding proteins for subsequent analysis via tandem mass spectrometry. Protein mass spectrometry identified several members of the family of small GTPases as specific OTA- and OTB-binding proteins. Moreover, a 3D molecular modeling approach integrating docking and molecular dynamics simulations was applied to study the mycotoxin-protein complex stability over time, providing mechanistic insights from an atomistic point of view. Ras superfamily GTPases, which were previously demonstrated to be transcriptionally deregulated in the presence of OTA, play crucial roles in various cellular functions, including DNA replication, mitosis, protein transport and cell adhesion, thus offering plausible links to cellular effects observed in response to OTA. In summary, results from this study for the first time identify small GTPases as direct molecular targets of OTA and suggest a potential role of small GTPases in OTA toxicity.