Amblyomin‐X is a complex Kunitz‐type protease inhibitor from the tick Amblyomma sculptum , with selective pro‐apoptotic effects in tumor cells. While promising when applied exogenously, its large‐scale recombinant production is hindered by structural complexity. This study evaluated the feasibility of expressing Amblyomin‐X via a suicide gene therapy approach and investigated its intracellular fate following gene delivery in human melanoma and nontumoral cell lines. Amblyomin‐X transcripts were equally detected in both cell types; however, protein detection was restricted to nontumoral cells. Thus, we examined the mechanisms underlying Amblyomin‐X degradation in melanoma cells. Mutagenesis of predicted ubiquitination/SUMOylation sites failed to restore protein detection, whereas inhibition of proteasome activity with MG132 restored Amblyomin‐X detection. This confirmed the involvement of a proteasome‐dependent mechanism that limits protein stability and consequently its cytotoxic activity when intracellularly produced in melanoma cells. These findings indicate that proteasomal degradation represents a tumor‐specific barrier to the intracellular expression of Amblyomin‐X in human cancer cells. Addressing this limitation could enable the potent activity of Amblyomin‐X to be harnessed through gene‐based anticancer strategies. Furthermore, these findings advance the understanding of how structurally complex Kunitz‐type proteins with unstructured regions may behave in the context of gene therapy.
Cinel et al. (Thu,) studied this question.