We have demonstrated that the peptide L-2 designed from an alanine scanning of the Limulus-derived LALF32-51 region is a potential candidate for the anticancer therapy and its cell-penetrating capacity is an associated useful property. By the modification in the primary structure of L-2, a second-generation peptide (CIGB-552) was developed. However, the molecular mechanism underlying its cytotoxic activity remains partially unknown. In this study, it was shown that CIGB-552 increases the levels of COMMD1, a protein involved in copper homeostasis, sodium transport, and the NF-κB signaling pathway. We found that CIGB-552 induces ubiquitination of RelA and inhibits the antiapoptotic activity regulated by NF-κB, whereas the knockdown of COMMD1 blocks this effect. We also found that CIGB-552 decreases the antioxidant capacity and induces the peroxidation of proteins and lipids in the tumor cells. Altogether, this study provides new insights into the mechanism of action of the peptide CIGB-552, which could be relevant in the design of future anticancer therapies. 1. Introduction In previous work, a peptide-based approach was used to identify peptides devoid of LPS-binding capacity from LALF residues 32–51. Two peptides (L-2 and L-20) lost their ability to bind LPS and exhibited a differential cytotoxic activity, although a similar cell penetrating capacity was demonstrated for both peptides [1]. We introduced a chemical modification in the primary structure of the peptide L-2 to improve the biological activity and specificity. The chemical modification included the substitution of a natural amino acid residue by an unnatural amino acid (D-configuration) and blocked N-terminal by acylation. This modification led to the development of a second-generation peptide (CIGB-552) with increased cytotoxic activity on murine and human tumor cells [2]. Although the antitumor effects of the peptides involve an increase in the apoptosis and a negative regulation of cell-cycle progression, little is known regarding this mechanism of action. In this study, two complementary approaches were used: a yeast two-hybrid search for molecules that specifically interact with the peptides and a pull-down technique to validate the interaction. COMMD1 was identified as a peptide-binding protein. Furthermore, the specificity peptide/COMMD1 complexes were corroborated by related synthetic peptides with a differential cytotoxic activity (L-2, L-20, and CIGB-552) in cells expressing endogenous COMMD1. COMM domain—containing 1 (COMMD1), the first COMMD family member to be identified, is a
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