In vivo tumor growth inhibition by Solanum tuberosum aspartic protease 3 (StAP3) treatment.

Solanum tuberosum aspartic Proteases (StAPs) show selective plasma membrane permeabilization, inducing cytotoxicity of cancer cells versus normal cells in vitro. Herein, we aimed to evaluate both StAP3 systemic toxicity and antitumoral activity against human melanoma in vivo. The toxicity of a single high dose of StAP3 (10 µg/g body weight, intraperitoneally) was assessed in a Balb/c mice model. Subcutaneous A375 human melanoma xenografts in athymic nude (nu/nu) mice were induced. Once tumors developed (mean larger dimension = 3.8 ± 0.09 mm), mice were StAP3-treated (6 µg/g body weight, subcutaneously under the tumor at a single dose). For both models, controls were treated with physiologic saline solution. StAP3-treated mice showed a significant inhibition of tumor growth (p < 0.05) compared with controls. No signs of toxicity were detected in StAP3-treated mice in both models. These results suggest the potential of these plant proteases as anticancer agents.

The swaposin-like domain of potato aspartic protease (StAsp-PSI) exerts antimicrobial activity on plant and human pathogens.

Plant-specific insert domain (PSI) is a region of approximately 100 amino acid residues present in most plant aspartic protease (AP) precursors. PSI is not a true saposin domain; it is the exchange of the N- and C-terminal portions of the saposin like domain. Hence, PSI is called a swaposin domain. Here, we report the cloned, heterologous expression and purification of PSI from StAsp 1 (Solanum tuberosum aspartic protease 1), called StAsp-PSI. Results obtained here show that StAsp-PSI is able to kill spores of two potato pathogens in a dose-dependent manner without any deleterious effect on plant cells. As reported for StAPs (S. tuberosum aspartic proteases), the StAsp-PSI ability to kill microbial pathogens is dependent on the direct interaction of the protein with the microbial cell wall/or membrane, leading to increased permeability and lysis. Additionally, we demonstrated that, like proteins of the SAPLIP family, StAsp-PSI and StAPs are cytotoxic to Gram-negative and Gram-positive bacteria in a dose dependent manner. The amino acid residues conserved in SP_B (pulmonary surfactant protein B) and StAsp-PSI could explain the cytotoxic activity exerted by StAsp-PSI and StAPs against Gram-positive bacteria. These results and data previously reported suggest that the presence of the PSI domain in mature StAPs could be related to their antimicrobial activity.

Roles of glycosylation on the antifungal activity and apoplast accumulation of StAPs (Solanum tuberosum aspartic proteases).

Specific roles of glycosylation appear to be protein-dependent. Plant aspartic proteases (APs) contain two or more consensus N-glycosylation sites; however, the importance of them is not well understood. StAPs (Solanum tuberosum aspartic proteases) are bifunctional proteins with both proteolytic and antimicrobial activities. These proteins are accumulated into the intercellular washing fluid of potato tubers and leaves after wounding or infection. In this paper we investigated the importance of glycosylation on the StAPs apoplast accumulation, biochemical parameters, and fungicidal activity. Assays to evaluate the importance of StAPs glycosylation groups by using glycosylation inhibitors demonstrate that carbohydrate portions are essential to StAPs accumulation into the apoplast of tubers and leaves after wounding or detachment, respectively. Bifunctional activity of StAPs is differentially affected by this post-translational modification. Results obtained show that not significant changes were produced in the physicochemical properties after StAPs deglycosylation (pH and thermal-optimum activity and index of protein surface hydrophobicity). Otherwise, StAPs antifungal activity is affected by deglycosylation. Deglycosylated StAPs (dgStAPs) fungicidal activity is lower than native StAPs at all concentrations and times assayed. In summary, glycosylation has not a significant role on the StAPs conformational structure. However, it is involved in the StAPs subcellular accumulation and antifungal activity suggesting that it could be necessary for StAPs membrane and/or protein interactions and subsequently its biological function(s).

Antimicrobial activity of potato aspartic proteases (StAPs) involves membrane permeabilization.

Solanum tuberosum aspartic proteases (StAPs) with antimicrobial activity are induced after abiotic and biotic stress. In this study the ability of StAPs to produce a direct antimicrobial effect was investigated. Viability assays demonstrated that StAPs are able to kill spores of Fusarium solani and Phytophthora infestans in a dose-dependent manner. Localization experiments with FITC-labelled StAPs proved that the proteins interact directly with the surface of spores and hyphae of F. solani and P. infestans. Moreover, incubation of spores and hyphae with StAPs resulted in membrane permeabilization, as shown by the uptake of the fluorescent dye SYTOX Green. It is concluded that the antimicrobial effect of StAPs against F. solani and P. infestans is caused by a direct interaction with the microbial surfaces followed by membrane permeabilization.