Biological action of two tomato cysteine-rich miniproteins

Natural products are known since the past as disease remedies. In recent years, natural products have received great attention for disease prevention owing to their various health benefits, including low toxicity and diminished side effects. Cystine-knot miniproteins (CKMs) are members of a large family of small cysteine-rich proteins widespread in plant and animal species. CKMs display a broad spectrum of therapeutically useful natural biological activities and several CKMs are marketed as therapeutics or are in clinical development. CKMs typically consist of less than 50 amino acids and have a unique three-dimensional structure characterized by three intra-molecular disulfide bonds forming a cystine-knot and a small tripled stranded beta-sheet. The cystine-knot acts as structural scaffold for the protein conferring to the cystine-knot miniproteins a compact and remarkably stable structure towards extreme pH, chemical and thermal denaturation, and proteolytic attack. This scaffold was first discovered in 1982 in a carboxypeptidase inhibitor from potato (PCI). PCI inhibits mammalian pancreatic carboxypeptidase and can act as antithrombotic agent. Moreover, it has been shown that PCI also function as inhibitor of pancreatic adenocarcinoma cell lines growth acting as antagonist of the human epidermal growth factor by binding to the epidermal growth factor receptor (EGFR). The activity of the EGFR, that is a tyrosine kinase receptor of the ErbB family, is abnormally elevated in most human solid tumors and has been associated with progression and poor prognosis. Nowadays, it has become clear that the EGFR signaling pathway also plays a key role in angiogenesis, the formation of new blood vessels from pre-existing vessels to form capillary networks. Angiogenesis is a fundamental event of physiological processes, like organogenesis, wound healing and muscle growth, but it also takes place in pathological situations and promotes tumor growth and progression, metastasis as well as diabetic retinopathy and psoriasis. Therefore, anti-angiogenic treatment is considered a very effective strategy in cancer therapy and inhibition of EGFR seems to represent a novel and promising approach. This research aims to investigate the biological action on human cells of two tomato miniproteins which are normal components of our diet. The motivation for this research stems from the discovery that two tomato cystine-knot miniproteins (TCMPs), one expressed in the flower (TCMP-1) and the other in the mature fruit (TCMP-2), are similar in sequence and structure to a potato carboxypeptidase inhibitor (PCI) that acts as EGF antagonist. Since EGF is one of the signalling molecules participating in the formation of new vessels, we have investigated whether, TCMP-1 and TCMP-2, that can bind to the surface of human cells, could exert anti-angiogenic activity with limited cell toxicity. We have demonstrated that tomato cystine-knot carboxypeptidase inhibitors both native and recombinant, possess anti-angiogenic activity in vitro. The tomato miniproteins act as an anti-angiogenic factor in the nanomolar concentration range, which is of particular biological relevance. Furthermore, our experiments have proved that the two miniproteins do not exert toxic effects on endothelial cell proliferation and viability. Their anti-angiogenic activity is associated with an inhibitory effect on ERK1/2 activation, suggesting that TCMPs interfere with MAPK signaling pathway. Since tomato cystine-knot carboxypeptidase inhibitors are present in edible fruits, these compounds should be endowed with low toxicity when tested in animal models of disease and in humans. Tumorigenic angiogenesis could represent a target for therapeutic intervention with tomato miniproteins, and their anti-angiogenic activity could be tested as well for the prevention of other diseases. On these bases, miniproteins from tomato may be of potential pharmacological interest and deserve further investigation.