ABSTRACT
Both ribosome-inactivating proteins (RIPs) and plant proteinase inhibitors, belong to protein families known to regulate cellular homeostasis and likely involved in plant defense. Nevertheless the interest in these protein classes is due to their potential use for the treatment of several important human diseases such as cancer. Thus, in the present study, type 1 ribosome-inactivating protein and wheat subtilisin/chymotrypsin inhibitor, were engineered into a chimeric protein with cytotoxic action selective for murine tumor cells, while lacking any appreciable toxicity on murine normal cells. This chimeric protein selectively sensitizes to apoptotic death cells derived from Simian-virus-40-transformed mouse fibroblasts (SVT2 cells). The cytotoxicity of this new recombinant product has been detected also on three different human malignant cells. Therefore action on tumor cells of this protein could represent a potentially very attractive novel tool for anticancer drug design.
Subject(s)
Antineoplastic Agents/pharmacology , Plant Proteins/pharmacology , Recombinant Fusion Proteins/pharmacology , Ribosome Inactivating Proteins, Type 1/pharmacology , Serine Proteinase Inhibitors/pharmacology , Animals , Antineoplastic Agents/metabolism , Apoptosis/drug effects , Cell Line, Tumor , Cloning, Molecular , Humans , Mice , Phytolacca/genetics , Plant Proteins/genetics , Plant Proteins/metabolism , Protein Transport , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Ribosome Inactivating Proteins, Type 1/genetics , Ribosome Inactivating Proteins, Type 1/metabolism , Serine Proteinase Inhibitors/genetics , Serine Proteinase Inhibitors/metabolismABSTRACT
A cDNA encoding the proteinase inhibitor WSCI (wheat subtilisin/chymotrypsin inhibitor) was isolated by RT-PCR. Degenerate oligonucleotide primers were designed based on the amino acid sequence of WSCI and on the nucleotide sequence of the two homologous inhibitors (CI-2A and CI-2B) isolated from barley. For large-scale production, wsci cDNA was cloned into the E. coli vector pGEX-2T. The fusion protein GST-WSCI was efficiently produced in the bacterial expression system and, as the native inhibitor, was capable of inhibiting bacterial subtilisin, mammalian chymotrypsins and chymotrypsin-like activities present in crude extracts of a number of insect larvae ( Helicoverpa armigera , Plodia interpunctella and Tenebrio molitor ). The recombinant protein produced was also able to interfere with chymotrypsin-like activity isolated from immature wheat caryopses. These findings support a physiological role for this inhibitor during grain maturation.
