A role for the intersubunit disulfides of seminal RNase in the mechanism of its antitumor action.
The dimeric structure of seminal ribonuclease (BS-RNase) is maintained by noncovalent interactions and by two intersubunit disulfide bridges. Another unusual feature of this enzyme is its antitumour action, consisting in a cytotoxic activity selective for malignant cells. This cytotoxic action is exerted when the protein reaches the cytosol of the affected cells, where it degrades ribosomal RNA, thus blocking protein synthesis and leading cells to death. The current model proposed for the mechanism of antitumour action of BS-RNase is based on the ability of the protein to resist the neutralizing action of the cytosolic RNase inhibitor, a resistance due to the dimeric structure of the enzyme. Monomeric RNases, and monomeric derivatives of BS-RNase, are strongly bound by the inhibitor and inactive as antitumor agents. Here we report on monomeric derivatives of BS-RNase that, although strongly inhibited by the cytosolic RNase inhibitor, are cytotoxic towards malignant cells. These monomers are produced by reductive cleavage of the intersubunit disulfides of the native, dimeric protein followed by linking the exposed sulfhydryls to small thiols through formation of mixed disulfides. We found that sulfhydryls from cell monolayers and cell membranes can attack these mixed disulfides in the monomeric derivatives, and reconstitute, through sulfhydryl-disulfide interchange reactions, the native dimeric protein, which is internalized as such, and displays its antitumour action.
Bracale
Aurora
A
Dipartimento di Chimica Biologica, Università di Napoli, Italy.
Castaldi
Francesco
F
Nitsch
Lucio
L
D'Alessio
Giuseppe
G
D016475:3T3 Cells; D000818:Animals; D000970:Antineoplastic Agents / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D002417:Cattle; D002461:Cell Line, Transformed; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism; D002470:Cell Survival / Q000187:drug effects; D019281:Dimerization; D004220:Disulfides / Q000737:chemistry*; D004354:Drug Screening Assays, Antitumor; D004722:Endoribonucleases / Q000737:chemistry* / Q000378:metabolism* / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000378:metabolism / Q000494:pharmacology; D005347:Fibroblasts; D007460:Iodoacetamide / Q000378:metabolism; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D012669:Seminal Vesicles / Q000201:enzymology
D000970:Antineoplastic Agents; D004220:Disulfides; D004791:Enzyme Inhibitors; D021122:Protein Subunits; D004722:Endoribonucleases; C032523:ribonuclease SPL; D007460:Iodoacetamide