Onconasetrade mark, a homolog of bovine pancreatic ribonuclease A (RNase A) with high conformational stability, is cytotoxic and has efficacy as a cancer chemotherapeutic agent. Unlike wild-type RNase A, the G88R variant is toxic to cancer cells. Here, variants in which disulfide bonds were removed from or added to G88R RNase A were used to probe the relationship between conformational stability and cytotoxicity in a methodical manner. The conformational stability of the C40A/G88R/C95A and C65A/C72A/G88R variants is less than that of G88R RNase A. In contrast, a new disulfide bond that links the N and C termini (residues 4 and 118) increases the conformational stability of G88R RNase A and C65A/C72A/G88R RNase A. These changes have little effect on the ribonucleolytic activity of the enzyme or on its ability to evade the cytosolic ribonuclease inhibitor protein. The changes do, however, have a substantial effect on toxicity toward human erythroleukemia cells. Specifically, conformational stability correlates directly with cytotoxicity as well as with resistance to proteolysis. These data indicate that conformational stability is a key determinant of RNase A cytotoxicity and suggest that cytotoxicity relies on avoiding proteolysis. This finding suggests a means to produce new cancer chemotherapeutic agents based on mammalian ribonucleases. Study holds ProTherm entries: 8519, 8520, 8521, 8522, 8523, 8524, 8525 Extra Details: conformational stability; cytotoxicity; disulfide bond;,cancer chemotherapeutic agents
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:36 p.m.
|Number of data points||12|
|Proteins||Ribonuclease pancreatic ; Ribonuclease pancreatic|
|Assays/Quantities/Protocols||Experimental Assay: Tm ; Derived Quantity: dTm|
|Libraries||Mutations for sequence KETAAAKFERQHMDSSTSAASSSNYCNQMMKSRNLTKDRCKPVNTFVHESLADVQAVCSQKNVACKNGQTNCYQSYSTMSITDCRETGSSKYPNCAYKTTQANKHIIVACEGNPYVPVHFDASV|