THE PROTEOLYTIC SPECIFICITY OF THE NATURAL ENEDIYNE-CONTAINING CHROMOPROTEINS IS UNIQUE TO EACH CHROMOPROTEIN

Background: Enediyne chromoproteins are potent antitumor antibiotic ag ents. They consist of a labile nine-membered enediyne chromophore non- covalently associated with a stabilizing acidic polypeptide. Studies i n vitvo on three members of this superfamily of natural produces - ked arcidin, maduropeptin and neocarzinostatin - demonstrated that their c hromophores cleave DNA at sites specific to each chromophore. Recently , we showed that these chromoproteins possess proteolytic activity aga inst histones in vitvo, with histone H1 as a preferred substrate. Base d on these results, we speculated that this selective proteolytic acti vity may be important in vivo in the delivery of the enediynes intact to the DNA in chromatin. Results: We show here that each chromoprotein generates a unique set of H1 fragments as revealed by gel analyses of the H1 cleavage reaction products. To probe the observed cleavage spe cificity, we synthesized a 24-amino-acid peptide representing a basic region of histone H1. This model peptide was incubated individually wi th similar concentrations of the kedarcidin, neocarzinostatin and madu ropeptin chromoproteins as well as the kedarcidin apoprotein. The reac tion produces were analyzed by electrospray liquid chromatography/mass spectrometry. Our results indicate that all proteins cleave the pepti de at selected backbone amides, and that these sites vary according to the chromoprotein used. Moreover, the kedarcidin apoprotein appears t o be less specific than the kedarcidin chromoprotein complex. Conclusi ons: The small size, unique architecture and very acidic nature of the enediyne chromoproteins are highly unusual. These natural products ex hibit the dual functionalities of specific DNA cleavage and selective proteolytic activity. This observation adds to the fascinating propert ies of these molecules and suggests that it is possible not only to de sign small. moieties to cleave DNA but also to conceive of small prote ins to deliver these moieties intact to defined areas of chromatin.