N Zein1, P Reiss, M Bernatowicz, M Bolgar. 1. Bristol-Myers Squibb Company, Pharmaceutical Research Institute, Princeton, NJ 08543-4000, USA.
Abstract
BACKGROUND: Enediyne chromoproteins are potent antitumor antibiotic agents. They consist of a labile nine-membered enediyne chromophore non-covalently associated with a stabilizing acidic polypeptide. Studies in vitro on three members of this superfamily of natural products--kedarcidin, maduropeptin and neocarzinostatin--demonstrated that their chromophores cleave DNA at sites specific to each chromophore. Recently, we showed that these chromoproteins possess proteolytic activity against histones in vitro, with histone H1 as a preferred substrate. Based on these results, we speculated that this selective proteolytic activity 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 specificity, we synthesized a 24-amino-acid peptide representing a basic region of histone H1. This model peptide was incubated individually with similar concentrations of the kedarcidin, neocarzinostatin and maduropeptin chromoproteins as well as the kedarcidin apoprotein. The reaction products were analyzed by electrospray liquid chromatography/mass spectrometry. Our results indicate that all proteins cleave the peptide at selected backbone amides, and that these sites vary according to the chromoprotein used. Moreover, the kedarcidin apoprotein appears to be less specific than the kedarcidin chromoprotein complex. CONCLUSIONS: The small size, unique architecture and very acidic nature of the enediyne chromoproteins are highly unusual. These natural products exhibit the dual functionalities of specific DNA cleavage and selective proteolytic activity. This observation adds to the fascinating properties of these molecules and suggests that it is possible not only to design small moieties to cleave DNA but also to conceive of small proteins to deliver these moieties intact to defined areas of chromatin.
BACKGROUND: Enediyne chromoproteins are potent antitumor antibiotic agents. They consist of a labile nine-membered enediyne chromophore non-covalently associated with a stabilizing acidic polypeptide. Studies in vitro on three members of this superfamily of natural products--kedarcidin, maduropeptin and neocarzinostatin--demonstrated that their chromophores cleave DNA at sites specific to each chromophore. Recently, we showed that these chromoproteins possess proteolytic activity against histones in vitro, with histone H1 as a preferred substrate. Based on these results, we speculated that this selective proteolytic activity 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 specificity, we synthesized a 24-amino-acid peptide representing a basic region of histone H1. This model peptide was incubated individually with similar concentrations of the kedarcidin, neocarzinostatin and maduropeptin chromoproteins as well as the kedarcidin apoprotein. The reaction products were analyzed by electrospray liquid chromatography/mass spectrometry. Our results indicate that all proteins cleave the peptide at selected backbone amides, and that these sites vary according to the chromoprotein used. Moreover, the kedarcidin apoprotein appears to be less specific than the kedarcidin chromoprotein complex. CONCLUSIONS: The small size, unique architecture and very acidic nature of the enediyne chromoproteins are highly unusual. These natural products exhibit the dual functionalities of specific DNA cleavage and selective proteolytic activity. This observation adds to the fascinating properties of these molecules and suggests that it is possible not only to design small moieties to cleave DNA but also to conceive of small proteins to deliver these moieties intact to defined areas of chromatin.
Authors: Mingkun Fu; Sen Li; Enada Archibold; Michael J Yurkovich; John J Nash; Hilkka I Kenttämaa Journal: J Am Soc Mass Spectrom Date: 2010-06-25 Impact factor: 3.109