Abstract
Previous work in our laboratory (Montague, J., Gaido, M., Frye, C., and Cidlowski, J. (1994) J. Biol. Chem. 269, 18877-18880) has shown that human recombinant cyclophilins A, B, and C have sequence homology with the apoptotic nuclease NUC18 and that denatured cyclophilins can degrade DNA. We have now evaluated the nucleolytic activity of recombinant cyclophilins under native conditions. We show that nuclease activity inherent to cyclophilins is distinct from cis-trans-peptidylprolyl isomerase activity and is similar to that described for apoptotic nucleases. Cyclophilin nucleolytic activity is stimulated by Ca2+ and/or Mg2+, with a combination of the two being optimal for cyclophilins A and B. Mg2+ alone is sufficient for cyclophilin C nuclease activity. pH optimums are in the range of pH 7.5-9.5. Cyclophilins can degrade both single-stranded and double-stranded DNA. Additionally, cyclophilins produce 3'-OH termini in linear double-stranded substrates, suggesting the cuts produced are similar to those of apoptotic cells. Cyclophilins also display endonucleolytic activity, demonstrated by their ability to degrade supercoiled DNA. In the absence of ions, cyclophilins bind linearized DNA. When added to nuclei from nonapoptotic cells, cyclophilin C induces 50-kilobase pair DNA fragmentation but not internucleosomal fragmentation. Together, these data suggest that cyclophilins are involved in degradation of the genome during apoptosis.
Highlights
From the ‡Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, North Carolina 27599 and ¶NIEHS, National Institutes of Health, Research Triangle Park, North Carolina 27709
We show that nuclease activity inherent to cyclophilins is distinct from cis-trans-peptidylprolyl isomerase activity and is similar to that described for apoptotic nucleases
Recombinant CypA, -B, and -C Degrade DNA under Nondenaturing Conditions—Previously, we used a radioactive gel nuclease assay to demonstrate that three members of the Cyp family, CypA, -B, and -C, are able to degrade DNA [29]; this assay used a protein denaturation and renaturation step prior to analysis of nuclease activity, and it was unclear if native Cyps have the same nucleolytic activity
Summary
Cyclophilin; CypA, -B, and -C, cyclophilins A, B, and C, respectively; CsA, cyclosporin A; TBE, Tris/ borate/EDTA; TE, Tris/EDTA; MES, 4-morpholineethanesulfonic acid; CAPS, 3-(cyclohexylamino)propanesulfonic acid. The folding rates of human carbonic anhydrase II [15], RNase T1, [16], and collagen [17] have been shown to be increased by Cyp in vitro Whether this isomerase activity is physiologically significant is still not clear. While the characteristics of these apoptotic nuclease activities have been extensively described [32,33,34,35], the actual enzymes responsible have not been identified. In this manuscript, we have evaluated and characterized the nuclease activity of Cyps under native conditions and assessed their potential role in apoptosis. CypC is shown to produce 50-kb fragments in chromatin, further suggesting a nucleolytic role for Cyps in apoptosis
Sign-in/Register to view highlights & summary 
Published Version (
Free)
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call