Evidence that non-caspase proteases are required for chromatin degradation during apoptosis.
Chromatin degradation into oligonucleosomal and approximately 30-50 Kb fragments is a hallmark of apoptosis. Crude nuclear extract from apoptotic rat thymocytes is able to recapitulate both types of DNA fragmentation in an assay using HeLa cell nuclei as an exogenous substrate. Using size exclusion chromatography we have identified a novel activity (approximately 260 Kd) that produces only approximately 30-50 Kb DNA fragments, and a 25 Kd activity that generates both approximately 30-50 Kb and oligonucleosomal fragments. Both activities produced DNA fragments with 3'-OH termini, are dependent on Ca2+ and Mg2+ and are inhibited by N-ethyl-maleimide, sodium tetrathionate, aurintricarboxylic acid and sodium chloride, similar to other nucleases implicated in apoptosis. These activities were inhibited by the serine protease inhibitors N-tosyl-L-phenylalanine chloromethyl ketone and N alpha-p-tosyl-L-lysine chloromethyl ketone, but not by the serine protease inhibitor diisopropyl fluorophosphate, or by calpain inhibitors I or II, or the capsase inhibitors Ac-Asp-Glu-Val-Asp-aldehyde, Ac-Tyr-Val-Ala-Asp-aldehyde, or Z-Val-Ala-Asp-fluoromethyl ketone. Both activities were insensitive to protease inhibitors when extracts were incubated with naked linear DNA, indicating the presence of both nuclease and protease activities in the preparation. Together, these observations suggest the involvement of non-caspase proteases in apoptosis which perhaps function by altering chromatin substructure and exposing it to nucleolytic attack.
Hughes
F M
FM
Laboratory of Signal Transduction, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina 27709, USA.
Evans-Storms
R B
RB
Cidlowski
J A
JA
D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D000818:Animals; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D002118:Calcium / Q000378:metabolism; D002154:Calpain / Q000037:antagonists & inhibitors / Q000378:metabolism; D020170:Caspase 1 / Q000378:metabolism; D053148:Caspase 3; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism; D002466:Cell Nucleolus / Q000737:chemistry / Q000201:enzymology; D002843:Chromatin / Q000378:metabolism*; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D053938:DNA Fragmentation / Q000502:physiology*; D016521:Electrophoresis, Gel, Pulsed-Field; D004720:Endonucleases / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D007531:Isoflurophate / Q000494:pharmacology; D008274:Magnesium / Q000378:metabolism; D008297:Male; D009842:Oligopeptides / Q000494:pharmacology; D010446:Peptide Fragments / Q000032:analysis / Q000378:metabolism; D011480:Protease Inhibitors / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D012697:Serine Endopeptidases / Q000378:metabolism*; D015842:Serine Proteinase Inhibitors / Q000494:pharmacology; D013950:Thymus Gland / Q000166:cytology; D014107:Tosyllysine Chloromethyl Ketone / Q000494:pharmacology; D014108:Tosylphenylalanyl Chloromethyl Ketone / Q000494:pharmacology
D000590:Amino Acid Chloromethyl Ketones; D061945:Caspase Inhibitors; D002843:Chromatin; D015853:Cysteine Proteinase Inhibitors; D009842:Oligopeptides; D010446:Peptide Fragments; D011480:Protease Inhibitors; D015842:Serine Proteinase Inhibitors; C108637:aspartyl-glutamyl-valyl-aspartal; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; D007531:Isoflurophate; C096429:L 709049; D014107:Tosyllysine Chloromethyl Ketone; D014108:Tosylphenylalanyl Chloromethyl Ketone; D004720:Endonucleases; D012697:Serine Endopeptidases; C505407:CASP3 protein, human; D002154:Calpain; C505410:Casp3 protein, rat; D053148:Caspase 3; D020169:Caspases; D020170:Caspase 1; D008274:Magnesium; D002118:Calcium