Abstract
Apoptosis is an important physiological process that involves the deletion of specific cells in a controlled and timely manner. A biochemical hallmark typifying apoptosis in normal lymphocytes is DNA cleavage caused by a calcium-dependent nuclease. We have previously identified and purified an 18-kDa nuclease (NUC18) from glucocorticoid-treated rat thymocytes whose activity is associated with this apoptotic DNA fragmentation. Partial protein sequencing of pure NUC18 has generated two peptide sequences that have a remarkable similarity to rat cyclophilin A and other members of the cyclophilin family. We report here that recombinant cyclophilins A, B, and C have a calcium/magnesium-dependent nuclease activity with biochemical and pharmacological properties similar to those of NUC18. Our results raise the intriguing possibility that cyclophilin or a cyclophilin-related protein may play a role in lymphocyte apoptosis.
Highlights
Apoptosis is an important physiological process that DNA degradation that occurs during apoptosis in these cells involves the deletion of specific cells in a controlled and (15)and is regulatedby several activatorsof apoptosis in lymtimely manner.A biochemical hallmark typifying apop- phocytes, including glucocorticoids, calcium ionophores (161, tosis in normal lymphocytesis DNA cleavage caused by and inhibitorsof protein synthesis (17).NUC18 enzymatic aca calcium-dependentnuclease
This known inhibitorof apoptosis clearly abolished the nucleus (17,23-26). This family of proteins are well charthe activity derived from samplesof bacteria transformed with acterized peptidylprolyl cis-tmns-isomerases that hind to the the cyclophilinAexpressionplasmid.,aurintricarimmunosuppressive drug CsA
Thenucle- identified 18-kDa nuclease (NUC18, isolated from purified apase activityof bacterially expressed cyclophilin A displays the optotic rat thymocytenuclei (15) suggestthatNUC18may same sensitivity to inhibitors observed previously for NUC18 purified from rat thymocyte nuclei (15)
Summary
Apoptosis is an important physiological process that DNA degradation that occurs during apoptosis in these cells involves the deletion of specific cells in a controlled and (15)and is regulatedby several activatorsof apoptosis in lymtimely manner.A biochemical hallmark typifying apop- phocytes, including glucocorticoids, calcium ionophores (161, tosis in normal lymphocytesis DNA cleavage caused by and inhibitorsof protein synthesis (17).NUC18 enzymatic aca calcium-dependentnuclease. Par- polyacrylamide gels,and we show here thatNUC18 is remarktial protein sequencing of pure NUC18 has generated ably similar to thecyclophilin family of proteins. Two peptide sequences that have a remarkable similarity to rat cyclophilin A and other members of the cyclophilin family.We report here that recombinantcyclophilins A, B, and C have a calciudmagnesium-dependent nuclease activity with biochemicaland pharmacological. Since being identifiedin mammals,cyclophilin and cyclophilin-related peptides havebeen isolated from a wide variety properties similar to those of NUCl8. Cyclophilin has been found in philin-related protein mayplaya role in lymphocyte every tissue type studied to date in humans (19, 24) and exapoptosis. Hibits varied cellular localization, including cytoplasmic forms (18)and nuclear forms (24, 25), aswell as endoplasmic reticulum-directed (26)and even forms associated with thesecretory twiecpcentb(aaolaDadhnyh1aoxxspcphiluohNyoomhselAcOtihooo,eclosoicgmio2Ayipscplvpptbsuchbcier)eyttcoeahoaioite.rniooadnttiaattcopmnilmAcsasoiteceltcyaioctieocidctdegcoissbotmynchicwtaerirsbddinoteabetorasipivebiie-ulsrlobesyntofdsipylila,mupanemsaltc,aeeatycnototdthztbtohvsewaihnaaihnrduieoturnaeronytliusemcepycinvtntescnmineeradirbst(nehintu.gdnssooiicos1ldacoomsetobeiwagTcea0olsonttndbileeryhhpplnhgfct,seeaiayaetolomaoiingeenemvasrln(rfitpntrdsn)9saoatSdhtemttoeae.uotugpidho)heeydsx(sullrmceoeaesilve7eaptisilsalipndowensltnee,acseepeetneDdfoorgoihgoc9nicodtiatsftfseeanN,scdnitapihloianDlipvreteoutvalAs1mahltloresrnarN.nehldt2oceeeiltisshaudpdynyTiAeut)gvpsdclceere.tmtacmroheoceoorlrgaTtchecdienlhirgohdmcreesaithaetdiarcaaa,seltatosDregimlriiieydtrcgoessaonncaserbatNtahcrcsensaced(iotonatociavdtAs8dthtioxfdetauueovihoa)hnytwrfn(arcriec.(nyta3diseiAnlltdi6ecozziieti-roettcileo,ocf7deeeailh9ingnsflts)nddsarseolih)rda.nsetpvmfa,aeimsmcoheuatosTapaxdieaiDhwoacicsdtopoalonanhasiottlntNrstnnompihetuhser(oupvlsienyAiote1ocsooatshgpoitiesqrlwp3tiuisonoiolsehyasovuh,ettnfgfillattaimogyseosamyi1ehsgttrnrlmgnhhse4dbaathuieooidiiiodnee)elst--d-ft-t-.thepcicopminatDietb2matssmhnhyyfulpcuahi9eNouaoseeptcogeIt)kiamidmmgrht.lnlAnepgvcapoihtenwustntRoaireehpeioteayfiondxsssroayoehanrsisfdhaotoidynanclinhiihcsodoltsasguiiedtadieiem.trowguinnrmmbuvisept(eypottaicinme2moseNi-iothpcnttsso7padisnsionutaroaUosssoftn)oietetvneuiihv.unsmnConpdCbnsq.eodiaeo’seslstsie1uCvsoiesyeftiAntser8uevhfWeseyhl(dasrnypttetle3acoocsk,eoouhecamppl0ewolnnepsoaenhearo)Csnfipotrtpneo.advfhpossmfhwsnhesGcwieootitidAtossctioudhm.onwhrelieuicttceiviauonva(iytnciinlroic3thsnmhleeetttasthfaetie2yreaebodhbi,rtmd)srClabnhieye.hcbeteiehtcuhiasysseRayyCtatotAryntecavteiocfeCuvhsvhalmocpdexycAopeanist(asroicenipoA3ttcudtovfulniitshmp0oioopeietpthtpsiiteple)nrfnhplmayocoyu.pfnihitcre.anne,wAnhertumiee(ncilewTxso3lccfdsnlibtnlutnapeluss1heyrhilenomslietegn)ioleolovohtn,phcsrthdsnaeiuselio,hreaalaetioxromgyoaemvntedce(ghmidhlfScegeevniyifipbiaenoc4bnaetnalrittstepsaucl9intrehhoahtltrslsatin.hcayicem,oe1etrbitnCiycicoCtn)wtsnsirictymamsefrgttn-oslnloAeiitoeAetccloemseoarrsoofayitsdcctulnuu(hwhvo-chctyh2nfirmlhlbeedeanaotaeooet8sedlyreess-rrf-t-/--l, lecular weight protein, designatedNUC18, correlates with the
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