Human Caspase-7 Activity and Regulation by Its N-terminal Peptide

Central to the execution phase of apoptosis are the two closely related caspase-3 and -7. They share common substrate specificity and structure, but differ completely in the sequence of their respective N-terminal regions including their N-peptides, a 23–28 residue segment that are removed during zymogen activation. We show that the N-peptide of caspase-7 plays no role in the fundamental activation or properties of the active protease in vitro. However, the N-peptide modifies the properties of caspase-7 in vivo. In ectopic expression experiments, caspase-7 constructs with no N-peptide are far more lethal than constructs that have an uncleavable peptide. Moreover, the N-peptide of caspase-7 must be removed before efficient activation of the zymogen can occur in vivo. These disparate requirements for the N-peptide argue that it serves to physically sequester the caspase-7 zymogen in a cytosolic location that prevents access by upstream activators (caspase-8, -9, and -10). The N-peptide must first be removed, probably by caspase-3, before efficient conversion and activation of the zymogen can occur in vivo. Central to the execution phase of apoptosis are the two closely related caspase-3 and -7. They share common substrate specificity and structure, but differ completely in the sequence of their respective N-terminal regions including their N-peptides, a 23–28 residue segment that are removed during zymogen activation. We show that the N-peptide of caspase-7 plays no role in the fundamental activation or properties of the active protease in vitro. However, the N-peptide modifies the properties of caspase-7 in vivo. In ectopic expression experiments, caspase-7 constructs with no N-peptide are far more lethal than constructs that have an uncleavable peptide. Moreover, the N-peptide of caspase-7 must be removed before efficient activation of the zymogen can occur in vivo. These disparate requirements for the N-peptide argue that it serves to physically sequester the caspase-7 zymogen in a cytosolic location that prevents access by upstream activators (caspase-8, -9, and -10). The N-peptide must first be removed, probably by caspase-3, before efficient conversion and activation of the zymogen can occur in vivo. Apoptosis is an orchestrated series of cellular events in which a doomed cell has its cellular component dismantled and packaged into smaller bodies more easily removed by neighboring cells or macrophages. At the core of the death process is a family of cytosolic cysteine proteases, caspases, with a specificity for aspartic acid residues (reviewed in Refs. 1Denault J.B. Salvesen G.S. Chem. Rev. 2002; 102: 4489-4500Crossref PubMed Scopus (273) Google Scholar, 2Nicholson D.W. Cell Death Differ. 1999; 6: 1028-1042Crossref PubMed Scopus (1306) Google Scholar, 3Thornberry N.A. Lazebnik Y. Science. 1998; 281: 1312-1316Crossref PubMed Scopus (6182) Google Scholar). Stimuli from death receptor ligands (extrinsic pathway) or a variety of chemotherapeutic agents, drugs or cellular stresses (intrinsic pathway) cause the activation of apical (initiator) caspase-8, -9, or -10. Once activated, these initiators are able to process and directly activate downstream executioner caspase-3 and -7. The limited proteolytic activity of the executioners on a set of cellular protein substrates is responsible for the hallmark phenotype of apoptosis (reviewed in Ref. 2Nicholson D.W. Cell Death Differ. 1999; 6: 1028-1042Crossref PubMed Scopus (1306) Google Scholar). For the executioner caspases, proteolysis between the large and small subunit is thought to be the fundamental activating event (4Chai J. Wu Q. Shiozaki E. Srinivasula S.M. Alnemri E.S. Shi Y. Cell. 2001; 107: 399-407Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar, 5Kang B.H. Ko E. Kwon O.K. Choi K.Y. Biochem. J. 2002; 364: 629-634Crossref PubMed Scopus (17) Google Scholar, 6Riedl S.J. Fuentes-Prior P. Renatus M. Kairies N. Krapp R. Huber R. Salvesen G.S. Bode W. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 14790-14795Crossref PubMed Scopus (196) Google Scholar, 7Bose K. Clark A.C. Biochemistry. 2001; 40: 14236-14242Crossref PubMed Scopus (56) Google Scholar, 8Stennicke H.R. Salvesen G.S. Biochim. Biophys. Acta. 2000; 1477: 299-306Crossref PubMed Scopus (296) Google Scholar). All caspases possess N-terminal extensions, and in the case of the in initiators these are required for recruitment to the respective activation complexes. In contrast, a consensus role for the short N-terminal extensions of the executioners (Fig. 1A) has yet to be established. Initial findings suggest that the N-terminal peptides of caspase-3 and -7 have no effect on activity or the ability to be activated in vitro. However, in vivo both caspases seem to require the removal of the N-peptides for efficient activation. Indeed, in some cells caspase-3 removes the N-peptide of caspase-7 before the latter is activated by an initiator granzyme B, a serine protease that activates caspases during T-cell-mediated killing (9Yang X. Stennicke H.R. Wang B. Green D.R. Janicke R.U. Srinivasan A. Seth P. Salvesen G.S. Froelich C.J. J. Biol. Chem. 1998; 273: 34278-34283Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar). Other roles attributed to the N-peptide are the silencing of caspase-3 (10Meergans T. Hildebrandt A.K. Horak D. Haenisch C. Wendel A. Biochem. J. 2000; 349: 135-140Crossref PubMed Google Scholar) and caspase-6 (11Cowling V. Downward J. Cell Death Differ. 2002; 9: 1046-1056Crossref PubMed Scopus (209) Google Scholar) and prevention of nuclear import of Xenopus caspase-7 (12Yaoita Y. Biochem. Biophys. Res. Commun. 2002; 291: 79-84Crossref PubMed Scopus (17) Google Scholar). The later hypothesis is appealing, but reports are conflicting regarding the subcellular localization of caspase-7. Although it is generally agreed that the zymogen of caspase-7 is cytosolic, subcellular fractionation experiments have suggested that active caspase-7 relocalizes to the nucleus (12Yaoita Y. Biochem. Biophys. Res. Commun. 2002; 291: 79-84Crossref PubMed Scopus (17) Google Scholar), microsomes (13Zhivotovsky B. Samali A. Gahm A. Orrenius S. Cell Death Differ. 1999; 6: 644-651Crossref PubMed Scopus (303) Google Scholar), or mitochondria (14Chandler J.M. Cohen G.M. MacFarlane M. J. Biol. Chem. 1998; 273: 10815-10818Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar) during apoptosis. Some of those discrepancies may be artifacts of the experimental procedure given the fact that the pI of caspase-7 lacking the N-peptide is very different than the one of the zymogen (pI = 8.0 versus 5.5) and may cause the protein to precipitate upon N-peptide removal. Secondly and most importantly, apoptosis is associated with pH and ionic strength changes as well as nuclear-cytoplasmic barrier disruption (15Faleiro L. Lazebnik Y. J. Cell Biol. 2000; 151: 951-959Crossref PubMed Scopus (209) Google Scholar) and may result in mixing and/or relocalization of compartment specific markers used in subcellular fractionation experiments. Finally, usage of EGFP fusion protein as a tracker for caspase localization (12Yaoita Y. Biochem. Biophys. Res. Commun. 2002; 291: 79-84Crossref PubMed Scopus (17) Google Scholar, 16Shikama Y.U.M. Miyashita T. Yamada M. Exp. Cell Res. 2001; 264: 315-325Crossref PubMed Scopus (56) Google Scholar) may also be a source of experimental variation because of potential dimerization of green fluorescent proteins, natural tendency to localize to the nucleus and increase in molecular weight of the caspase complex. In the present study we have tested a number of these hypotheses for the function of the enigmatic N-peptide of caspase-7. Cell Culture and Transfection—293A cells (QBI-293A, Quantum Biotechnology Inc., Montréal, Canada) and COS-7 cells (ATCC, Manassas, VA) were cultivated in DME medium supplemented with 10% heat-inactivated bovine serum (Irvine Scientific, Irvine, CA), 2 mml-glutamine, penicillin/streptomycin (Invitrogen) and routinely passed every 3 days. For ectopic expression, cells at 40–60% confluence were transfected using FuGENE 6 (Roche Applied Science) as suggested by the manufacturer using 3 μl of transfectant reagent per 1.0 μg of DNA in 0.1 ml of DME medium. Cells were treated and harvested as indicated for each experiment. Mammalian Expression Constructs—The cDNA for human caspase-7 (GenBank™ acc. no. NM_001227) was use as a template for all constructs. All pcDNA3 constructs were subcloned into KpnI and XhoI by with the the at the and the at the The was to the (Fig. of each by with the sequence The was by with that residues by a sequence All were using with the and the cysteine residue also as in the All constructs were to DNA sequence Expression and caspases were in using as fusion using the expression All constructs were subcloned into and XhoI of using the as for pcDNA3 constructs. The was the used in the were in E. and by as H.R. Salvesen G.S. 1999; PubMed Scopus Google Scholar). The and were as and the was with the and its were in pH with protease and were required used granzyme and were at for at to cellular were from the of the by in and for DNA was by a from 2 cells were on to in pH and 10% at for as P. J. Biol. Chem. Full Text PDF PubMed Google Scholar). were to with the and and Chem. for were using an pH and protease as C. S.J. J. 2001; PubMed Scopus Google Scholar). The were caspase-7 and and protein Inc., cells in were in μl of on for and at for was using protein using bovine serum as a of protein were for caspase activity using of substrate Inc., of in caspase pH 10% and H.R. Salvesen G.S. 1999; PubMed Scopus Google Scholar). activity was on a at = = were to the active as H.R. Salvesen G.S. 1999; PubMed Scopus Google Scholar) and were in caspase using or the substrate using or in were in caspase at in μl was and were by with reagent and In was using the in an of substrate was used for each were transfected in with the indicated constructs was with Irvine, and for at Cells were and in ml of with the protease were with or μl of of S. at for μl were and for at was by and with were on and to as were using in Inc., and DNA cells in were and in ml of pH and and on for was by at for and was to and at for was to and to 0.1 and at for were with and with DNA was in and on a K. S. 2000; PubMed Google Scholar). were in and transfected with the Cells were with and with for 2 were with in for at and were using and a were with and with of the the of to into the role of the N-peptide of caspase-7 we two one the first residues and to an N-peptide and one a acid the N-peptide These were with caspase-7 for their ability to death in human Expression of caspase-7 in cells is to cause some cell death but of the N-peptide the effect prevention of N-peptide removal it (Fig. effect is at very expression μg upon which all caspase-7 constructs were able to cell death In all experiments the of cell by from the was with than or is to in protein of caspase-7 constructs from cell expression (Fig. the of present to be as by a caspase-7 (Fig. is that of the N-peptide of caspase-7 during the expression The no effect on cell and is caspase activation by the constructs we a using different of transfected cells were harvested at and the caspase activity in cell was using substrate (Fig. and In more than are We used DNA to μg to changes in the activation and for and μg are activity was at for both but to a at before at expression activity at as with a activation (Fig. were using μg of transfected DNA was of DNA were used (Fig. and in the latter cell death was by for and as with is the fact that no more activity was and that removal of the N-peptide is for the caspase-7 killing a of the of caspase activity that is caspase-7 we the with cell death by ectopic expression of the of caspase-7 in a increase of activity in cell as with the activity in cells that the activity is attributed to caspase-7 the of caspase-7 is required for its activation (4Chai J. Wu Q. Shiozaki E. Srinivasula S.M. Alnemri E.S. Shi Y. Cell. 2001; 107: 399-407Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar, 6Riedl S.J. Fuentes-Prior P. Renatus M. Kairies N. Krapp R. Huber R. Salvesen G.S. Bode W. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 14790-14795Crossref PubMed Scopus (196) Google Scholar, Q. Salvesen G.S. Biochem. J. PubMed Scopus Google Scholar, Renatus M. S. Green D.R. Salvesen G.S. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). 2 that each result in activation of the zymogen (Fig. we at each for expression in cells (Fig. using and that of of the of (Fig. and is that removal of the N-peptide was for all constructs probably the to ability of caspase-7 to activate caspase-7 a was with the that to or was active or an we an to active caspases Renatus M. S. Green D.R. Salvesen G.S. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). cells were with the cell was with and were with (Fig. is was for that a of caspase-7 was that caspase-7 activation as a of is a event of to at the caspase-7 is generally to be a downstream caspase (9Yang X. Stennicke H.R. Wang B. Green D.R. Janicke R.U. Srinivasan A. Seth P. Salvesen G.S. Froelich C.J. J. Biol. Chem. 1998; 273: 34278-34283Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar, K. K. Salvesen G.S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) its to the activation of caspases, in which case cell death be a of executioner and activity and that of caspase-7. most caspases, and caspase-3, substrate specificity with caspase-7 H.R. Renatus M. M. Salvesen G.S. Biochem. J. 2000; PubMed Scopus Google Scholar, N.A. T. M. S. D.W. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) the of activity in to caspase-3 or in cell of transfected cells However, because active caspase by may be in cell death L. J. D. K. S. D.W. P. J.M. A. 2002; PubMed Scopus Google Scholar), we used of caspases as a more all caspase-7 constructs were (Fig. However, caspase-3 (Fig. and caspase-6 (Fig. were from transfected cell no was The of the caspase-3 and in a to caspase activation by that of those caspases were activated by the caspase-7. the of caspase-3 and activation of caspase-7 that of the apical caspases (caspase-8, -9, or able to activate executioner Cell Death by that caspase-7 to the activation of caspases, we for of death substrates to be caspase-3 or -7 We S. PubMed Scopus Google Scholar) as a nuclear substrate and Y. Y. S. M. T. R. R. J. PubMed Scopus Google Scholar) as a cytosolic Expression of caspase-7 in of to the and a of apoptosis is from (Fig. In both was more in the is that in more than ectopic expression of that caspase-7 can in a caspase access to by nuclear We a substrate of caspase-6 A. Alnemri E.S. Lazebnik T. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, K. M. Froelich C.J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), and no the of is in that caspase-7 We DNA (Fig. a hallmark of apoptosis M. K. A. S. 1998; PubMed Scopus Google Scholar). The of DNA was to cells with and with and from cells transfected with an or caspase-7 apoptosis by caspase-7 the we also for of from mitochondria in transfected increase in cytosolic (Fig. or was as with cells transfected with of mitochondria in cell death by caspase-7. a the N-peptide of caspase-7 is a of residues of a nuclear localization of the N-peptide during apoptosis and cause of caspase-7 as suggested for the Xenopus (12Yaoita Y. Biochem. Biophys. Res. Commun. 2002; 291: 79-84Crossref PubMed Scopus (17) Google Scholar). We the subcellular localization of caspase-7. However, because cell for by caspase-7 is by changes we the subcellular localization of the respective in COS-7 cells (Fig. in was for of the or all were from the is with subcellular fractionation experiments that both and were cytosolic that a small of caspase-7 may its to but the that removal of the N-peptide an active or of human caspase-7 in the of the of apoptosis to N-peptide removal may an increase in the activity of caspase-7. we the of in E. were as and to by as H.R. Salvesen G.S. 1999; PubMed Scopus Google Scholar). active we that is to or Indeed, and for and on the substrate and the substrate were experimental is that in experiments using the are to be to in activity but to some of the N-peptide of caspase-7 are of 2 different of experiments with the of from a different and/or different substrate were of the were using of a given of substrate to a or with a as by the of each were using of a given of substrate to a or with a as by the of each are of 2 different of experiments with the of from a different and/or different substrate were of the were using of a given of substrate to a or with a as by the of each in a We that during of the protein from E. was present as a zymogen of expression was that the N-peptide may activation of the of caspase-7 activation in E. to the conversion of the zymogen that is by (Fig. of with of the at (Fig. as by as as to large and a small (Fig. In conversion of was by 2 In both a in the of the small subunit at These that the N-peptide plays a role in the at in the during expression in E. However, it is that in expression or of expression we an in which the of by caspase-7 be more We used the of and to from activity of the These were in in the of and used to the ability of caspase-7 to process the The of a at was by the its to removed the but the of the in the experiments We that of the zymogen was more efficient than for the zymogen for the of active caspase-7 (Fig. that the N-peptide with conversion of the zymogen and the of cell death is in cells for (Fig. We that removal of the N-peptide the activity of the but the of zymogen it is as a caspase-7 as an that is by the The N-peptide of in but in is thought to be the to caspase-7 activation used in cells to apoptosis. is thought to be the role of apical and (reviewed in Refs. 1Denault J.B. Salvesen G.S. Chem. Rev. 2002; 102: 4489-4500Crossref PubMed Scopus (273) Google Scholar, 2Nicholson D.W. Cell Death Differ. 1999; 6: 1028-1042Crossref PubMed Scopus (1306) Google Scholar, 3Thornberry N.A. Lazebnik Y. Science. 1998; 281: 1312-1316Crossref PubMed Scopus (6182) Google Scholar). We the role of the N-peptide in the activation of by in (Fig. of were used to process of and We the to a the are in E. is to properties of caspase-7 it is in or in the have an at of caspase-7 zymogen by two large and two small subunit to proteolysis of the at the two activation and no was in the of of the two of caspase-7 (Fig. was using as an was required to process the of zymogen the N-peptide the ability of apical caspases to activate in vitro. the N-peptide zymogen activation in vivo we caspase-7 constructs in as a of in the were and with in of the subunit related from the (Fig. are to the in the in and was for is and activated, far more than We that the N-peptide the activation of caspase-7 by the in but in vitro. The for are The role of caspase-7 as an executioner in the of is on its with the most from a is that both have specificity on small substrates N.A. T. M. S. D.W. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), the of are for caspase-7 H.R. Renatus M. M. Salvesen G.S. Biochem. J. 2000; PubMed Scopus Google Scholar). Moreover, are the to be by the of the caspase (reviewed in Refs. 1999; PubMed Scopus Google and G.S. Rev. Cell. Biol. 2002; PubMed Scopus Google Scholar). are two during the the for the of both is that a that of one is by of the is the phenotype in in the caspase-3 on a and cell with K. S. D. P. PubMed Scopus Google Scholar). However, on a have a phenotype C. J. Exp. 2002; PubMed Scopus Google Scholar) that the latter of caspase-3 caspase-7 is a the human cell that caspase-3 R.U. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar) apoptosis activation of caspase-7 Y. C. 2001; PubMed Scopus Google Scholar). the silencing of caspase-3 expression in caspases, including caspase-7 S. K. T. Srinivasan A. D.W. Lazebnik Y. 2000; 6: PubMed Scopus (273) Google Scholar). However, the most between the in the of their N-peptides and the short sequence that the N-peptide before the of the These may activity of caspase-3 and -7 in vivo. The and of the N-terminal peptides different caspase as caspase-8, -9, and peptides that or caspase recruitment required for their recruitment to complexes. In these the apical caspases able to activate executioner caspase-3 and -7 by limited The of the executioners short N-peptides a and the function of these N-peptides is The caspase-3 and -7 N-peptides are removed during apoptosis and are from to For human and Xenopus share limited acid their respective N-terminal both the for the N-peptide and the sequence in a function in all of caspase-7. for the N-peptide of caspase-7 is that it a nuclear localization the segment (12Yaoita Y. Biochem. Biophys. Res. Commun. 2002; 291: 79-84Crossref PubMed Scopus (17) Google Scholar). may be the case with Xenopus with which the study was it seem to be the case for human we were to nuclear of or N-peptide caspase-7 in COS-7 of caspase-7 can result in some nuclear but is to be an of the function of the N-peptide in nuclear has in role that has for the N-peptide is the silencing of the zymogen of caspase-3 (10Meergans T. Hildebrandt A.K. Horak D. Haenisch C. Wendel A. Biochem. J. 2000; 349: 135-140Crossref PubMed Google Scholar). The from was that the caspase-3 N-peptide the protease in an At first it that a function be to the caspase-7 its cells more to death by caspase-7 However, we are of apoptosis is at the caspase-7 of upstream initiator caspases and on death by ectopic expression of caspase-7 that is a in vivo. They that the N-peptide of caspase-7 the properties of as a substrate of but as a substrate of the initiator or suggest that in the of activation of and are to the as substrates for by but the activators and in vitro. that the of the N-peptide access of caspase-7 to the zymogen or as a of caspase-7 The latter is by that show for each of the active of caspase-7. The that the N-peptide the of the zymogen in a However, the for caspase-7 or for residues to because is no or because of with to the of the in the (4Chai J. Wu Q. Shiozaki E. Srinivasula S.M. Alnemri E.S. Shi Y. Cell. 2001; 107: 399-407Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar, 6Riedl S.J. Fuentes-Prior P. Renatus M. Kairies N. Krapp R. Huber R. Salvesen G.S. Bode W. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 14790-14795Crossref PubMed Scopus (196) Google Scholar, Y. T. J. J.M. L. Chem. Biol. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). In are no between the N-peptide and the the hypothesis that the N-peptide can directly caspase-7 structure, activity or and must be for the function of the is a in the of caspase-3 and -7 that on the function of their respective both in and natural by by N-peptide removal S.J. Shi L. P. G.M. Green D.R. J. PubMed Scopus (273) Google Scholar). In contrast, in the the activation of caspase-7 first by N-peptide and it has that caspase-3 removes the N-peptide of caspase-7 before it is activated by the initiator granzyme in cells (9Yang X. Stennicke H.R. Wang B. Green D.R. Janicke R.U. Srinivasan A. Seth P. Salvesen G.S. Froelich C.J. J. Biol. Chem. 1998; 273: 34278-34283Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar). we show that it is also for by caspase-8, prevention of N-peptide removal caspase-7 in vivo. importantly, we also show that removal of the N-peptide is for the efficient activation of caspase-7 in cell experiments, and also in vivo. is no of the for the of N-peptide removal in caspase-7 activation. However, the are with a of the zymogen from its apical that is the N-peptide is removed from which it can be activated by the of of the may in the properties of the it is a Indeed, the of the zymogen is than the one of the protein lacking the protein to a protein or The N-peptide and the a However, most subcellular fractionation and caspase-7 as cytosolic in cells (13Zhivotovsky B. Samali A. Gahm A. Orrenius S. Cell Death Differ. 1999; 6: 644-651Crossref PubMed Scopus (303) Google Scholar, J.M. Cohen G.M. MacFarlane M. J. Biol. Chem. 1998; 273: 10815-10818Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar), it is the the of study the of subcellular localization in the In including in cytosolic (9Yang X. Stennicke H.R. Wang B. Green D.R. Janicke R.U. Srinivasan A. Seth P. Salvesen G.S. Froelich C.J. J. Biol. Chem. 1998; 273: 34278-34283Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar) is activated by and granzyme in an N-peptide but in cells these initiators are in their access the N-peptide is removed, by The study that caspase-7 is a to require a activation in in (9Yang X. Stennicke H.R. Wang B. Green D.R. Janicke R.U. Srinivasan A. Seth P. Salvesen G.S. Froelich C.J. J. Biol. Chem. 1998; 273: 34278-34283Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar). is of most of the but is on the activity of caspase-3 to the to the N-peptide before the activation process are required to the molecular by which the N-peptide prevents and of caspase-7. the of the N-peptide on both and on the role of caspase-7 in the We and for and Stennicke for

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