Title: Defects in the Ubiquitin Pathway Induce Caspase-independent Apoptosis Blocked by Bcl-2
Abstract: Apoptosis requires the activation of caspases (formerly interleukin 1β-converting enzyme-like proteases), in particular those related to the caspase-3/7/6 subfamily. Recent data, however, revealed that, although caspase-specific inhibitors delay apoptosis, they are often incapable of preventing it. To obtain evidence for caspase-independent steps of apoptosis, we artificially created a high amount of short-lived or aberrant proteins by blocking the ubiquitin degradation pathway. A temperature-sensitive defect in the ubiquitin-activating enzyme E1 induced apoptosis independent of the activation of caspase-3 and -6 and the cleavage of their respective substrates poly(ADP-ribose) polymerase and lamin A. In addition, neither the caspase 3/7-specific inhibitorN-benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethylketone nor the general caspase inhibitorN-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone were capable of blocking this type of cell death. By contrast, Bcl-2 overexpression effectively protected cells from apoptosis induced by a defect in the E1 enzyme at the nonpermissive temperature. Bcl-2 acted downstream of the accumulation of short-lived or aberrant proteins because it did not prevent the overexpression of the short-lived proteins p53, p27 kip1, and cyclins D1 and B1 under conditions of decreased ubiquitination. These results suggest the existence of short-lived proteins that may serve the role of caspase-independent effectors of apoptosis and attractive targets of the death-protective action of Bcl-2. Apoptosis requires the activation of caspases (formerly interleukin 1β-converting enzyme-like proteases), in particular those related to the caspase-3/7/6 subfamily. Recent data, however, revealed that, although caspase-specific inhibitors delay apoptosis, they are often incapable of preventing it. To obtain evidence for caspase-independent steps of apoptosis, we artificially created a high amount of short-lived or aberrant proteins by blocking the ubiquitin degradation pathway. A temperature-sensitive defect in the ubiquitin-activating enzyme E1 induced apoptosis independent of the activation of caspase-3 and -6 and the cleavage of their respective substrates poly(ADP-ribose) polymerase and lamin A. In addition, neither the caspase 3/7-specific inhibitorN-benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethylketone nor the general caspase inhibitorN-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone were capable of blocking this type of cell death. By contrast, Bcl-2 overexpression effectively protected cells from apoptosis induced by a defect in the E1 enzyme at the nonpermissive temperature. Bcl-2 acted downstream of the accumulation of short-lived or aberrant proteins because it did not prevent the overexpression of the short-lived proteins p53, p27 kip1, and cyclins D1 and B1 under conditions of decreased ubiquitination. These results suggest the existence of short-lived proteins that may serve the role of caspase-independent effectors of apoptosis and attractive targets of the death-protective action of Bcl-2. Programmed cell death (apoptosis) is an essential process to maintain homeostasis in multicellular organisms. It is triggered by a variety of physiological and nonphysiological agents that generate a common set of morphological alterations such as cell shrinkage, surface blebbing, chromatin condensation, DNA and nuclear fragmentations, and the formation of membrane-enclosed apoptotic bodies that are phagocytosed by neighboring cells (1Duvall E. Wyllie A.H. Immunol. Today. 1986; 7: 115-119Abstract Full Text PDF PubMed Scopus (626) Google Scholar).The apoptotic process can be divided into three, functionally distinct phases: initiation, effector, and degradation (2Kroemer G. Zamzami N. Susin S.A. Immunol. Today. 1997; 18: 44-51Abstract Full Text PDF PubMed Scopus (1379) Google Scholar). The initiation phase involves the activation of heterogeneous intracellular signaling pathways that are distinct (“private”) for each death stimulus. These pathways converge on a common effector phase that, in turn, executes death by degrading various cellular components (3Fraser A. Evan G. Cell. 1996; 85: 781-784Abstract Full Text Full Text PDF PubMed Scopus (611) Google Scholar). The crucial molecular players in these phases are a series of cysteine proteases that are homologues of the interleukin 1β-converting enzyme (4Yuan J. Curr. Opin. Cell Biol. 1995; 7: 211-214Crossref PubMed Scopus (87) Google Scholar). Unlike other cysteine proteases, these enzymes cleave their substrates following aspartate residues and have therefore been named caspases (5Alnemri E.S. Livingston D.J. Nicholson D.W. Salvesen G. Thornberry N.A. Wong W.W. Yuan J. Cell. 1996; 87: 171Abstract Full Text Full Text PDF PubMed Scopus (2129) Google Scholar). All caspases exist as zymogens that require cleavage at internal aspartate residues to generate two-subunit active enzymes (6Martin S.J. Green D.R. Cell. 1995; 82: 349-352Abstract Full Text PDF PubMed Scopus (1258) Google Scholar). Because of this property, they can activate each other and form an amplified protease cascade similar to that seen in clotting and complement activation (6Martin S.J. Green D.R. Cell. 1995; 82: 349-352Abstract Full Text PDF PubMed Scopus (1258) Google Scholar). Most caspases are organized in “private” signaling pathways because their deletion or inhibition by specific inhibitors (Ac-YVAD-CHO, cytokine response modifier A (CrmA)) does not interfere with all sorts of apoptosis (7Kuida K. Lippke J.A. Ku G. Harding M.W. Livingston D.J. Su M.S.-S. Flavell R.A. Science. 1995; 267: 2000-2002Crossref PubMed Scopus (1443) Google Scholar, 8Jacobson M.D. Weil M. Raff M.C. J. Cell Biol. 1996; 133: 1041-1051Crossref PubMed Scopus (365) Google Scholar, 9White D.W. Gilmore T.D. Oncogene. 1996; 13: 891-899PubMed Google Scholar). However, at least three caspases, caspase-3, -6, and -7, seem to participate in the common death effector phase of apoptosis (5Alnemri E.S. Livingston D.J. Nicholson D.W. Salvesen G. Thornberry N.A. Wong W.W. Yuan J. Cell. 1996; 87: 171Abstract Full Text Full Text PDF PubMed Scopus (2129) Google Scholar, 10Nicholson D.W. Ali A. Thornberry N.A. Vaillancourt J.P. Ding C.K. Gallant M. Gareau Y. Griffin P.R. Labelle M. Lazebnik Y.A. 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Moreover, specific peptide inhibitors of caspase-3 and -7 such as Z-DEVD-fmk 1The abbreviations used are: Z-DEVD-fmk, N-benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethylketone; CHX, cycloheximide; DAPI, 4′,6′-diamidino-2-phenylindole; MG132, carbobenzoxyl-leucinyl-leucinyl-leucinal-H; PARP, poly(ADP-ribose) polymerase; PT, permeability transition; PVDF, polyvinylidene difluoride; Ub, ubiquitin; Z-VAD-fmk, N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone; PAGE, polyacrylamide gel electrophoresis; Pipes, 1,4-piperazinediethanesulfonic acid; E1, ubiquitin-activating enzyme; E2, ubiquitin carrier protein; E3, ubiquitin-protein isopeptide ligase. 1The abbreviations used are: Z-DEVD-fmk, N-benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethylketone; CHX, cycloheximide; DAPI, 4′,6′-diamidino-2-phenylindole; MG132, carbobenzoxyl-leucinyl-leucinyl-leucinal-H; PARP, poly(ADP-ribose) polymerase; PT, permeability transition; PVDF, polyvinylidene difluoride; Ub, ubiquitin; Z-VAD-fmk, N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone; PAGE, polyacrylamide gel electrophoresis; Pipes, 1,4-piperazinediethanesulfonic acid; E1, ubiquitin-activating enzyme; E2, ubiquitin carrier protein; E3, ubiquitin-protein isopeptide ligase. interfere with most, if not all, forms of mammalian apoptosis. This suggests that the caspases of the death effector phase have been evolutionarily conserved, but that higher organisms have adopted variations of these enzymes to regulate “private” initiation phases as well. Numerous substrates of caspase-3, -7, and -6 have been identified in mammalian cells. Among them are poly(ADP-ribose) polymerase (PARP), a repair enzyme preferentially cleaved by caspase-3 and -7 (14Lazebnik Y.A. Kaufmann S.H. Desnoyers S. Poirier G.G. Earnshaw W.C. Nature. 1994; 371: 346-347Crossref PubMed Scopus (2338) Google Scholar), and lamin A, a component of the nuclear lamina, preferentially cleaved by caspase-6 (15Orth K. O'Rourke K. Salvesen G.S. Dixit V.M. J. Biol. Chem. 1996; 271: 20977-20980Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar, 16Takahashi A. Alnemri E. Lazebnik Y. Fernandes-Alnemri T. Litwack G. Moir R.D. Goldman R.D. Poirier G.G. Kaufmann S.H. Earnshaw W.C. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 8395-8400Crossref PubMed Scopus (469) Google Scholar).Genetic studies in C. elegans further uncovered a gene product called ced-9, which has an inhibitory effect on apoptosis during nematodal development (13Shaham S. Horvitz H.R. Genes Dev. 1996; 10: 578-591Crossref PubMed Scopus (202) Google Scholar). The mammalian homologue of ced-9 is Bcl-2, a proto-oncogene product that was originally identified at a t(14;18) chromosomal breakpoint common to human follicular lymphomas (17Hengartner M.O. Horvitz H.R. Cell. 1994; 76: 665-676Abstract Full Text PDF PubMed Scopus (1043) Google Scholar). Bcl-2 can functionally substitute for ced-9 and prevent nematodal (17Hengartner M.O. Horvitz H.R. Cell. 1994; 76: 665-676Abstract Full Text PDF PubMed Scopus (1043) Google Scholar) as well as mammalian apoptosis induced by various, often unrelated death stimuli (18Reed J.C. J. Cell Biol. 1994; 124: 1-6Crossref PubMed Scopus (2385) Google Scholar). Both, genetic and biochemical studies revealed that ced-9 and Bcl-2 act upstream of ced-3 and caspase 3, respectively, to block caspase activation and apoptosis (13Shaham S. Horvitz H.R. Genes Dev. 1996; 10: 578-591Crossref PubMed Scopus (202) Google Scholar, 19Monney L. Otter I. Olivier R. Ravn U. Mirzasaleh H. Fellay I. Poirier G.G. Borner C. Biochem. Biophys. Res. Commun. 1996; 221: 340-345Crossref PubMed Scopus (63) Google Scholar, 20Boulakia C.A. Chen G. Ng F.W.H. Teodoro J.G. Branton P.E. Nicholson D.W. Poirier G.G. Shore G.C. Oncogene. 1996; 12: 529-535PubMed Google Scholar, 21Chinnaiyan A.M. Orth K. O'Rourke K. Duan H. Poirier G.G. Dixit V.M. J. Biol. Chem. 1996; 271: 4573-4576Abstract Full Text Full Text PDF PubMed Scopus (597) Google Scholar). However, neither ced-9 nor Bcl-2 directly interact with their respective caspases. The link has been provided by ced-4, another gene product essential for apoptosis in C. elegans (13Shaham S. Horvitz H.R. Genes Dev. 1996; 10: 578-591Crossref PubMed Scopus (202) Google Scholar). ced-4 directly binds to and activates ced-3/caspases and triggers nematodal and mammalian apoptosis in a ced-3-dependent manner (13Shaham S. Horvitz H.R. Genes Dev. 1996; 10: 578-591Crossref PubMed Scopus (202) Google Scholar, 22Chinnaiyan A.M. O'Rourke K. Lane B.R. Dixit V.M. Science. 1997; 275: 1122-1126Crossref PubMed Scopus (553) Google Scholar, 23Seshagiri S. Miller L.K. Curr. Biol. 1997; 7: 455-460Abstract Full Text Full Text PDF PubMed Google Scholar). Simultaneously, ced-4 interacts with ced-9/Bcl-2 and allows the latter to block the activation of ced-3/caspases (22Chinnaiyan A.M. O'Rourke K. Lane B.R. Dixit V.M. Science. 1997; 275: 1122-1126Crossref PubMed Scopus (553) Google Scholar, 23Seshagiri S. Miller L.K. Curr. Biol. 1997; 7: 455-460Abstract Full Text Full Text PDF PubMed Google Scholar, 24Wu D. Wallen H.D. Nunez G. Science. 1997; 275: 1126-1129Crossref PubMed Scopus (285) Google Scholar). Thus, ced-4 has a double impact on the effector phase of apoptosis; it can act as a caspase activator and as an adapter for Bcl-2 to inhibit caspases. A further characterization of the function of ced-4 has recently become available through the cloning of a mammalian homologue, called Apaf-1 (25Zou H. Henzel W.J. Liu X. Lutschg A. Wang W. Cell. 1997; 90: 405-413Abstract Full Text Full Text PDF PubMed Scopus (2724) Google Scholar).On the search for mammalian activators of caspases such as ced-4 homologues, we made the following assumption. If these activators were constitutively expressed, as it had been proposed (26Weil M. Jacobson M.D. Coles H.S.R. Davies T.J. Gardner R.L. Raff K.D. Raff M.C. J. Cell Biol. 1996; 133: 1053-1059Crossref PubMed Scopus (354) Google Scholar), their death-promoting activities should be dormant in surviving cells. Any death stimulus would have to reactivate them by posttranslational modifications or the binding or release of regulatory proteins. An attractive, but often overlooked alternative is changes in the protein half-lives of the caspase activators. Since the ubiquitin system is a prominent regulator of protein turnover, we tested whether defects in this system would lead to caspase activation or apoptosis.Ubiquitin-dependent degradation is necessary to eliminate damaged, misfolded proteins as well as proteins whose impact on biologic processes has to be of short duration (short-lived proteins) (27Jennissen H.P. Eur. J. Biochem. 1995; 231: 1-30Crossref PubMed Scopus (112) Google Scholar). In this pathway, ubiquitin (Ub) is first activated in an ATP-dependent manner by a thiolester linkage to the Ub-activating enzyme, E1. Activated Ub is then transferred to one of several Ub-conjugating enzymes, E2, to form another thiolester intermediate. From E2, Ub is ligated to lysine residues of target substrates sometimes with the help of specific E3 Ub protein ligases. Further ubiquitination of the mono-Ub protein substrates leads to the formation of multi-Ub chains that are recognized by the 26 S proteasome for degradation. An essential protease of the 26 S proteasome has recently been found to have a threonine residue in its active center. This site has subsequently been identified as the target for the specific proteasomal inhibitor lactacystin (28Fenteany G. Standaert R.F. Lane W.S. Choi S. Corey E.J. Schreiber S.L. Science. 1995; 268: 726-731Crossref PubMed Scopus (1492) Google Scholar). While several E2 enzymes exist, only one E1 enzyme has been found thus far in mammalian cells (27Jennissen H.P. Eur. J. Biochem. 1995; 231: 1-30Crossref PubMed Scopus (112) Google Scholar). Thus, inactivation of the E1 enzyme abrogates ubiquitination and leads to an accumulation of aberrant or short-lived protein (29Chowdary D.R. Dermody J.J. Jha K.K. Ozer H.L. Mol. Cell. Biol. 1994; 14: 1997-2003Crossref PubMed Scopus (266) Google Scholar). This is accentuated by a further blockage of the proteasome by lactacystin.We show here by using a cellular system of diminished E1 activity and simultaneous proteasomal inhibition that the turnover of short-lived proteins plays a crucial role in the apoptotic response. When present in high amounts, these proteins seem to activate a caspase-independent type of apoptosis that is still effectively impeded by Bcl-2 overexpression.DISCUSSIONThe present study describes a novel, caspase-independent form of apoptosis that can be effectively delayed by Bcl-2 overexpression. The molecular players in this process appear to be short-lived or aberrant proteins that are usually degraded by the ubiquitin/proteasome system. Due to diminished ubiquitination, these proteins accumulate to levels that are probably sufficient to trigger apoptosis without the participation of caspases. Because Bcl-2 overexpression still allows these proteins to accumulate but not to trigger apoptosis, they represent attractive targets for the death-protective action of Bcl-2.Ubiquitin-dependent degradation mainly serves to rid the cell of abnormal and misfolded proteins and to limit the time and amounts of availability of critical regulatory proteins (short-lived proteins) (27Jennissen H.P. Eur. J. Biochem. 1995; 231: 1-30Crossref PubMed Scopus (112) Google Scholar). This is essential for numerous cellular processes such as cell cycle control (38Glotzer M. Murray A.W. Kirschner M.W. Nature. 1991; 349: 132-138Crossref PubMed Scopus (1886) Google Scholar), gene transcription (47Scheffner M. Werness B.A. Huibregtse J.M. Levine A.J. Howley P.M. Cell. 1990; 63: 1129-1136Abstract Full Text PDF PubMed Scopus (3427) Google Scholar), chromatin maintenance (48Hunt L.T. Dayhoff M.O. Biochem. Biophys. Res. 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Osborne B.A. EMBO J. 1996; 15: 3835-3844Crossref PubMed Scopus (300) Google Scholar).Short-lived Proteins as Death Effectors or Inhibitors of Survival FactorsWhat might be the effector molecules that trigger apoptosis in response to decreased ubiquitination? Whereas aberrant proteins may interfere with vital cellular functions in rather nonspecific ways, accumulated short-lived proteins may have selective targets. Two mechanisms of action can be envisaged. First, short-lived proteins may be death effectors at high levels. They would be present in minute, nontoxic amounts in normal cells but accumulate to levels that activate some unknown “death substrates” in cells with a defective E1 enzyme. If apoptosis is induced by other stimuli such as staurosporine, tumor necrosis factor α, or Fas/APO, the short-lived proteins may accumulate due to a posttranslational modification that prevents their ubiquitin-dependent degradation. Various transcription factors such as p53, Myc, Jun, E2F, and cell cycle components such as the cyclins and cdc25 are rapidly degraded by the ubiquitin degradation pathway following phosphorylation, dephosphorylation, or other modifications (41Glotzer M. Curr. Biol. 1995; 9: 970-972Abstract Full Text Full Text PDF Scopus (27) Google Scholar, 62Ciechanover A. Digiuseppe J.A. Bercovich B. Orian A. Richter J.D. Schwartz A.L. Brodeur G.M. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 139-143Crossref PubMed Scopus (315) Google Scholar, 63Nefsky B. Beach D. EMBO J. 1996; 15: 1301-1312Crossref PubMed Scopus (95) Google Scholar, 64Murray A. Cell. 1995; 81: 149-152Abstract Full Text PDF PubMed Scopus (274) Google Scholar, 65Isaksson A. Musti A.M. Bohmann D. Biochim. Biophys. Acta. 1996; 1288: F21-F29PubMed Google Scholar, 66Hateboer G. Kerkhoven R.M. Shvarts A. Bernards R. Beijersbergen R.L. Genes Dev. 1996; 10: 2960-2970Crossref PubMed Scopus (189) Google Scholar). 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It is, however, unknown how their usual function as cell cycle or transcriptional regulators is converted into an apoptotic activity. According to the data here, this may be done by changing their half-lives. A similar mechanism has already been suggested for the conversion of these proteins into oncogene products (65Isaksson A. Musti A.M. Bohmann D. Biochim. Biophys. Acta. 1996; 1288: F21-F29PubMed Google Scholar) and indicates that the ubiquitin system may be an attractive, common regulator of tumorigenesis and apoptosis.As an alternative to a direct death effector function, the short-lived proteins may inhibit crucial survival factors. A good example is the case of the transcription factor NFκB. This protein has recently been shown to protect cells from tumor necrosis factor α-induced apoptosis (72Liu Z. Hsu H. Goeddel D.V. Karin M. Cell. 1996; 87: 565-576Abstract Full Text Full Text PDF PubMed Scopus (1778) Google Scholar, 73Van Antwerp D.J. Martin S.J. Kafri T. Green D.R. 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Three mechanisms can be envisaged.(i) Bcl-2 directs accumulating proteins to a ubiquitin-independent proteolytic degradation system. Such “antizymes” have been identified for p53 (E6) (47Scheffner M. Werness B.A. Huibregtse J.M. Levine A.J. Howley P.M. Cell. 1990; 63: 1129-1136Abstract Full Text PDF PubMed Scopus (3427) Google Scholar), cyclin A/B2 (p34 cdc2) (74Stewart E. Kobayashi H. Harrison D. Hunt T. EMBO J. 1994; 13: 584-594Crossref PubMed Scopus (91) Google Scholar), and ODC (ODC antizyme) (75Murakami Y. Matsufuji S. Kameji T. Hayashi S. Igarashi K. Tamura T. Tanaka K. Ichihara A. Nature. 1992; 360: 597-600Crossref PubMed Scopus (665) Google Scholar) and serve to accelerate the degradation of the respective short-lived protein. The death substrates that may be “antizymed” by Bcl-2 are, however, distinct from p53, p27 kip1, or the cyclins D1 and B1 because the latter accumulate to similar levels in vector control and Bcl-2-overexpressing cells. Moreover, the degradation machinery of these substrates are neither the proteasome nor lysosomes. Proteasomal inhibitors as well as lysosomotropic agents enhance the accumulation of short-lived proteins and provoke apoptosis much faster than decreased ubiquitination alone, but Bcl-2 is still effectively death-protective. These data do not, however, exclude the possibility that Bcl-2 exploits another proteolytic system to degrade specific short-lived death effectors. Moreover, Bcl-2 may sequester short-lived proteins into lysosomes where they are not degraded but unable to trigger apoptosis.(ii) Alternatively, Bcl-2 converts aberrant or short-lived proteins into an apoptosis-incompetent conformation. This could be best explained by a chaperoning function of Bcl-2. Chaperones like the well known heat shock proteins are ATPases that hold aberrant, denatured proteins in a nontoxic conformation. 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Thus, neither Bax nor p53 seem to be the targets of Bcl-2 and mediators of the apoptotic response induced by decreased ubiquitination.(iii) Finally, Bcl-2 could replace a survival factor whose activity is blocked by decreased ubiquitination. As discussed above, the activity of the survival factor c-Rel/NFκB is controlled by the ubiquitination of its inhibitor IκB. Interestingly, a temperature-sensitive c-Rel induces apoptosis at the nonpermissive temperature that can be blocked by Bcl-2 overexpression (9White D.W. Gilmore T.D. Oncogene. 1996; 13: 891-899PubMed Google Scholar). This indicates that Bcl-2 may compensate for the loss of anti-apoptotic gene products that are usually induced by c-Rel/NFκB.Cycloheximide Effects Support the Involvement of Short-lived Proteins in ApoptosisIt has become widely accepted that the apoptotic effector machinery is constitutively expressed in mammalian cells (26Weil M. Jacobson M.D. Coles H.S.R. Davies T.J. Gardner R.L. Raff K.D. Raff M.C. J. 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