Cyclopentenone Prostaglandins of the J Series Inhibit the Ubiquitin Isopeptidase Activity of the Proteasome Pathway

Abstract

Electrophilic eicosanoids of the J series, with their distinctive cross-conjugated α,β-unsaturated ketone, inactivate genetically wild type tumor suppressor p53 in a manner analogous to prostaglandins of the A series. Like the prostaglandins of the A series, prostaglandins of the J series have a structural determinant (endocyclic cyclopentenone) that confers the ability to impair the conformation, the phosphorylation, and the transcriptional activity of the p53 tumor suppressor with equivalent potency and efficacy. However, J series prostaglandins have a unique structural determinant (exocyclic α,β-unsaturated ketone) that confers unique efficacy as an apoptotic agonist. In seeking to understand how J series prostaglandins cause apoptosis despite their inactivation of p53, we discovered that they inhibit the ubiquitin isopeptidase activity of the proteasome pathway. In this regard, J series prostaglandins were more efficacious inhibitors than representative members of the A, B, or E series prostaglandins. Disruption of the proteasome pathway with proteasome inhibitors can cause apoptosis independently of p53. Therefore, this finding helps reconcile the p53 transcriptional independence of apoptosis caused by Δ12-prostaglandin J2. This discovery represents a novel mechanism for proteasome pathway inhibition in intact cells. Furthermore, it identifies isopeptidases as novel targets for the development of antineoplastic agents. Electrophilic eicosanoids of the J series, with their distinctive cross-conjugated α,β-unsaturated ketone, inactivate genetically wild type tumor suppressor p53 in a manner analogous to prostaglandins of the A series. Like the prostaglandins of the A series, prostaglandins of the J series have a structural determinant (endocyclic cyclopentenone) that confers the ability to impair the conformation, the phosphorylation, and the transcriptional activity of the p53 tumor suppressor with equivalent potency and efficacy. However, J series prostaglandins have a unique structural determinant (exocyclic α,β-unsaturated ketone) that confers unique efficacy as an apoptotic agonist. In seeking to understand how J series prostaglandins cause apoptosis despite their inactivation of p53, we discovered that they inhibit the ubiquitin isopeptidase activity of the proteasome pathway. In this regard, J series prostaglandins were more efficacious inhibitors than representative members of the A, B, or E series prostaglandins. Disruption of the proteasome pathway with proteasome inhibitors can cause apoptosis independently of p53. Therefore, this finding helps reconcile the p53 transcriptional independence of apoptosis caused by Δ12-prostaglandin J2. This discovery represents a novel mechanism for proteasome pathway inhibition in intact cells. Furthermore, it identifies isopeptidases as novel targets for the development of antineoplastic agents. prostaglandin(s) acetyl-Asp-Glu-Val-Asp-methylcoumarin-7-amide inhibitor of NFκB nuclear factor κB carbobenzoxy-l-leucyl-l-leucyl-l-glutamate α-(4-methyl-coumaryl-7-amide) carbobenzoxy-l-leucyl-l-leucyl-l-leucine α-(4-methyl-coumaryl-7-amide) succinyl-l-leucyl-l-leucyl-l-valyl-l-tyrosine α-(4-methyl-coumaryl-7-amide) carbobenzoxy-l-leucyl-l-arginyl-l-glycyl-l-glycine α-(4-methyl-coumaryl-7-amide) carbobenzyloxy-l-leucyl-l-leucyl-norvaline carbobenzoxy-valinyl-phenylalaninal 4-hydroxynonenal polyacrylamide gel electrophoresis 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid analysis of variance Certain electrophilic prostaglandins (PG)1 can repress transactivation by NFκB and p53 (1Rossi A. Elia G. Santoro M.G. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 746-750Crossref PubMed Scopus (183) Google Scholar, 2Rossi A. Kapahi P. Natoli G. Takahashi T. Chen Y. Karin M. 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Ushikubi F. Physiol. Rev. 1999; 79: 1193-1226Crossref PubMed Scopus (0) Google Scholar). If the endocyclic α,β-unsaturated ketone shared by A and J series PG confers activity, as hypothesized (1Rossi A. Elia G. Santoro M.G. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 746-750Crossref PubMed Scopus (183) Google Scholar, 2Rossi A. Kapahi P. Natoli G. Takahashi T. Chen Y. Karin M. Santoro M.G. Nature. 2000; 403: 103-108Crossref PubMed Scopus (1204) Google Scholar, 4Moos P.J. Edes K. Fitzpatrick F.A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 9215-9220Crossref PubMed Scopus (65) Google Scholar, 9Honn K.V. Marnett L.J. Biochem. Biophys. Res. Commun. 1985; 129: 34-40Crossref PubMed Scopus (92) Google Scholar, 10Narumiya S. Ohno K. Fukushima M. Fujiwara M. J. Pharmacol. Exp. Ther. 1987; 242: 306-311PubMed Google Scholar), then two predictions should be valid. First, individual A series and J series PG should act rather uniformly on the cellular processes they affect. Second, their cellular effects should be self-consistent with established models of NFκB and p53 function. However, not all experiments affirm these predictions. For example, the A and J series PG both repress NFκB transcription and inhibit IκB kinase (1Rossi A. Elia G. Santoro M.G. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 746-750Crossref PubMed Scopus (183) Google Scholar, 2Rossi A. Kapahi P. Natoli G. Takahashi T. Chen Y. Karin M. Santoro M.G. Nature. 2000; 403: 103-108Crossref PubMed Scopus (1204) Google Scholar); however, only Δ12-PGJ2 is anti-inflammatory (11Gilroy D.W. Colville-Nash P.R. Willis D. Chivers J. Paul-Clark M.J. Willoughby D.A. Nat. Med. 1999; 5: 698-701Crossref PubMed Scopus (1123) Google Scholar). Likewise, the A and J series PG both repress p53 transcription; however, only the A series PG antagonize p53-dependent apoptosis (Ref. 4Moos P.J. Edes K. Fitzpatrick F.A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 9215-9220Crossref PubMed Scopus (65) Google Scholar and see below). Herein we report the discovery of a molecular mechanism that clarifies the distinctive cellular effects of cyclopentenone PG. Namely, J series PG preferentially inhibit the ubiquitin isopeptidase activity (ubiquitin-specific protease) of the proteasome pathway. This pathway is the major nonlysosomal degradation pathway in cells (12Hochstrasser M. Annu. Rev. Genet. 1996; 30: 405-439Crossref PubMed Scopus (1461) Google Scholar, 13Hershko A. Ciechanover A. Annu. Rev. Biochem. 1998; 67: 425-479Crossref PubMed Scopus (6959) Google Scholar). The degradation of target proteins via this pathway largely depends on their covalent modification with a ubiquitin polymer. This polymer consists of ubiquitin (8.5 kDa) subunits that are covalently attached via isopeptide bonds between the COOH terminus of one ubiquitin and the lysine ε-amino group of another ubiquitin. In turn, this polymer is covalently attached to a lysine ε-amino group on the target protein via another isopeptide bond. Ubiquitin isopeptidases are a large family of cysteine hydrolases that preferentially cleave these isopeptide bonds (14Wilkinson K.D. Tashayev V.L. O'Connor L.B. Larsen C.N. Kasperek E. Pickart C.M. Biochemistry. 1995; 34: 14535-14546Crossref PubMed Scopus (264) Google Scholar). We demonstrate that inhibition of ubiquitin isopeptidase activity by PG depends on nuances of olefin-ketone conjugation. J series PG with a cross-conjugated α,β-unsaturated dienone are more efficacious inhibitors compared with A series PG with a single α,β-unsaturated ketone (Fig. 1). We further demonstrate that J series PG cause apoptosis, concurrently with inhibition of isopeptidase activity, and independently of p53-mediated gene transactivation. Our discovery has particular implications for the development of novel antineoplastic agents that are effective against cancers with dysfunctional p53 pathways. We used Dulbecco's minimum essential medium and media supplements (Life Technologies, Inc.); Δ12-PGJ2, PGA1, PGA2, PGB1, PGE2, and 15-keto-PGE2(Cayman Chemicals, Ann Arbor, MI); etoposide (Sigma); complete protease inhibitor mixture and FuGene-6 transfection reagent (Roche Molecular Biochemicals); enhanced chemiluminescence reagents (Amersham Pharmacia); luciferase reporter lysis buffer and reporter detection reagents (Promega); antibodies directed against p53, PAb240, FL393, and DO-1 (Santa Cruz Biotechnology), Phospho-p53 (Ser15) (Cell Signaling Technology), and PAb1620 (Oncogene Research Products); antibodies against p21WAF1/CIP1 (C-19, Santa Cruz Biotechnology) and ubiquitin (Ubi-1, Zymed Laboratories Inc.); horseradish peroxidase-conjugated secondary antibodies; protein A/G PLUS-Agarose (Santa Cruz Biotechnology); z-LLE-MCA, z-LLL-MCA, MG115, Ac-DEVD-MCA (Peptides International); LLVY-MCA, z-LRGG-MCA (Alexis Corporation); MDL 28170 (z-VF-CHO, Calbiochem); Ub-125I-lysozyme (gift from Dr. Martin Rechsteiner, Department of Biochemistry, University of Utah); annexin V, Alexa Fluor® 488 conjugate (Molecular Probes, Inc.); a luciferase p53 cis reporter construct (p53-Luc, Stratagene); β-galactosidase expression vector (pSV-β; Promega); and pC53-SN3 (wild type) and pC53-SCX3 (V143A mutant) p53 constructs ((15Baker S.J. 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We maintained Ts20 cells at 32 °C (permissive temperature) but placed them in a 40 °C incubator (nonpermissive temperature) to inactivate the E1 ubiquitin-activating enzyme in experiments designed to observe disassembly of polyubiquitinated proteins (17Chowdary D.R. Dermody J.J. Jha K.K. Ozer H.L. Mol. Cell. Biol. 1994; 14: 1997-2003Crossref PubMed Scopus (266) Google Scholar). All other cells were maintained at 37 °C. We measured p53-DNA binding activity via electrophoretic mobility shift assay. We isolated nuclei and generated protein-DNA complexes as described previously (18Wang P. Wu P. Siegel M.I. Egan R.W. Billah M.M. J. Biol. Chem. 1995; 270: 9558-9563Abstract Full Text Full Text PDF PubMed Scopus (632) Google Scholar). 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DO-1 p53 in or as a We the at to the We the with 1 of We then measured p53 or ubiquitin by analysis with antibodies for p53 and ubiquitin that and are despite their We on a with the for the and then the cells with We of buffer and and of Alexa Fluor® annexin and for at We the cells with of buffer and them in of We the cells with and and them by We activity and as described previously P.J. Fitzpatrick F.A. 1998; Google Scholar). We p21WAF1/CIP1 expression from experiments P.J. Edes K. Fitzpatrick F.A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 9215-9220Crossref PubMed Scopus (65) Google Scholar). We proteasome activity by the of for the and and of the we cells with the for the We cells in of with 2 to We and of a We of proteasome in lysis to 50 We the at 37 °C for and by the at and the at We the proteasome activity by the of as described previously M. J. Biol. Chem. Full Text PDF PubMed Google Scholar). We cells with the for the proteasome activity, we of of an 1 and at 37 we a and of bovine bovine in We then isolated the acid by acid on and for at We the in the by and the of to the of We isopeptidase activity by of a that the terminus of is in isopeptide We that this to degradation by the we cell in with to inhibit proteasome activity inhibition were used to activity and to of isopeptidase We of prostaglandins to isopeptidase were for on to We the at 37 °C for and as described were for proteasome activity to that the isopeptidase with the prostaglandins not to proteasome and can repress transcription by p53, p53-dependent apoptosis P.J. Edes K. Fitzpatrick F.A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 9215-9220Crossref PubMed Scopus (65) Google Scholar). If their electrophilic cyclopentenone confers this activity, as we then Δ12-PGJ2 should act to and with we that cells to Δ12-PGJ2 p53 that as by a of Δ12-PGJ2 p53 protein in cells from wild a mutant This mutant p53 is a for of this on p53 is an in p53 and p53 in cells to the (Fig. However, with B, and The of this are in the ability of p53. transcription of a p53-dependent reporter gene by Δ12-PGJ2 (Fig. 2 p53 to we ability to an a p53 binding In this etoposide the of complexes (Fig. 2 that be with (Fig. 2 and by mutant only the of complexes However, (Fig. 2 or with etoposide (Fig. 2 the of in of their effects on p53, J series PG are and from A series PG. PG have effects on wild type p53 in cell other than and cells not In to their effects on p53 A and J series PG effects on for agents that impair p53 and not cause apoptosis (Fig. However, apoptosis, by of and cellular of annexin (Fig. The of apoptosis by Δ12-PGJ2, with of p53 in cells is to reconcile with the of p53 tumor C. Hall P.A. J. Pathol. 1999; 187: 112-126Crossref PubMed Scopus (1234) Google Scholar, 7Ljungman M. Neoplasia (Bratisl.). 2000; 2: 208-225Crossref PubMed Scopus (186) Google Scholar). transcription of by p53, by in cells with Δ12-PGJ2 However, we that these cells to p21WAF1/CIP1 protein (Fig. B, This that Δ12-PGJ2 the of This is distinctive for and protein of p21WAF1/CIP1 in in cells with or P.J. Edes K. Fitzpatrick F.A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 9215-9220Crossref PubMed Scopus (65) Google Scholar) (Fig. A B, 2 and via the proteasome pathway the activity of transcription tumor and p21WAF1/CIP1 and p53 A. Ciechanover A. A. Nat. Med. 2000; PubMed Scopus Google Mol. Sci. 2000; PubMed Scopus Google Scholar). of the proteasome activity can cause apoptosis J. J. A. J. S. P.J. Cancer Res. 1999; Google Scholar). Therefore, we hypothesized that Δ12-PGJ2 the proteasome pathway. We used and RKO-E6 cells as a to this RKO-E6 cells are from cells with the The is an enzyme that degradation of the p53 tumor suppressor protein M. 1999; PubMed Scopus Google Scholar). that RKO-E6 cells p53 to a than cells from to In one that both cell p53 to a Δ12-PGJ2 the proteasome pathway. with the of p53 protein in RKO-E6 cells in cells with Δ12-PGJ2 A, that RKO-E6 cells with etoposide p53 compared with cells (Fig. that the the degradation of p53. that RKO-E6 cells with and of p53 compared with the (Fig. A, that these cyclopentenone PG not inhibit degradation of p53 as as and RKO-E6 cells with Δ12-PGJ2 p53 protein compared with A, 1). A that the proteasome MG115, and RKO-E6 cells in a manner analogous to Δ12-PGJ2 (Fig. A, p53 proteins are for degradation by with S. Mol. Cell. Biol. 2000; PubMed Scopus Google Scholar). with Δ12-PGJ2 caused and RKO-E6 cells to molecular p53 by (Fig. B, with by and analysis with that these molecular p53 were polyubiquitinated of p53 (Fig. and The of in that Δ12-PGJ2 the proteasome pathway in a This is not to p53 or to Δ12-PGJ2 caused a of in cells that p53 not conjugate in cells with J series PG (Fig. compared with PGA1, PGA2, or J series PG a cross-conjugated ketone, the other PG not have this that not it is to Δ12-PGJ2 in F.A. J. Biol. Chem. Full Text PDF PubMed Google the proteasome pathway at a from the A, not inhibit the or of the cells were for at 37 °C with MG115, or were for of and and a are the with B, despite a proteasome Δ12-PGJ2 is at cells were for at 37 °C with MG115, or of polyubiquitinated proteins by analysis with Δ12-PGJ2 the of molecular of The in by to the molecular of polyubiquitinated that in cells with Δ12-PGJ2 or the molecular of the is that Δ12-PGJ2 the or other ubiquitin ubiquitin-activating or cells with Δ12-PGJ2 This that these are in their of ubiquitin Δ12-PGJ2 potency and efficacy as an apoptotic in RKO-E6 cells not This is with that apoptosis via a that is of p53 The proteasome consists of two subunits that and polyubiquitinated protein and a A. Ciechanover A. A. Nat. Med. 2000; PubMed Scopus Google Scholar). 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We Dr. Martin and University of Dr. Harvey Ozer, Jersey of and Dr. Mark Meuth, University of Sheffield, United and Dr. Bert Vogelstein, Johns Hopkins of for reagents and cell We Dr. for and on this