Confinement of Caspase-12 Proteolytic Activity to Autoprocessing

Corrections

BIOCHEMISTRY CHEMISTRY Correction for “Confinement of caspase-12 proteolytic activity to Correction for “Direct asymmetric vinylogous Michael addition autoprocessing,” by Sophie Roy, Jeffrey R. Sharom, Caroline of cyclic enones to nitroalkenes via dienamine catalysis,” by Houde, Thomas P. Loisel, John P. Vaillancourt, Wei Shao, Maya Giorgio Bencivenni, Patrizia Galzerano, Andrea Mazzanti, Saleh, and Donald W. Nicholson, which appeared in issue 11, Giuseppe Bartoli, and Paolo Melchiorre, which appeared in issue March 18, 2008, of Proc Natl Acad Sci USA (105:4133–4138; first 48, November 30, 2010, of Proc Natl Acad Sci USA (107:20642– published March 10, 2008; 10.1073/pnas.0706658105). 20647; first published June 21, 2010; 10.1073/pnas.1001150107). The authors note that Fig. 6 appeared incorrectly. There was The authors note that they incorrectly assigned the structure an error in the alignment of the molecular mass markers, and of the reaction product reported in Scheme 4. The published minor adjustments have been made to the assignment of caspase- structure represents the γ-aminated adduct 8, when it should 12 bands. The authors also note that the source of the anti- instead be the α-analogue arising from an α-site selective path- caspase-12 antibody for Fig. 6 was Sigma (clone 14F7). This way. As a result of this, Scheme 4 and its related comments error does not affect the conclusions of the article. The corrected should be removed from the article. figure and its corresponding legend appear below. On page 20642, left column, within the Abstract, lines 16–18, “Finally, we describe the extension of the dienamine catalysis- induced vinylogous nucleophilicity to the asymmetric γ-amination of cyclohexene carbaldehyde” should be removed from the article. On page 20645, right column, third full paragraph, lines 1–8, to page 20646, left column, first paragraph, lines 1–2, “Finally, to explore the potential of the chiral primary amine-induced vinylogous nucleophilicity, we wondered whether this unique re- kDa IP: Anti-Flag-caspase-1 activity concept may be translated to an aldehyde derivative 150 Western: Anti-Csp-12 adorned with a six-membered ring scaffold, reminiscent of the 100 β-substituted cyclohexanone framework. Although the vinylogous Michael addition of 1-cyclohexene-1-carboxaldehyde 7 to ni- 75 trostyrene 2a did not proceed at all, the combination with tert- proCaspase-12 50 butylazodicarboxylate under the catalysis of A furnished the γ 8 37 * -amination product with perfect regio- and enantioselectivity Auto-processing products (Scheme 4)” should be removed from the article. 25 IgG light chain These errors do not affect the conclusions of the article of the 20 vinylogous Michael addition of cyclic enones to nitroalkenes. The 15 ability of primary amine catalysis to address the synthetic issue connected with the enantioselective carbon–carbon bond formation gamma to a carbonyl group, promoting vinylogous nucleophilicity upon selective activation of unmodified cyclic unsaturated 12345 ketones, is fully supported by the separated results presented in Tables 1, 2, and 3, and Schemes 2 and 3. Total IP www.pnas.org/cgi/doi/10.1073/pnas.1302980110 Fig. 6. Procaspase-12 forms a complex with caspase-1 and is partially autoprocessed in the complex. HEK293T cells were cotransfected with expression vectors harboring Flag-tagged procaspase-1 (all lanes) plus either procaspase-12 (lanes 1 and 4) or the catalytically incapacitated C299A mutant (lanes 2 and 5). After 24 h, cells were harvested and lysed. One tenth of the lysate was directly applied to SDS/PAGE (lanes 1 and 2), and the remainder was immunoharvested with antibodies directed against the caspase-1 Flag epitope tag (lanes 4 and 5; lane 3 was processed in the same way, except that only lysis buffer was used). Immunoblotting for the large subunit (p20) of caspase-12 revealed that procaspase-12 and the resulting autocleavage product were both immunoharvested with caspase-1. The asterisk indicates a band of unknown identity that is detected by prebleed control serum.

www.pnas.org/cgi/doi/10.1073/pnas.1302753110

4852–4853 | PNAS | March 19, 2013 | vol. 110 | no. 12 www.pnas.org Downloaded by guest on September 25, 2021 MICROBIOLOGY Correction for “Commensal bacteria play a role in mating identical in design to those we reported. In the table below, preference of Drosophila melanogaster,” by Gil Sharon, Daniel we combined the results of those additional replicate experi- Segal, John M. Ringo, Abraham Hefetz, Ilana Zilber-Rosenberg, ments with those already reported. From the new analysis, we and Eugene Rosenberg, which appeared in issue 46, November now find that experiment 4, in which flies were infected with 16, 2010, of Proc Natl Acad Sci USA (107:20051–20056; first pub- amixtureofLactobacillus spp., assortative mating was not lished November 1, 2010; 10.1073/pnas.1009906107). restored. Otherwise, the conclusions of the article were not The authors note the following: “The mating frequencies changed by our reanalysis. We acknowledge the statistical ad- reported in Table 1 of this paper did not follow a multinomial vice of Dan Yekutieli and thank Tal Lahav for calculating the distribution, making the statistical analysis inapplicable. This odds ratios and their 95% confidence intervals, and for per- problem was obviated by considering only the first matings forming the chi-squared tests presented in the corrected in each experimental unit and computing odds ratios. After Table 1.” submitting the paper, we continued to perform experiments The corrected Table 1 appears below.

Table 1. The role of bacteria in diet-induced mating preference of D. melanogaster Experiment Fly treatment* N† OR‡ 95% CI P value‡

− 1 Starch-grown × CMY-grown 18 3.21 2.14–4.81 1.8 × 10 8 2 Experiment 1 after antibiotics 11 1.04 0.63–1.71 0.9888 3 Experiment 2 after infection of starch-grown ﬂies with homologous bacteria§ 6 2.68 1.40–5.11 0.0477 4 Experiment 3 with Lactobacillus spp. replacing homologous bacteria 4 1.76 0.74–4.19 0.2912 5 Experiment 3 with Lactobacillus plantarum replacing homologous bacteria 7 2.14 1.35–3.39 0.0019 6 Infection control (no added bacteria) 4 1.26 0.53–3.00 0.7712

*After all treatments, the ﬂies were grown for one generation in CMY medium before performing the mating preference test. † N is the number of replicate experiments. ‡ Cochran-Mantel-Haenszel Odds Ratio and P value are from the Cochran-Mantel-Haenszel Chi-squared test (34, 35). §Antibiotic-treated starch- and CMY-grown ﬂies were infected with bacteria isolated from their respective growth medium (before antibiotic treatment).

34. Mantel N, Haenszel W (1959) Statistical aspects of the analysis of data from retrospective studies of disease. J Natl Cancer Inst 22(4):719–748. 35. Cochran WD (1954) Some methods for strengthening the common χ2 tests. Biometrics 10:417–451.

www.pnas.org/cgi/doi/10.1073/pnas.1302326110

MEDICAL SCIENCES SYSTEMS BIOLOGY Correction for “Interaction of intracellular β amyloid peptide Correction for “Expanded methyl-sensitive cut counting reveals with chaperone proteins,” by Virginia Fonte, Vadim Kapulkin, hypomethylation as an epigenetic state that highlights functional Andrew Taft, Amy Fluet, David Friedman, and Christopher sequences of the genome,” by Alejandro Colaneri, Nickolas Staffa, D. Link, which appeared in issue 14, July 9, 2002, of Proc Natl David C. Fargo, Yuan Gao, Tianyuan Wang, Shyamal D. Peddada, Acad Sci USA (99:9439–9444; ﬁrst published June 27, and Lutz Birnbaumer, which appeared in issue 23, June 7, 2011, – ﬁ 2002; 10.1073/pnas.152313999). of Proc Natl Acad Sci USA (108:9715 9720; rst published The authors note that the author name Vadim Kapulkin should May 20, 2011; 10.1073/pnas.1105713108). instead appear as Wadim Jan Kapulkin. The corrected author line The authors note that, within the corresponding author foot- note on page 9715, the email address “[email protected]” appears below. The online version has been corrected. should instead appear as “[email protected]”.

Virginia Fonte, Wadim Jan Kapulkin, Andrew Taft, www.pnas.org/cgi/doi/10.1073/pnas.1302473110 Amy Fluet, David Friedman, and Christopher D. Link

www.pnas.org/cgi/doi/10.1073/pnas.1302545110 CORRECTIONS

PNAS | March 19, 2013 | vol. 110 | no. 12 | 4853 Downloaded by guest on September 25, 2021 Corrections

BIOCHEMISTRY Correction for “Enhancing nuclear receptor-induced transcrip- Sci USA (105:19199–19204; first published December 3, 2008; tion requires nuclear motor and LSD1-dependent gene net- 10.1073/pnas.0810634105). working in interchromatin granules,” by Qidong Hu, Young-Soo The authors wish to note, “The legend of Fig. 2B should Kwon, Esperanza Nunez, Maria Dafne Cardamone, Kasey R. state that the experiment was performed in HMEC cells, rather Hutt, Kenneth A. Ohgi, Ivan Garcia-Bassets, David W. Rose, than in MCF7 cells, similar to the confirmatory experiment Christopher K. Glass, Michael G. Rosenfeld, and Xiang-Dong Fu, presented in Fig. 2C.” The figure and its corrected legend which appeared in issue 49, December 9, 2008, of Proc Natl Acad appear below.

Fig. 2. Rapid induction of interchromosomal interactions by nuclear hormone signaling. (A) 3D-FISH confirmation of E2-induced (60 min) TFF1:GREB1 interchromosomal interactions in HMECs with the distribution of loci distances measured (box plot with scatter plot) and quantification of colocalization (bar graph) before and after E2 treatment. Cells exhibiting mono- or biallelic interactions were combined for comparison with cells showing no colocalization; statistical sig- nificance in the bar graph was determined by χ2 test (**, P < 0.001). (B) 2D FISH confirmation of the interchromosomal interactions in HMEC cells by combining chromosome paint (aqua) and specific DNA probes (green and red). (Upper) Illustrates two examples of mock-treated cells. (Lower) Shows the biallelic interactions/ nuclear reorganization after E2 treatment for 60 min, exhibiting kissing events between chromosome 21 and chromosome 2. (C) Similar analysis on HMECs, but in this case using 3D FISH to paint chromosome 2 (red) and chromosome 21 (green), showing E2-induced chromosome 2–chromosome 21 interaction. Both assays revealed neither chromosome 21–chromosome 21 nor chromosome 2–chromosome 2 interactions in response to E2. (D) Temporal kinetics of GREB1:TFF1 interactions 2 by 3D FISH in HMECs (**, P < 0.001 by χ ). (E–G) Nuclear microinjection of siRNA against ERα, CBP/p300,orSRC1/pCIP prevented E2-induced interchromosomal interactions, counting both mono- and biallelic interactions (**, P < 0.001 by χ2). The injection of siER and siDLC1 were done in the same experiment, sharing the same control group. (H) Nuclear microinjection of siRNA against LSD1, which was shown to be required for estrogen-induced gene expression (22), did not block

E2-induced interchromosomal interactions. The injection of siLSD1 and SRC1/pCIP were done in a single experiment, sharing the same control group. www.pnas.org/cgi/doi/10.1073/pnas.1400054111

2046–2047 | PNAS | February 4, 2014 | vol. 111 | no. 5 www.pnas.org BIOCHEMISTRY IMMUNOLOGY Update to Correction for “Confinement of caspase-12 proteolytic Correction for “MicroRNA-directed program of cytotoxic CD8+ activity to autoprocessing,” by Sophie Roy, Jeffrey R. Sharom, T-cell differentiation,” by Sara Trifari, Matthew E. Pipkin, Caroline Houde, Thomas P. Loisel, John P. Vaillancourt, Wei Hozefa S. Bandukwala, Tarmo Äijö, Jed Bassein, Runqiang Shao, Maya Saleh, and Donald W. Nicholson, which appeared in Chen, Gustavo J. Martinez, and Anjana Rao, which appeared issue 11, March 18, 2008, of Proc Natl Acad Sci USA (105:4133– in issue 46, November 12, 2013, of Proc Natl Acad Sci USA 4138; first published March 10, 2008; 10.1073/pnas.0706658105). (110:18608–18613; first published October 25, 2013; 10.1073/ The authors wish to note: “We recently published corrections pnas.1317191110). to two articles that describe the functional role (1) and enzy- The authors note that the accession number for the GEO mology (2) of caspase-12. The figures in these two articles have database is GSE51393. been thoroughly investigated by a committee at McGill Univer- sity. With regard to Fig. 6 in the article in PNAS, two findings www.pnas.org/cgi/doi/10.1073/pnas.1400120111 were determined: First, that republication of the data was a consequence of miscommunication among co-authors working in different locations, and second, that the molecular weight markers were unintentionally mislabeled. The latter issue has recently been corrected (3). The former issue has recently been MEDICAL SCIENCES, ENGINEERING rectified because figure 4 of the Nature article was replaced with Correction for “Generation of functionally competent and du- a de novo independent experiment (4). Interpretation of the rable engineered blood vessels from human induced pluripotent experiment and the conclusions of both the PNAS article and the stem cells,” by Rekha Samuel, Laurence Daheron, Shan Liao, Nature article are unaffected by these changes. The authors Trupti Vardam, Walid S. Kamoun, Ana Batista, Christa Buecker, apologize for any confusion.” Richard Schäfer, Xiaoxing Han, Patrick Au, David T. Scadden, Dan G. Duda, Dai Fukumura, and Rakesh K. Jain, which appeared in issue 31, July 30, 2013, of Proc Natl Acad Sci USA (110:12774– 1. Saleh M, et al. (2006) Enhanced bacterial clearance and sepsis resistance in caspase-12- deficient mice. Nature 440(7087):1064–1068. 12779; first published July 16, 2013; 10.1073/pnas.1310675110). 2. Roy S, et al. (2008) Confinement of caspase-12 proteolytic activity to autoprocessing. The authors note that the following statement should be Proc Natl Acad Sci USA 105(11):4133–4138. added to the Acknowledgments: “This work is in part supported 3. Roy S, et al. (2013) Correction: Confinement of caspase-12 proteolytic activity to au- by Department of Defense (DoD) Breast Cancer Research In- toprocessing. Proc Natl Acad Sci USA 110(12):4852. ” 4. Saleh M, et al. (2013) Corrigendum: Enhanced bacterial clearance and sepsis resistance novator Award W81XWH-10-1-0016 (to R.K.J.). in caspase-12-deficient mice. Nature, 10.1038/nature12181. www.pnas.org/cgi/doi/10.1073/pnas.1400494111 www.pnas.org/cgi/doi/10.1073/pnas.1323789111 CORRECTIONS

PNAS | February 4, 2014 | vol. 111 | no. 5 | 2047 Confinement of caspase-12 proteolytic activity to autoprocessing

Sophie Roy*, Jeffrey R. Sharom†, Caroline Houde‡, Thomas P. Loisel§, John P. Vaillancourt¶, Wei Shaoʈ, Maya Salehʈ, and Donald W. Nicholson**††

*Department of Cardiovascular Diseases, ‡Apoptosis Research Group, and **Bone, Respiratory, Immunology, and Endocrine Franchise, Merck Research Laboratories, 126 East Lincoln Avenue, Rahway, NJ 07065-0900; †Department of Medical Genetics and Microbiology, University of Toronto, Medical Sciences Building, 1 King’s College Circle, No. 4396, Toronto, ON, Canada M5S 1A8; §Department of Biology, Enobia Pharma Inc., 2901 Rachel Street East, Suite 23, Montreal, QC, Canada H1W 4A4; ¶Department of Biochemistry, Merck Frosst Centre for Therapeutic Research, 16711 Trans Canada Highway, Kirkland, QC, Canada H9H 3L1; and ʈDepartment of Medicine, McGill University, 687 Pine Avenue West, Montreal, QC, Canada H3A 1A1

Edited by Richard A. Flavell, Yale University School of Medicine, New Haven, CT, and approved January 31, 2008 (received for review July 16, 2007) Caspase-12 is a dominant-negative regulator of caspase-1 (IL- tokine production in whole blood and is overrepresented in 1␤-converting enzyme) and an attenuator of cytokine respon- individuals with severe sepsis and septic mortality (3). In mice, siveness to septic infections. This molecular role for caspase-12 caspase-12 gene deletion confers resistance to peritonitis and appears to be akin to the role of cFLIP in regulating caspase-8 in septic shock in surgical models of human sepsis, and the resulting the extrinsic cell death pathway; however, unlike cFLIP/Usurpin, survival advantage is conferred by enhanced bacterial clearance we demonstrate here that caspase-12 is catalytically competent. in caspase-12-deficient animals (4). Collectively, these data in To examine these catalytic properties, rat caspase-12 was humans and rodents demonstrate that the presence of caspase-12 cloned, and the recombinant enzyme was used to examine the attenuates responsiveness to bacterial invasion and confers a cleavage of macromolecular and synthetic fluorogenic sub- survival disadvantage. In neither case was an effect of apoptosis strates. Although caspase-12 could mediate autoproteolytic observed (including ER stress). Mechanistically, it was found maturation of its own proenzyme, in both cis and trans,itwas that caspase-12 directly associated with caspase-1 and precluded not able to cleave any other polypeptide substrate, including normal caspase-1 activation. This interaction results in reduced other caspase proenzymes, apoptotic substrates, cytokine pre- cytokine processing, especially those processed by caspase-1, cursors, or proteins in the endoplasmic reticulum that normally including IL-1␤ and IL-18 (IGIF, IFN-␥-inducing factor), and undergo caspase-mediated proteolysis. The dearth of potential enhanced susceptibility to bacterial invasion as a consequence. substrates for caspase-12 also was confirmed by whole-cell Surprisingly, the catalytic activity of caspase-12 was not neces- diagonal-gel analysis. Autolytic cleavage within the caspase-12 sary for this inhibitory effect on the caspase-1 cytokine pathway proenzyme was mapped to a single site at the large–small (4), suggesting that, in the same way that cFLIP is a dominant- 319 subunit junction, ATAD , and this motif was recognized by negative regulator of caspase-8 and the extrinsic cell death caspase-12 when incorporated into synthetic fluorogenic sub- pathway (5, 6), caspase-12 is its counterpart for regulating the strates. The specific activity of caspase-12 with these substates inflammatory caspase cascade. Unlike cFLIP, however, was several orders of magnitude lower than caspases-1 and -3, caspase-12 does not contain critical lesions in the catalytic highlighting its relative catalytic paucity. In intact cells, machinery that would preclude it from proteolytic activity. caspase-12 autoproteolysis occurred in the inhibitory complex Although several laboratories have struggled to detect proteo- containing caspase-1. We propose that the proteolytic activity of lytic activity from caspase-12, it is possible that its catalytic caspase-12 is confined to its own proenzyme and that auto- capabilities are important for its role in caspase-1 modulation or cleavage within the caspase-1 complex may be a means for other cellular functions. To this end, we have characterized the temporal limitation of the inhibitory effects of caspase-12 on proteolytic properties of caspase-12. proinflammatory cytokine maturation. Results apoptosis ͉ inflammation ͉ sepsis Caspase-12 Contains Key Features Necessary for Proteolysis. For these studies, rat procaspase-12 was cloned and expressed in he caspases are a family of cysteinyl proteases that play a bacteria to produce active enzyme (National Center for Biotech- Tcritical role in apoptotic cell death as well as in inflammatory nology Information accession no. AF317633.1). The deduced pro- cytokine processing (1). The last and most recently discovered tein sequence was found to be 86% identical (95% similar) to mouse caspase is caspase-12, which is highly related to the cytokine procaspase-12 and 48% identical (61% similar) to human ␤ processing group-I caspases that include caspase-1 (ICE, IL-1 - caspase-12 (a 79-residue segment missing in the human sequence converting enzyme) (Fig. 1). Early studies suggested a potential accounts for much of the rodent/human sequence nonidentity). A role for caspase-12 in endoplasmic reticulum (ER) stress- phylogenetic analysis clearly placed caspase-12 from all three induced apoptosis, including the cell death response to ␤ species in the group-I inflammatory caspase cluster, which includes amyloid- deposition and, therefore, the pathogenesis of Alz- caspase-1, -4, -5, -12, and -14 (caspase-11 in the mouse and rat is the heimer’s disease (2). Recent studies from our laboratories, orthologue to human caspase-4 and -5) (Fig. 1A). Detailed analysis however, are not consistent with these earlier findings (3, 4). of the key residues contained within caspases that are known to be

Instead, caspase-12 was shown to play a role in modulating the BIOCHEMISTRY inflammatory caspase activation pathway by a direct dominant- negative effect on caspase-1 and to have no detectable effect on Author contributions: S.R. and J.R.S. contributed equally to this work; S.R., J.R.S., C.H., T.P.L., apoptotic pathways. In humans, for example, an SNP results in J.P.V., M.S., and D.W.N. designed research; J.R.S., C.H., T.P.L., J.P.V., W.S., and M.S. per- the synthesis of either a full-length caspase-12 proenzyme or a formed research; S.R., J.R.S., C.H., T.P.L., M.S., and D.W.N. analyzed data; and C.H. and substantially truncated protein containing only the prodomain, D.W.N. wrote the paper. but not the large and small subunits of the active mature enzyme. The authors declare no conﬂict of interest. The presence of the long polymorph, which is rare in the human This article is a PNAS Direct Submission. population and largely confined to individuals of African de- ††To whom correspondence should be addressed. E-mail: [email protected]. scent, confers hyporesponsiveness to endotoxin-stimulated cy- © 2008 by The National Academy of Sciences of the USA www.pnas.org͞cgi͞doi͞10.1073͞pnas.0706658105 PNAS ͉ March 18, 2008 ͉ vol. 105 ͉ no. 11 ͉ 4133–4138 Fig. 2. Caspase-12 is capable of both cis and trans autocatalytic processing. (A) Bacterial expression of either wild-type procaspase-12 or the catalytically inactivated C299A mutant were induced by IPTG for the indicated time. Anti- caspase-12 immunoblotting of total bacterial lysates showed cis autoprocessing occurred in the samples containing wild-type, but not C299A, procaspase- 12. (B) Bacterial lysates containing either the inactive form of caspase-12 (C299A) or the autoprocessed wild-type form were used as enzymes for in vitro processing of [35S]procaspase-12 C299A. Trans cleavage with wild-type caspase-12 yielded 36- and 12-kDa fragments.

Fig. 1. Caspase-12 features. (A) Human protein sequence alignment using sion systems are ideal for studying these events in vitro because the Merck multiclustal algorithm (14) places caspase-12 in the caspase-1 (ICE) processing does not occur during the early stages of induction, group-I gene family (boxed). Mouse and rat phylogenic trees differ from human by the consolidation of human caspase-4 and -5 into rodent caspase- but then rapidly occurs as media acidifies (9). Rat caspase-12 11. (B) An alignment of all known active mammalian caspases shows that all expression in Escherichia coli was induced, and samples of the residues within the caspase enzymes that form the catalytic dyad and tether culture were harvested at varying time points and analyzed by the P1 carboxylate side chain of substrates (blue) are absolutely conserved with SDS/PAGE and Western blotting using antisera raised against the exception of Arg[ICE:341] (ICE numbering system), which is Lys in rodent the large subunit of recombinant rat caspase-12. As occurs with caspase-12s (red). other caspases in this system, procaspase-12 accumulated as its full-length proenzyme form for the first hour and then was rapidly converted to its mature form (Fig. 2A). That the matu- essential for proteolytic activity (based on the 3D x-ray structures ration of caspase-12 was autolytic and not mediated by a of caspase-1 and -3) (7, 8) showed absolute conservation in rat bacterial protease was confirmed in parallel samples in which the caspase-12, with one exception (Fig. 1B). These conserved elements 299 (ICE:285) (ICE:237) essential catalytic Cys was mutated to Ala (C A). To test include the catalytic diad [Cys and His ], the con- whether caspase-12 could also autoprocess in trans, mature served QxCxG pentapeptide motif that harbors the catalytic cys- active caspase-12 was generated in bacteria and then was used to teine residue (QACRG for rat caspase-12, like the majority of all test for cleavage of [35S]procaspase-12. Mature active caspase-12 other caspases), and three of the four residues that stabilize the Asp from bacterial expression was able to efficiently process [35S]pro- (ICE:179) carboxylate side chain within the S1 subsite [Arg , caspase-12 (Fig. 2B), and this activity also was ablated if the (ICE:283) (ICE:347) Gln , and Ser ]. The lone exception is a conservative bacterial-expressed caspase-12 was inactivated through the (ICE:341) substitution of Arg for Lys in rat caspase-12. This same C299A mutation. Together these data demonstrate that rat substitution is present in mouse, but not human, caspase-12. With caspase-12 is proteolytically competent and is able to mediate its the exception that the effect of a conservative substitution in one of own maturation through autoproteolysis in both cis and trans. the four residues that stabilize the P1 Asp of caspase substrates is unknown, rodent caspase-12 appears to have the needed elements Caspase-12 Mediates Discrete Autoproteolysis at Asp319. In vitro, for catalytic activity. This finding is in contrast to cFLIP, where most proteolytically active caspases are able to segregate their these critical residues are substantially changed, resulting in no large subunits from their small subunits through autolytic pro- catalytic ability (6). teolysis and to remove their N-terminal prodomains as well. If both events were occurring for rat caspase-12, then the accu- Autolytic Processing of Caspase-12 in Cis and Trans. All caspases with mulated product would be Ϸ20 kDa. Instead, the accumulating catalytic activity are capable of autolytic processing between species after expression in bacteria (or trans cleavage of [35S]pro- their large and small subunits when generated in heterologous caspase-12) was 36 kDa (Fig. 2), indicating that one event was expression systems (cis activation). Inducible bacterial expres- occurring, but not the other. This finding is indicative of either

4134 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0706658105 Roy et al. Fig. 3. Identiﬁcation of procaspase-12 autoprocessing site. (A) The rat caspase-12 proenzyme has a predicted molecular mass of 48 kDa and is represented by the bar with its tripartite constituents: the N-terminal prodomain, the 20-kDa large subunit that harbors the catalytic Cys residue within the QACRG pentapeptide motif, and the 12-kDa small subunit. Potential caspase cleavage motifs are identiﬁed above the bar, with the corresponding fragment sizes indicated below the bar. (B)[35S]procaspase-12 mutants were used as substrates for in vitro cleavage assays with bacterial lysates containing Fig. 4. Macromolecular cleavage by caspase-12 does not extend to other either the inactive form of caspase-12 (C299A; left lanes) or the wild-type form known caspase substrates. Bacterial lysates containing either the inactive form (right lanes). Only the conversion of the ATAD319 (Left) to ATAA319 (Right) of caspase-12 (C299A or CA) or the autoprocessed wild-type form of caspase-12 resulted in the loss of autoprocessing activity. (wt) were used as enzyme for in vitro processing of the indicated [35S]precursor proteins. (A) Recombinant caspase-12 (rCaspase-12) titration shows concentration responsiveness in the reaction mixtures (Left), and the maximum a highly selective substrate requirement for caspase-12 or the concentration was used to test for rCaspase-12 processing of procaspase-9 unavailability of suitable automaturation sites within the (proC9) (Right). (B) Multiple caspase substrates were tested by the same caspase-12 proenzyme. To map the cleavage site to resolve this procedure as described for A, with incubation times of 90 min. Only pro- issue and to determine what the preferred motif for caspase-12 caspase-12, used as a positive control, was cleaved. recognition might be, Asp residues across the proenzyme were systematically changed to Ala by site-directed mutagenesis (Fig. 3A) (data not shown). The resulting [35S]procaspase-12 polypep- caspase-9 (Fig. 4A) among multiple known caspase substrates tides were used as substrate for cleavage by active caspase-12 that (Fig. 4B), including other caspase proenzymes (caspase-1, -4, and was generated in bacteria. A single mutation, D319A, prevented -5 from the group-I inflammatory subfamily, caspase-3 from the all processing activity (Fig. 3B), demonstrating that caspase-12 group-II effector subfamily, and caspase-8 and -9 from the mediates automaturation by a single cleavage event between the group-III intrinsic and extrinsic pathways). We also examined large and small subunits at D319, but does not further process the caspase-1 substrates (IL-1␤ and IL-18/IGIF), representative large subunit from the prodomain. The sequence at that site apoptotic substrates (PARP and ICAD), proteins cleaved in the (ATAD319T) also could shed light on the preferred sequence ER during apoptosis (Bap31 and Golgin), and the amyloid-␤ motif for caspase-12. Surprisingly, this motif is relatively fea- precursor protein (APP). Each cleavage reaction was performed tureless, so it is possible that the active site cleft of caspase-12 on the respective [35S]polypeptide and included a parallel incu- may not tolerate more elaborate side chains on the residues bation with catalytically inactivated C299A caspase-12. Even at upstream of the P1 Asp. However, predictions made by combi- high concentrations for extended incubation times (up to 6 h), natorial positional scanning would suggest that this site in none of these proteins was cleaved by caspase-12 except the 35 caspase-12 would be amenable to efficient cleavage by both [ S]procaspase-12 positive control (Fig. 4B). To comprehen- BIOCHEMISTRY group-I (inflammatory) and group-III (apoptotic initiator) sively extend the number of potential substrates examined, caspases (10). In limited in vitro testing, we detected such recombinant caspase-12 was used in a whole-proteome cleavage cleavages (data not shown). analysis using diagonal-gel PAGE coupled with MALDI-TOF mass spectroscopy (data not shown). This procedure has been Caspase-12 Proteolytic Activity Is Confined to Self. Although more applied successfully to the identification of mitochondrial recent data do not support a role for caspase-12 in mediating ER caspase-3 substrates (12). Although this procedure yielded hun- stress-induced apoptosis, earlier work suggested that caspase-12 dreds of putative substrates for caspase-1 and -3, only 11 were could cleave and activate caspase-9, the principle initiator identified for caspase-12. All 11 of these potential substrates caspase of the intrinsic cell death pathway (11). We examined turned out to be false positives. Validation experiments included

Roy et al. PNAS ͉ March 18, 2008 ͉ vol. 105 ͉ no. 11 ͉ 4135 digestion of cell lysates with caspase-12 and detection of cleavage by Western analysis, in addition to in vitro transcription/ translation of the putative substrate and cleavage of the resulting product with caspase-12. Collectively, these data indicate that, although caspase-12 is very effective at macromolecular recognition and cleavage of itself after D319, it does not effectively recognize any other cellular polypeptide that was examined. There are many caveats to these studies (our inability to examine potential substrates of low abundance, the potential of cofactor requirements, and the possibility that assay conditions are not appropriate), yet it is clear that caspase-12, under in vitro conditions that are highly permissive for other caspases, does not recognize polypeptide substrates other than itself, which it cleaves with high efficiency. This finding suggests the possibility that self-cleavage, as a potential mechanism for self-regulation, may be the primary function of caspase-12’s catalytic abilities.

Caspase-12 Recognition of Synthetic Substrates and Inhibitors. Within the spectrum of macromolecular substrates tested, caspase-12 only recognized its own proenzyme as a substrate. Therefore, we wanted to profile the caspase-12 enzyme for its ability to cleave fluorogenic ligands that are recognized by other caspase family members. We used a subset of four tetrapeptide- aminomethylcoumarins that were determined to represent the preferred substrate motifs for other caspases by positional scanning combinatorial libraries (10). These included the preferred group-I, -II, and -III substrates (Ac-WEHD-AMC, Ac- IETD-AMC, and Ac-DEVD-AMC, respectively), as well as the canonical caspase-1 (ICE) substrate (Ac-YVAD-AMC). Re- combinant rat caspase-12 was unable to cleave any of these ligands despite robust cleavage by other caspases (data not shown). Owing to the conservative change in rodent caspase-12s Fig. 5. Activity of caspase-12 and K357R-modified caspase-12 on fluorogenic (ICE:341) of Arg to Lys and the important role that this residue synthetic substrates. (A) Recombinant caspase-12 was generated by expres- plays in forming the S1 subsite that tethers the carboxylate side sion in bacteria and tested for cleavage of [35S]procaspase-12 (C299A) in vitro chain of the P1 Asp, we mutated this residue to Arg [K357R by using the indicated volumes of either wild-type caspase-12 (Left)orthe mutant; residue 357 being the equivalent to Arg(ICE:341) in the K357R-modified caspase-12 (Right). No difference in cleavage was observed. preferred ICE nomenclature system]. This K357R-modified form (B) Various forms of recombinant caspase-12 were tested for activity against of caspase-12 displayed enhanced catalytic activity toward syn- the canonical caspase-1 substrate, Ac-YVAD-AMC. No activity was detected with wild-type or catalytically incapacitated C299A caspase-12, whereas the thetic tetrapeptide substrates (Fig. 5A) without concomitant 357 changes in proteolytic efficiency toward its macromolecular K R modification restored activity against this synthetic substrate. (C) The K357R-modified caspase-12 was tested for cleavage activity against a panel of substrate, procaspase-12 (Fig. 5B). In profiling the four canon- fluorogenic substrates that are able to detect all known caspases. The pre- 357 ical fluorogenic caspase substrates, K R-modified caspase-12 ferred recognition pattern is consistent with other group-I caspases. (D) retained a group-I (caspase-1/ICE-like) substrate preference Indicated fluorogenic ligands corresponding to the autocleavage motif within (Fig. 5C). This substrate preference is consistent with the procaspase-12 were prepared by solid-phase synthesis. Unmodified wild-type phylogenetic association of caspase-12 with other members of caspase-12 was expressed in bacteria and tested for fluorogenic activity the group-I caspase subfamily (caspase-1, -4, and -5) (see Fig. against these and other fluorogenic ligands. Caspase-12 specific activity is Ϫ Ϫ 1A). To study the native enzyme without a corrective modifi- 0.0652 AFU⅐min 1⅐mg 1. cation, however, we synthesized fluorogenic peptides corresponding to the self-recognition sequence within procaspase-12 at the junction of the large and small subunits (see Fig. 3A). sponding proenzyme at the macromolecular level and to the These peptides included the tetrapeptide, Ac-ATAD-AMC, and corresponding tetrapeptide motif in synthetic substrates. This two extended peptides, Ac-IATAD-AMC and Ac-SKGIATAD- high degree of specificity is further supported by the inability of AMC. All three peptides were cleaved by wild-type rat canonical peptide-aldehyde caspase inhibitors to block in vitro 35 caspase-12 at approximately the same rate and yielded the same cleavage of [ S]procaspase-12 by recombinant active caspase-12 specific activity of 65.2 AFU⅐minϪ1⅐mgϪ1 (Fig. 5D) (data not [group-I (Ac-YVAD-CHO and Ac-WEHD-CHO), group-II shown). In comparing the efficiency of Ac-ATAD-AMC cleav- (Ac-DEVD-CHO), and group-III (Ac-IETD-CHO) inhibitors ␮ age to that of caspase-3 on its preferred substrate (Ac-DEVD- were each tested up to 100 M without effect] (data not shown). AMC), the specific activity was approximately four to five orders of magnitude lower (3,940 AFU⅐minϪ1⅐mgϪ1). As one of the Caspase-12 Autoproteolysis Occurs in the Caspase-1 Complex in Intact most catalytically efficient caspases, caspase-3 is a benchmark for Cells. We have previously demonstrated that caspase-12 forms a other proteases in this gene family. For comparison, the Kcat/Km complex with caspase-1 in intact cells and prevents caspase-1 for caspase-3 (vs. preferred substrate) is comparable to activation and subsequent cytokine processing (4). We therefore caspase-1, 10-fold higher than caspase-4 and -5, 900-fold higher examined whether caspase-12 autocleavage was occurring in than caspase-2, and 3,600-fold higher than caspase-9 (13). There- these complexes (Fig. 6) by cotransfecting HEK293T cells with fore, the approximated catalytic efficiency of caspase-12 places procaspase-1 plus procaspase-12. Caspase-1 was immunohar- it as the least catalytically active caspase known. In summary, vested after cell lysis, and bound species of caspase-12 were these data show that caspase-12 is catalytically competent, but detected by immunoblotting with specific anticaspase-12 anti- that its specificity is highly confined to cleavage of its corre- sera. The caspase-12 species that were associated with caspase-1

4136 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0706658105 Roy et al. tandem gene duplication. It has been catalytically inactivated by multiple incapacitating mutations in residues that are necessary for catalysis, but not for dominant-negative recognition of caspase-8. This catalytic incompetency may be manifested in caspase-12 by another route; namely, the highly restricted substrate specificity and low catalytic efficiency of this enzyme. Alternatively, the catalytic activity may be important for events in the caspase-1 cascade that are not evident within the experiments conducted here. For example, at early stages of complex formation, caspase-12 may hold caspase-1 in check via its dominant-negative effect, but eventually release its inhibition through autoproteolysis. This built-in latency may be important to prevent excessively rapid activation of the inflammatory caspase cascade under normal circumstances, but may become a liability under severe pathogenic conditions, such as in sepsis. We are currently exploring these possibilities by gener- ating gene-modified mice harboring the C299A catalytically inactivated caspase-12 and by humanizing caspase-12-deficient mice with either the long form of human caspase-12 or its C299A counterpart. In summary, caspase-12 is highly unique in that it functions as a dominant-negative regulator of the inflammatory caspase cascade, yet it possesses proteolytic activity of unknown function or conse- Fig. 6. Procaspase-12 forms a complex with caspase-1 and is partially auto- quences. Furthermore, caspase-12 is unique in its high degree of processed in the complex. HEK293T cells were cotransfected with expression macromolecular specificity, a feature that distinguishes it from all vectors harboring Flag-tagged procaspase-1 (all lanes) plus either pro- other caspases. An understanding of the role of this proteolysis may caspase-12 (lanes 1 and 4) or the catalytically incapacitated C299A mutant (lanes 2 and 5). After 24 h, cells were harvested and lysed. One tenth of the be important to provide an opportunity to modulate caspase-12 lysate was directly applied to SDS/PAGE (lanes 1 and 2), and the remainder was given the role of this caspase in conferring vulnerability to sepsis. immunoharvested with antibodies directed against the caspase-1 Flag epitope tag (lanes 4 and 5; lane 3 was processed in the same way, except that Methods only lysis buffer was used). Immunoblotting for the large subunit (p20) of Cloning and Mutagenesis of Rat Caspase-12. The 1,263-bp ORF for rat procaspase-12 revealed that procaspase-12 and the resulting autocleavage prod- caspase-12 was PCR-amplified from a rat lung cDNA library (Clontech) and uct were both immunoharvested with caspase-1. The asterisk indicates a band inserted into pBluescript II SKϩ (Promega). The full sequence was deposited to of unknown identity that is detected by prebleed control serum. the National Center for Biotechnology Information (accession no. AF317633.1). The insert was subcloned into the prokaryotic expression vectors pET11d and pET20b(ϩ) (Novagen) to generate a C-terminal His-tagged fusion included both the full-length caspase-12 proenzyme and its protein. Where indicated, the cDNA for rat procaspase-12 in pET11d was autoproteolytic product (the 36-kDa prodomain/large-subunit mutagenized by using the PCR-based QuikChange site-directed mutagenesis fragment; the small subunit would not be detected if associated kit (Stratagene) to convert residues as indicated. The constructs’ sequence integrity was confirmed by sequencing. with the complex because the large subunit is the epitope for the detecting antibody). That the caspase-12 cleavage product was Preparation of Bacterial Lysates Expressing Rat Caspase-12. The cDNA for generated by autoproteolysis and not by other proteases, such as wild-type or mutant procaspase-12, subcloned in pET11d, was transformed the associated caspase-1, was confirmed in parallel samples in into the protease-deficient strain of E. coli BL21 (DE3) pLysS (Novagen). which catalytically inactivated C299A caspase-12 was used. No Protein expression was induced in cultures exponentially growing in M9 processed caspase-12 was detectable in these complexes with medium with 100 ␮g/ml carbenicillin by adding 1 mM isopropyl-D- caspase-1, demonstrating that processing is autolytic. Together thiogalactoside (IPTG) and persuing incubation at either 28°C or 37°C. For these data demonstrate that caspase-12 undergoes autocatalyic soluble fraction samples, 2.5-ml aliquots of bacterial culture were removed at ␮ cleavage in the caspase-1 complex and that the resulting cleavage various time points and centrifuged. Pellets were resuspended in 500 l of cold ICE buffer III [50 mM Hepes/KOH (pH 7.0), 2 mM EDTA, 0.1% (wt/vol) CHAPS, fragment remains associated with caspase-1, although we have 10% (wt/vol) sucrose, and 5 mM DTT], lysed by sonication on ice, and cleared not explored potential differences in dissociation kinetics by centrifugation at 4°C. For total fraction samples, 1-ml aliquots of bacterial between cleaved versus uncleaved caspase-12. culture were removed at various time points and centrifuged. Pellets were lysed by resuspension in 200 ␮lof1ϫ Laemmli SDS loading buffer and Discussion sonicated to shear DNA. In both humans and mice, the presence of full-length caspase-12 (manifest by the infrequent read-through polymorphism in humans Generation of Rat Caspase-12 Antiserum. E. coli BL21(DE3) pLysS was trans- and by wild-type caspase-12 in mice) results in the attenuation of formed with the cDNA for rat caspase-12 p20 subcloned into pET11d. Protein inflammatory responsiveness and enhanced vulnerability to septic expression was induced in exponentially growing cells by a 5-h induction at 37°C in M9 medium containing 1.5 mM IPTG. Under such conditions, rat infection and mortality (3, 4). It appears that the primary mecha- caspase-12 p20 was primarily localized in the inclusion body fraction, where it nism by which caspase-12 confers hyporesponsiveness to infection was the only protein detectable by SDS/PAGE. Inclusion bodies were purified, is by dampening the formation of key cytokines [IL-1␤ and IL-18 denatured in 6 M guanidine HCl and 25 mM Tris (pH 7.4), and used directly for ␥

(and INF- as a consequence)] through a dominant-negative effect rabbit immunization. BIOCHEMISTRY on caspase-1 (ICE), the protease that mediates proteolytic maturation of these cytokines. In this regard, the mechanism is similar SDS/PAGE and Immunoblotting. For Coomassie blue staining, 10 ␮l of bacterial to the dominant-negative effect of cFLIP on caspase-8 in the lysates were fractionated by SDS/4–20% PAGE (Novex) and stained with extrinsic cell death pathway (5). Although caspase-12 possesses Simply Blue Safestain (Invitrogen) according to the manufacturer’s instruc- ␮ catalytic activity as demonstrated here, this activity is dispensable tions. For Western blotting, 1 l of bacterial lysate was fractionated by SDS/4–20% PAGE (Novex) and electrophoretically transferred to a nitrocellu- for the dominant-negative effect on caspase-1. This finding begs the lose membrane. Blots were incubated in blocking buffer [Tris-buffered saline question as to whether the catalytic activity is relevant in vivo or is (pH 7.4) with 0.05% Tween-20 (0.05% TBST), 5% milk (blotting grade blocker an inconsequential evolutionary remnant. For example, cFLIP is a nonfat dry milk from Bio-Rad)] for 1 h and in primary antibody diluted member of the caspase-8/caspase-10 gene cluster and arose from 1/20,000 in blocking buffer for 1 h. Blots were washed three times in 0.1% TBST

Roy et al. PNAS ͉ March 18, 2008 ͉ vol. 105 ͉ no. 11 ͉ 4137 for 5 min. They were then further incubated for1hatroom temperature in methylcoumarin (AMC). Reactions contained 50 ␮l of soluble bacterial secondary IgG coupled to horseradish peroxidase diluted 1/3,000 in blocking lysate and 10 ␮M substrate in a final volume of 200 ␮l of ICE buffer III. AMC buffer. After washing three times in 0.3% TBST for 5 min, followed by three release was measured in a Cytofluor 96-well plate reader by using an times in 0.1% TBST for 5 min, detection was performed by using ECL (Amer- excitation wavelength of 380 nm and an emission wavelength of 460 nm. sham). Synthetic inhibitors and fluorogenic substrates were synthesized in our laboratories, but are broadly available (without license) through several In Vitro Cleavage of Radiolabeled Proteins. [35S]Methionine-labeled proteins vendors. were obtained by coupled in vitro transcription/translation using the Promega TNT reticulocyte lysate system as per the manufacturer’s instructions. Cleav- Coimmunoprecipitation Experiments. HEK293T cells were cotransfected by age of the radiolabeled product was performed by incubation at 37°C in the using Lipofectamine Plus reagent (Invitrogen) with Flag-tagged caspase-1 presence of soluble bacterial lysate in a final reaction volume of 25 ␮l. Unless C[ICE:285]A and caspase-12 in either the wild-type form or C[ICE:285]A(C299A) otherwise indicated, cleavage mixtures were incubated for 90 min. Cleavage catalytically inactivated mutant. At 24 h after transfection, cell lysates were reactions were terminated by the addition of 5ϫ SDS Laemmli loading buffer, prepared in buffer B [20 mM Tris⅐HCl (pH 8.0), 150 mM KCl, 10% glycerol, 5 mM resolved by SDS/10–20% PAGE (Novex), and visualized by autoradiography MgCl2, 0.1% Nonidet P-40, and a protease inhibitor mixture] and caspase-1, -5, (BioMax MR film; Kodak). and -9 were immunoprecipitated by using Flag M2 agarose beads (Sigma– Aldrich) at 4°C for 2 h, followed by three washes of the beads with lysis buffer. Caspase Fluorogenic Enzyme Enzymatic Assays. Caspase catalytic activity was Immunoconjugates were eluted from the beads by using Flag peptides measured by using a continuous fluorometric assay based on the proteo- (Sigma–Aldrich) and were processed for Western blot analysis by using anti- lytic cleavage of a substrate with the general structure 7-amino-4- caspase-12 antiserum.

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4138 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0706658105 Roy et al.