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• W2162263320 abstract "Extracellular serpins such as antithrombin and α1-antitrypsin are the quintessential regulators of proteolytic pathways. In contrast, the biological functions of the intracellular serpins remain obscure. We now report that the C. elegans intracellular serpin, SRP-6, exhibits a prosurvival function by blocking necrosis. Minutes after hypotonic shock, srp-6 null animals underwent a catastrophic series of events culminating in lysosomal disruption, cytoplasmic proteolysis, and death. This newly defined hypo-osmotic stress lethal (Osl) phenotype was dependent upon calpains and lysosomal cysteine peptidases, two in vitro targets of SRP-6. By protecting against both the induction of and the lethal effects from lysosomal injury, SRP-6 also blocked death induced by heat shock, oxidative stress, hypoxia, and cation channel hyperactivity. These findings suggest that multiple noxious stimuli converge upon a peptidase-driven, core stress response pathway that, in the absence of serpin regulation, triggers a lysosomal-dependent necrotic cell death routine. Extracellular serpins such as antithrombin and α1-antitrypsin are the quintessential regulators of proteolytic pathways. In contrast, the biological functions of the intracellular serpins remain obscure. We now report that the C. elegans intracellular serpin, SRP-6, exhibits a prosurvival function by blocking necrosis. Minutes after hypotonic shock, srp-6 null animals underwent a catastrophic series of events culminating in lysosomal disruption, cytoplasmic proteolysis, and death. This newly defined hypo-osmotic stress lethal (Osl) phenotype was dependent upon calpains and lysosomal cysteine peptidases, two in vitro targets of SRP-6. By protecting against both the induction of and the lethal effects from lysosomal injury, SRP-6 also blocked death induced by heat shock, oxidative stress, hypoxia, and cation channel hyperactivity. These findings suggest that multiple noxious stimuli converge upon a peptidase-driven, core stress response pathway that, in the absence of serpin regulation, triggers a lysosomal-dependent necrotic cell death routine. Serpins are the quintessential regulators of extracellular serine peptidase pathways, where they prevent the deleterious effects of excessive peptide bond hydrolysis (Law et al., 2006Law R.H. Zhang Q. McGowan S. Buckle A.M. Silverman G.A. Wong W. Rosado C.J. Langendorf C.G. Pike R.N. Bird P.I. et al.An overview of the serpin superfamily.Genome Biol. 2006; 7: 216-226Crossref PubMed Scopus (438) Google Scholar, Silverman et al., 2001Silverman G.A. Bird P.I. Carrell R.W. Coughlin P.B. Gettins P.G. Irving J.I. Lomas D.A. Luke C.J. Moyer R.W. Pemberton P.A. et al.The serpins are an expanding superfamily of structurally similar but functionally diverse proteins: Evolution, mechanism of inhibition, novel functions, and a revised nomenclature.J. Biol. Chem. 2001; 276: 33293-33296Crossref PubMed Scopus (1027) Google Scholar). For example, thrombin, which converts fibrinogen to fibrin, is generated by activated upstream peptidases including coagulation factors X (fXa) and fIXa (Church et al., 2007Church F.C. Pike R.N. Tollefsen D.M. Buckle A.M. Ciaccia A.V. Olson S.T. Regulation of hemostasis by heparin-binding serpins.in: Silverman G.A. Lomas D.A. Molecular and Cellular Aspects of the Serpinopathies and Disorders of Serpin Activity. World Scientific Publishing Co. Pte. Ltd., Singapore2007: 509-554Crossref Scopus (8) Google Scholar). Antithrombin prevents excessive thrombosis by inhibiting fXa, fIXa, and a thrombin-mediated amplification loop that perpetuates zymogen conversion. Thus, serpins regulate proteolytic pathways by inhibiting multiple peptidases and by employing a unique suicide-substrate-like mechanism to irreversibly terminate enzymatic activity. Serpins, with over 1500 representatives in Archaea, Prokarya, and Eukarya as well as some viruses, are the largest and most widely dispersed family of peptidase inhibitors (Law et al., 2006Law R.H. Zhang Q. McGowan S. Buckle A.M. Silverman G.A. Wong W. Rosado C.J. Langendorf C.G. Pike R.N. Bird P.I. et al.An overview of the serpin superfamily.Genome Biol. 2006; 7: 216-226Crossref PubMed Scopus (438) Google Scholar). The 37 human serpins are divided into 9 clades (A–I) (Silverman et al., 2001Silverman G.A. Bird P.I. Carrell R.W. Coughlin P.B. Gettins P.G. Irving J.I. Lomas D.A. Luke C.J. Moyer R.W. Pemberton P.A. et al.The serpins are an expanding superfamily of structurally similar but functionally diverse proteins: Evolution, mechanism of inhibition, novel functions, and a revised nomenclature.J. Biol. Chem. 2001; 276: 33293-33296Crossref PubMed Scopus (1027) Google Scholar). Most of these serpins are secreted and constitute ∼10% of the plasma proteins. In contrast, the vertebrate clade B serpins belong to a larger intracellular subfamily (Silverman et al., 2004Silverman G.A. Whisstock J.C. Askew D.J. Pak S.C. Luke C. Cataltepe S. Irving J.A. Bird P.I. Human clade B serpins (ov-serpins) belong to a cohort of evolutionarily-dispersed intracellular proteinase inhibitor clades that protect cells from promiscuous proteolysis.Cell. Mol. Life Sci. 2004; 61: 301-325Crossref PubMed Scopus (150) Google Scholar). The intracellular serpins (serpinsIC) lack cleavable N-terminal signal peptides and dwell within the nucleo-cytosolic compartment. Several serpinsIC also neutralize papain-like lysosomal cysteine peptidases, but the biological significance of this activity is unknown (Silverman et al., 2004Silverman G.A. Whisstock J.C. Askew D.J. Pak S.C. Luke C. Cataltepe S. Irving J.A. Bird P.I. Human clade B serpins (ov-serpins) belong to a cohort of evolutionarily-dispersed intracellular proteinase inhibitor clades that protect cells from promiscuous proteolysis.Cell. Mol. Life Sci. 2004; 61: 301-325Crossref PubMed Scopus (150) Google Scholar). Based on their subcellular location, we postulate that serpinsIC provide an important survival function by limiting the collateral damage induced by endogenous lysosomal cysteine or serine peptidases. If this hypothesis is correct, then the loss of lysosomal integrity must be a common occurrence during cellular stress. However, the role of lysosomes in cellular injury is uncertain. Initially considered “suicide bags” that provoked programmed cell death and tissue involution, the inability to show a causal relationship between lysosomal fragility and cell death led to the supposition that lysosmes were durable structures, which released their powerful complement of acid hydrolases into the cytosol only during postmortem autolysis (de Duve, 1983de Duve C. Lysosomes revisited.Eur. J. Biochem. 1983; 137: 391-397Crossref PubMed Scopus (330) Google Scholar). Subsequently, caspase-dependent apoptosis (type I death) was considered the predominant form of programmed cell death with lysosomes relegated to an injurious role only in cases where autophagy precipitated injury (type II death) (Clarke, 1990Clarke P.G. Developmental cell death: morphological diversity and multiple mechanisms.Anat. Embryol. (Berl.). 1990; 181: 195-213Crossref PubMed Scopus (1509) Google Scholar). More recently, lysosomes were implicated, albeit marginally, in the induction or execution of the three major cell death routines, including necrosis (type III death) (Golstein and Kroemer, 2007Golstein P. Kroemer G. Cell death by necrosis: towards a molecular definition.Trends Biochem. Sci. 2007; 32: 37-43Abstract Full Text Full Text PDF PubMed Scopus (733) Google Scholar, Zong and Thompson, 2006Zong W.X. Thompson C.B. Necrotic death as a cell fate.Genes Dev. 2006; 20: 1-15Crossref PubMed Scopus (677) Google Scholar). Indeed, studies suggest that increasing lysosomal membrane permeability by exposure to oxygen-free radicals, photo-oxidation products, or calpains shifts the cell death routine from apoptosis (ordered and programmed) to necrosis (unordered and unprogrammed) (Golstein and Kroemer, 2007Golstein P. Kroemer G. Cell death by necrosis: towards a molecular definition.Trends Biochem. Sci. 2007; 32: 37-43Abstract Full Text Full Text PDF PubMed Scopus (733) Google Scholar, Zong and Thompson, 2006Zong W.X. Thompson C.B. Necrotic death as a cell fate.Genes Dev. 2006; 20: 1-15Crossref PubMed Scopus (677) Google Scholar). Since there is little data suggesting that eukaryotic serpinsIC protect against caspase-dependent apoptosis (Silverman et al., 2004Silverman G.A. Whisstock J.C. Askew D.J. Pak S.C. Luke C. Cataltepe S. Irving J.A. Bird P.I. Human clade B serpins (ov-serpins) belong to a cohort of evolutionarily-dispersed intracellular proteinase inhibitor clades that protect cells from promiscuous proteolysis.Cell. Mol. Life Sci. 2004; 61: 301-325Crossref PubMed Scopus (150) Google Scholar), these inhibitors may play a role by preventing lysosomal peptidases from exacerbating caspase-independent apoptosis or necrosis. Experimental support for this hypothesis, in vivo, has yet to come from targeted deletions of serpinsIC in mice. Although Serpinb9 nulls show increased loss of cytotoxic lymphocytes, Serpinb2 or -b6 nulls show normal development, survival, fertility, wound healing, and resistance to infection (Dougherty et al., 1999Dougherty K.M. Pearson J.M. Yang A.Y. Westrick R.J. Baker M.S. Ginsburg D. The plasminogen activator inhibitor-2 gene is not required for normal murine development or survival.Proc. Natl. Acad. Sci. USA. 1999; 96: 686-691Crossref PubMed Scopus (96) Google Scholar, Scarff et al., 2004Scarff K.L. Ung K.S. Nandurkar H. Crack P.J. Bird C.H. Bird P.I. Targeted disruption of SPI3/Serpinb6 does not result in developmental or growth defects, leukocyte dysfunction, or susceptibility to stroke.Mol. Cell. Biol. 2004; 24: 4075-4082Crossref PubMed Scopus (24) Google Scholar, Zhang et al., 2006Zhang M. Park S.M. Wang Y. Shah R. Liu N. Murmann A.E. Wang C.R. Peter M.E. Ashton-Rickardt P.G. Serine protease inhibitor 6 protects cytotoxic T cells from self-inflicted injury by ensuring the integrity of cytotoxic granules.Immunity. 2006; 24: 451-461Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). Expansion of the serpinIC cluster to 28 genes in the mouse, as compared to 13 in humans, as well as functional overlaps between paralogs may confound this experimental approach (Silverman et al., 2004Silverman G.A. Whisstock J.C. Askew D.J. Pak S.C. Luke C. Cataltepe S. Irving J.A. Bird P.I. Human clade B serpins (ov-serpins) belong to a cohort of evolutionarily-dispersed intracellular proteinase inhibitor clades that protect cells from promiscuous proteolysis.Cell. Mol. Life Sci. 2004; 61: 301-325Crossref PubMed Scopus (150) Google Scholar). Members of the serpinIC family are also conserved in the genomes of simpler multicellular organisms, such as Caenorhabditis elegans. This nematode harbors 9 serpinsIC of which 5 are bona fide peptidase inhibitors (Luke et al., 2006Luke C.J. Pak S.C. Askew D.J. Askew Y.S. Smith J.E. Silverman G.A. Selective conservation of the RSL-encoding, proteinase inhibitory-type, clade l serpins in Caenorhabditis species.Front. Biosci. 2006; 11: 581-594Crossref PubMed Scopus (13) Google Scholar). We sought to determine whether a reverse genetic approach in a model organism with a smaller serpinIC repertoire could yield insight into the biologic function of this evolutionarily well-conserved, yet enigmatic family of proteins. We identified a surprisingly virulent necrotic phenotype (Osl) in animals (srp-6(ok319)) with homozygous loss of the inhibitory-type serpinIC, srp-6. Interestingly SRP-6 protected animals from necrosis by blocking calpain-associated lysosomal lysis and by neutralizing lysosomal cysteine peptidases released from injured organelles. In addition, SRP-6 protected against necrosis induced by heat stress, hypoxia hyperoxia, and cation channel hyperactivity. Taken together, these studies showed that massive cellular necrosis was neither unregulated nor unprogrammed but was triggered by an ordered, SRP-6-regulated, peptidase-driven stress response pathway targeting the lysosomal compartment. Thus, serpinsIC may serve as prosurvival factors by protecting against both the induction and sequelae of lysosomal injury. An interstitial deletion encompassing the reactive site loop (RSL) and 85% of the srp-6 coding region occurred in srp-6(ok319) animals (Supplemental Results; Figure S1). srp-6 nulls were similar to wild-type Bristol N2 (srp-6(+)) animals in terms of anatomy, pharyngeal pumping, life span, fecundity, and movement (not shown). At 20°C–25°C, however, the survival of the srp-6(ok319) animals was decreased markedly when they were collected in water rather than isotonic M9 buffer (Figure 1A). DIC microscopy showed that the srp-6(ok319) animals had suffered a grim fate (Figures 1B–1K). srp-6(ok319) larvae thrashed about for a few minutes and became rod-like with multiple vacuoles and refractile cell corpses (Figures 1D–1K; Movies S1A and S1B). Animals were unresponsive to light touch and never recovered movement upon transfer to NGM growth plates. Adult animals shared a similar fate and frequently extruded their uteri and intestines through the vulval opening or anus after one or two jack-knife-like contractions (Figures 1B and 1C; Movies S2A and S2B). Typically, the median survival of srp-6(ok319) animals was ∼1 min (Figure 1A), whereas >90% of the srp-6(+) animals were alive after 30 min, and most were viable after immersion for ∼24 hr (not shown). Intestinal cells of srp-6(ok319) animals lost membrane integrity within ∼60 s of water immersion as shown by uptake of propidium iodide (Figures 1L–1Q). Consistent with the site of injury, a srp-6::GFP transgene showed expression within intestinal cells (Figure S2). Death did not occur by drowning as srp-6(ok319) animals died rapidly upon transfer to 2% agarose pads prepared with water. The phenotype of srp-6(ok319) animals in water, which was designated hypo-osmotic stress lethal (Osl) by the C. elegans nomenclature committee (http://biosci.umn.edu/CGC/Nomenclature/genes.htm), was autosomal recessive (data not shown). A second srp-6 deletion mutant, srp-6(tm1994), also was Osl (data not shown). Of note, animals with enhanced fibroblast growth factor signaling amassed fluid in their pseudocoelomic space under isotonic conditions (Kokel et al., 1998Kokel M. Borland C.Z. DeLong L. Horvitz H.R. Stern M.J. clr-1 encodes a receptor tyrosine phosphatase that negatively regulates an FGF receptor signaling pathway in Caenorhabditis elegans.Genes Dev. 1998; 12: 1425-1437Crossref PubMed Scopus (91) Google Scholar). Under normal growth conditions, this Clear (Clr) phenotype was distinct morphologically from that of Osl. Moreover clr-1(e1745) mutants, unlike srp-6(ok319) animals with the Osl phenotype, survived when placed in water (not shown). The secretory-excretory system, which consists of the excretory, gland, duct, and pore cells, is responsible for osmoregulation in C. elegans (Nelson and Riddle, 1984Nelson F.K. Riddle D.L. Functional study of the Caenorhabditis elegans secretory-excretory system using laser microsurgery.J. Exp. Zool. 1984; 231: 45-56Crossref PubMed Scopus (146) Google Scholar). srp-6(ok319) animals expressing a clh-4::GFP transgene showed an apparently normal excretory system (Figure S3). More importantly, srp-6(RNAi) phenocopied Osl at a time when C. elegans development was complete (Figure S4). We concluded that Osl was due to a physiological rather than an anatomical defect. In response to hypotonic shock, cells undergo a regulatory volume decrease (RVD) to restore cell volume and osmolality (Jakab et al., 2002Jakab M. Furst J. Gschwentner M. Botta G. Garavaglia M.L. Bazzini C. Rodighiero S. Meyer G. Eichmueller S. Woll E. et al.Mechanisms sensing and modulating signals arising from cell swelling.Cell. Physiol. Biochem. 2002; 12: 235-258Crossref PubMed Scopus (116) Google Scholar). RVD involves a rapid rise in intracellular Ca2+ concentrations ([Ca2+]i) followed by an efflux of inorganic ions, organic osmolytes, and water. In C. elegans, changes in cellular size and [Ca2+]i can be assessed by approximating the volume of the intact organism and by measuring cytoplasmic fluorescence with the calcium indicator, Fluo-4, respectively (Lamitina and Strange, 2005Lamitina S.T. Strange K. Transcriptional targets of DAF-16 insulin signaling pathway protect C. elegans from extreme hypertonic stress.Am. J. Physiol. Cell Physiol. 2005; 288: C467-C474Crossref PubMed Scopus (113) Google Scholar). Both srp-6(ok319) and srp-6(+) animals showed a comparable increase in volume and [Ca2+]i minutes after hypotonic shock at 25°C (Figures 2A and 2C). However, the [Ca2+]i of srp-6(ok319) animals did not return to baseline and volume never decreased. We concluded that mutant animals were either RVD defective or that RVD was normal but could not be completed due to the concomitant activation of a cell death routine. We noticed that srp-6(ok319) animals exposed to water at 10°C were protected from death. Since this temperature does not appreciably alter water influx and cell swelling, we repeated the experiments at this temperature. Both srp-6(+) and srp-6(ok319) animals demonstrated a comparable increase and subsequent decrease in both cell volume and [Ca2+]i (Figures 2B and 2D). These findings suggested (1) srp-6(ok319) animals could undergo an RVD, (2) cell swelling and the increased turgor pressure per se did not kill the animals, and (3) hypotonic shock triggered a temperature-sensitive, SRP-6-regulated cell death routine. In C. elegans, apoptosis occurs in embryonic and larval development and typically involves the caspase, CED-3, which is regulated by pro-apoptotic (CED-4, EGL-1) and anti-apoptotic (CED-9) factors (Lettre and Hengartner, 2006Lettre G. Hengartner M.O. Developmental apoptosis in C. elegans: a complex CEDnario.Nat. Rev. Mol. Cell Biol. 2006; 7: 97-108Crossref PubMed Scopus (217) Google Scholar). However, neither the ced-3(n717), ced-4(n1162), nor ced-9(1950) mutation suppressed srp-6(RNAi)-induced Osl (Figure 3A). Also, the caspase inhibitors DEVD-CHO or YVAD-CHO could not block Osl (Figure 3B). In C. elegans, genes associated with the autophagic pathway are critical for normal dauer diapause (an adaptive stress response) but have not yet been linked to cell death (Melendez et al., 2003Melendez A. Talloczy Z. Seaman M. Eskelinen E.L. Hall D.H. Levine B. Autophagy genes are essential for dauer development and life-span extension in C. elegans.Science. 2003; 301: 1387-1391Crossref PubMed Scopus (975) Google Scholar). srp-6(ok319) animals exposed to bec-1(RNAi) (orthologous to yeast APG6) showed no increase in survival (Figure 3C) but did show abnormal dauer formation (data not shown). Necrotic cell death has been described in C. elegans (Driscoll and Gerstbrein, 2003Driscoll M. Gerstbrein B. Dying for a cause: invertebrate genetics takes on human neurodegeneration.Nat. Rev. Genet. 2003; 4: 181-194Crossref PubMed Scopus (136) Google Scholar). This necrotic or degenerin pathway mimics that associated with excitotoxic injury and occurs in neurons expressing gain-of-function mutations in degenerin/epithelial Na+ channels (deg-1, mec-4, mec-10, unc-8, and unc-105), a GTP-binding protein subunit (gsa-1, a Gαs), and a nicotinic acetylcholine receptor (deg-3) (Berger et al., 1998Berger A.J. Hart A.C. Kaplan J.M. G alphas-induced neurodegeneration in Caenorhabditis elegans.J. Neurosci. 1998; 18: 2871-2880Crossref PubMed Google Scholar, Chalfie and Wolinsky, 1990Chalfie M. Wolinsky E. The identification and suppression of inherited neurodegeneration in Caenorhabditis elegans.Nature. 1990; 345: 410-416Crossref PubMed Scopus (251) Google Scholar, Yassin et al., 2001Yassin L. Gillo B. Kahan T. Halevi S. Eshel M. Treinin M. Characterization of the deg-3/des-2 receptor: a nicotinic acetylcholine receptor that mutates to cause neuronal degeneration.Mol. Cell. Neurosci. 2001; 17: 589-599Crossref PubMed Scopus (58) Google Scholar). Hyperactivation of ion channels in C. elegans leads to a rise in [Ca2+]i, and necrosis can be suppressed by mutations that block the release of Ca2+ from intracellular stores (Xu et al., 2001Xu K. Tavernarakis N. Driscoll M. Necrotic cell death in C. elegans requires the function of calreticulin and regulators of Ca(2+) release from the endoplasmic reticulum.Neuron. 2001; 31: 957-971Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar). We performed srp-6(RNAi) on animals lacking the ryanodine (RyR/unc-68(kh30)) or inositol-1,4,5-trisphosphate (IP3R/itr-1(sa73)) receptor. These receptor mutants and null or loss-of-function mutations of the ER Ca2+-binding protein, calreticulin (crt-1(bz31)), significantly suppressed death induced by srp-6(RNAi) (Figure 3D). Similarly, srp-6(ok319) animals treated with an intracellular Ca2+ chelator, EGTA-AM or BAPTA-AM, were protected from death (Figure S5A). srp-6(ok319) animals were not more sensitive to elevations in [Ca2+]i, as treatment with thapsigargin did not induce cell death despite a rate of rise in [Ca2+]i comparable to that observed with Osl (not shown). These data suggested that the rise in [Ca2+]i associated with a normal RVD was required, but not sufficient, to activate Osl. Aspartic (asp-3 and asp-4) and cysteine (calpains, clp-1, and tra-3/clp-5) peptidases are involved in the degenerin pathway (Syntichaki et al., 2002Syntichaki P. Xu K. Driscoll M. Tavernarakis N. Specific aspartyl and calpain proteases are required for neurodegeneration in C. elegans.Nature. 2002; 419: 939-944Crossref PubMed Scopus (246) Google Scholar). Interestingly, asp-1(RNAi), which is expressed predominantly in the lysosomes and brush border of intestinal cells (Tcherepanova et al., 2000Tcherepanova I. Bhattacharyya L. Rubin C.S. Freedman J.H. Aspartic proteases from the nematode Caenorhabditis elegans. Structural organization and developmental and cell-specific expression of asp-1.J. Biol. Chem. 2000; 275: 26359-26369Crossref PubMed Scopus (55) Google Scholar), and to a lesser extent asp-3(RNAi), but not asp-4(RNAi), protected srp-6(ok319) animals from Osl (Figure 3E). Inhibition of calpains by tra-3/clp-5(RNAi) or W05G11.4(RNAi), but not clp-1(RNAi), also showed protection (Figure 3F). The C. elegans genome harbors at least 31 lysosomal cysteine peptidases and several are expressed in intestinal cells. RNAi with cpl-1 (catL-like) and cpr-6 (catB-like) protected srp-6(ok319) animals from death (Figure 3G). Consistent with the RNAi studies, pre-treatment of srp-6(ok319) animals with the papain-like cysteine peptidase inhibitor, E-64d, protected against Osl (Figure 3B). Although E-64d protected against death, this treatment did not block the Osl-induced rise in [Ca2+]i, suggesting that Ca2+ release occurred upstream of cysteine peptidase activation (Figure S5B). Taken together, these studies suggested that necrosis was the manner of death in Osl. Since Osl was partially dependent upon cysteine peptidases, we determined whether SRP-6 might serve as their inhibitor. Like human SERPINB3, recombinant SRP-6 inhibited papain-like lysosomal cysteine peptidases (clan CA, family C1) such as catL and catK, but not any of the serine peptidases within a broad array of purified enzymes (Table S1). Interestingly, SRP-6 also inhibited calpain-2 (clan CA, family C2). The inhibition of these enzymes was characteristic of serpin-target peptidase interactions as shown by typical stoichiometries of inhibition (SI) (Figures 4A and 4B), second-order rate constants (Figures 4C and 4D), covalent complex formation (Figures 4E and 4F), and peptidase-mediated RSL cleavages (Figure 4G). To confirm that the loss of cysteine peptidase inhibitory activity of SRP-6 was the cause of Osl, we performed transgenic rescue experiments of srp-6(ok319) animals using a srp-6 gene with either a wild-type or mutated RSL. The mutations were placed at the P14 hinge region (T337R) or the reactive center P1 and P2 residues (L339A;T340A). The classical P14 mutation blocks covalent complex formation with any target peptidase. The P2 and P1 mutations block papain-like lysosomal cysteine peptidase binding, as these enzymes prefer hydrophobic residues at the P2 position and bind cooperatively to residues at the P1 position (Schick et al., 1998Schick C. Bromme D. Bartuski A.J. Uemura Y. Schechter N.M. Silverman G.A. The reactive site loop of the serpin SCCA1 is essential for cysteine proteinase inhibition.Proc. Natl. Acad. Sci. USA. 1998; 95: 13465-13470Crossref PubMed Scopus (76) Google Scholar). Each of these constructs was coinjected with a Pnhx-2::GFP reporter plasmid (the nhx-2 promoter drives expression exclusively within intestinal cells; Nehrke, 2003Nehrke K. A reduction in intestinal cell pHi due to loss of the Caenorhabditis elegans Na+/H+ exchanger NHX-2 increases life span.J. Biol. Chem. 2003; 278: 44657-44666Crossref PubMed Scopus (91) Google Scholar) into the gonads of young adult srp-6(ok319) animals. Transgenic lines with the wild-type srp-6 gene (VK249) showed nearly complete suppression of Osl (Figure 4H). In contrast, srp-6(ok319) animals harboring the srp-6 transgene with either the P14 mutation (VK248) or the P2-P1 mutations (VK247) were still Osl (Figure 4I). To determine whether intestinal cell expression was sufficient to rescue srp-6(ok319) animals, we replaced the srp-6 promoter with that from nhx-2. The nhx-2-driven wild-type srp-6, but not the P14 mutant, also rescued srp-6 nulls (VK611 and VK618, Figures 3H and 3I, respectively). We concluded that the loss of intestinal cell SRP-6 cysteine peptidase inhibitory activity caused Osl. By transmission electron microscopy (TEM), the membranes of lysosomal-like gut granule membranes were disrupted several minutes after hypotonic shock (Figures 5A–5F). At this stage, most mitochondria and other cellular structures were still intact. To confirm that lysosomal instability and release of cathepsins was an early feature of Osl, confocal microscopy was used to follow the fate of gut granules labeled coincidently with the endocytic marker, tetramethylrhodamine (TMR)-dextran, and the general fluorescent cysteine peptidase substrate, (Z-FR)2-R110 (Figures 5G–5R). Dual labeling confirmed that the majority of gut granules appeared to be lysosomes containing endocytosed molecules and active cysteine peptidases. The labeled granules of only live srp-6(ok319) animals began to lyse minutes after hypotonic shock (Figures 5G–5R; Movies S3A and S3B, S4A and S4B, and S5A and S5B). Widefield fluorescence microscopy revealed that gut granule lysis was followed by a wave of intense cytoplasmic (Z-FR)2-R110 fluorescence, indicating the presence of diffuse cysteine peptidase activity prior to death (Figures 5S–5AD; Movies S6A and S6B). These studies showed that the disintegration of lysosomal-like gut granules and subsequent cytoplasmic cysteine peptidase activity were active components of the cell death routine and not a post-mortem event. To test this hypothesis, we generated srp-6(ok319);glo-1(zu437) double mutants. glo-1 is a Rab-like GTPase required for the biogenesis of acidic, lysosomal-like gut granules (Hermann et al., 2005Hermann G.J. Schroeder L.K. Hieb C.A. Kershner A.M. Rabbitts B.M. Fonarev P. Grant B.D. Priess J.R. Genetic analysis of lysosomal trafficking in Caenorhabditis elegans.Mol. Biol. Cell. 2005; 16: 3273-3288Crossref PubMed Scopus (172) Google Scholar). glo-1(zu437) mutants lack these lysosomes and appear to direct their contents into the intestinal lumen. srp-6(ok319);glo-1(zu437) animals exposed to hypotonic shock were protected from death (Figure 3H). Consistent with these findings, the lysosomes of srp-6(ok319);tra-3(RNAi) (Figures 5AE–5AL) and EGTA-AM-treated srp-6(ok319) animals, two treatments that block Osl, remained intact after hypotonic shock (Figure S5C). Taken together, these data demonstrated that lysosomal-like gut granule instability was required for Osl. Since lysosomes did not lyse in srp-6(+) animals, we concluded that SRP-6 protected against lysosomal rupture. To determine whether SRP-6 also protected the cell after lysosomal disruption, we compared the survival of srp-6(ok319) to srp-6(+) animals after inducing direct lysosomal injury with acridine orange (AO). Lysosomes trap this weak basic dye and lyse upon exposure to blue light (Hiruma et al., 2007Hiruma H. Katakura T. Takenami T. Igawa S. Kanoh M. Fujimura T. Kawakami T. Vesicle disruption, plasma membrane bleb formation, and acute cell death caused by illumination with blue light in acridine orange-loaded malignant cells.J. Photochem. Photobiol. B. 2007; 86: 1-8Crossref PubMed Scopus (19) Google Scholar). As expected, most of the gut granules from both animals lysed minutes after exposure to blue light (Figures 6A–6H). However, the srp-6(ok319) animals showed markedly increased intestinal cell vacuolization and mortality (Figure 6M). Surprisingly, when we examined the surviving srp-6(+) animals ∼30 min after treatment, their lysosomal compartment was reconstituted as shown by the reappearance of granular AO staining (Figures 6I–6L). These data showed that SRP-6 also protected the cell from injury after lysosomal rupture. To determine whether the protective role of SRP-6 was restricted to Osl or could be extended to other stressors, we compared the survival rate of srp-6(ok319) to srp-6(+) animals after exposure to heat-shock (Lithgow et al., 1995Lithgow G.J. White T.M. Melov S. Johnson T.E. Thermotolerance and extended life-span conferred by single-gene mutations and induced by thermal stress.Proc. Natl. Acad. Sci. USA. 1995; 92: 7540-7544Crossref PubMed Scopus (660) Google Scholar), hypoxia (sodium azide) (Scott et al., 2002Scott B.A. Avidan M.S. Crowder C.M. Regulation of hypoxic death in C. elegans by the insulin/IGF receptor homolog DAF-2.Science. 2002; 296: 2388-2391Crossref PubMed Scopus (196) Google Scholar), and" @default.
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