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Age-related macular degeneration (AMD), the leading cause of irreversible blindness among Americans over 50, is characterized by dysfunction and death of retinal pigment epithelial (RPE) cells. The RPE accumulates iron in AMD, and iron overload triggers RPE cell death in vitro and in vivo. However, the mechanism of RPE iron accumulation in AMD is unknown. We show that high-fat-diet-induced obesity, a risk factor for AMD, drives systemic and local inflammatory circuits upregulating interleukin-1β (IL-1β). IL-1β upregulates RPE iron importers and downregulates iron exporters, causing iron accumulation, oxidative stress, and dysfunction. We term this maladaptive, chronic activation of a nutritional immunity pathway the cellular iron sequestration response (CISR). RNA sequencing (RNA-seq) analysis of choroid and retina from human donors revealed that hallmarks of this pathway are present in AMD microglia and macrophages. Together, these data suggest that inflamed adipose tissue, through the CISR, can lead to RPE pathology in AMD.

To evaluate patient-reported quality of life pertaining to gastroesophageal reflux disease symptoms in patients undergoing upper airway surgery for comorbid obstructive sleep apnea.A prospective survey-based study was conducted on patients with gastroesophageal reflux disease and comorbid obstructive sleep apnea receiving surgery from July 2020-December 2020. Patients completed the Gastroesophageal Reflux Disease-Health Related Quality of Life Questionnaire at two time-points: one week before surgery and at 6 months following surgery. Disease-related symptoms were rated from 0 (no symptoms) to 5 (incapacitating symptoms). Patient survey scores, demographics, medications, and sleep study parameters were collected for analysis. A p-value0.05 indicated statistical significance.Twenty-two patients completed the baseline preoperative and 6-month postoperative questionnaires. Median baseline vs. 6-month survey scores significantly decreased for symptoms including heartburn in general (3.0 vs. 2.0, p = 0.006), when lying down (2.5 vs. 1.5, p = 0.046), when standing (2.0 vs 1.0, p = 0.003), following meals (2.0 vs. 2.0, p = 0.042), and cumulative survey score (15.5 vs. 11.0, p = 0.029). Heartburn altering diet or sleep, odynophagia, dysphagia, and medication burden did not change following surgery (p 0.05). More patients were satisfied with their postoperative condition compared to baseline, however this did not reach statistical significance (40.9% vs. 18.2%, p = 0.18).Our results suggest that upper airway surgery to treat obstructive sleep apnea may have a positive impact on patient-reported symptoms of gastroesophageal reflux disease, and further investigation into the role of surgery in this setting for improvement of both quality of life and true clinical disease severity is merited.

α-synuclein (α-syn) aggregation and accumulation drive neurodegeneration in Parkinson's disease (PD). The substantia nigra of patients with PD contains excess iron, yet the underlying mechanism accounting for this iron accumulation is unclear. Here, we show that misfolded α-syn activates microglia, which release interleukin 6 (IL-6). IL-6, via its trans-signaling pathway, induces changes in the neuronal iron transcriptome that promote ferrous iron uptake and decrease cellular iron export via a pathway we term the cellular iron sequestration response, or CISR. The brains of patients with PD exhibit molecular signatures of the IL-6-mediated CISR. Genetic deletion of IL-6, or treatment with the iron chelator deferiprone, reduces pathological α-syn toxicity in a mouse model of sporadic PD. These data suggest that IL-6-induced CISR leads to toxic neuronal iron accumulation, contributing to synuclein-induced neurodegeneration.

Apolipoprotein E (APOE) is a component of lipid-transporting particles and a recognition ligand for receptors, which bind these particles. The APOE isoform ε2 is a risk factor for age-related macular degeneration; nevertheless, APOE absence in humans and mice does not significantly affect the retina. We found that retinal cholesterol biosynthesis and the levels of retinal cholesterol were increased in Apoe(−/−) mice, whereas cholesterol elimination by metabolism was decreased. No focal cholesterol deposits were observed in the Apoe(−/−) retina. Retinal proteomics identified the most abundant cholesterol-related proteins in WT mice and revealed that, of these cholesterol-related proteins, only APOA4 had increased expression in the Apoe(−/−) retina. In addition, there were changes in retinal abundance of proteins involved in proinflammatory and antiinflammatory responses, cellular cytoskeleton maintenance, vesicular traffic, and retinal iron homeostasis. The data obtained indicate that when APOE is absent, particles containing APOA1, APOA4, and APOJ still transport cholesterol in the intraretinal space, but these particles are not taken up by retinal cells. Therefore, cholesterol biosynthesis inside retinal cells increase, whereas metabolism to oxysterols decreases to prevent cells from cholesterol depletion. These and other compensatory changes underlie only a minor retinal phenotype in Apoe(−/−) mice.

Apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), an essential component of the inflammasome complex, is frequently silenced by epigenetic methylation in many tumor cells. Here, we demonstrate that restoration of ASC expression in human colorectal cancer DLD-1 cells, in which ASC is silenced by aberrant methylation, potentiated cell death mediated by DNA damaging agent. Contrarily, ASC knockdown in HT-29 cells rendered cells less susceptible to etoposide toxicity. The increased susceptibility of ASC-expressing DLD-1 cells to genotoxic stress was independent of inflammasome or caspase activation, but partially dependent on mitochondrial ROS production and JNK activation. Thus, our data suggest that ASC expression in cancer cells is an important factor in determining their susceptibility to chemotherapy.

Human pyrin with gain-of-function mutations in its B30.2/SPRY domain causes the autoinflammatory disease familial Mediterranean fever by assembling an ASC-dependent inflammasome that activates caspase-1. Wild-type human pyrin can also form an inflammasome complex with ASC after engagement by autoinflammatory PSTPIP1 mutants. How the pyrin inflammasome is activated in the absence of disease-associated mutations is not yet known. We report here that ribotoxic stress triggers the assembly of the human pyrin inflammasome, leading to ASC oligomerization and caspase-1 activation in THP-1 macrophages and in a 293T cell line stably reconstituted with components of the pyrin inflammasome. Knockdown of pyrin and selective inhibition of p38 MAPK greatly attenuated caspase-1 activation by ribotoxic stress, whereas expression of the conditional mutant ΔMEKK3:ER* allowed the activation of caspase-1 without ribotoxic stress. Disruption of microtubules by colchicine also inhibited pyrin inflammasome activation by ribotoxic stress. Together, our results indicate that ribotoxic stress activates the human pyrin inflammasome through a mechanism that requires p38 MAPK signaling and microtubule stability.

The NLRP3 inflammasome is a key component of the innate immune response to pathogenic infection and tissue damage. It is also involved in the pathogenesis of a number of human diseases, including gouty arthritis, silicosis, atherosclerosis, and type 2 diabetes. The assembly of the NLRP3 inflammasome requires a priming signal derived from pattern recognition or cytokine receptors, followed by a second signal derived from extracellular ATP, pore-forming toxins, or crystalline materials. How these two signals activate the NLRP3 inflammasome is not yet clear. Here, we show that in mouse macrophages, signaling by the pattern recognition receptor TLR4 through MyD88 can rapidly and non-transcriptionally prime NLRP3 by stimulating its deubiquitination. This process is dependent on mitochondrial reactive oxygen species production and can be inhibited by antioxidants. We further show that signaling by ATP can also induce deubiquitination of NLRP3 by a mechanism that is not sensitive to antioxidants. Pharmacological inhibition of NLRP3 deubiquitination completely blocked NLRP3 activation in both mouse and human cells, indicating that deubiquitination of NLRP3 is required for its activation. Our findings suggest that NLRP3 is activated by a two-step deubiquitination mechanism initiated by Toll-like receptor signaling and mitochondrial reactive oxygen species and further potentiated by ATP, which could explain how NLRP3 is activated by diverse danger signals.

Omi/HtrA2 is a nuclear encoded mitochondrial serine protease with dual and opposite functions that depend entirely on its subcellular localization. During apoptosis, Omi/HtrA2 is released into the cytoplasm where it participates in cell death. While confined in the inter-membrane space of the mitochondria, Omi/HtrA2 has a pro-survival function that may involve the regulation of protein quality control (PQC) and mitochondrial homeostasis. Loss of Omi/HtrA2's protease activity causes the neuromuscular disorder of the mnd2 ( m otor n euron d egeneration 2 ) mutant mice. These mice develop multiple defects including neurodegeneration with parkinsonian features. Loss of Omi/HtrA2 in non-neuronal tissues has also been shown to cause premature aging. The normal function of Omi/HtrA2 in the mitochondria and how its deregulation causes neurodegeneration or premature aging are unknown. Here we report that the mitochondrial Mulan E3 ubiquitin ligase is a specific substrate of Omi/HtrA2. During exposure to H2O2, Omi/HtrA2 degrades Mulan, and this regulation is lost in cells that carry the inactive protease. Furthermore, we show accumulation of Mulan protein in various tissues of mnd2 mice as well as in Omi/HtrA2(−/−) mouse embryonic fibroblasts (MEFs). This causes a significant decrease of mitofusin 2 (Mfn2) protein, and increased mitophagy. Our work describes a new stress-signaling pathway that is initiated in the mitochondria and involves the regulation of Mulan by Omi/HtrA2 protease. Deregulation of this pathway, as it occurs in mnd2 mutant mice, causes mitochondrial dysfunction and mitophagy, and could be responsible for the motor neuron disease and the premature aging phenotype observed in these animals.

The molecular mechanism by which mutations in the cytoskeleton-organizing protein PSTPIP1 cause the autoinflammatory PAPA syndrome is still elusive. Here, we demonstrate that PSTPIP1 requires the familial Mediterranean fever protein pyrin to assemble the ASC pyroptosome, a molecular platform that recruits and activates caspase-1. We provide evidence that pyrin is a cytosolic receptor for PSTPIP1. Pyrin exists as a homotrimer in an autoinhibited state due to intramolecular interactions between its pyrin domain (PYD) and B-box. Ligation by PSTPIP1, which is also a homotrimer, activates pyrin by unmasking its PYD, thereby allowing it to interact with ASC and facilitate ASC oligomerization into an active ASC pyroptosome. Because of their high binding affinity to pyrin's B-box, PAPA-associated PSTPIP1 mutants were found to be more effective than WT PSTPIP1 in inducing pyrin activation. Therefore, constitutive ligation and activation of pyrin by mutant PSTPIP1 proteins explain the autoinflammatory phenotype seen in PAPA syndrome.

Presenilin mutations are responsible for most cases of autosomal dominant inherited forms of early onset Alzheimer disease. Presenilins play an important role in amyloid beta-precursor processing, NOTCH receptor signaling, and apoptosis. However, the molecular mechanisms by which presenilins regulate apoptosis are not fully understood. Here, we report that presenilin-1 (PS1) regulates the proteolytic activity of the serine protease Omi/HtrA2 through direct interaction with its regulatory PDZ domain. We show that a peptide corresponding to the cytoplasmic C-terminal tail of PS1 dramatically increases the proteolytic activity of Omi/HtrA2 toward the inhibitor of apoptosis proteins and beta-casein and induces cell death in an Omi/HtrA2-dependent manner. Consistent with these results, ectopic expression of full-length PS1, but not PS1 lacking the C-terminal PDZ binding motif, potentiated Omi/HtrA2-induced cell death. Our results suggest that the C terminus of PS1 is an activation peptide ligand for the PDZ domain of Omi/HtrA2 and may regulate the protease activity of Omi/HtrA2 after its release from the mitochondria during apoptosis. This mechanism of Omi/HtrA2 activation is similar to the mechanism of activation of the related bacterial DegS protease by the outer-membrane porins.

The mature serine protease Omi/HtrA2 is released from the mitochondria into the cytosol during apoptosis. Suppression of Omi/HtrA2 by RNA interference in human cell lines reduces cell death in response to TRAIL and etoposide. In contrast, ectopic expression of mature wildtype Omi/HtrA2, but not an active site mutant, induces potent caspase activation and apoptosis. In vitro assays demonstrated that Omi/HtrA2 could degrade inhibitor of apoptosis proteins (IAPs). Consistent with this observation, increased expression of Omi/HtrA2 in cells increases degradation of XIAP, while suppression of Omi/HtrA2 by RNA interference has an opposite effect. Combined, our data demonstrate that IAPs are substrates for Omi/HtrA2, and their degradation could be a mechanism by which the mitochondrially released Omi/HtrA2 activates caspases during apoptosis.

Inhibitors of apoptosis proteins (IAPs) interact with caspases and inhibit their protease activity, whereas the IAP-inhibitory proteins Smac/DIABLO in mammals and Reaper, Hid, and Grim in flies relieve IAP-mediated inhibition [1–5] to induce cell death. Here we describe the functional characterization of the novel Drosophila cell death protein Sickle (Skl), which binds to IAPs and neutralizes their apoptotic inhibitory activity. Skl exhibits no sequence homology to Reaper, Hid, Grim, or Smac/DIABLO, except within the 4 residue N-terminal IAP binding motif. Skl interacts with Drosophila and mammalian IAPs and can promote caspase activation in the presence of IAPs. Consistent with these findings, expression of Skl in Drosophila and mammalian cell lines or in Drosophila embryos induces apoptosis. Skl can also synergize with Grim to induce cell death in the Drosophila eye imaginal disc. Based on biochemical and structural data, the N terminus of Skl, like that of the mammalian Smac/DIABLO, is absolutely required for its apoptotic and caspase-promoting activities and its ability to interact with IAPs. These findings point to conservation in the structure and function of the IAP-inhibitory proteins across species and suggest the existence of other family members.

To identify human proteins that bind to the Smac and caspase-9 binding pocket on the baculoviral inhibitor of apoptosis protein (IAP) repeat 3 (BIR3) domain of human XIAP, we used BIR3 as an affinity reagent, followed by elution with the BIR3 binding peptide AVPIA, microsequencing, and mass spectrometry. The mature serine protease Omi (also known as HtrA2) was identified as a mitochondrial direct BIR3-binding protein and a caspase activator. Like mature Smac (also known as Diablo), mature Omi contains a conserved IAP-binding motif (AVPS) at its N terminus, which is exposed after processing of its N-terminal mitochondrial targeting sequence upon import into the mitochondria. Mature Omi is released together with mature Smac from the mitochondria into the cytosol upon disruption of the outer mitochondrial membrane during apoptosis. Finally, mature Omi can induce apoptosis in human cells in a caspase-independent manner through its protease activity and in a caspase-dependent manner via its ability to disrupt caspase-IAP interaction. Our results provide clear evidence for the involvement of a mitochondrial serine protease in the apoptotic pathway, emphasizing the critical role of the mitochondria in cell death.

Apoptosis involves the cessation of cellular processes, the breakdown of intracellular organelles, and, finally, the nonphlogistic clearance of apoptotic cells from the body. Important for these events is a family of proteases, caspases, which are activated by a proteolytic cleavage cascade and drive apoptosis by targeting key proteins within the cell. Here, we demonstrate that serum response factor (SRF), a transcription factor essential for proliferative gene expression, is cleaved by caspases and that this cleavage occurs in proliferating murine fibroblasts and can be induced in the human B-cell line BJAB. We identify the two major sites at which SRF cleavage occurs as Asp(245) and Asp(254), the caspases responsible for the cleavage and generate a mutant of SRF resistant to cleavage in BJAB cells. Investigation of the physiological and functional significance of SRF cleavage reveals that it correlates with the loss of c-fos expression, whereby neither SRF cleavage fragment retains transcriptional activity. Moreover, the expression of a noncleavable SRF in BJAB cells suppresses apoptosis induced by Fas cross-linking. These results suggest that for apoptosis to proceed, the transcriptional events promoting cell survival and proliferation, in which SRF is involved, must first be inactivated.

Procaspase-9 contains an NH2-terminal caspase-associated recruitment domain (CARD), which is essential for direct association with Apaf-1 and activation. Procaspase-1 also contains an NH2-terminal CARD domain, suggesting that its mechanism of activation, like that of procaspase-9, involves association with an Apaf-1-related molecule. Here we describe the identification of a human Apaf-1-related protein, named Ipaf that contains an NH2-terminal CARD domain, a central nucleotide-binding domain, and a COOH-terminal regulatory leucine-rich repeat domain (LRR). Ipaf associates directly and specifically with the CARD domain of procaspase-1 through CARD-CARD interaction. A constitutively active Ipaf lacking its COOH-terminal LRR domain can induce autocatalytic processing and activation of procaspase-1 and caspase-1-dependent apoptosis in transfected cells. Our results suggest that Ipaf is a specific and direct activator of procaspase-1 and could be involved in activation of caspase-1 in response to pro-inflammatory and apoptotic stimuli.

To understand the mechanism of activation of the IκB kinase (IKK) complex in the tumor necrosis factor (TNF) receptor 1 pathway, we examined the possibility that oligomerization of the IKK complex triggered by ligand-induced trimerization of the TNF receptor 1 complex is responsible for activation of the IKKs. Gel filtration analysis of the IKK complex revealed that TNFα stimulation induces a large increase in the size of this complex, suggesting oligomerization. Substitution of the C-terminal region of IKKγ, which interacts with RIP, with a truncated DR4 lacking its cytoplasmic death domain, produced a molecule that could induce IKK and NF-κB activation in cells in response to TRAIL. Enforced oligomerization of the N terminus of IKKγ or truncated IKKα or IKKβ lacking their serine-cluster domains can also induce IKK and NF-κB activation. These data suggest that IKKγ functions as a signaling adaptor between the upstream regulators such as RIP and the IKKs and that oligomerization of the IKK complex by upstream regulators is a critical step in activation of this complex.

Activation of procaspase-9 by Apaf-1 in the cytochrome c/dATP–dependent pathway requires proteolytic cleavage to generate the mature caspase molecule. To elucidate the mechanism of activation of procaspase-9 by Apaf-1, we designed an in vitro Apaf-1–procaspase-9 activation system using recombinant components. Here, we show that deletion of the Apaf-1 WD-40 repeats makes Apaf-1 constitutively active and capable of processing procaspase-9 independent of cytochrome c and dATP. Apaf-1-mediated processing of procaspase-9 occurs at Asp-315 by an intrinsic autocatalytic activity of procaspase-9 itself. We provide evidence that Apaf-1 can form oligomers and may facilitate procaspase-9 autoactivation by oligomerizing its precursor molecules. Once activated, caspase-9 can initiate a caspase cascade involving the downstream executioners caspase-3, -6, and -7.

A human receptor for the cytotoxic ligand TRAIL (TRAIL receptor-1, designated DR4) was identified recently as a member of the tumor necrosis factor receptor family. In this report we describe the identification of two additional human TRAIL receptors, TRAIL receptor-2 and TRAIL receptor-3, that belong to the tumor necrosis factor receptor family. Interestingly, TRAIL receptor-2 but not TRAIL receptor-3 contains a cytoplasmic "death domain" necessary for induction of apoptosis and is hence designated death receptor-5 (DR5). Like DR4, DR5 engages the apoptotic pathway independent of the adaptor molecule FADD/MORT1. Because of its lack of a death domain, TRAIL receptor-3 is not capable of inducing apoptosis. However, by competing for TRAIL, it is capable of inhibiting TRAIL-induced apoptosis. Thus, TRAIL receptor-3 may function as an antagonistic decoy receptor to attenuate the cytotoxic effect of TRAIL in most tissues that are TRAIL+, DR4+, and DR5+.

We identified and cloned a novel human protein that contains FADD/Mort1 death effector domain homology regions, designated FLAME-1. FLAME-1, although most similar in structure to Mch4 and Mch5, does not possess caspase activity but can interact specifically with FADD, Mch4, and Mch5. Interestingly, FLAME-1 is recruited to the Fas receptor complex and can abrogate Fas/TNFR-induced apoptosis upon expression in FasL/tumor necrosis factor-sensitive MCF-7 cells, possibly by acting as a dominant-negative inhibitor. These findings identify a novel endogenous control point that regulates Fas/TNFR1-mediated apoptosis.

Biochemical analyses of nuclear apoptosis in vitro have revealed the existence of multiple active interleukin-1beta-converting enzyme-related proteases (caspases) with distinct substrate recognition properties in extracts of preapoptotic chicken DU249 cells (S/M extracts). Previously we demonstrated that the activity of a caspase that cleaves lamins is required for the disintegration of nuclei in the late stages of apoptosis, despite the presence of a second active caspase that cleaves poly(ADP-ribose) polymerase (PARP). One simple explanation for this observation was that the lamin-cleaving caspase is sufficient to drive the nuclear events of apoptotic execution. Here, we report that phenylarsine oxide (PAO) inhibits the protease activities of recombinant human caspases as well as endogenous chicken caspases that are active in S/M extracts. PAO at 100 microM blocks the morphological changes of nuclear apoptosis in vitro and internucleosomal DNA fragmentation in S/M extracts without interfering with PARP or lamin A cleavage. Thus, lamin cleavage is not sufficient to drive the changes in nuclear morphology characteristic of apoptosis. Affinity labeling with YV(bio)KD-aomk shows that the degree of sensitivity to PAO differs among active caspases in S/M extracts. These results suggest that a PAO-sensitive caspase that is distinct from the PARP- or lamin-cleaving enzymes is required for the initiation of apoptotic morphological changes and for the activation of endonuclease(s). Taken together, our results suggest that two or more caspases are required for proteolytic events that are essential for the initiation and completion of nuclear apoptotic changes. The observation that PAO is an inhibitor of caspases and nuclear apoptotic events should be useful for the biochemical dissection of apoptosis in vitro and in vivo.

Recent evidence suggests that CPP32 is an essential component of an aspartate-specific cysteine protease (ASCP) cascade responsible for apoptosis execution in mammalian cells. Activation of CPP32 could lead to activation of other downstream ASCPs, resulting in late morphological changes such as lamin cleavage and DNA fragmentation, observed in cells undergoing apoptosis. Here we describe the identification and cloning of a novel human ASCP named Mch6 from Jurkat T lymphocytes. We demonstrate that the pro-enzymes of Mch6 and the lamin-cleaving enzyme Mch2alpha are substrates for mature CPP32. Site-directed mutagenesis revealed that CPP32 processes pro-Mch6 preferentially at Asp330 to generate two subunits of molecular masses 37 kDa (p37) and 10 kDa (p10). However, CPP32 processes pro-Mch2alpha at three aspartate processing sites (Asp23, Asp179, and Asp193) to produce the large (p18) and small (p11) subunits of the mature Mch2alpha enzyme. The CPP32-processed Mch2alpha is capable of cleaving the VEIDN lamin cleavage site, indicating that CPP32 can, in fact, activate pro-Mch2alpha. Granzyme B at a concentration that allows processing and activation of CPP32 failed to process pro-Mch2alpha. However, incubation of pro-Mch2alpha with granzyme B in the presence of a cellular extract containing pro-CPP32 resulted in activation of pro-CPP32 and subsequent processing of pro-Mch2alpha. Interestingly, granzyme B can also process pro-Mch6 but at a site N-terminal to that cleaved by CPP32. These data suggest that Mch2alpha and Mch6 are downstream proteases activated in CPP32- and granzyme B-mediated apoptosis. This is the first demonstration of a protease cascade involving granzyme B, CPP32, Mch2alpha, and Mch6 and evidence that the lamin-cleaving enzyme Mch2 is a target of mature CPP32.

Apoptosis may involve a specialized proteolytic cascade catalyzed by interleukin-1beta-converting enzyme-like proteases. We have recently identified three new members of this family (CPP32, MCH2, MCH3) and shown that they play an important role in promoting cell death. Here we report the chromosomal mapping of CPP32 to 4q34, MCH2 to 4q25, and MCH3 to 10q25.

To understand the mechanism of interleukin-1 beta converting enzyme (ICE) activation in apoptosis, we analyzed the expression of ICE mRNA in two human cell lines by reverse transcription-polymerase chain reaction technique. This resulted in the identification and cloning of four alternatively spliced ICE mRNA isoforms. Although all the alternative splicing events were within the coding sequence of ICE, the four ICE isoforms maintained open reading frames and were designated as ICE beta, gamma, delta, and epsilon. In ICE gamma, most of the propeptide (amino acids 20-112) is deleted, which suggests that it may function as a catalyst for ICE autoprocessing in vivo. In ICE delta, amino acids 288-335, which contain the cleavage sites between the p20 and p10 subunits of ICE, are deleted thus resulting in its inactivation. Intriguingly, in ICE epsilon amino acids 20-335, which encompass most of the propeptide and the p20 subunit, are deleted resulting in the formation of a molecule that is homologous to the p10 subunit. Examination of the ability of these four ICE isoforms to cause apoptosis revealed that only the parental ICE alpha and isoforms beta and gamma, but not isoforms delta and epsilon, can induce apoptosis when overexpressed in Sf9 insect cells. In addition, coexpression of the p20 and p10 but not the p20 and ICE epsilon in Sf9 cells results in apoptosis. Interestingly, expression of ICE epsilon and to a lesser degree ICE delta resulted in extension of the survival of baculovirus-infected cells in a manner similar to expression of BCL2. The ability of ICE epsilon to extend the survival of Sf9 cells suggests that baculovirus-induced apoptosis in these cells is mediated by an ICE-like protease. We show that ICE epsilon can bind to the p20 subunit of ICE and potentially may compete with the p10 subunit to form an inactive ICE complex. Therefore, by acting as a dominant inhibitor of ICE activity, ICE epsilon may regulate ICE activation in vivo.

Recently, we identified a 59-kDa nuclear phosphoprotein that is associated with a recombinant mouse FKBP-52 (Alnemri, E. S., Fernandes-Alnemri, T., Nelki, D. S., Dudley, K., DuBois, G. C., and Litwack, G. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 6839-6843). Here we describe the cloning, overexpression, and characterization of this protein from Spodoptera frugiperda insect cells (Sf9 cells). The cloned cDNA codes for an acidic protein of 412 amino acids with distinct structural domains. Starting with the N terminus, the first 218 amino acids contain two highly acidic domains separated by a short basic domain. Following the second large acidic domain is another basic domain of 87 amino acids with significant sequence and structural homology to HMG1 and HMG2 DNA binding proteins. The two basic domains contain several nuclear targeting signals. The last 108 C-terminal amino acids contain a binding domain for immunosuppressive drugs FK506 and rapamycin, which makes this protein a new member of the immunophilin family. We provide evidence that the new immunophilin (FKBP46) is a DNA binding protein that can bind immunosuppressive drug FK506 and possesses peptidylprolyl isomerase activity. FKBP46 is localized in the nucleus and is associated with a nuclear kinase that specifically phosphorylates it in the presence of Mg2+ and ATP. Upon subsequent sequence analysis of the mouse FKBP52 cDNA used in our previous study, it was observed that a spermatid nuclear transition protein 2 (TP2) sequence is fused in frame with the C terminus of the recombinant FKBP52 probably as a result of a cloning artifact. We demonstrate that the FKBP46 does not form a complex with the FKBP52 but rather with the highly basic nuclear protein TP2. Our data suggest that interaction of FKBP46 with TP2 is mediated by the N-terminal acidic domains of FKBP46. This implies that the acidic domains of FKBP46 are involved in protein-protein interaction between nuclear FKBP46 and other basic chromatin proteins.

Proteolytic activation of initiator procaspases is a crucial step in the cellular commitment to apoptosis. Alternative models have been postulated for the activation mechanism, namely the oligomerization or induced proximity model and the allosteric regulation model. While the former holds that procaspases become activated upon proper oligomerization by an adaptor protein, the latter states that the adaptor is an allosteric regulator for procaspases. The allosteric regulation model has been applied for the activation of procaspase-9 by apoptotic protease-activating factor (Apaf-1) in an oligomeric complex known as the apoptosome. Using approaches that allow for controlled oligomerization, we show here that aggregation of multiple procaspase-9 molecules can induce their activation independent of the apoptosome. Oligomerization-induced procaspase-9 activation, both within the apoptosome and in artificial systems, requires stable homophilic association of the protease domains, raising the possibility that the function of Apaf-1 is not only to oligomerize procaspase-9 but also to maintain the interaction of the caspase-9 protease domain after processing. In addition, we provide biochemical evidence that other apoptosis initiator caspases (caspase-2 and -10) as well as a procaspase involved in inflammation (murine caspase-11) are also activated by oligomerization. Thus, oligomerization of precursor molecules appears to be a general mechanism for the activation of both apoptosis initiator and inflammatory procaspases.

Cleavage of Bid has been shown to promote apoptosis by inducing mitochondrial membrane permeabilization with the resultant release of apoptosis-inducing proteins from the intermembrane space into the cytosol. However, direct visualization of the Bid-induced release of various proteins from the highly compartmentalized intermembrane space and the changes in the mitochondrial metabolic machinery remain elusive. Using green fluorescent protein fusion proteins and immunostaining in individual permeabilized HepG2 cells, first we demonstrated that truncated Bid (15.5-kDa C-terminal fragment, tBid) evoked a rapid and essentially complete release of cytochrome c and Smac/DIABLO from every mitochondrion. To establish at a resolution of seconds the kinetics of tBid-induced cytochrome c and Smac/DIABLO release and depolarization, we monitored the mitochondrial membrane potential (DeltaPsi(m)) fluorimetrically in permeabilized cells and applied a rapid filtration method to obtain cytosolic fractions for Western blotting. We found that subnanomolar doses of tBid were sufficient to evoke cytochrome c release and mitochondrial depolarization, whereas full-length Bid was 100-fold less effective. Bcl-x(L) prevented tBid-induced cytochrome c release and depolarization. In response to 2.5 nm tBid, cytochrome c release started after a 10 s delay, displayed rapid progression, and was complete at 50-70 s. Release of Smac/DIABLO was synchronized with cytochrome c release, whereas the loss of DeltaPsi(m) lagged slightly behind cytochrome c release. Furthermore, tBid-induced cytochrome c release was insensitive to changes in substrate composition, but tBid-induced depolarization did not occur in the presence of extramitochondrial ATP supply. Thus, tBid-induced permeabilization of the outer membrane permits rapid release of cytochrome c and Smac/DIABLO from all domains of the intermembrane space. The tBid-induced loss of DeltaPsi(m) occurs after cytochrome c release and reflects impairment of oxidative metabolism.

Apoptosis is primarily executed by active caspases, which are derived from the inactive procaspase zymogens through proteolytic cleavage. Here we report the crystal structures of a caspase zymogen, procaspase-7, and an active caspase-7 without any bound inhibitors. Compared to the inhibitor-bound caspase-7, procaspase-7 zymogen exhibits significant structural differences surrounding the catalytic cleft, which precludes the formation of a productive conformation. Proteolytic cleavage between the large and small subunits allows rearrangement of essential loops in the active site, priming active caspase-7 for inhibitor/substrate binding. Strikingly, binding by inhibitors causes a 180° flipping of the N terminus in the small subunit, which interacts with and stabilizes the catalytic cleft. These analyses reveal the structural mechanisms of caspase activation and demonstrate that the inhibitor/substrate binding is a process of induced fit.

Tumor necrosis factor-alpha (TNFalpha) mediates cytochrome c release from mitochondria, loss of mitochondrial membrane potential (DeltaPsim) and apoptosis in sensitive leukemic cells. In the present study, by using the human leukemic U937 cell line, we demonstrate that the cytochrome c release is caspase-8-dependent and can be blocked by an inhibitor of caspase-8, Z-Ile-Glu (OMe)-Thr-Asp(OMe)-fluoromethyl ketone (Z-IETD.fmk), or a pan caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (Z-VAD.fmk). However, TNFalpha-mediated loss of DeltaPsim was not inhibited by caspase inhibitors. The apoptotic process was blocked by either Z-IETD.fmk or Z-VAD.fmk in cells with lower DeltaPsim. U937 cells with stable transfection of the cellular inhibitor of apoptosis protein 1 (c-IAP1) are resistant to TNFalpha-induced activation of caspases, Bid cleavage, cytochrome c release and DeltaPsim collapse. In addition, both c-IAP1 and XIAP were not up-regulated upon prolonged exposure to TNFalpha. In contrast, there was a caspase-dependent cleavage of XIAP, but not c-IAP1, during treatment with TNFalpha for 7 days. These results demonstrate that c-IAP1 blocks TNFalpha signaling at a level controlling both activation of caspase-8 and a signal to cause loss of DeltaPsim. The sensitive U937 cell line failed to acquire resistance and gain a self-protecting advantage against apoptosis, upon induction of c-IAP1 expression.