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The inflammasome is a critical molecular complex that activates interleukin-1 driven inflammation in response to pathogen- and danger-associated signals. Germline mutations in the inflammasome sensor NLRP1 cause Mendelian systemic autoimmunity and skin cancer susceptibility, but its endogenous regulation remains less understood. Here we use a proteomics screen to uncover dipeptidyl dipeptidase DPP9 as a novel interacting partner with human NLRP1 and a related inflammasome regulator, CARD8. DPP9 functions as an endogenous inhibitor of NLRP1 inflammasome in diverse primary cell types from human and mice. DPP8/9 inhibition via small molecule drugs and CRISPR/Cas9-mediated genetic deletion specifically activate the human NLRP1 inflammasome, leading to ASC speck formation, pyroptotic cell death, and secretion of cleaved interleukin-1β. Mechanistically, DPP9 interacts with a unique autoproteolytic domain (Function to Find Domain (FIIND)) found in NLRP1 and CARD8. This scaffolding function of DPP9 and its catalytic activity act synergistically to maintain NLRP1 in its inactive state and repress downstream inflammasome activation. We further identified a single patient-derived germline missense mutation in the NLRP1 FIIND domain that abrogates DPP9 binding, leading to inflammasome hyperactivation seen in the Mendelian autoinflammatory disease Autoinflammation with Arthritis and Dyskeratosis. These results unite recent findings on the regulation of murine Nlrp1b by Dpp8/9 and uncover a new regulatory mechanism for the NLRP1 inflammasome in primary human cells. Our results further suggest that DPP9 could be a multifunctional inflammasome regulator involved in human autoinflammatory diseases., (© 2018 Zhong et al.)

B cell lymphoma gene 2 (Bcl-2) family proteins are key regulators of programmed cell death and important targets for drug discovery. Pro-apoptotic and anti-apoptotic Bcl-2 family proteins reciprocally modulate their activities in large part through protein interactions involving a motif known as BH3 (Bcl-2 homology 3). Nur77 is an orphan member of the nuclear receptor family that lacks a BH3 domain but nevertheless binds certain anti-apoptotic Bcl-2 family proteins (Bcl-2, Bfl-1, and Bcl-B), modulating their effects on apoptosis and autophagy. We used a combination of NMR spectroscopy-based methods, mutagenesis, and functional studies to define the interaction site of a Nur77 peptide on anti-apoptotic Bcl-2 family proteins and reveal a novel interaction surface. Nur77 binds adjacent to the BH3 peptide-binding crevice, suggesting the possibility of cross-talk between these discrete binding sites. Mutagenesis of residues lining the identified interaction site on Bcl-B negated the interaction with Nur77 protein in cells and prevented Nur77-mediated modulation of apoptosis and autophagy. The findings establish a new protein interaction site with the potential to modulate the apoptosis and autophagy mechanisms governed by Bcl-2 family proteins., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Autophagy is a catabolic cellular mechanism for entrapping cellular macromolecules and organelles in intracellular vesicles and degrading their contents by fusion with lysosomes. Important roles for autophagy have been elucidated for cell survival during nutrient insufficiency, eradication of intracellular pathogens, and counteracting aging through clearance of senescent proteins and mitochondria. Autophagic vesicles become decorated with LC3, a protein that mediates their fusion with lysosomes. LC3 is a substrate of the cysteine protease ATG4B (Autophagin-1), where cleavage generates a C-terminal glycine required for LC3 conjugation to lipids in autophagosomes. ATG4B both cleaves pro-LC3 and also hydrolyzes lipids from cleaved LC3. We show here that phosphorylation of ATG4B at Ser-383 and Ser-392 increases its hydrolyase activity as measured using LC3 as a substrate. Reconstituting atg4b(-/-) cells with phosphorylation-deficient ATG4B showed a role of ATG4B phosphorylation in LC3 delipidation and autophagic flux, thus demonstrating that the cellular activity of ATG4B is modulated by phosphorylation. Proteolytic conversion of pro-LC3 to LC3-I was not significantly impacted by ATG4B phosphorylation in cells. Phosphorylation-deficient ATG4B also showed reduced interactions with the lipid-conjugated LC3 but not unconjugated LC3. Taken together, these findings demonstrate a role for Ser-383 and Ser-392 phosphorylation of ATG4B in control of autophagy., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

The cytosolic protein c-FLIP (cellular Fas-associated death domain-like interleukin 1β-converting enzyme inhibitory protein) is an inhibitor of death receptor-mediated apoptosis that is up-regulated in a variety of cancers, contributing to apoptosis resistance. Several compounds found to restore sensitivity of cancer cells to TRAIL, a TNF family death ligand with promising therapeutic potential, act by targeting c-FLIP ubiquitination and degradation by the proteasome. The generation of reactive oxygen species (ROS) has been implicated in c-FLIP protein degradation. However, the mechanism by which ROS post-transcriptionally regulate c-FLIP protein levels is not well understood. We show here that treatment of prostate cancer PPC-1 cells with the superoxide generators menadione, paraquat, or buthionine sulfoximine down-regulates c-FLIP long (c-FLIP(L)) protein levels, which is prevented by the proteasome inhibitor MG132. Furthermore, pretreatment of PPC-1 cells with a ROS scavenger prevented ubiquitination and loss of c-FLIP(L) protein induced by menadione or paraquat. We identified lysine 167 as a novel ubiquitination site of c-FLIP(L) important for ROS-dependent degradation. We also identified threonine 166 as a novel phosphorylation site and demonstrate that Thr-166 phosphorylation is required for ROS-induced Lys-167 ubiquitination. The mutation of either Thr-166 or Lys-167 was sufficient to stabilize c-FLIP protein levels in PPC-1, HEK293T, and HeLa cancer cells treated with menadione or paraquat. Accordingly, expression of c-FLIP T166A or K167R mutants protected cells from ROS-mediated sensitization to TRAIL-induced cell death. Our findings reveal novel ROS-dependent post-translational modifications of the c-FLIP protein that regulate its stability, thus impacting sensitivity of cancer cells to TRAIL.

Type 1 diabetes (T1D) is expected to cause significant changes in the serum proteome; however, few studies have systematically assessed the proteomic profile change associated with the disease. In this study, a semiquantitative spectral counting-based two dimensional liquid chromatography mass spectrometry platform was used to analyze serum samples from T1D patients and controls. In this discovery phase, significant differences were found for 21 serum proteins implicated in inflammation, oxidation, metabolic regulation, and autoimmunity. To assess the validity of these findings, six candidate proteins including adiponectin, insulin-like growth factor binding protein 2, serum amyloid protein A, C-reactive protein, myeloperoxidase, and transforming growth factor beta induced were selected for subsequent immune assays for 1139 T1D patients and 848 controls. A series of statistical analyses using cases and controls matched for age, sex, and genetic risk confirmed that T1D patients have significantly higher serum levels for four of the six proteins: adiponectin (odds ratio (OR) = 1.95, p = 10(-27)), insulin-like growth factor binding protein 2 (OR = 2.02, p < 10(-20)), C-reactive protein (OR = 1.13, p = 0.007), serum amyloid protein A (OR = 1.51, p < 10(-16)); whereas the serum levels were significantly lower in patients than controls for the two other proteins: transforming growth factor beta induced (OR = 0.74, p < 10(-5)) and myeloperoxidase (OR = 0.51, p < 10(-41)). Compared with subjects in the bottom quartile, subjects in the top quartile for adiponectin (OR = 6.29, p < 10(-37)), insulin-like growth factor binding protein 2 (OR = 7.95, p < 10(-46)), C-reactive protein (OR = 1.38, p = 0.025), serum amyloid protein A (OR = 3.36, p < 10(-16)) had the highest risk of T1D, whereas subjects in the top quartile of transforming growth factor beta induced (OR = 0.41, p < 10(-11)) and myeloperoxidase (OR = 0.10, p < 10(-43)) had the lowest risk of T1D. These findings provided valuable information on the proteomic changes in the sera of T1D patients.

In multicellular organisms, apoptosis is a powerful method of host defense against viral infection. Apoptosis is mediated by a cascade of caspase-family proteases that commit infected cells to a form of programmed cell death. Therefore, to replicate within host cells, viruses have developed various strategies to inhibit caspase activation. In the mitochondrial cell-death pathway, release of cytochrome c from mitochondria into the cytosol triggers assembly of the oligomeric apoptosome, resulting in dimerization and activation of the apical caspase-9 (C9), and in turn its downstream effector caspases, leading to apoptosis. We previously showed that the vaccinia virus-encoded Bcl-2-like protein, F1L, which suppresses cytochrome c release by binding Bcl-2 family proteins, is also a C9 inhibitor. Here, we identify a novel motif within the flexible N-terminal region of F1L that is necessary and sufficient for interaction with and inhibition of C9. Based on functional studies and mutagenesis, we developed an atomic model of the complex in which F1L inhibits C9 by engaging the active site in the reverse orientation with respect to substrate peptides, in a manner analogous to that of XIAP-mediated inhibition of caspases-3 and -7. These studies offer new insights into the mechanism of apoptosome inhibition by F1L as well as novel probes to understand the molecular bases of apoptosome regulation and turnover. They also suggest how the two distinct functionalities of F1L (inhibition of C9 and suppression of pro-apoptotic Bcl-2 family proteins) may operate in a cellular setting.

Accumulation of misfolded proteins in the endoplasmic reticulum (ER) causes ER stress and activates inositol-requiring protein-1 (IRE1), among other ER-associated signaling proteins of the unfolded protein response (UPR) in mammalian cells. IRE1 signaling becomes attenuated under prolonged ER stress. The mechanisms by which this occurs are not well understood. An ER resident protein, Bax inhibitor-1 (BI-1), interacts with IRE1 and directly inhibits IRE1 activity. However, little is known about regulation of the BI-1 protein. We show here that bifunctional apoptosis regulator (BAR) functions as an ER-associated RING-type E3 ligase, interacts with BI-1, and promotes proteasomal degradation of BI-1. Overexpression of BAR reduced BI-1 protein levels in a RING-dependent manner. Conversely, knockdown of endogenous BAR increased BI-1 protein levels and enhanced inhibition of IRE1 signaling during ER stress. We also found that the levels of endogenous BAR were reduced under prolonged ER stress. Our findings suggest that post-translational regulation of the BI-1 protein by E3 ligase BAR contributes to the dynamic control of IRE1 signaling during ER stress.

The unfolded protein response (UPR) or endoplasmic reticulum (ER) stress response is a physiological process enabling cells to cope with altered protein synthesis demands. However, under conditions of obesity, prolonged activation of the UPR has been shown to have deteriorating effects on different metabolic pathways. Here we identify Bax inhibitor-1 (BI-1), an evolutionary conserved ER-membrane protein, as a novel modulator of the obesity-associated alteration of the UPR. BI-1 partially inhibits the UPR by interacting with IRE1alpha and inhibiting IRE1alpha endonuclease activity as seen on the splicing of the transcription factor Xbp-1. Because we observed a down-regulation of BI-1 expression in liver and muscle of genetically obese ob/ob and db/db mice as well as in mice with diet-induced obesity in vivo, we investigated the effect of restoring BI-1 expression on metabolic processes in these mice. Importantly, BI-1 overexpression by adenoviral gene transfer dramatically improved glucose metabolism in both standard diet-fed mice as well as in mice with diet-induced obesity and, critically, reversed hyperglycemia in db/db mice. This improvement in whole body glucose metabolism and insulin sensitivity was due to dramatically reduced gluconeogenesis as shown by reduction of glucose-6-phosphatase and phosphoenolpyruvate carboxykinase expression. Taken together, these results identify BI-1 as a critical regulator of ER stress responses in the development of obesity-associated insulin resistance and provide proof of concept evidence that gene transfer-mediated elevations in hepatic BI-1 may represent a promising approach for the treatment of type 2 diabetes.

Apoptosis plays important roles in host defense, including the elimination of virus-infected cells. The executioners of apoptosis are caspase family proteases. We report that vaccinia virus-encoded F1L protein, previously recognized as anti-apoptotic viral Bcl-2 family protein, is a caspase-9 inhibitor. F1L binds to and specifically inhibits caspase-9, the apical protease in the mitochondrial cell death pathway while failing to inhibit other caspases. In cells, F1L inhibits apoptosis and proteolytic processing of caspases induced by overexpression of caspase-9 but not caspase-8. An N-terminal region of F1L preceding the Bcl-2-like fold accounts for caspase-9 inhibition and significantly contributes to the anti-apoptotic activity of F1L. Viral F1L thus provides the first example of caspase inhibition by a Bcl-2 family member; it functions both as a suppressor of proapoptotic Bcl-2 family proteins and as an inhibitor of caspase-9, thereby neutralizing two sequential steps in the mitochondrial cell death pathway.

Macrophages detect pathogen infection via the activation of their plasma membrane-bound Toll-like receptor proteins (TLRs). The heterotypic interaction between the Toll/interleukin-1 receptor (TIR) domains of TLRs and adaptor proteins, like Myeloid differentiation primary response gene 88 (MyD88), is the first intracellular step in the signaling pathway of the mammalian innate immune response. The hetero-oligomerization of the TIRs of the receptor and adaptor brings about the activation of the transcription factor NF-kappaB, which regulates the synthesis of pro-inflammatory cytokines. Here, we report the first crystal structure of a bacterial TIR domain solved at 2.5 A resolution. The three-dimensional fold of Paracoccus denitrificans TIR is identical to that observed for the TIR of human TLRs and MyD88 proteins. The structure shows a unique dimerization interface involving the DD-loop and EE-loop residues, whereas leaving the BB-loop highly exposed. Peptide amide hydrogen-deuterium exchange mass spectrometry also reveals that the same region is used for dimerization in solution and in the context of the full-length protein. These results, together with a functional interaction between P. denitrificans TIR and MyD88 visualized in a co-immunoprecipitation assay, further substantiate the model that bacterial TIR proteins adopt structural mimicry of the host active receptor TIR domains to interfere with the signaling of TLRs and their adaptors to decrease the inflammatory response.

The accumulation of unfolded proteins in the endoplasmic reticulum (ER) is caused by many disease-relevant conditions, inducing conserved signaling events collectively known as the unfolded protein response. When ER stress is excessive or prolonged, cell death (usually occurring by apoptosis) is triggered. We undertook a chemical biology approach for investigating mechanisms of ER stress-induced cell death. Using a cell-based high throughput screening assay to identify compounds that rescued a neuronal cell line from thapsigargin-induced cell death, we identified benzodiazepinones that selectively inhibit cell death caused by inducers of ER stress (thapsigargin and tunicamycin) but not by inducers of extrinsic (tumor necrosis factor) or intrinsic (mitochondrial) cell death pathways. The compounds displayed activity in several cell lines and primary cultured neurons. Mechanism of action studies revealed that these compounds inhibit ER stress-induced activation of p38 MAPK and kinases responsible for c-Jun phosphorylation. Active benzodiazepinones suppressed cell death at the level of apoptotic signal kinase-1 (ASK1) within the IRE1 pathway but without directly inhibiting the kinase activity of ASK1 or >400 other kinases tested. Rather, active compounds enhanced phosphorylation of serine 967 of ASK1, promoting ASK1 binding to 14-3-3, an event associated with suppression of ASK1 function. Reducing ASK1 protein expression using small interfering RNA also protected cells from ER stress-induced apoptosis, confirming the importance of this protein kinase. Taken together, these findings demonstrate an essential role for ASK1 in cell death induced by ER stress. The compounds identified may prove useful for revealing endogenous mechanisms that regulate inhibitory phosphorylation of ASK1.

The pathogenesis of Huntington disease (HD) is attributed to the misfolding of huntingtin (htt) caused by an expanded polyglutamine (polyQ) domain. Considerable effort has been devoted to identifying molecules that can prevent or reduce htt misfolding and the associated neuropathology. Although overexpression of chaperones is known to reduce htt cytotoxicity in cellular models, only modest protection is seen with Hsp70 overexpression in HD mouse models. Because the activity of Hsp70 is modulated by co-chaperones, an interesting issue is whether the in vivo effects of chaperones on polyQ protein toxicity are dependent on other modulators. In the present study, we focused on BAG1, a co-chaperone that interacts with Hsp70 and regulates its activity. Of htt mice expressing the N171-82Q mutant, we found that male N171-82Q mice show a greater deficit in rotarod performance than female N171-82Q mice. This sex-dependent motor deficit was improved by crossing N171-82Q mice with transgenic mice overexpressing BAG1 in neurons. Transgenic BAG1 also reduces the levels of mutant htt in synaptosomal fraction of male HD mice. Overexpression of BAG1 augmented the effects of Hsp70 by reducing aggregation of mutant htt in cultured cells and improving neurite outgrowth in htt-transfected PC12 cells. These findings suggest that the effects of chaperones on HD pathology are influenced by both their modulators and sex-dependent factors.

In this study, Bax inhibitor-1 (BI-1) overexpression reduces the ER pool of Ca(2+) released by thapsigargin. Cells overexpressing BI-1 also showed lower intracellular Ca(2+) release induced by the Ca(2+) ionophore ionomycin as well as agonists of ryanodine receptors and inositol trisphosphate receptors. In contrast, cells expressing carboxyl-terminal deleted BI-1 (CDelta-BI-1 cells) displayed normal intracellular Ca(2+) mobilization. Basal Ca(2+) release rates from the ER were higher in BI-1-overexpressing cells than in control or CDelta-BI-1 cells. We determined that the carboxyl-terminal cytosolic region of BI-1 contains a lysine-rich motif (EKDKKKEKK) resembling the pH-sensing domains of ion channels. Acidic conditions triggered more extensive Ca(2+) release from ER microsomes from BI-1-overexpressing cells and BI-1-reconstituted liposomes. Acidic conditions also induced BI-1 protein oligomerization. Interestingly subjecting BI-1-overexpressing cells to acidic conditions induced more Bax recruitment to mitochondria, more cytochrome c release from mitochondria, and more cell death. These findings suggest that BI-1 increases Ca(2+) leak rates from the ER through a mechanism that is dependent on pH and on the carboxyl-terminal cytosolic region of the BI-1 protein. The findings also reveal a cell death-promoting phenotype for BI-1 that is manifested under low pH conditions.

BI-1 (Bax inhibitor-1) is an evolutionarily conserved multitransmembrane protein that resides in the endoplasmic reticulum (ER) and that has documented cytoprotective functions in both animals and plants. Recent studies indicate that BI-1 shares in common with Bcl-2/Bax family proteins the ability to regulate the amounts of Ca(2+) that can be released from the ER by agents, such as the ER-Ca(2+)-ATPase (SERCA) inhibitor thapsigargin (TG). Using an ER-targeted, Ca(2+) indicator (cameleon), with characteristics optimized for measuring ER Ca(2+) ([Ca(2+)](er)), we studied the effects of BI-1 on [Ca(2+)](er) in resting and TG-treated cells. Similar to cells overexpressing antiapoptotic Bcl-2 or Bcl-X(L), overexpression of BI-1 resulted in lower resting [Ca(2+)](er), with concomitantly less Ca(2+) released into the cytosol upon stimulation by TG and with a higher rate of Ca(2+) leakage from the ER. Co-expression of SERCA restored levels of [Ca(2+)](er) to normal, showing opposing actions of the ER-Ca(2+)ATPase and BI-1 on ER Ca(2+) homeostasis. Conversely, cells with deficient BI-1 have increased [Ca(2+)](er), and release more Ca(2+) into the cytosol when challenged with TG. In BI-1-deficient cells, Bcl-X(L) fails to reduce [Ca(2+)](er), indicating that BI-1 functions downstream of Bcl-X(L). In bax(-/-)bak(-/-) double knock-out cells, both BI-1 and Bcl-X(L) retained their ability to reduce [Ca(2+)](er), suggesting that BI-1 and Bcl-X(L) operate downstream of or parallel to Bax/Bak. The findings reveal a hierarchy of functional interactions of BI-1 with Bcl-2/Bax family proteins in regulating ER Ca(2+) homeostasis.

The pro-apoptotic members of the Bcl-2 family include initiator proteins that contain only BH3 domains and downstream effector multi-BH domain-containing proteins, including Bax and Bak. In this report, we compared the ability of the six human anti-apoptotic Bcl-2 family members to suppress apoptosis induced by overexpression of Bax or Bak, correlating findings with protein interactions measured by three different methods: co-immunoprecipitation, glutathione S-transferase pulldown, and fluorescence polarization assays employing synthetic BH3 peptides from Bax and Bak. Bcl-B and Mcl-1 showed strong preferences for binding to and suppression of Bax and Bak, respectively. In contrast, the other anti-apoptotic Bcl-2 family proteins (Bcl-2, Bcl-X(L), Bcl-W, and Bfl-1) suppressed apoptosis induced by overexpression of either Bax or Bak, and they displayed an ability to bind both Bax and Bak by at least one of the three protein interaction methods. Interestingly, however, full-length Bax and Bak proteins and synthetic Bax and Bak BH3 peptides exhibited discernible differences in their interactions with some anti-apoptotic members of the Bcl-2 family, cautioning against reliance on a single method for detecting protein interactions of functional significance. Altogether, the findings reveal striking distinctions in the behaviors of Bcl-B and Mcl-1 relative to the other anti-apoptotic Bcl-2 family members, where Bcl-B and Mcl-1 display reciprocal abilities to bind and neutralize Bax and Bak.

Hepatitis B virus infection is associated with hepatocellular carcinoma, claiming 1 million lives annually worldwide. To understand the carcinogenic mechanism of hepatitis B virus-encoded oncoprotein HBx, we explored the function of HBx interaction with its cellular target HBXIP. Previously, we demonstrated that viral HBx and cellular HBXIP control mitotic spindle formation, regulating centrosome splitting. By using various fragments of HBx, we determined that residues (137)CRHK(140) within HBx are necessary for binding HBXIP. Mutation of the (137)CRHK(140) motif in HBx abolished its ability to bind HBXIP and to dysregulate centrosome dynamics in HeLa and immortal diploid RPE-1 cells. Unlike wild-type HBx, which targets to centrosomes as determined by subcellular fractionation and immunofluorescence microscopy, HBx mutants failed to localize to centrosomes. Overexpression of viral HBx wild-type protein and knockdown of endogenous HBXIP altered centrosome assembly and induced modifications of pericentrin and centrin-2, two essential proteins required for centrosome formation and function, whereas HBXIP nonbinding mutants of HBx did not. Overexpression of HBXIP or fragments of HBXIP that bind HBx neutralized the effects of viral HBx on centrosome dynamics and spindle formation. These results suggest that HBXIP is a critical target of viral HBx for promoting genetic instability through formation of defective spindles and subsequent aberrant chromosome segregation.

The Bax inhibitor-1 (BI-1) is an anti-apoptotic protein that is located in endoplasmic reticulum (ER) membranes and protects cells from ER stress-induced apoptosis. The ER is associated with generation of reactive oxygen species (ROS) through oxidative protein folding. This study examined the role of BI-1 in the regulation of ER stress-induced accumulation of ROS and expression of unfolded protein response-associated proteins. BI-1 reduced the expression levels of glucose response protein 78, C/EBP homologous protein, phospho-eukaryotic initiation factor 2alpha, IRE1alpha, XBP-1, and phospho-JNK and inhibited the cleavage of ATF-6alpha p-90, leading to the inhibition of ROS. Although ROS scavengers offer some protection against ER stress-induced apoptosis, the expression of pro-apoptotic ER stress proteins was not affected. This study shows that the response of unfolded proteins is followed by ROS accumulation under ER stress, which is regulated in BI-1 cells. The mechanism for these BI-1-associated functions involves the expression of heme oxygenase-1 (HO-1) through nuclear factor erythroid 2-related factor 2. In BI-1 cells, the transfection of HO-1 small interfering RNA completely abolished the BI-1-induced protection. The endogenous expression of HO-1 through ER stress-initiated ROS is believed to be as a protection signal. In conclusion, these observations suggest that BI-1 can inhibit the ER stress proteins as well as the accumulation of ROS, thereby protecting the cells. Moreover, HO-1 plays an important role in the BI-1-associated protection against ER stress.

Inhibitor of apoptosis proteins (IAPs) regulate apoptosis primarily by inhibiting caspase-family proteases. However, many IAPs also possess E3 ligase (ubiquitin-protein isopeptide ligase) activities implicated in both caspase-dependent and -independent functions of these proteins. Here, we compared the structural features of cIAP1 responsible for its interactions with two known target proteins, TRAF2 and SMAC. The N-terminal (BIR1) and C-terminal (BIR3) BIR domains of cIAP1 were determined to be necessary and sufficient for binding TRAF2 and SMAC, respectively. Mutational analysis of the BIR1 and BIR3 domains identified critical residues required for TRAF2 and SMAC binding. Using these mutants, cIAP1-mediated ubiquitination of TRAF2 and SMAC in vitro was determined to be correspondingly dependent on intact binding sites on BIR1 and BIR3. Because TRAF2 regulates NF-kappaB activation, the effects of cIAP1 on TRAF2-mediated induction of NF-kappaB transcriptional activity were studied using reporter gene assays. Expression of a fragment of cIAP1 encompassing the three BIR domains (but not full-length cIAP1) greatly enhanced TRAF2-induced increases in NF-kappaB activity, providing a convenient assay for monitoring BIR-dependent effects of cIAP1 on TRAF2 in cells. BIR1 mutants of the BIR1-3 fragment of cIAP1 that failed to bind TRAF2 lost the ability to modulate NF-kappaB activity, demonstrating a requirement for BIR1-mediated interactions with TRAF2. Altogether, these findings demonstrate the modularity and diversification of BIR domains, showing that a single cIAP can direct its E3 ligase activity toward different substrates and can alter the cellular functions of different protein targets in accordance with differences in the specificity of individual BIR domains.

Siah1 is the central component of a multiprotein E3 ubiquitin ligase complex that targets beta-catenin for destruction in response to p53 activation. The E3 complex comprises, in addition to Siah1, Siah-interacting protein (SIP), the adaptor protein Skp1, and the F-box protein Ebi. Here we show that SIP engages Siah1 by means of two elements, both of which are required for mediating beta-catenin destruction in cells. An N-terminal dimerization domain of SIP sits across the saddle-shaped upper surface of Siah1, with two extended legs packing against the sides of Siah1 by means of a consensus PXAXVXP motif that is common to a family of Siah-binding proteins. The C-terminal domain of SIP, which binds to Skp1, protrudes from the lower surface of Siah1, and we propose that this surface provides the scaffold for bringing substrate and the E2 enzyme into apposition in the functional complex.

Epstein-Barr virus is a human herpesvirus that causes infectious mononucleosis and lymphoproliferative malignancies. LMP1 (latent membrane protein-1), which is encoded by this virus and which is essential for transformation of B lymphocytes, acts as a constitutively active mimic of the tumor necrosis factor receptor (TNFR) CD40. LMP1 is an integral membrane protein containing six transmembrane segments and a cytoplasmic domain at the C terminus that binds to intracellular TNFR-associated factors (TRAFs). TRAFs are intracellular co-inducers of downstream signaling from CD40 and other TNFRs, and TRAF3 is required for activation of B lymphocytes by LMP1. Cytoplasmic C-terminal activation region 1 of LMP1 bears a motif (PQQAT) that conforms to the TRAF recognition motif PVQET in CD40. In this study, we report the crystal structure of this portion of LMP1 C-terminal activation region-1 (204PQQATDD210) bound in complex with TRAF3. The PQQAT motif is bound in the same binding crevice on TRAF3 where CD40 is bound, providing a molecular mechanism for LMP1 to act as a CD40 decoy for TRAF3. The LMP1 motif is presented in the TRAF3 crevice as a close structural mimic of the PVQET motif in CD40, and the intermolecular contacts are similar. However, the viral protein makes a unique contact: a hydrogen bond network formed between Asp210 in LMP1 and Tyr395 and Arg393 in TRAF3. This intermolecular contact is not made in the CD40-TRAF3 complex. The additional hydrogen bonds may stabilize the complex and strengthen the binding to permit LMP1 to compete with CD40 for binding to the TRAF3 crevice, influencing downstream signaling to B lymphocytes and contributing to dysregulated signaling by LMP1.

Recently, we discovered that Humanin (HN), a small endogenous peptide of 24 amino acids, binds to and inhibits the proapoptotic protein Bax. We show here that HN also interacts with the BH3-only Bcl-2/Bax family protein, Bid, as well as a truncated form of Bid (tBid) associated with protease-mediated activation of this proapoptotic protein. Synthetic HN peptide binds purified Bid and tBid in vitro and blocks tBid-induced release of cytochrome c and SMAC from isolated mitochondria, whereas mutant peptides that fail to bind Bid or tBid lack this activity. Moreover, HN peptide also retained protective activity on bax-/-mitochondria, indicating that HN can block tBid-induced release of apoptogenic proteins from these organelles in a Bax-independent manner. HN peptide inhibits tBid-induced oligomerization of Bax and Bak in mitochondrial membranes, as shown by experiments with chemical cross-linkers or gel filtration. Gene transfection experiments showed that HN (but not an inactive mutant of HN) also protects intact cells from apoptosis induced by overexpression of tBid. We conclude that Bid represents an additional cellular target of HN, and we propose that HN-mediated suppression of Bid contributes to the antiapoptotic activity of this endogenous peptide.

Humanin (HN) is a recently identified endogenous peptide that protects cells against cytotoxicity induced by various stimuli. Recently, we showed that HN binds to and inhibits Bax, a proapoptotic Bcl-2 family protein, suggesting a mechanism for HN action. In this study, we identified Bim, a Bcl-2 homology 3-only member of the Bcl-2/Bax family, as an additional HN target protein. Using in vitro protein binding, immunoprecipitation, and coimmunolocalization assays, we demonstrated that HN binds directly to the extra long isoform of Bim (BimEL) but not the long (BimL) or short (BimS) isoforms. HN also protects cells against apoptosis induced by BimEL but not BimL and BimS in gene transfection studies. In contrast, mutants of HN which failed to bind BimEL failed to protect from BimEL-induced cell death. Moreover, HN inhibited BimEL-induced release of SMAC and cytochrome c from mitochondria isolated from bax-/-cells, indicating that HN can suppress BimEL independently of its effect on Bax. Finally, we demonstrate that HN prevents BimEL-induced oligomerization of Bak using isolated mitochondria. Taken together, our results indicate that the inhibition of BimEL may contribute to the antiapoptotic properties of the HN peptide.

Recent work has shown that Bcl-2 and other anti-apoptotic proteins partially deplete the endoplasmic reticulum (ER) Ca(2+) store and that this alteration of Ca(2+) signaling reduces cellular sensitivity to apoptotic stimuli. We expressed in HeLa cells Bcl-2, Bax, and Bcl-2/Bax chimeras in which the putative pore-forming domains of the two proteins (alpha 5-alpha 6) were mutually swapped, comparing the effects on Ca(2+) signaling of the two proteins and relating them to defined molecular domains. The results showed that only Bcl-2 reduces ER Ca(2+) levels and that this effect does not depend on the alpha 5-alpha 6 helices of this oncoprotein. Soon after its expression, Bax increased ER Ca(2+) loading, with ensuing potentiation of mitochondrial Ca(2+) responses. Then the cells progressed into an apoptotic phenotype (which included drastic reductions of cytosolic and mitochondrial Ca(2+) responses and alterations of organelle morphology). These results provide a coherent scenario that high-lights a primary role of Ca(2+) signals in deciphering apoptotic stimuli.

Members of the tumor necrosis factor (TNF) superfamily of receptors such as Fas/CD95 and the TNF-related apoptosis-inducing ligand (TRAIL) receptors DR4 and DR5 induce apoptosis by recruiting adaptor molecules and caspases. The central adaptor molecule for these receptors is a death domain-containing protein, FADD, which binds to the activated receptor via death domain-death domain interactions. Here, we show that in addition to the death domain, the C-terminal tails of DR4 and DR5 positively regulate FADD binding, caspase activation and apoptosis. In contrast, the corresponding region in the Fas receptor has the opposite effect and inhibits binding to the receptor death domain. Replacement of wild-type or mutant DR5 molecules into DR5-deficient BJAB cells indicates that some agonistic antibodies display an absolute requirement for the C-terminal tail for FADD binding and signaling while other antibodies can function in the absence of this mechanism. These data demonstrate that regions outside the death domains of DR4 and DR5 have opposite effects to that of Fas in regulating FADD recruitment and show that different death receptor agonists can use distinct molecular mechanisms to activate signaling from the same receptor.

Genes encoding proteins with PYRIN/PAAD/DAPIN domains, a nucleotide binding fold (NACHT), and leucine rich repeats have recently been recognized as important mediators in autoimmune inflammatory disorders. Here we characterize the expression and function of a member of the PYRIN and NACHT domain (PAN) family, PAN1 (also known as NALP2 and PYPAF2). PAN1 protein expression is regulated by lipopolysaccharide (LPS) and interferons (IFNbeta and IFNgamma) in THP-1 macrophage cells. In gene transfection studies PAN1 manifests an inhibitory influence on NF-kappaB activation induced by various pro-inflammatory stimuli, including tumor necrosis factor TNFalpha and interleukin-1beta (IL-1beta). Gene transfer-mediated elevations in PAN1 protein also suppressed activation of IkappaB kinases induced by inflammatory cytokines. Conversely, reducing endogenous levels of PAN1 using small interfering RNA enhanced LPS-induced production of ICAM-1 (intercellular adhesion molecule 1), an NF-kappaB-dependent gene. We also show here that PAN1 binds via its PYRIN domain to ASC, an adapter protein involved in caspase-1 activation. This binding is disrupted by mutation of the alpha1 helix of ASC. In gene transfer experiments PAN1 enhances caspase-1 activation and IL-1beta secretion in collaboration with ASC. Conversely, reducing endogenous levels of PAN1 using small interfering RNA significantly reduced LPS-induced secretion of IL-1beta in monocytes. We propose that PAN1 functions as a modulator of the activation of NF-kappaB and pro-caspase-1 in macrophages.

XIAP is member of the IAP family of anti-apoptotic proteins and is known for its ability to bind and suppress caspase family cell death proteases. A phenylurea series of chemical inhibitors of XIAP was recently generated by our laboratories (Schimmer, A. D., Welsh, K., Pinilla, C., Bonneau, M., Wang, Z., Pedersen, I. M., Scott, F. L., Glinsky, G. V., Scudiero, D. A., Sausville, E., Salvesen, G., Nefzi, A., Ostresh, J. M., Houghten, R. A., and Reed, J. C. (2004) Cancer Cell 5, 25-35). We examined the mechanisms of action of these chemical compounds using biochemical, molecular biological, and genetic methods. Active phenylurea-based compounds dissociated effector protease caspase-3 but not initiator protease caspase-9 from XIAP in vitro and restored caspase-3 but not caspase-9 enzymatic activity. When applied to tumor cell lines in culture, active phenylurea-based compounds induced apoptosis in a rapid, concentration-dependent manner, associated with activation of cellular caspases. Apoptosis induced by active phenylurea-based compounds was blocked by chemical inhibitors of caspases, with inhibitors of downstream effector caspases displaying more effective suppression than inhibitors of upstream initiator caspases. Phenylurea-based XIAP antagonists induced apoptosis (defined by annexin V staining) prior to mitochondrial membrane depolarization, in contrast to cytotoxic anticancer drugs. Consistent with these findings, apoptosis induced by phenylurea-based compounds was not altered by genetic alterations in the expression of Bcl-2 family proteins that control mitochondria-dependent cell death pathways, including over-expression of anti-apoptotic proteins Bcl-2 or Bcl-X(L) and genetic ablation of pro-apoptotic proteins Bax and Bak. Conversely, conditional over-expression of an active fragment of XIAP or genetic ablation of XIAP expression altered the apoptosis dose-response of the compounds. Altogether, these findings indicate that phenylurea-based XIAP antagonists block interaction of downstream effector caspases with XIAP, thus inducing apoptosis of tumor cell lines through a caspase-dependent, Bcl-2/Bax-independent mechanism.

Detachment of adherent epithelial cells from the extracellular matrix induces apoptosis, known as anoikis. Integrin stimulation protects cells from anoikis, but the responsible mechanisms are not well known. Here, we demonstrated that a pro-apoptotic GTP-binding protein, DAP3 (death-associated protein 3), is critical for induction of anoikis. Down-regulation of DAP3 expression by antisense oligonucleotides inhibited anoikis. Conversely, overexpression of DAP3 augmented cell death and caspase activation induced by cell detachment. Furthermore, the association of DAP3 with FADD and the activation of caspase-8 were induced by cell detachment. We also showed that DAP3 is phosphorylated by kinase Akt (PKB), and active Akt can nullify apoptosis induction by DAP3. Mutation of a consensus Akt phosphorylation site in DAP3 renders it resistant to suppression by active Akt in cells. Integrin ligation stimulates Akt activation and phosphorylation of DAP3 in intact cells, as well as suppresses the ability of DAP3 overexpression to augment anoikis. Involvement of DAP3 in anoikis signaling demonstrates a novel role for this GTP-binding protein in apoptosis induction caused by cell detachment.

Regulators of apoptosis are thought to work in concert, but the molecular interactions of this process are not understood. Here, we show that in response to cell death stimulation, survivin, a member of the inhibitor of apoptosis (IAP) gene family, associates with another IAP protein, XIAP, via conserved baculovirus IAP repeats. Formation of a survivin-XIAP complex promotes increased XIAP stability against ubiquitination/proteasomal destruction and synergistic inhibition of apoptosis, which is abolished in XIAP(-/-) cells. Therefore, orchestration of an IAP-IAP complex regulates apoptosis.

Members of the tumor necrosis factor superfamily of receptors induce apoptosis by recruiting adaptor molecules through death domain interactions. The central adaptor molecule for these receptors is the death domain-containing protein Fas-associated death domain (FADD). FADD binds a death domain on a receptor or additional adaptor and recruits caspases to the activated receptor. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) signals apoptosis through two receptors, DR4 and DR5. Although there is much interest in TRAIL, the mechanism by which FADD is recruited to the TRAIL receptors is not clear. Using a reverse two-hybrid system we previously identified mutations in the death effector domain of FADD that prevented binding to Fas/CD95. Here we show that these mutations also prevent binding to DR5. FADD-deficient Jurkat cells stably expressing these FADD mutations did not transduce TRAIL or Fas/CD95 signaling. Second site compensating mutations that restore binding to and signaling through Fas/CD95 and DR5 were also in the death effector domain. We conclude that in contrast to current models where the death domain of FADD functions independently of the death effector domain, the death effector domain of FADD comes into direct contact with both TRAIL and Fas/CD95 receptors.

The Chlamydia protein CADD (Chlamydia protein associating with death domains) has been implicated in the modulation of host cell apoptosis via binding to the death domains of tumor necrosis factor family receptors. Transfection of CADD into mammalian cells induces apoptosis. Here we present the CADD crystal structure, which reveals a dimer of seven-helix bundles. Each bundle contains a di-iron center adjacent to an internal cavity, forming an active site similar to that of methane mono-oxygenase hydrolase. We further show that CADD mutants lacking critical metal-coordinating residues are substantially less effective in inducing apoptosis but retain their ability to bind to death domains. We conclude that CADD is a novel redox protein toxin unique to Chlamydia species and propose that both its redox activity and death domain binding ability are required for its biological activity.

The proapoptotic Bcl-2 family protein Bid is cleaved by caspase-8 to release the C-terminal fragment tBid, which translocates to the outer mitochondrial membrane and induces massive cytochrome c release and cell death. In this study, we have characterized the conformation of tBid in lipid membrane environments, using NMR and CD spectroscopy with lipid micelle and lipid bilayer samples. In micelles, tBid adopts a unique helical conformation, and the solution NMR (1)H/(15)N HSQC spectra have a single well resolved resonance for each of the protein amide sites. In lipid bilayers, tBid associates with the membrane with its helices parallel to the membrane surface and without trans-membrane helix insertion, and the solid-state NMR (1)H/(15)N polarization inversion with spin exchange at the magic angle spectrum has all of the amide resonances centered at (15)N chemical shift (70-90 ppm) and (1)H-(15)N dipolar coupling (0-5 kHz) frequencies associated with NH bonds parallel to the bilayer surface, with no intensity at frequencies associated with NH bonds in trans-membrane helices. Thus, the cytotoxic activity of tBid at mitochondria may be similar to that observed for antibiotic polypeptides, which bind to the surface of bacterial membranes as amphipathic helices and destabilize the bilayer structure, promoting the leakage of cell contents.

Increasing evidence suggests that lysosomal proteases are actively involved in apoptosis. Using HeLa cells as the model system, we show that selective lysosome disruption with L-leucyl-L-leucine methyl ester results in apoptosis, characterized by translocation of lysosomal proteases into the cytosol and by the cleavage of a proapoptotic Bcl-2-family member Bid. Apoptosis and Bid cleavage, but not translocation of lysosomal proteases to the cytosol, could be prevented by 15 microM L-trans-epoxysuccinyl(OEt)-Leu-3-methylbutylamide, an inhibitor of papain-like cysteine proteases. Incubation of cells with 15 microM N-benzoyloxycarbonyl-VAD-fluoromethyl ketone prevented apoptosis but not Bid cleavage, suggesting that cathepsin-mediated apoptosis in this system is caspase-dependent. In vitro experiments performed at neutral pH showed that papain-like cathepsins B, H, L, S, and K cleave Bid predominantly at Arg(65) or Arg(71). No Bid cleavage was observed with cathepsins C and X or the aspartic protease cathepsin D. Incubation of full-length Bid treated with cathepsins B, H, L, and S resulted in rapid cytochrome c release from isolated mitochondria. Thus, Bid may be an important mediator of apoptosis induced by lysosomal disruption.

Lymphotoxin-beta receptor (LTbetaR) and CD40 are members of the tumor necrosis factor family of signaling receptors that regulate cell survival or death through activation of NF-kappaB. These receptors transmit signals through downstream adaptor proteins called tumor necrosis factor receptor-associated factors (TRAFs). In this study, the crystal structure of a region of the cytoplasmic domain of LTbetaR bound to TRAF3 has revealed an unexpected new recognition motif, 388IPEEGD393, for TRAF3 binding. Although this motif is distinct in sequence and structure from the PVQET motif in CD40 and PIQCT in the regulator TRAF-associated NF-kappaB activator (TANK), recognition is mediated in the same binding crevice on the surface of TRAF3. The results reveal structurally adaptive "hot spots" in the TRAF3-binding crevice that promote molecular interactions driving specific signaling after contact with LTbetaR, CD40, or the downstream regulator TANK.

We cloned a novel cDNA derived from the CARD6 gene locus on chromosome 5p12 of 311 amino acids in length. By immunoprecipitation we detected specific binding of this CARD6-encoding protein to Nod1 (CARD4), Cardiak (Rip2/Rick), NAC (NALP1/DEFCAP/CARD7), and TUCAN (CARD8/Cardinal/NDPP/Dakar), caspase recruitment domain (CARD)-containing proteins implicated in NF-kappa B and caspase-1 activation but not to other CARD family proteins. Cardiak and Nod1 (but not other CARD proteins) also exhibited opposing effects on CARD6 protein phosphorylation and expression, providing further evidence of functional interactions among these proteins in cells. In transfection experiments, the CARD6 protein suppressed NF-kappa B induction by Nod1 or Cardiak but did not interfere with NF-kappa B activation by the CARD-containing adapter protein Bcl10 or the cytokine tumor necrosis factor-alpha, demonstrating specificity of CARD6 for Nod-1 and Cardiak-dependent pathways. In contrast to its effects on Nod1- and Cardiak-dependent NF-kappa B activation, CARD6 did not interfere with caspase-1-dependent interleukin-1 beta secretion induced by Cardiak or Nod1. CARD6 also did not affect caspase activation and apoptosis induced by overexpression of Fas, Bax, or other pro-apoptotic stimuli. Thus, CARD6 represents a selective modulator of NF-kappa B activation by Cardiak and Nod1, adding to the repertoire of CARD-family proteins implicated in inflammatory responses and innate immunity.

Here we report on the identification of peptides targeting the X-inhibitor of apoptosis protein (XIAP). XIAP functions as a caspase inhibitor and is a member of the inhibitors of apoptosis (IAP) family of proteins. IAPs are often overexpressed in cancers and leukemias and are associated with an unfavorable clinical prognosis. We have selected peptides from a phage library by using recombinant full-length human XIAP or a fragment containing only the baculovirus IAP repeat 2 (BIR2) domain. A consensus motif, C(D/E/P)(W/F/Y)-acid/basic-XC, was recovered from two independent screenings by using different libraries. Phage-displaying variations of the consensus sequence bound specifically to the BIR2 domain of XIAP but not to other IAPs. The interaction was specific as it could be blocked by the cognate synthetic peptides in a dose-dependent manner. Phage displaying the XIAP-binding motif CEFESC bound to the BIR2 domain of XIAP with an estimated dissociation constant of 1.8 nm as determined by surface plasmon resonance. Protein-protein interaction assays revealed that caspase-3 and caspase-7 (but not caspase-8) blocked the binding of the CEFESC phage to XIAP, indicating that this peptide targets a domain within XIAP that is related to the caspase-binding site. In fact, the sequence EFES is homologous to a loop unique to the executioner caspase-3 and caspase-7 that are targeted by XIAP. Finally, we demonstrated that an internalizing version of the XIAP-binding peptide identified in our screenings (PFKQ) can induce programmed cell death in leukemia cells. Peptides interacting with XIAP could serve as prototypes for the design of low molecular weight modulators of apoptosis.

Seven in absentia homologue (Siah) family proteins bind ubiquitin-conjugating enzymes and target proteins for proteasome-mediated degradation. Recently we identified a novel Siah-interacting protein (SIP) that is a Sgt1-related molecule that provides a physical link between Siah family proteins and the Skp1-Cullin-F-box ubiquitin ligase component Skp1. In the present study, a structure-based approach was used to identify interacting residues in Siah that are required for association with SIP. In Siah1 a large concave surface is formed across the dimer interface. Analysis of the electrostatic surface potential of the Siah1 dimer reveals that the beta-sheet concavity is predominately electronegative, suggesting that the protein-protein interactions between Siah1 and SIP are mediated by ionic contacts. The structural prediction was confirmed by site-directed mutagenesis of these electronegative residues, resulting in loss of binding of Siah1 to SIP in vitro and in cells. The results also provide a structural basis for understanding the mechanism by which Siah family proteins interact with partner proteins such as SIP.

PAAD domains are found in diverse proteins of unknown function and are structurally related to a superfamily of protein interaction modules that includes death domains, death effector domains, and Caspase activation and recruitment domains. Using bioinformatics strategies, cDNAs were identified that encode a novel protein of 110 kDa containing a PAAD domain followed by a putative nucleotide-binding (NACHT) domain and several leucine-rich repeat domains. This protein thus resembles Cryopyrin, a protein implicated in hereditary hyperinflammation syndromes, and was termed PAN2 for PAAD and NACHT-containing protein 2. When expressed in HEK293 cells, PAN2 suppressed NF-kappaB induction by the cytokines tumor necrosis factor-alpha (TNFalpha) and interleukin-1beta (IL-1beta), suggesting that this protein operates at a point of convergence in these two cytokine signaling pathways. This PAN2-mediated suppression of NF-kappaB was evident both in reporter gene assays that measured NF-kappaB transcriptional activity and electromobility shift assays that measured NF-kappaB DNA binding activity. PAN2 also suppressed NF-kappaB induction resulting from overexpression of several adapter proteins and protein kinases involved in the TNF or IL-1 receptor signal transduction, including TRAF2, TRAF6, RIP, IRAK2, and NF-kappaB-inducing kinase as well as the IkappaB kinases IKKalpha and IKKbeta. PAN2 also inhibited the cytokine-mediated activation of IKKalpha and IKKbeta as measured by in vitro kinase assays. Furthermore, PAN2 association with IKKalpha was demonstrated by co-immunoprecipitation assays, suggesting a direct effect on the IKK complex. These observations suggest a role for PAN2 in modulating NF-kappaB activity in cells, thus providing the insights into the potential functions of PAAD family proteins and their roles in controlling inflammatory responses.

Ubiquitous calpains (mu- and m-calpain) have been repeatedly implicated in apoptosis, but the underlying mechanism(s) remain(s) to be elucidated. We examined ionomycin-induced cell death in LCLC 103H cells, derived from a human large cell lung carcinoma. We detected hallmarks of apoptosis such as membrane blebbing, nuclear condensation, DNA ladder formation, caspase activation, and poly-(ADP-ribose)polymerase cleavage. Apoptosis was prevented by preincubation of the cells with the calpain inhibitor acetyl-calpastatin 27-peptide and the caspase inhibitor Z-DEVD-fmk, implicating both the calpains and caspases in the apoptotic process. The apoptotic events correlated in a calpastatin-inhibitable manner with Bid and Bcl-2 decrease and with activation of caspases-9, -3, and -7. In vitro both ubiquitous calpains cleaved recombinant Bcl-2, Bid, and Bcl-x(L) at single sites truncating their N-terminal regions. Binding studies revealed diminished interactions of calpain-truncated Bcl-2 and Bid with immobilized intact Bcl-2 family proteins. Moreover, calpain-cleaved Bcl-2 and Bid induced cytochrome c release from isolated mitochondria. We conclude that ionomycin-induced calpain activation promotes decrease of Bcl-2 proteins thereby triggering the intrinsic apoptotic pathway.

TRAIL (Apo2 ligand) is a member of the tumor necrosis factor (TNF) family of cytokines that induces apoptosis. Because TRAIL preferentially kills tumor cells, sparing normal tissues, interest has emerged in applying this biological factor for cancer therapy in humans. However, not all tumors respond to TRAIL, raising questions about resistance mechanisms. We demonstrate here that a variety of natural and synthetic ligands of peroxisome proliferator-activated receptor-gamma (PPAR gamma) sensitize tumor but not normal cells to apoptosis induction by TRAIL. PPAR gamma ligands selectively reduce levels of FLIP, an apoptosis-suppressing protein that blocks early events in TRAIL/TNF family death receptor signaling. Both PPAR gamma agonists and antagonists displayed these effects, regardless of the levels of PPAR gamma expression and even in the presence of a PPAR gamma dominant-negative mutant, indicating a PPAR gamma-independent mechanism. Reductions in FLIP and sensitization to TRAIL-induced apoptosis were also not correlated with NF-kappa B, further suggesting a novel mechanism. PPAR gamma modulators induced ubiquitination and proteasome-dependent degradation of FLIP, without concomitant reductions in FLIP mRNA. The findings suggest the existence of a pharmacologically regulated novel target of this class of drugs that controls FLIP protein turnover, and raise the possibility of combining PPAR gamma modulators with TRAIL for more efficacious elimination of tumor cells through apoptosis.

We report here the identification of a bacterial protein capable of interacting with mammalian death receptors in vitro and in vivo. The protein is encoded in the genome of Chlamydia trachomatis and has homologues in other Chlamydia species. This protein, which we refer to as "Chlamydia protein associating with death domains" (CADD), induces apoptosis in a variety of mammalian cell lines when expressed by transient gene transfection. Apoptosis induction can be blocked by Caspase inhibitors, indicating that CADD triggers cell death by engaging the host apoptotic machinery. CADD interacts with death domains of tumor necrosis factor (TNF) family receptors TNFR1, Fas, DR4, and DR5 but not with the respective downstream adaptors. In infected epithelial cells, CADD is expressed late in the infectious cycle of C. trachomatis and co-localizes with Fas in the proximity of the inclusion body. The results suggest a role for CADD modulating the apoptosis pathways of cells infected, revealing a new mechanism of host-pathogen interaction.

A novel Death Effector Domain-containing protein was identified, DEDD2, which is closest in amino acid sequence homology to death effector domain-containing DNA-binding protein, DEDD. DEDD2 mRNA is expressed widely in adult human tissues with highest levels in liver, kidney, and peripheral blood leukocytes. DEDD2 interacts with FLIP, but not with Fas-associated death domain (FADD) or caspase-8. Overexpression of DEDD2 induces moderate apoptosis and results in substantial sensitization to apoptosis induced by Fas (CD95/APO-1), tumor necrosis factor-related apoptosis-inducing ligand (TRAIL, Apo2L), or FADD. In contrast, Bax- or staurosporine-mediated cell death is not affected by expression of DEDD2. Fluorescence microscopy showed that overexpressed DEDD2 translocates to the nucleus, which is dependent on the presence of a bipartite nuclear localization signal in the DEDD2 protein. Mutagenesis studies revealed that the translocation of the DED of DEDD2 to the nucleus is essential for its pro-apoptotic activity. These findings suggest that DEDD2 is involved in the regulation of nuclear events mediated by the extrinsic apoptosis pathway.

Apoptosis induction through CD95 (APO-1/Fas) critically depends on generation of active caspase-8 at the death-inducing signaling complex (DISC). Depending on the cell type, active caspase-8 either directly activates caspase-3 (type I cells) or relies on mitochondrial signal amplification (type II cells). In MCF7-Fas cells that are deficient for pro-caspase-3, even high amounts of caspase-8 produced at the DISC cannot directly activate downstream effector caspases without mitochondrial help. Overexpression of Bcl-x(L) in these cells renders them resistant to CD95-mediated apoptosis. However, activation of caspase-8 in control (vector) and Bcl-x(L) transfectants of MCF7-Fas cells proceeds with similar kinetics, resulting in a complete processing of cellular caspase-8. Most of the cytosolic caspase-8 substrates are not cleaved in the Bcl-x(L) protected cells, raising the question of how Bcl-x(L)-expressing MCF7-Fas cells survive large amounts of potentially cytotoxic caspase-8. We now demonstrate that active caspase-8 is initially generated at the DISC of both MCF7-Fas-Vec and MCF7-Fas-Bcl-x(L) cells and that the early steps of CD95 signaling such as caspase-8-dependent cleavage of DISC bound c-FLIP(L), caspase-8-dependent clustering, and internalization of CD95, as well as processing of pro-caspase-8 bound to mitochondria are very similar in both transfectants. However, events downstream of mitochondria, such as release of cytochrome c, only occur in the vector-transfected MCF7-Fas cells, and no in vivo caspase-8 activity can be detected in the Bcl-x(L)-expressing cells. Our data suggest that, in Bcl-x(L)-expressing MCF7-Fas cells, active caspase-8 is sequestered on the outer mitochondrial surface presumably by association with the protein "bifunctional apoptosis regulator" in a way that does not allow substrates to be cleaved, identifying a novel mechanism of regulation of apoptosis sensitivity by mitochondrial Bcl-x(L).

The production of bio-active interleukin-1beta (IL-1beta), a pro-inflammatory cytokine, is mediated by activated caspase-1. One of the known molecular mechanisms underlying pro-caspase-1 processing and activation involves binding of the caspase-1 prodomain to a caspase recruitment domain (CARD)-containing serine/threonine kinase known as RIP2/CARDIAK/RICK. We have identified a novel protein, COP (CARD only protein), which has a high degree of sequence identity to the caspase-1 prodomain. COP binds to both RIP2 and the caspase-1 prodomain and inhibits RIP2-induced caspase-1 oligomerization. COP inhibits caspase- 1-induced IL-1beta secretion as well as lipopolysaccharide-induced IL-1beta secretion in transfected cells. Our data indicate that COP can regulate IL-1beta secretion, implying that COP may play a role in down-regulating inflammatory responses analogous to the CARD protein ICEBERG.

There are many very effective methods to introduce transcriptionally active DNA into viable cells but approaches to deliver functional proteins are limited. We have developed a lipid-mediated delivery system that can deliver functional proteins or other bioactive molecules into living cells. This delivery system is composed of a new trifluoroacetylated lipopolyamine (TFA-DODAPL) and dioleoyl phosphatidylethanolamine (DOPE). This cationic formulation successfully delivered antibodies, dextran sulfates, phycobiliproteins, albumin, and enzymes (beta-galactosidase and proteases) into the cytoplasm of numerous adherent and suspension cells. Two systems were used to demonstrate that the proteins were delivered in a functionally active form. First, intracellular beta-galactosidase activity was clearly demonstrated within X-gal-stained cells after TFA-DODAPL:DOPE-mediated delivery of the enzyme. Second, the delivery system mediated delivery of several caspases (caspase 3, caspase 8, and granzyme B) into cultured cell lines and primary cells triggering apoptosis. Mechanistic studies showed that up to 100% of the protein mixed with the lipid formulation was captured into a lipid-protein complex, and up to 50% of the input protein associated with cells. This lipid-mediated transport system makes protein delivery into cultured cells as convenient, effective, and reliable as DNA transfection.

Caspase-associated recruitment domains (CARDs) are protein interaction domains that participate in activation or suppression of CARD-carrying members of the caspase family of apoptosis-inducing proteases. A novel CARD-containing protein was identified that is overexpressed in some types of cancer and that binds and suppresses activation of procaspase-9, which we term TUCAN (tumor-up-regulated CARD-containing antagonist of caspase nine). The CARD domain of TUCAN selectively binds itself and procaspase-9. TUCAN interferes with binding of Apaf1 to procaspase-9 and suppresses caspase activation induced by the Apaf1 activator, cytochrome c. Overexpression of TUCAN in cells by stable or transient transfection inhibits apoptosis and caspase activation induced by Apaf1/caspase-9-dependent stimuli, including Bax, VP16, and staurosporine, but not by Apaf1/caspase-9-independent stimuli, Fas and granzyme B. High levels of endogenous TUCAN protein were detected in several tumor cell lines and in colon cancer specimens, correlating with shorter patient survival. Thus, TUCAN represents a new member of the CARD family that selectively suppresses apoptosis induced via the mitochondrial pathway for caspase activation.

Reperfusion after myocardial ischemia is associated with a rapid influx of calcium, leading to activation of various enzymes including calpain. Isolated perfused adult rabbit hearts subjected to global ischemia and reperfusion were studied. Calpain or a calpain-like activity was activated within 15 min after reperfusion, and preconditioning suppressed calpain activation. In contrast, caspase activation was not detected although cytochrome c was released after ischemia and reperfusion. The pro-apoptotic BH3-only Bcl-2 family member, Bid, was cleaved during ischemia/reperfusion in the adult rabbit heart. Recombinant Bid was cleaved by calpain to a fragment that was able to mediate cytochrome c release. The calpain cleavage site was mapped to a region within Bid that is extremely susceptible to proteolysis. These findings suggest that there is cross-talk between apoptotic and necrotic pathways in myocardial ischemia/reperfusion injury.

The inhibitor of apoptosis proteins (IAP) regulates cell death by inhibiting caspases. The region of X-linked (X) IAP containing the second baculovirus IAP repeat domain (BIR2) is sufficient for inhibiting caspase-3 and -7. In this study, we found that the modes of inhibition of these two caspases were different: caspase-3 is inhibited in a competitive manner whereas caspase-7 inhibition occurs through a mixed competitive and noncompetitive mechanism. Binding assays revealed that the inhibition of caspase-3 by XIAP was totally dependent on the interaction between the active site of caspase-3 and the linker region between the BIR1 and BIR2 domains of XIAP. In contrast, the active site and the NH(2)-terminal region of caspase-7 bound to the linker region and the BIR2, respectively. Moreover the BIR2 with a mutated linker region, which inhibited caspase-3 very weakly, still bound to and inhibited caspase-7. Furthermore, a chimeric caspase-7/3 comprising the NH(2)-terminal portion of caspase-7 and COOH-terminal portion of caspase-3 was inhibited by XIAP by a mixed competitive and noncompetitive mechanism. Our results suggest that the linker region between BIR1 and BIR2 domains is responsible for active site-directed, competitive inhibition of both caspase-3 and -7, whereas the BIR2 itself is involved in noncompetitive inhibition of caspase-7.

Smac, a second mitochondria-derived activator of caspases, promotes caspase activation in the cytochrome c (cyto-c)/Apaf-1/caspase-9 pathway. Here, we show that treatment of multiple myeloma (MM) cells with dexamethasone (Dex) triggers the release of Smac from mitochondria to cytosol and activates caspase-9 without concurrent release of cyto-c and Apaf-1 oligomerization. Smac binds to XIAP (an inhibitor of apoptosis protein) and thereby, at least in part, eliminates its inhibitory effect on caspase-9. Interleukin-6, a growth factor for MM, blocks Dex-induced apoptosis and prevents release of Smac. Taken together, these findings demonstrate that Smac plays a functional role in mediating Dex-induced caspase-9 activation and apoptosis in MM cells.

We have identified three new tumor necrosis factor-receptor associated factor (TRAF) domain-containing proteins in humans using bioinformatics approaches, including: MUL, the product of the causative gene in Mulibrey Nanism syndrome; USP7 (HAUSP), an ubiquitin protease; and SPOP, a POZ domain-containing protein. Unlike classical TRAF family proteins involved in TNF family receptor (TNFR) signaling, the TRAF domains (TDs) of MUL, USP7, and SPOP are located near the NH(2) termini or central region of these proteins, rather than carboxyl end. MUL and USP7 are capable of binding in vitro via their TDs to all of the previously identified TRAF family proteins (TRAF1, TRAF2, TRAF3, TRAF4, TRAF5, and TRAF6), whereas the TD of SPOP interacts weakly with TRAF1 and TRAF6 only. The TD of MUL also interacted with itself, whereas the TDs of USP7 and SPOP did not self-associate. Analysis of various MUL and USP7 mutants by transient transfection assays indicated that the TDs of these proteins are necessary and sufficient for suppressing NF-kappaB induction by TRAF2 and TRAF6 as well as certain TRAF-binding TNF family receptors. In contrast, the TD of SPOP did not inhibit NF-kappaB induction. Immunofluorescence confocal microscopy indicated that MUL localizes to cytosolic bodies, with targeting to these structures mediated by a RBCC tripartite domain within the MUL protein. USP7 localized predominantly to the nucleus, in a TD-dependent manner. Data base searches revealed multiple proteins containing TDs homologous to those found in MUL, USP7, and SPOP throughout eukaryotes, including yeast, protists, plants, invertebrates, and mammals, suggesting that this branch of the TD family arose from an ancient gene. We propose the moniker TEFs (TD-encompassing factors) for this large family of proteins.

Bag1 is a regulator of heat shock protein 70 kDa (Hsp70/Hsc70) family proteins that interacts with steroid hormone receptors. Four isoforms of Bag1 have been recognized: Bag1, Bag1S, Bag1M (RAP46/HAP46), and Bag1L. Although Bag1L, Bag1M, and Bag1 can bind the androgen receptor (AR) in vitro, only Bag1L enhanced AR transcriptional activity. Bag1L was determined to be a nuclear protein by immunofluorescence microscopy, whereas Bag1, Bag1S, and Bag1M were predominantly cytoplasmic. Forced nuclear targeting of Bag1M, but not Bag1 or Bag1S, resulted in potent AR coactivation, indicating that Bag1M possesses the necessary structural features provided it is expressed within the nucleus. The ability of Bag1L to enhance AR activity was reduced with the removal of an NH(2)-terminal domain of Bag1L, which was found to be required for efficient nuclear localization and/or retention. In contrast, deletion of a conserved ubiquitin-like domain from Bag1L did not interfere with its nuclear targeting or AR regulatory activity. Thus, both the unique NH(2)-terminal domain and the COOH-terminal Hsc70-binding domain of Bag1L are simultaneously required for its function as an AR regulator, whereas the conserved ubiquitin-like domain is expendable.