Multiple-interactions among EMILIN1 and EMILIN2 N- and C-terminal domains

EMILIN1 and EMILIN2 belong to a family of extracellular matrix glycoproteins characterized by the N-terminal cysteine-rich EMI domain, a long segment with high probabilty for coiled-coil structure formation and a C-terminal gC1q domain. To study EMILIN1 and EMILIN2 interaction and assembly we have applied qualitative and quantitative two hybrid systems using constructs corresponding to the gC1q and EMI domains. The identified interactions were further confirmed in yeast extracts of co-transfected cells followed by co-immunoprecipitation. The data indicated that gC1q domains are able to self-interact as well as to interact one each other and with the EMI domains, but no self interactions were detected between the EMI domains. Furthermore EMILINs interactions were studied in 293-EBNA cells co-transfected with full lenght EMILIN1 and EMILIN2 constructs. Specific antibodies were able to co-immunoprecipitate EMILINs, indicating that also full-lenght proteins can give rise to non-covalent homo- and hetero-multimers even if reduced and alkylated before mixing. Immunofluorescence analysis on mouse cell cultures and tissues sections with specific antibodies showed co-distribution of EMILIN1 and EMILIN2. Thus, we can hypothesize that EMILINs multimers are formed by head-to-tail interaction between C-terminal and N-terminal domains of EMILIN1 and/or EMILIN2 but also by tail-to-tail interaction between gC1q domains. These multiple interactions may regulate homo-typic and/or hetero-typic linear and eventually lateral branching assemblies of EMILIN1 and EMILIN2 in tissues.     

Regulation of the extrinsic apoptotic pathway by the extracellular matrix glycoprotein EMILIN2

Elastin microfibril interface-located proteins (EMILINs) constitute a family of extracellular matrix (ECM) glycoproteins characterized by the presence of an EMI domain at the N terminus and a gC1q domain at the C terminus. EMILIN1, the archetype molecule of the family, is involved in elastogenesis and hypertension etiology, whereas the function of EMILIN2 has not been resolved. Here, we provide evidence that the expression of EMILIN2 triggers the apoptosis of different cell lines. Cell death depends on the activation of the extrinsic apoptotic pathway following EMILIN2 binding to the TRAIL receptors DR4 and, to a lesser extent, DR5. Binding is followed by receptor clustering, colocalization with lipid rafts, death-inducing signaling complex assembly, and caspase activation. The direct activation of death receptors by an ECM molecule that mimics the activity of the known death receptor ligands is novel. The knockdown of EMILIN2 increases transformed cell survival, and overexpression impairs clonogenicity in soft agar and three-dimensional growth in natural matrices due to massive apoptosis. These data demonstrate an unexpected direct and functional interaction of an ECM constituent with death receptors and discloses an additional mechanism by which ECM cues can negatively affect cell survival.     

Overlapping, complementary and site-specific expression pattern of genes of the EMILIN/Multimerin family.

The EDEN gene superfamily comprises genes that contain the EMI domain, a structural motif recently identified in proteins of the extracellular matrix. We report here the detailed expression pattern of genes of the EMILIN/Multimerin family, the most numerous group of EDEN superfamily, during mouse development. In situ hybridization has revealed that the EMILIN/Multimerin genes are particularly expressed in the cardio-vascular system and in mesenchymal cells. In general, the territories of expression of each gene are partially overlapping or complementary with that of other members of the family and, usually, more than one gene of the family is active in different tissues, consistent with the possibility of functional compensation. The analysis is particularly relevant in the interpretation of gene targeting experiments.     

Isolation and characterization of EMILIN-2, a new component of the growing EMILINs family and a member of the EMI domain-containing superfamily

EMILIN (elastin microfibril interfase located Protein) is an elastic fiber-associated glycoprotein consisting of a self-interacting globular C1q domain at the C terminus, a short collagenous stalk, an extended region of potential coiled-coil structure, and an N-terminal cysteine-rich domain (EMI domain). Using the globular C1q domain as a bait in the yeast two-hybrid system, we have isolated a cDNA encoding a novel protein. Determination of the entire primary structure demonstrated that this EMILIN-binding polypeptide is highly homologous to EMILIN. The domain organization is superimposable, one important difference being a proline-rich (41%) segment of 56 residues between the potential coiled-coil region and the collagenous domain absent in EMILIN. The entire gene (localized on chromosome 18p11.3) was isolated from a BAC clone, and it is structurally almost identical to that of EMILIN (8 exons, 7 introns with identical phases at the exon/intron boundaries) but much larger (about 40 versus 8 kilobases) than that of EMILIN. Given these findings we propose to name the novel protein EMILIN-2 and the prototype member of this family EMILIN-1 (formerly EMILIN). The mRNA expression of EMILIN-2 is more restricted compared with that of EMILIN-1; highest levels are present in fetal heart and adult lung, whereas, differently from EMILIN-1, adult aorta, small intestine, and appendix show very low expression, and adult uterus and fetal kidney are negative. Finally, the EMILIN-2 protein is secreted extracellularly by in vitro-grown cells, and in accordance with the partial coexpression in fetal and adult tissues, the two proteins shown extensive but not absolute immunocolocalization in vitro.     

C. elegans MAC-1, an essential member of the AAA family of ATPases, can bind CED-4 and prevent cell death

In the nematode Caenorhabditis elegans, CED-4 plays a central role in the regulation of programmed cell death. To identify proteins with essential or pleiotropic activities that might also regulate cell death, we used the yeast two-hybrid system to screen for CED-4-binding proteins. We identified MAC-1, a member of the AAA family of ATPases that is similar to Smallminded of Drosophila. Immunoprecipitation studies confirm that MAC-1 interacts with CED-4, and also with Apaf-1, the mammalian homologue of CED-4. Furthermore, MAC-1 can form a multi-protein complex that also includes CED-3 or CED-9. A MAC-1 transgene under the control of a heat shock promoter prevents some natural cell deaths in C. elegans, and this protection is enhanced in a ced-9(n1950sd)/+ genetic background. We observe a similar effect in mammalian cells, where expression of MAC-1 can prevent CED-4 and CED-3 from inducing apoptosis. Finally, mac-1 is an essential gene, since inactivation by RNA-mediated interference causes worms to arrest early in larval development. This arrest is similar to that observed in Smallminded mutants, but is not related to the ability of MAC-1 to bind CED-4, since it still occurs in ced-3 or ced-4 null mutants. These results suggest that MAC-1 identifies a new class of proteins that are essential for development, and which might regulate cell death in specific circumstances.     

Human CED-6 encodes a functional homologue of the Caenorhabditis elegans engulfment protein CED-6

The rapid engulfment of apoptotic cells is a specialized innate immune response used by organisms to remove apoptotic cells. In mammals, several receptors that recognize apoptotic cells have been identified; molecules that transduce signals from these receptors to downstream cytoskeleton molecules have not been found, however [1] [2] [3]. Our previous analysis of the engulfment gene ced-6 in Caenorhabditis elegans has suggested that CED-6 is an adaptor protein that participates in a signal transduction pathway that mediates the specific recognition and engulfment of apoptotic cells [1]. Here, we describe our isolation and characterization of a human cDNA encoding a protein, hCED-6, with strong sequence similarity to C. elegans CED-6. As is the case with the worm protein, hCED-6 contains a phosphotyrosine-binding (PTB) domain and potential Src-homology domain 3 (SH3) binding sites. Both CED-6 and hCED-6 contain a predicted coiled-coil domain in the middle region. The hCED-6 protein lacks the extended carboxyl terminus found in worm CED-6; this carboxy-terminal extension appears not to be essential for CED-6 function in C. elegans, however. Overexpression of hCED-6 rescues the engulfment defect of ced-6 mutants in C. elegans significantly, suggesting that hCED-6 is a functional homologue of C. elegans CED-6. Human ced-6 is expressed widely in most human tissues. Thus, CED-6, and the CED-6 signal transduction pathway, might be conserved from C. elegans to humans and are present in most, if not all, human tissues.     

Disruption of the CED-9.CED-4 complex by EGL-1 is a critical step for programmed cell death in Caenorhabditis elegans

In the nematode Caenorhabditis elegans, the apoptotic machinery is composed of four basic elements: the caspase CED-3, the Apaf-1 homologue CED-4, and the Bcl-2 family members CED-9 and EGL-1. The ced-9(n1950) gain-of-function mutation prevents most, if not all, somatic cell deaths in C. elegans. It encodes a CED-9 protein with a glycine-to-glutamate substitution at position 169, which is located within the highly conserved Bcl-2 homology 1 domain. We performed biochemical analyses with the CED-9G169E protein to gain insight into the mechanism of programmed cell death. We find that CED-9G169E retains the ability to bind both EGL-1 and CED-4, although its affinity for EGL-1 is reduced. In contrast to the behavior of wild-type CED-9, the interaction between CED-9G169E and CED-4 is not disrupted by expression of EGL-1. Furthermore, CED-4 and CED-9G169E co-localizes with EGL-1 to the mitochondria in mammalian cells, and expression of EGL-1 does not induce translocation of CED-4 to the cytosol. Finally, the ability of EGL-1 to promote apoptosis is impaired by the replacement of wild-type CED-9 with CED-9G169E, and this effect is correlated with the inability of EGL-1 to induce the displacement of CED-4 from the CED-9.CED-4 complex. These studies suggest that the release of CED-4 from the CED-9.CED-4 complex is a necessary step for induction of programmed cell death in C. elegans.     

Identification and characterization of a dimerization domain in CED-6, an adapter protein involved in engulfment of apoptotic cells

Phagocytosis of apoptotic cells is a key step in the completion of programmed cell death that occurs throughout life in multicellular organisms. The molecular events involved in clearance of apoptotic cells are just beginning to be elucidated. Recently, CED-6, an adapter protein involved in engulfment has been cloned in Caenorhabditis elegans and in humans. CED-6 is composed of a phosphotyrosine-binding (PTB) domain and a proline-rich C-terminal domain with no apparent catalytic domain. Since PTB domains, originally identified in Shc, mediate intracellular signaling downstream of cell surface receptors, CED-6 has also been proposed to mediate intracellular signals leading to engulfment. In this report, we demonstrate that CED-6 dimerizes through a leucine zipper domain that is immediately adjacent to the PTB domain. Several lines of evidence based on co-immunoprecipitation studies, yeast two-hybrid assays, and gel filtration studies suggest that CED-6 exists as a dimer in vivo. Through mutational analyses, we show that the leucine zipper is necessary and sufficient for CED-6 dimerization and that this dimerization is conserved among C. elegans, rodent, and human CED-6 proteins. We propose that dimerization may have unique implications for ligand binding via CED-6 and its function during the phagocytosis of apoptotic cells.     

RNA aptamers targeting the cell death inhibitor CED-9 induce cell killing in Caenorhabditis elegans

Bcl-2 family proteins include anti- and proapoptotic factors that play important roles in regulating apoptosis in diverse species. Identification of compounds that can modulate the activities of Bcl-2 family proteins will facilitate development of drugs for treatment of apoptosis-related human diseases. We used an in vitro selection method named systematic evolution of ligands by exponential enrichment (SELEX) to isolate RNA aptamers that bind the Caenorhabditis elegans Bcl-2 homolog CED-9 with high affinity and specificity and tested whether these aptamers modulate programmed cell death in C. elegans. Five CED-9 aptamers were isolated and classified into three groups based on their predicted secondary structures. Biochemical analyses indicated that two of these aptamers, R9-2 and R9-7, and EGL-1, an endogenous CED-9-binding proapoptotic protein, bound to distinct regions of CED-9. However, these two aptamers shared overlapping CED-9 binding sites with CED-4, another CED-9-binding proapoptotic factor. Importantly ectopic expression of these two aptamers in touch receptor neurons induced efficient killing of these neurons largely in a CED-3 caspase-dependent manner. These findings suggest that RNA aptamers can be used to modulate programmed cell death in vivo and can potentially be used to develop drugs to treat human diseases caused by abnormal apoptosis.     

EMILIN-3, peculiar member of elastin microfibril interface-located protein (EMILIN) family, has distinct expression pattern, forms oligomeric assemblies, and serves as transforming growth factor β (TGF-β) antagonist

EMILIN-3 is a glycoprotein of the extracellular matrix belonging to a family that contains a characteristic N-terminal cysteine-rich EMI domain. Currently, EMILIN-3 is the least characterized member of the elastin microfibril interface-located protein (EMILIN)/Multimerin family. Using RNA, immunohistochemical, and protein chemistry approaches, we carried out a detailed characterization of the expression and biochemical properties of EMILIN-3 in mouse. During embryonic and postnatal development, EMILIN-3 showed a peculiar and dynamic pattern of gene expression and protein distribution. EMILIN-3 mRNA was first detected at E8.5-E9.5 in the tail bud and in the primitive gut, and at later stages it became abundant in the developing gonads and osteogenic mesenchyme. Interestingly and in contrast to other EMILIN/Multimerin genes, EMILIN-3 was not found in the cardiovascular system. Despite the absence of the globular C1q domain, immunoprecipitation and Western blot analyses demonstrated that EMILIN-3 forms disulfide-bonded homotrimers and higher order oligomers. Circular dichroism spectroscopy indicated that the most C-terminal part of EMILIN-3 has a substantial α-helical content and forms coiled coil structures involved in EMILIN-3 homo-oligomerization. Transfection experiments with recombinant constructs showed that the EMI domain contributes to the higher order self-assembly but was dispensable for homotrimer formation. EMILIN-3 was found to bind heparin with high affinity, a property mediated by the EMI domain, thus revealing a new function for this domain that may contribute to the interaction of EMILIN-3 with other extracellular matrix and/or cell surface molecules. Finally, in vitro experiments showed that EMILIN-3 is able to function as an extracellular regulator of the activity of TGF-β ligands.     

Regulation of apoptosis by C. elegans CED-9 in the absence of the C-terminal transmembrane domain

Bcl-2 proteins regulate apoptosis in organisms as diverse as mammals and nematodes. These proteins are often localized at mitochondria by a C-terminal transmembrane domain. Although the transmembrane domain and mitochondrial localization are centrally involved in specific cases of vertebrate Bcl-2 activity, the significance of this localization is not clear for all species. Studying the Caenorhabditis elegans Bcl-2 homolog CED-9, we found that the transmembrane domain was both necessary and sufficient for localization at mitochondrial outer membranes. Furthermore, we found that in our assays, ced-9 transgenes lacking the transmembrane domain, although somewhat less active than equivalent transgenes derived from wild-type ced-9, rescued embryonic lethality of ced-9(lf) animals and responded properly to upstream signals in controlling the fate of Pn.aap neurons. Both of these apoptotic activities were retained in a construct where CED-9 lacking the transmembrane domain was targeted to the cytosolic surface of the endoplasmic reticulum and derived organelles, suggesting that in wild-type animals, accumulation at mitochondria is not essential for CED-9 to either inhibit or promote apoptosis in C. elegans. Taken together, these data are consistent with a multimodal character of CED-9 action, with an ability to regulate apoptosis through interactions in the cytosol coexisting with additional evolutionarily conserved role(s) at the membrane.     

Crystal structure of the cell corpse engulfment protein CED-2 in Caenorhabditis elegans

In the nematode Caenorhabditis elegans, the cell corpse engulfment proteins CED-2, CED-5, and CED-12 act in the same pathway to regulate the activation of the Rac small GTPase, CED-10, leading to the rearrangement of the actin cytoskeleton for engulfing apoptotic cells. Nevertheless, it is not well understood how these proteins act together. Here we report the crystal structures of the CED-2 protein as determined by X-ray crystallography. The full-length CED-2 protein and its truncated form containing the N-terminal SH2 domain and the first SH3 domain show similar three-dimensional structures. A CED-2 point mutation (F125G) disrupting its interaction with the PXXP motif of CED-5 did not affect its rescuing activity. However, CED-2 was found to interact with the N-terminal region of CED-5. Our findings suggest that CED-2 may regulate cell corpse engulfment by interacting with CED-5 through the N-terminal region rather than the PXXP motif.     

The Caenorhabditis elegans sex determination protein FEM-1 is a CED-3 substrate that associates with CED-4 and mediates apoptosis in mammalian cells

Sex-specific elimination of cells by apoptosis plays a role in sex determination in Caenorhabditis elegans. Recently, a mammalian pro-apoptotic protein named F1Aalpha has been identified. F1Aalpha shares extensive homology throughout the entire protein with the C. elegans protein, FEM-1, which is essential for achieving all aspects of the male phenotype in the nematode. In this report, the role of FEM-1 in apoptosis was investigated. Overexpression of FEM-1 induces caspase-dependent apoptosis in mammalian cells. FEM-1 is cleaved in vitro by the C. elegans caspase, CED-3, generating an N-terminal cleavage product that corresponds to the minimal effector domain for apoptosis. Furthermore, CED-4 associates with FEM-1 in vitro and in vivo in mammalian cells and potentiates FEM-1-mediated apoptosis. Similarly, Apaf-1, the mammalian homologue of CED-4 was found to associate with F1Aalpha. These data suggest that FEM-1 and F1Aalpha may mediate apoptosis by communicating directly with the core machinery of apoptosis.     

Structural, biochemical, and functional analyses of CED-9 recognition by the proapoptotic proteins EGL-1 and CED-4

Programmed cell death in Caenorhabditis elegans is initiated by the binding of EGL-1 to CED-9, which disrupts the CED-4/CED-9 complex and allows CED-4 to activate the cell-killing caspase CED-3. Here we demonstrate that the C-terminal half of EGL-1 is necessary and sufficient for binding to CED-9 and for killing cells. Structure of the EGL-1/CED-9 complex revealed that EGL-1 adopts an extended alpha-helical conformation and induces substantial structural rearrangements in CED-9 upon binding. EGL-1 interface mutants failed to bind to CED-9 or to release CED-4 from the CED-4/CED-9 complex, and were unable to induce cell death in vivo. A surface patch on CED-9, different from that required for binding to EGL-1, was identified to be responsible for binding to CED-4. These data suggest a working mechanism for the release of CED-4 from the CED-4/CED-9 complex upon EGL-1 binding and provide a mechanistic framework for understanding apoptosis activation in C. elegans.     

CED-4 forms a 2 : 2 heterotetrameric complex with CED-9 until specifically displaced by EGL-1 or CED-13

The pathway to cell death in Caenorhabditis elegans is well established. In cells undergoing apoptosis, the Bcl-2 homology domain 3 (BH3)-only protein EGL-1 binds to CED-9 at the mitochondrial membrane to cause the release of CED-4, which oligomerises and facilitates the activation of the caspase CED-3. However, despite many studies, the biophysical features of the CED-4/CED-9 complex have not been fully characterised. Here, we report the purification of a soluble and stable 2 : 2 heterotetrameric complex formed by recombinant CED-4 and CED-9 coexpressed in bacteria. Consistent with previous studies, synthetic peptides corresponding to the BH3 domains of worm BH3-only proteins (EGL-1, CED-13) dissociate CED-4 from CED-9, but not from the gain-of-function CED-9 (G169E) mutant. Surprisingly, the ability of worm BH3 domains to dissociate CED-4 was specific since mammalian BH3-only proteins could not do so.     

Evidence that CED-9/Bcl2 and CED-4/Apaf-1 localization is not consistent with the current model for C. elegans apoptosis induction

In C. elegans, the BH3-only domain protein EGL-1, the Apaf-1 homolog CED-4 and the CED-3 caspase are required for apoptosis induction, whereas the Bcl-2 homolog CED-9 prevents apoptosis. Mammalian B-cell lymphoma 2 (Bcl-2) inhibits apoptosis by preventing the release of the Apaf-1 (apoptotic protease-activating factor 1) activator cytochrome c from mitochondria. In contrast, C. elegans CED-9 is thought to inhibit CED-4 by sequestering it at the outer mitochondrial membrane by direct binding. We show that CED-9 associates with the outer mitochondrial membrane within distinct foci that do not overlap with CED-4, which is predominantly perinuclear and does not localize to mitochondria. CED-4 further accumulates in the perinuclear space in response to proapoptotic stimuli such as ionizing radiation. This increased accumulation depends on EGL-1 and is abrogated in ced-9 gain-of-function mutants. CED-4 accumulation is not sufficient to trigger apoptosis execution, even though it may prime cells for apoptosis. Our results suggest that the cell death protection conferred by CED-9 cannot be solely explained by a direct interaction with CED-4.     

Establishing a blueprint for CED-3-dependent killing through identification of multiple substrates for this protease

Genetic studies have established that the cysteine protease CED-3 plays a central role in coordinating programmed cell death in Caenorhabditis elegans. However, it remains unclear how CED-3 activation results in cell death because few substrates for this protease have been described. We have used a global proteomics approach to seek substrates for CED-3 and have identified 22 worm proteins that undergo CED-3-dependent proteolysis. Proteins that were found to be substrates for CED-3 included the cytoskeleton proteins actin, myosin light chain, and tubulin, as well as proteins involved in ATP synthesis, cellular metabolism, and chaperone function. We estimate that approximately 3% of the C. elegans proteome is susceptible to CED-3-dependent proteolysis. Notably, the endoplasmic reticulum chaperone calreticulin, which has been implicated in the recognition of apoptotic cells by phagocytes, was cleaved by CED-3 and was also cleaved by human caspases during apoptosis. Inhibitors of caspase activity blocked the appearance of calreticulin on the surface of apoptotic cells, suggesting a mechanism for the surface display of calreticulin during apoptosis. Further analysis of these substrates is likely to yield important insights into the mechanism of killing by CED-3 and its human caspase counterparts.     

Apaf1 and the apoptotic machinery

The molecular characterization of the Caenorhabditis elegans cell death genes has been crucial in revealing some of the biochemical mechanisms underlying apoptosis in all animals. Four C. elegans genes, egl-1, ced-9, ced-4 and ced-3 are required for all somatic programmed cell death to occur. This genetic network is highly conserved during evolution. The pro-death gene egl-1 and the anti-death gene ced-9 have structural and functional similarities to the vertebrate Bcl2 gene family. The killer gene ced-3 encodes a cystein-aspartate protease (caspase), which is the archetype of a family of conserved proteins known as effectors of apoptosis in mammals. Zou and collaborators1 reported the biochemical identification of an apoptotic protease activating factor (Apaf1), a human homolog of C. elegans CED-4, providing important clues to how CED-4 and its potential relatives could work. A number of proteins have been shown to interact with Apaf1 or to be determinant for its activity as an apoptotic adapter. The aim of this review is to provide an overview of the recent progress made in the field of developmental apoptosis by means of the murine Apaf1 targeted mutations. The central role of Apaf1 in the cell death machinery (apoptosome) and its involvement in different apoptotic pathways will also be discussed.     

Fate of the nuclear lamina during Caenorhabditis elegans apoptosis

Invertebrates and in Drosophila, lamins and lamin-associated proteins are primary targets for cleavage by caspases. Eliminating mammalian lamins causes apoptosis, whereas expressing mutant lamins that cannot be cleaved by caspase-6 delay apoptosis. Caenorhabditis elegans has a single lamin protein, Ce-lamin, and a caspase, CED-3, that is responsible for most if not all somatic apoptosis. In this study we show that in C. elegans embryos induced to undergo apoptosis Ce-lamin is degraded surprisingly late. In such embryos CED-4 translocated to the nuclear envelope but the cytological localization of Ce-lamin remained similar to that in wild-type embryos. TUNEL labeling indicated that Ce-lamin was degraded only after DNA is fragmented. Ce-lamin, Ce-emerin, or Ce-MAN1 were not cleaved by recombinant CED-3, showing that these lamina proteins are not substrates for CED-3 cleavage. These results suggest that lamin cleavage probably is not essential for apoptosis in C. elegans.