Genome sequence comparison reveals independent inactivation of the caspase-15 gene in different evolutionary lineages of mammals

We have recently demonstrated that placental mammalian species such as pig and dog express a novel proapoptotic protease, caspase-15, whereas mouse and humans lack this enzyme. Here we investigated the evolutionary fate of the caspase-15 gene in different mammalian lineages by analyzing whole-genome shotgun sequences of 30 mammalian species for the presence of caspase-15 orthologs. Caspase-15 gene sequences were found in representatives of all major mammalian clades except for the superorders Afrotheria (tenrec, rock hyrax, and elephant) and Euarchontoglires (rodents, rabbit, tree shrew, and primates), which either lacked any caspase-15-like sequences or contained mutated remnants of the caspase-15 gene. Polymerase chain reaction screenings confirmed the results of the database searches and showed that the caspase-15 gene is expressed not only in various placental mammals but also in the marsupial, Monodelphis domestica. The observed species distribution implies that caspase-15 has originated in an early ancestor of modern mammals and has been conserved, over more than 180 Myr, in marsupials and many placental mammals, whereas it was independently lost in 2 phylogenetically distant clades of placental mammals, that is, Afrotheria and Euarchontoglires. Our data suggest that the inactivation of the caspase-15 gene was not counteracted by, and may even have been driven by, evolutionary constraints in these clades, and therefore, caution against the uncritical use of gene absence for the inference of phylogenetic relationships.     

Ordering the Cytochrome c–initiated Caspase Cascade: Hierarchical Activation of Caspases-2, -3, -6, -7, -8, and -10 in a Caspase-9–dependent Manner

Exit of cytochrome c from mitochondria into the cytosol has been implicated as an important step in apoptosis. In the cytosol, cytochrome c binds to the CED-4 homologue, Apaf-1, thereby triggering Apaf-1–mediated activation of caspase-9. Caspase-9 is thought to propagate the death signal by triggering other caspase activation events, the details of which remain obscure. Here, we report that six additional caspases (caspases-2, -3, -6, -7, -8, and -10) are processed in cell-free extracts in response to cytochrome c, and that three others (caspases-1, -4, and -5) failed to be activated under the same conditions. In vitro association assays confirmed that caspase-9 selectively bound to Apaf-1, whereas caspases-1, -2, -3, -6, -7, -8, and -10 did not. Depletion of caspase-9 from cell extracts abrogated cytochrome c–inducible activation of caspases-2, -3, -6, -7, -8, and -10, suggesting that caspase-9 is required for all of these downstream caspase activation events. Immunodepletion of caspases-3, -6, and -7 from cell extracts enabled us to order the sequence of caspase activation events downstream of caspase-9 and reveal the presence of a branched caspase cascade. Caspase-3 is required for the activation of four other caspases (-2, -6, -8, and -10) in this pathway and also participates in a feedback amplification loop involving caspase-9.     

The genomes of the South American opossum (Monodelphis domestica) and platypus (Ornithorhynchus anatinus) encode a more complete purine catabolic pathway than placental mammals

The end product of purine catabolism varies amongst vertebrates and is a consequence of independent gene inactivation events that have truncated the purine catabolic pathway. Mammals have traditionally been grouped into two classes based on their end product of purine catabolism: most mammals, whose end product is allantoin due to an ancient loss of allantoinase (ALLN), and the hominoids, whose end product is uric acid due to recent inactivations of urate oxidase (UOX). However little is known about purine catabolism in marsupials and monotremes. Here we report the results of a comparative genomics study designed to characterize the purine catabolic pathway in a marsupial, the South American opossum (Monodelphis domestica), and a monotreme, the platypus (Ornithorhynchus anatinus). We found that both genomes encode a more complete set of genes for purine catabolism than do eutherians and conclude that a near complete purine catabolic pathway was present in the common ancestor of all mammals, and that the loss of ALLN is specific to placental mammals. Our results therefore provide a revised history for gene loss in the purine catabolic pathway and suggest that marsupials and monotremes represent a third class of mammals with respect to their end products of purine catabolism.     

Caspase-3 mediated feedback activation of apical caspases in doxorubicin and TNF-α induced apoptosis

Aberrant apoptosis has been associated with the development and therapeutic resistance of cancer. Recent studies suggest that caspase deficiency/downregulation is frequently detected in different cancers. We have previously shown that caspase-3 reconstitution significantly sensitized MCF-7 cells to doxorubicin and etoposide. In contrast to the well established role of caspase-3 as an effector caspase, the focus of this study is to delineate caspase-3 induced feedback activation of the apical caspases-2, -8, -9 and -10A in doxorubicin and TNF-α induced apoptosis. Using cell-free systems we show that caspases-9 and 2 are the most sensitive, caspase-8 is less sensitive and caspase-10A is the least sensitive to caspase-3 mediated-cleavage. When apoptosis is induced by doxorubicin or TNF-α in an intact cell model, cleavage of caspases-8 and -9, but not caspase-2, was markedly enhanced by caspase-3. Caspase-3 mediated-feedback and activation of caspase-8 and -9 in MCF-7/C3 cells is further supported by an increase in the cleavage of caspase-8 and 9 substrates and cytochrome c release. These data indicate that, in addition to its function as an effector caspase, caspase-3 plays an important role in maximizing the activation of apical caspases and crosstalk between the two major apoptotic pathways. The significant impact of caspase-3 on both effector and apical caspases suggests that modulation of caspase-3 activity would be a useful approach to overcome drug resistance in clinical oncology. XiaoHe Yang: This work was supported in part by the Career Development Award DAMD17-99-1-9180 from Department of Defense to X.H.Y.     

Marsupials and Eutherians reunited: genetic evidence for the Theria hypothesis of mammalian evolution

The three living monophyletic divisions of Class Mammalia are the Prototheria (monotremes), Metatheria (marsupials), and Eutheria (`placental' mammals). Determining the sister relationships among these three groups is the most fundamental question in mammalian evolution. Phylogenetic comparison of these mammals by either anatomy or mitochondrial DNA has resulted in two conflicting hypotheses, Theria and Marsupionta, and has fueled a ``genes versus morphology' controversy. We have cloned and analyzed a large nuclear gene, the mannose 6-phosphate/insulin-like growth factor II receptor (M6P/IGF2R), from representatives of all three mammalian groups, including platypus, echidna, opossum, wallaby, hedgehog, mouse, rat, rabbit, cow, pig, bat, tree shrew, colugo, ringtail lemur, and human. Statistical analysis of this nuclear gene unambiguously supports the morphology-based Theria hypothesis that excludes monotremes from a clade of marsupials and eutherians. The M6P/IGF2R was also able to resolve the finer structure of the eutherian mammalian family tree. In particular, our analyses support sister group relationships between lagomorphs and rodents, and between the primates and Dermoptera. Statistical support for the grouping of the hedgehog with Feruungulata and Chiroptera was also strong. Received: 8 December 2000 / Accepted: 01 February 2001     

Effect of Hypoxia on Caspase-3, -8, and -9 Activity and Expression in the Cerebral Cortex of Newborn Piglets

Caspases play an important role in programmed cell death. Caspase-3 is a key executioner of apoptosis, whose activation is mediated by the initiator caspases, caspase-8 and caspase-9. The present study tested the hypothesis that cerebral hypoxia results in increased activation and expression of caspases-3, -8, and -9 in the cytosolic fraction of the cerebral cortex of newborn piglets. To test this hypothesis the activity and expression of caspases-3, -8, and -9 were determined in newborn piglets divided into normoxic and hypoxic groups. Caspase activity was determined spectrofluorometrically using enzyme specific substrates. The expression of caspase protein was assessed by Western blot analysis using enzyme specific antibody. Caspases-3, -8, and -9 activity and expression was significantly higher in the hypoxic group than in the normoxic group. These results demonstrate that hypoxia induces activation and increased expression of both the initiator caspases and the executioner caspase in the cerebral cortex of newborn piglets. We conclude that hypoxia results in stimulation of both the pathways of caspase-3 activation.     

Delineation of the caspase-9 signaling cascade

In the intrinsic apoptosis pathway, mitochondrial disruption leads to the release of multiple apoptosis signaling molecules, triggering both caspase-dependent and -independent cell death. The release of cytochrome c induces the formation of the apoptosome, resulting in caspase-9 activation. Multiple caspases are activated downstream of caspase-9, however, the precise order of caspase activation downstream of caspase-9 in intact cells has not been completely resolved. To characterize the caspase-9 signaling cascade in intact cells, we employed chemically induced dimerization to activate caspase-9 specifically. Dimerization of caspase-9 led to rapid activation of effector caspases, including caspases-3, -6 and -7, as well as initiator caspases, including caspases-2, -8 and -10, in H9 and Jurkat cells. Knockdown of caspase-3 suppressed caspase-9-induced processing of the other caspases downstream of caspase-9. Silencing of caspase-6 partially inhibited caspase-9-mediated processing of caspases-2, -3 and -10, while silencing of caspase-7 partially inhibited caspase-9-induced processing of caspase-2, -3, -6 and -10. In contrast, deficiency in caspase-2, -8 or -10 did not significantly affect the caspase-9-induced caspase cascade. Our data provide novel insights into the ordering of a caspase signaling network downstream of caspase-9 in intact cells during apoptosis.     

Caspase-2: controversial killer or checkpoint controller?

The caspases are an evolutionarily conserved family of cysteine proteases, with essential roles in apoptosis or inflammation. Caspase-2 was the second caspase to be cloned and it resembles the prototypical nematode caspase CED-3 more closely than any other mammalian protein. An absence of caspase-2-specific reagents and the subtle phenotype of caspase-2-deficient mice have hampered definition of the physiological role of caspase-2 and identification of factors regulating its activity. Although some data implicate caspase-2 in apoptotic pathways, a link with apoptosis has been less firmly established for caspase-2 than for some other caspases. Emerging evidence suggests that caspase-2 regulates the cell cycle and may act as a tumour suppressor. This article critically reviews the current state of knowledge regarding the biochemistry and biology of this controversial caspase.     

The major histocompatibility complex in monotremes: an analysis of the evolution of <Emphasis Type="Italic">Mhc</Emphasis> class I genes across all three mammalian subclasses

We report the isolation and characterization of cDNA clones of expressed, functional major histocompatibility complex class-I ( Mhc-I) genes from two species of monotremes: the duck-billed platypus and the short-beaked echidna. The cDNA clones were isolated from libraries constructed from spleen RNA, clearly establishing their expression in at least this one peripheral lymphoid organ. From the presence of conserved amino acid residues, it appears the expressed sequences encode molecules that likely function as classical Mhc-I. These clones were isolated using monotreme Mhc-I processed pseudogenes as probes. These processed pseudogenes were isolated from genomic DNA and, based on their structure, are likely independently derived in the platypus and echidna. When all the monotreme sequences were included in phylogenetic analyses, we found no apparent orthologous relationships between the platypus and echidna Mhc-I. Analyses that included a large number of Mhc-I sequences from other taxa support a separate monotreme Mhc-I clade, basal to a therian Mhc-I clade that is comprised of sequences from marsupial and placental mammals. The phylogenies also support the hypothesis that Mhc-I genes of placental mammals, marsupials, and monotremes are derived from three separate lineages of Mhc-I genes, best explained by two rounds of duplications and deletions. The first round would have occurred prior to the divergence of monotremes and therians, and the second prior to the divergence of marsupials and placental mammals. The sequences described here represent the first reported functional monotreme Mhc-I, as well as the first processed pseudogenes of any type from monotremes.     

The multiple sex chromosomes of platypus and echidna are not completely identical and several share homology with the avian Z

Background

Sex-determining systems have evolved independently in vertebrates. Placental mammals and marsupials have an XY system, birds have a ZW system. Reptiles and amphibians have different systems, including temperature-dependent sex determination, and XY and ZW systems that differ in origin from birds and placental mammals. Monotremes diverged early in mammalian evolution, just after the mammalian clade diverged from the sauropsid clade. Our previous studies showed that male platypus has five X and five Y chromosomes, no SRY, and DMRT1 on an X chromosome. In order to investigate monotreme sex chromosome evolution, we performed a comparative study of platypus and echidna by chromosome painting and comparative gene mapping.

Results

Chromosome painting reveals a meiotic chain of nine sex chromosomes in the male echidna and establishes their order in the chain. Two of those differ from those in the platypus, three of the platypus sex chromosomes differ from those of the echidna and the order of several chromosomes is rearranged. Comparative gene mapping shows that, in addition to bird autosome regions, regions of bird Z chromosomes are homologous to regions in four platypus X chromosomes, that is, X₁, X₂, X₃, X₅, and in chromosome Y₁.

Conclusion

Monotreme sex chromosomes are easiest to explain on the hypothesis that autosomes were added sequentially to the translocation chain, with the final additions after platypus and echidna divergence. Genome sequencing and contig anchoring show no homology yet between platypus and therian Xs; thus, monotremes have a unique XY sex chromosome system that shares some homology with the avian Z.     

The central role of initiator caspase-9 in apoptosis signal transduction and the regulation of its activation and activity on the apoptosome

Key structural and catalytic features are conserved across the entire family of cysteine-dependent aspartate-specific proteases (caspases). Of the caspases involved in apoptosis signal transduction, the initiator caspases-2, -8 and -9 are activated at multi-protein activation platforms, and activation is thought to involve homo-dimerisation of the monomeric zymogens. Caspase-9, the essential initiator caspase required for apoptosis signalling through the mitochondrial pathway, is activated on the apoptosome complex, and failure to activate caspase-9 has profound pathophysiological consequences. Here, we review the pertinent literature on which the currently prevalent understanding of caspase-9 activation is based, extend this view by insight obtained from recent structural and kinetic studies on caspase-9 signalling, and describe an emerging model for the regulation of caspase-9 activation and activity that arise from the complexity of multi-protein interactions at the apoptosome. This integrated view allows us to postulate and to discuss functional consequences for caspase-9 activation and apoptosis execution that may take centre stage in future experimental cell research on apoptosis signalling.     

Cloning and characterization of the platypus mitochondrial genome

The vertebrate mitochondrial genome is highly conserved in size and gene content. Among the chordates there appears to be one basic gene arrangement, but rearrangements in the mitochondrial gene order of the avian lineages have indicated that the mitochondrial genome may be more variable than once thought. Different gene orders in marsupials and eutherian mammals leave the ancestral mammalian order in some doubt. We have investigated the mitochondrial gene order in the platypus (Ornithorhynchus anatinus), a representative of the third major group of mammals, to determine which mitochondrial gene arrangement is ancestral in mammals. We have found that the platypus mtDNA conforms to the basic chordate gene arrangement, common to fish, amphibians, and eutherian mammals, indicating that this arrangement was the original mammalian arrangement, and that the unusual rearrangements observed in the avians and marsupials are probably lineage-specific. Correspondence to: N.J. Gemmell     

Direct cleavage by the calcium-activated protease calpain can lead to inactivation of caspases

Caspases, a unique family of cysteine proteases involved in cytokine activation and in the execution of apoptosis can be sub-grouped according to the length of their prodomain. Long prodomain caspases such as caspase-8 and caspase-9 are believed to act mainly as upstream caspases to cleave downstream short prodomain caspases such as caspases-3 and -7. We report here the identification of caspases as direct substrates of calcium-activated proteases, calpains. Calpains cleave caspase-7 at sites distinct from those of the upstream caspases, generating proteolytically inactive fragments. Caspase-8 and caspase-9 can also be directly cleaved by calpains. Two calpain cleavage sites in caspase-9 have been identified by N-terminal sequencing of the cleaved products. Cleavage of caspase-9 by calpain generates truncated caspase-9 that is unable to activate caspase-3 in cell lysates. Furthermore, direct cleavage of caspase-9 by calpain blocks dATP and cytochrome-c induced caspase-3 activation. Therefore our results suggest that calpains may act as negative regulators of caspase processing and apoptosis by effectively inactivating upstream caspases.     

Structure, expression, and function of the Xenopus laevis caspase family

Caspases, a family of cysteine proteases, have been recognized as the central executors of programmed cell death. Nonetheless, the information on the caspase family has been limited to mammals, Drosophila, and nematodes. To examine the structure and characterization of the Xenopus caspase family, we have cloned the cDNAs encoding caspase-2 and -6-10 in addition to caspase-1 and -3, which we characterized previously (Yaoita, Y., and Nakajima, K. (1997) J. Biol. Chem. 272, 5122-5127). First, the existence of these caspases in frog suggests that the caspase cascades clarified in mammals are conserved at least from Amphibia. Interestingly, Xenopus caspase-1, -8, and -10 (especially caspase-8) showed a lower degree of identity to human equivalents than the other caspases. Second, mRNAs of many caspases increased during the climax of metamorphosis in regressing organs, tail, and intestine, where programmed cell death occurs, but not in apoptotic tail-derived cultured cells (XLT-15-11) treated with thyroid hormone, showing that new RNA synthesis of caspases is dispensable to programmed cell death. Third, comparison of human and Xenopus caspase sequences implies that some proposed regulations of human caspases are not conserved in frog.     

Insights into the evolution of mammalian telomerase: Platypus TERT shares similarities with genes of birds and other reptiles and localizes on sex chromosomes

ABSTRACT: BACKGROUND: The TERT gene encodes the catalytic subunit of the telomerase complex and is responsible for maintaining telomere length. Vertebrate telomerase has been studied in placental mammals, fish, and the chicken, but less attention has been paid to other vertebrates. The platypus occupies an important evolutionary position, providing unique insight into the evolution of mammalian genes. We report the cloning of a platypus TERT (pTERT) ortholog, and provide a comparison with genes of other vertebrates. RESULTS: The pTERT encodes a protein with the high homology to marsupial TERT and avian TERT. Like the TERT of sauropsids and marsupials, as well as that of sharks and echinoderms, pTERT contains extended variable linkers in the N-terminal region suggesting that they were present already in basal vertebrates and lost independently in placental mammals and ray-finned fish. Several alternatively spliced pTERT variants structurally similar to avian TERT variants were identified. Telomerase activity is expressed in all platypus tissues similarly to cold-blooded animals and murine rodents. pTERT was localized on pseudoautosomal regions of sex chromosomes X3/Y2, expanding the homology between human chromosome 5 and platypus sex chromosomes. The synteny analysis suggests that TERT co-localized with sex-linked genes in the last common mammalian ancestor. Interestingly, female platypuses express higher levels of telomerase in heart and liver tissues than do males. CONCLUSIONS: pTERT shares many features with TERT of the reptilian outgroup, suggesting that pTERT represents the ancestral mammalian TERT. Features specific to TERT of eutherian mammals have, therefore, evolved more recently after the divergence of monotremes.     

Old,new and emerging functions of caspases

Caspases are proteases with a well-defined role in apoptosis. However, increasing evidence indicates multiple functions of caspases outside apoptosis. Caspase-1 and caspase-11 have roles in inflammation and mediating inflammatory cell death by pyroptosis. Similarly, caspase-8 has dual role in cell death, mediating both receptor-mediated apoptosis and in its absence, necroptosis. Caspase-8 also functions in maintenance and homeostasis of the adult T-cell population. Caspase-3 has important roles in tissue differentiation, regeneration and neural development in ways that are distinct and do not involve any apoptotic activity. Several other caspases have demonstrated anti-tumor roles. Notable among them are caspase-2, -8 and -14. However, increased caspase-2 and -8 expression in certain types of tumor has also been linked to promoting tumorigenesis. Increased levels of caspase-3 in tumor cells causes apoptosis and secretion of paracrine factors that promotes compensatory proliferation in surrounding normal tissues, tumor cell repopulation and presents a barrier for effective therapeutic strategies. Besides this caspase-2 has emerged as a unique caspase with potential roles in maintaining genomic stability, metabolism, autophagy and aging. The present review focuses on some of these less studied and emerging functions of mammalian caspases.     

Aberrant expression of caspase-14 in epithelial tumors

Cysteine-dependent aspartate-specific proteases (caspases) are the cellular executors of apoptosis. Caspase-14 is the most divergent member of the family of mammalian caspases and displays a variety of unique characteristics. It is expressed in a limited number of tissues and has the shortest amino acid sequence within the caspase protein family. During induction of apoptosis, it is not processed, whereas terminal differentiation in skin leads to cleavage of caspase-14. Here we show that 40% of lung squamous cell carcinomas, 22% of breast cancers, and about 80% of cervical carcinomas express caspase-14. Immunohistochemistry reveals that caspase-14 is localized in areas of ongoing differentiation close to necrotic sites but is not strictly associated with the differentiation markers keratin-1/-10. Caspase-14 is neither mutated nor alternatively spliced in the tumors analyzed. Furthermore, caspase-14 is not processed into a small and large subunit, a process critical for the proteolytic activation of known effector caspases. We conclude that conditions exist in tumors leading to re-expression of this normally silent gene.     

Caspases activation in hyperthermia-induced stimulation of TRAIL apoptosis

In leukemia cells, hyperthermia enhances tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis. The phenomenon is caspase-dependent and results in membrane changes leading to an increased recognition of TRAIL death receptors by TRAIL. Because either caspase-2 or an apical proteolytic event has been recently proposed to act as an initiator of the cell death mechanism induced by heat shock, we have investigated the hierarchy of caspase activation in cells exposed to the combined heat shock plus TRAIL treatment. We report here that caspases-2, -3, and -8 were the first caspases to be activated. As expected, caspase-8 is required and indispensable during the initiation of this death signaling. Caspase-2 may also participate in the phenomenon but, in contrast to caspase-8, its presence appears dispensable because its depletion by small interfering RNA is devoid of effects. Our observations also suggest a role of caspase-3 and of a particular cleaved form of this caspase during the early signals of heat shock plus TRAIL-induced apoptosis.     

Functional Diversity and Evolution of Bitter Taste Receptors in Egg-Laying Mammals

Egg-laying mammals (monotremes) are a sister clade of therians (placental mammals and marsupials) and a key clade to understand mammalian evolution. They are classified into platypus and echidna, which exhibit distinct ecological features such as habitats and diet. Chemosensory genes, which encode sensory receptors for taste and smell, are believed to adapt to the individual habitats and diet of each mammal. In this study, we focused on the molecular evolution of bitter taste receptors (TAS2Rs) in monotremes. The sense of bitter taste is important to detect potentially harmful substances. We comprehensively surveyed agonists of all TAS2Rs in platypus (Ornithorhynchus anatinus) and short-beaked echidna (Tachyglossus aculeatus) and compared their functions with orthologous TAS2Rs of marsupial and placental mammals (i.e., therians). As results, the agonist screening revealed that the deorphanized monotreme receptors were functionally diversified. Platypus TAS2Rs had broader receptive ranges of agonists than those of echidna TAS2Rs. While platypus consumes a variety of aquatic invertebrates, echidna mainly consumes subterranean social insects (ants and termites) as well as other invertebrates. This result indicates that receptive ranges of TAS2Rs could be associated with feeding habits in monotremes. Furthermore, some orthologous receptors in monotremes and therians responded to β-glucosides, which are feeding deterrents in plants and insects. These results suggest that the ability to detect β-glucosides and other substances might be shared and ancestral among mammals.