Evolution of placentally expressed cathepsins

Species and strain variants of a family of placentally expressed cathepsins (PECs) were cloned and sequenced in order to identify evolutionary conserved structural characteristics of this large family of cysteine proteases. Cathepsins M, P, Q, and R, are conserved in mice and rats but homologs of these genes are not found in human or rabbit placenta, showing that this family of proteases are probably restricted to rodents. Species-specific gene duplications have given rise to variants of cathepsin M in mice, and cathepsin Q in rats. Although the PECs have diverged at a greater rate than the other lysosomal cathepsins, residues around the specificity sub-sites of the individual enzymes are conserved. Strain-specific polymorphisms show that the evolutionary rate of divergence of cathepsins M and 3, the most recently duplicated pair of mouse genes, is even higher than the other PECs. In human placenta, critical functions of the PECs are probably performed by broader specificity proteases such as cathepsins B and L.     

PHLDA2 is an imprinted gene in cattle

Genomic imprinting is an epigenetic non-Mendelian phenomenon found predominantly in placental mammals. Imprinted genes display differential expression in the offspring depending on whether the gene is maternally or paternally inherited. Currently, some 100 imprinted genes have been reported in mammals, and while some of these genes are imprinted across most mammalian species, others have been shown to be imprinted in only a few species. The PHLDA2 gene that codes for a pleckstrin homology-like domain, family A (member 2), protein has to date been shown to be a maternally expressed imprinted gene in humans, mice and pigs. Genes subject to imprinting can have major effects on mammalian growth, development and disease. For instance, disruption of imprinted genes can lead to aberrant growth syndromes in cloned domestic mammals, and it has been demonstrated that PHLDA2 mRNA expression levels are aberrant in the placenta of somatic clones of cattle. In this study, we demonstrate that PHLDA2 is expressed across a range of cattle foetal tissues and stages and provide the first evidence that PHLDA2 is a monoallelically expressed imprinted gene in cattle foetal tissues, and also in the bovine placenta.     

Insulin is imprinted in the placenta of the marsupial, Macropus eugenii

Therian mammals (marsupials and eutherians) rely on a placenta for embryo survival. All mammals have a yolk sac, but while both chorio-allantoic and chorio-vitelline (yolk sac) placentation can occur, most marsupials only develop a yolk sac placenta. Insulin (INS) is unusual in that it is the only gene that is imprinted exclusively in the yolk sac placenta. Marsupials, therefore, provide a unique opportunity to examine the conservation of INS imprinting in mammalian yolk sac placentation. Marsupial INS was cloned and its imprint status in the yolk sac placenta of the tammar wallaby, Macropus eugenii, examined. In two informative individuals of the eight that showed imprinting, INS was paternally expressed. INS protein was restricted to the yolk sac endoderm, while insulin receptor, IR, protein was additionally expressed in the trophoblast. INS protein increased during late gestation up to 2 days before birth, but was low the day before and on the day of birth. The conservation of imprinted expression of insulin in the yolk sac placenta of divergent mammalian species suggests that it is of critical importance in the yolk sac placenta. The restriction of imprinting to the yolk sac suggests that imprinting of INS evolved in the chorio-vitelline placenta independently of other tissues in the therian ancestor of marsupials and eutherians.     

Targeted expression of ced-3 and Ice induces programmed cell death in Drosophila

CED-3 is a cysteine protease required for programmed cell death in the nematode, Caenorhabditis elegans, and shares a sequence similarity with mammalian ICE (interleukin-1beta converting enzyme) family proteases. Both CED-3 and ICE family proteases can induce programmed cell death in mammalian cells. Structural and functional similarities between CED-3 and ICE family proteases indicate that the mechanism of cell death is evolutionarily conserved, suggesting the presence of a similar mechanism involving CED-3/ICE-like proteases in Drosophila. Here we determined whether CED-3 or ICE functions to induce programmed cell death in Drosophila. We have generated transformant lines in which ced-3 or Ice is ectopically expressed using the GAL4-UAS system. Expression of CED-3 and ICE can elicit cell death in Drosophila and the cell death was blocked by coexpressing the p35 gene which encodes a viral inhibitor of CED-3/ICE proteases. Results support the idea that the mechanism of programmed cell death controlled by CED-3/ICE is conserved among widely divergent animal species including Drosophila, and the system described provides a tool to dissect cell death mechanism downstream of CED-3/ICE proteases.     

Drosophila TIMP is a potent inhibitor of MMPs and TACE: similarities in structure and function to TIMP-3

The four tissue inhibitors of metalloproteinases (TIMPs) are endogenous inhibitors that regulate the activity of matrix metalloproteinases (MMPs) and certain disintegrin and metalloproteinase (ADAM) family proteases in mammals. The protease inhibitory activity is present in the N-terminal domains of TIMPs (N-TIMPs). In this work, the N-terminal inhibitory domain of the only TIMP produced by Drosophila (dN-TIMP) was expressed in Escherichia coli and folded in vitro. The purified recombinant protein is a potent inhibitor of human MMPs, including membrane-type 1-MMP, although it lacks a disulfide bond that is conserved in all other known N-TIMPs. Titration with the catalytic domain of human MMP-3 [MMP-3(DeltaC)] showed that dN-TIMP prepared by this method is correctly folded and fully active. dN-TIMP also inhibits, in vitro, the activity of the only two MMPs of Drosophila, dm1- and dm2-MMPs, indicating that the Drosophila TIMP is an endogenous inhibitor of the Drosophila MMPs. dN-TIMP resembles mammalian N-TIMP-3 in strongly inhibiting human tumor necrosis factor-alpha-converting enzyme (TACE/ADAM17) but is a weak inhibitor of human ADAM10. Models of the structures of dN-TIMP and N-TIMP-3 are strikingly similar in surface charge distribution, which may explain their functional similarity. Although the gene duplication events that led to the evolutionary development of the four mammalian TIMPs might be expected to be associated with functional specialization, Timp-3 appears to have conserved most of the functions of the ancestral TIMP gene.     

Human tissue kallikreins: physiologic roles and applications in cancer

Tissue kallikreins are members of the S1 family (clan SA) of trypsin-like serine proteases and are present in at least six mammalian orders. In humans, tissue kallikreins (hK) are encoded by 15 structurally similar, steroid hormone-regulated genes (KLK) that colocalize to chromosome 19q13.4, representing the largest cluster of contiguous protease genes in the entire genome. hKs are widely expressed in diverse tissues and implicated in a range of normal physiologic functions from the regulation of blood pressure and electrolyte balance to tissue remodeling, prohormone processing, neural plasticity, and skin desquamation. Several lines of evidence suggest that hKs may be involved in cascade reactions and that cross-talk may exist with proteases of other catalytic classes. The proteolytic activity of hKs is regulated in several ways including zymogen activation, endogenous inhibitors, such as serpins, and via internal (auto)cleavage leading to inactivation. Dysregulated hK expression is associated with multiple diseases, primarily cancer. As a consequence, many kallikreins, in addition to hK3/PSA, have been identified as promising diagnostic and/or prognostic biomarkers for several cancer types, including ovarian, breast, and prostate. Recent data also suggest that hKs may be causally involved in carcinogenesis, particularly in tumor metastasis and invasion, and, thus, may represent attractive drug targets to consider for therapeutic intervention.     

Identification and structural analysis of four serine proteases in a monotreme, the platypus, Ornithorhynchus anatinus

To study the emergence of the major subfamilies of serine proteases during vertebrate evolution, we present here the primary structure of four serine proteases expressed in the spleen of a monotreme, the platypus, Ornithorhynchus anatinus. Partial cDNA clones for four serine proteases were isolated by a PCR-based strategy. This strategy is based on the high level of sequence identity between various members of the large gene family of trypsin-related serine proteases, over two highly conserved regions, those of the histidine and the serine of the catalytic triad. The partial cDNA clones were used to isolate full-length or almost full-length cDNA clones for three of these proteases from a platypus spleen cDNA library. By phylogenetic analysis, these three clones were identified as being the platypus homologues of human coagulation factor X, neutrophil elastase, and a protease distantly related to the T-cell granzymes. The remaining partial clone was found to represent a close homologue of human complement factor D (adipsin). The isolation of these four clones shows that several of the major subfamilies of serine proteases had evolved as separate subfamilies long before the radiation of the major mammalian lineages of today, the monotremes, the marsupials, and the placental mammals. Upon comparison of the corresponding proteases of monotremes and eutherian mammals, the coagulation and complement proteases were shown to display a higher degree of conservation compared to the hematopoietic proteases N-elastase and the T-cell granzymes. This latter finding indicates a higher evolutionary pressure to maintain specific functions in the complement and coagulation enzymes compared to many of the hematopoietic serine proteases.     

Calpain-related diseases

Calpains are calcium-modulated proteases which respond to Ca2+ signals by removing limited portions of protein substrates, thereby irreversibly modifying their function(s). Members of this protease family are present in a variety of organisms ranging from mammals to plants; some of them are ubiquitously expressed, while others are tissue specific. Although calpains are apparently involved in a multitude of physiological and pathological events, their functions are still poorly understood. In two cases, however, the alteration of a member of the calpain family has been clearly identified as being responsible for a human disease: the loss of function of calpain 3 causes limb girdle muscular dystrophy type 2A, and mutations in the gene coding for calpain 10 have been shown to correlate with non-insulin-dependent diabetes.     

Crystal structure of chagasin, the endogenous cysteine-protease inhibitor from Trypanosoma cruzi

Trypanosoma cruzi chagasin belongs to a recently discovered family of cysteine protease inhibitors found in lower eukaryotes and prokaryotes but not in mammals. Chagasin binds tightly to cruzain, the major lysosomal T. cruzi cysteine protease, involved with infectivity and survival of the parasite in mammalian host cells. In the scope of a project to characterize proteins diferentially expressed during T. cruzi metacyclogenesis, we have determined the crystal structure of chagasin, which is now the first X-ray structure of a chagasin-like cysteine protease inhibitor to be reported. The structure was solved by the SIRAS method and refined at 1.7A resolution and a comparison with the two NMR structures available revealed some differences in the loops involved in binding to cysteine proteases. The highly flexible loop 4 could be entirely modeled and residues 29-33 from loop 2 form a 3(10)-helix structure that may be important to stabilize the loop conformation. Chagasin crystal structure was docked to the highest resolution structure available of cruzain and a model of chagasin-cruzain interaction was analyzed. The knowledge of the chagasin crystal structure may contribute to the elucidation of the molecular mechanism involved in the inhibition of cruzain and other T. cruzi cysteine proteases.     

Caenorhabditis elegans reticulon interacts with RME-1 during embryogenesis

Reticulon (RTN) family proteins are localized in the endoplasmic reticulum (ER). At least four different RTN genes have been identified in mammals, but in most cases, the functions of the encoded proteins except mammalian RTN4-A and RTN4-B are unknown. Each RTN gene produces 1-3 proteins by different promoters and alternative splicing. In Caenorhabditis elegans, there is a single gene (rtn gene) encoding three reticulon proteins, nRTN-A, B, and C. mRNA of nRTN-C is expressed in germ cells and embryos. However, nRTN-C protein is only expressed during embryogenesis and rapidly disappears after hatch. By yeast two-hybrid screening, two clones encoding the same C-terminal region of RME-1, a protein functioning in the endocytic recycling, were isolated. These findings suggest that nRTN-C functions in the endocytic pathway during embryogenesis.     

Human tissue kallikreins: a new enzymatic cascade pathway?

Serine proteases are proteolytic enzymes with an active serine residue in their catalytic site. Kallikreins are a subgroup of the serine protease family which is known to have diverse physiological functions. The human kallikrein gene family has now been fully characterized and includes 15 members tandemly located on chromosome 19q13.4. Here we discuss the common structural features of kallikreins at the DNA, mRNA and protein levels and summarize their tissue expression and hormonal regulation patterns. Kallikreins are expressed in many tissues including the salivary gland, endocrine tissues such as testis, prostate, breast and endometrium, and in the central nervous system. Most genes appear to be under steroid hormone regulation. The occurrence of several splice variants is common among kallikreins, and some of the splice variants seem to be tissue-specific and might be related to certain pathological conditions. Kallikreins are secreted in an inactive 'zymogen' form which is activated by cleavage of an N-terminal peptide. Some kalikreins can undergo autoactivation while others may be activated by other kallikreins or other proteases. Most kallikreins are predicted to have trypsin-like enzymatic activity except three which are probably chymotrypsin-like. New, but mainly circumstantial evidence, suggests that at least some kallikreins may be part of a novel enzymatic cascade pathway which is turned-on in aggressive forms of ovarian and probably other cancers.     

A blood fluke serine protease inhibitor regulates an endogenous larval elastase

The larvae of Schistosoma mansoni invade their mammalian host by utilizing a serine protease, cercarial elastase (SmCE), to degrade macromolecular proteins in host skin. The catalytic activity of serine and cysteine proteases can be regulated after activation by serpins. SmSrpQ, one of two S. mansoni serpins found in larval secretions, is only expressed during larval development and in the early stages of mammalian infection. In vitro, (35)S-SmSrpQ was able to form an SDS-stable complex with a component of the larval lysate, but no complex was detected when (35)S-SmSrpQ was incubated with several mammalian host proteases. Formation of a complex was sensitive to the protease active site inhibitors PMSF, Z-AAPF-CMK, and Z-AAPL-CMK. Western blot analysis of parasite lysates from different life stages detected a complex of comparable size to SmCE bound to SmSrpQ using anti-SmSrpQ or anti-SmCE antibodies. SmSrpQ and SmCE are located in adjacent but discrete compartments in the secretion glands of the parasite. Fluorescence immunohistochemical analysis of simulated infection showed co-localization of SmCE and SmSrpQ in host tissue suggesting a post release regulation of parasite protease activity during skin transversal. The results of this study suggest that cercarial elastase degradation of skin tissue is carefully regulated by SmSrpQ.     

Type II transmembrane serine proteases in development and disease

Recent advances in the mammalian genome projects have resulted in the identification of a surprisingly large number of genes encoding putative new members and even entire new families of proteolytic enzymes. In the past few years, one of these new families, type II transmembrane serine proteases (TTSPs), underwent a particularly rapid transformation from a group of predicted DNA and protein sequences into an established family of cell surface-associated proteases with important roles in the development and homeostasis of mammalian tissues such as heart, skin, inner ear, placenta, and digestive tract. Additionally, aberrant expression of TTSP genes appears to be involved in the aetiology of several human disorders, including cancer. This review presents our current knowledge of the biological functions of the individual TTSPs in mouse and human tissue development and disease.     

A Kazal-like extracellular serine protease inhibitor from Phytophthora infestans targets the tomato pathogenesis-related protease P69B

The oomycetes form one of several lineages within the eukaryotes that independently evolved a parasitic lifestyle and consequently are thought to have developed alternative mechanisms of pathogenicity. The oomycete Phytophthora infestans causes late blight, a ravaging disease of potato and tomato. Little is known about processes associated with P. infestans pathogenesis, particularly the suppression of host defense responses. We describe and functionally characterize an extracellular protease inhibitor, EPI1, from P. infestans. EPI1 contains two domains with significant similarity to the Kazal family of serine protease inhibitors. Database searches suggested that Kazal-like proteins are mainly restricted to animals and apicomplexan parasites but appear to be widespread and diverse in the oomycetes. Recombinant EPI1 specifically inhibited subtilisin A among major serine proteases and inhibited and interacted with the pathogenesis-related P69B subtilisin-like serine protease of tomato in intercellular fluids. The epi1 and P69B genes were coordinately expressed and up-regulated during infection of tomato by P. infestans. Inhibition of tomato proteases by EPI1 could form a novel type of defense-counterdefense mechanism between plants and microbial pathogens. In addition, this study points to a common virulence strategy between the oomycete plant pathogen P. infestans and several mammalian parasites, such as the apicomplexan Toxoplasma gondii.     

The S8 serine, C1A cysteine and A1 aspartic protease families in Arabidopsis

The Arabidopsis thaliana genome has over 550 protease sequences representing all five catalytic types: serine, cysteine, aspartic acid, metallo and threonine (MEROPS peptidase database, http://merops.sanger.ac.uk/), which probably reflect a wide variety of as yet unidentified functions performed by plant proteases. Recent indications that the 26S proteasome, a T1 family-threonine protease, is a regulator of light and hormone responsive signal transduction highlight the potential of proteases to participate in many aspects of plant growth and development. Recent discoveries that proteases are required for stomatal distribution, embryo development and disease resistance point to wider roles for four additional multigene families that include some of the most frequently studied (yet poorly understood) plant proteases: the subtilisin-like, serine proteases (family S8), the papain-like, cysteine proteases (family C1A), the pepsin-like, aspartic proteases (family A1) and the plant matrixin, metalloproteases (family M10A). In this report, 54 subtilisin-like, 30 papain-like and 59 pepsin-like proteases from Arabidopsis, are compared with S8, C1A and A1 proteases known from other plant species at the functional, phylogenetic and gene structure levels. Examples of structural conservation between S8, C1A and A1 genes from rice, barley, tomato and soybean and those from Arabidopsis are noted, indicating that some common, essential plant protease roles were established before the divergence of monocots and eudicots. Numerous examples of tandem duplications of protease genes and evidence for a variety of restricted expression patterns suggest that a high degree of specialization exists among proteases within each family. We propose that comprehensive analysis of the functions of these genes in Arabidopsis will firmly establish serine, cysteine and aspartic proteases as regulators and effectors of a wide range of plant processes.     

Caspase-dependent inactivation of proteasome function during programmed cell death in Drosophila and man

The caspase family of cysteine proteases plays a conserved role in the coordinate demolition of cellular structures during programmed cell death from nematodes to man. Because cells undergoing programmed cell death in nematodes, flies, and mammals all share common features, this suggests that caspases target a common set of cellular structures in each of these organisms. However, although many substrates for mammalian caspases have been identified, few substrates for these proteases have been identified in invertebrates. To search for similarities between the repertoires of proteins targeted for proteolysis by caspases in flies and mammals, we have performed proteomics-based screens in Drosophila and human cell lines undergoing apoptosis. Here we show that several subunits of the proteasome undergo caspase-dependent proteolysis in both organisms and that this results in diminished activity of this multicatalytic protease complex. These data suggest that caspase-dependent proteolysis decreases protein turnover by the proteasome and that this is a conserved event in programmed cell death from Drosophila to mammals.     

The mitochondrial rhomboid protease: Its rise from obscurity to the pinnacle of disease-relevant genes

The Rhomboid proteases belong to a highly conserved family of proteins that are present in all branches of life. In Drosophila, the secretory pathway-localized rhomboid proteases are crucial for epidermal growth factor (EGF) signaling. The identification of a mitochondrial-localized rhomboid protease shed light on other functions of rhomboid proteases including the maintenance of mitochondrial morphology and the regulation of apoptosis. More recent work has revealed other functions of the mitochondrial rhomboid protease in mitochondrial and cellular biology, failure of which have been implicated in human diseases. In this review, we will summarize the current knowledge and disease relevance of the mitochondrial-localized rhomboid protease. This article is part of a Special Issue entitled: Intramembrane Proteases.