Simple primary structure, complex turnover regulation and multiple roles of hyaluronan

Hyaluronan is a major macromolecular polysaccharide component of the extra-cellular matrix that confers structural frameworks for cells. Despite its relatively simple chemical composition, hyaluronan mediates many other important functional aspects including signalling activity during embryonic morphogenesis, cellular regeneration and wound healing. Abnormalities in hyaluronan metabolism have been implicated in many diseases, such as inflammatory disorders, cardiovascular diseases and cancer. To date, it has become increasingly clear that hyaluronan production in vertebrates is tightly regulated by three hyaluronan synthases and that hyaluronan catabolism is regulated by an enzymatic degradation reaction involving several hyaluronidases. Together, these discoveries have provided key insights into the physiological roles of hyaluronan and a deeper understanding of the mechanisms underlying altered hyaluronan turnover in diseases. The central aim of this review article is therefore to highlight the multiple roles of hyaluronan in physiological and pathological states via its complex turnover regulation.     

Regulation of procollagen amino-propeptide processing during mouse embryogenesis by specialization of homologous ADAMTS proteases: insights on collagen biosynthesis and dermatosparaxis

Mutations in ADAMTS2, a procollagen amino-propeptidase, cause severe skin fragility, designated as dermatosparaxis in animals, and a subtype of the Ehlers-Danlos syndrome (dermatosparactic type or VIIC) in humans. Not all collagen-rich tissues are affected to the same degree, which suggests compensation by the ADAMTS2 homologs ADAMTS3 and ADAMTS14. In situ hybridization of Adamts2, Adamts3 and Adamts14, and of the genes encoding the major fibrillar collagens, Col1a1, Col2a1 and Col3a1, during mouse embryogenesis, demonstrated distinct tissue-specific, overlapping expression patterns of the protease and substrate genes. Adamts3, but not Adamts2 or Adamts14, was co-expressed with Col2a1 in cartilage throughout development, and with Col1a1 in bone and musculotendinous tissues. ADAMTS3 induced procollagen I processing in dermatosparactic fibroblasts, suggesting a role in procollagen I processing during musculoskeletal development. Adamts2, but not Adamts3 or Adamts14, was co-expressed with Col3a1 in many tissues including the lungs and aorta, and Adamts2(-/-) mice showed widespread defects in procollagen III processing. Adamts2(-/-) mice had abnormal lungs, characterized by a decreased parenchymal density. However, the aorta and collagen fibrils in the aortic wall appeared normal. Although Adamts14 lacked developmental tissue-specific expression, it was co-expressed with Adamts2 in mature dermis, which possibly explains the presence of some processed skin procollagen in dermatosparaxis. The data show how evolutionarily related proteases with similar substrate preferences may have distinct biological roles owing to tissue-specific gene expression, and provide insights into collagen biosynthesis and the pathobiology of dermatosparaxis.     

Expression of Dll4 during mouse embryogenesis suggests multiple developmental roles

Delta-Notch signalling regulates cell-fate decisions in a variety of tissues in diverse organisms, through cell-to-cell interactions. Here, we report the expression pattern of a Delta gene family member, Delta-like 4 (Dll4). Dll4 expression was analyzed in mouse embryos and selected adult organs by monitoring beta-galactosidase (beta-gal) expression from a lacZ reporter cassette inserted downstream of the Dll4 promoter, which allowed for high sensitivity and single cell resolution. Expression was detected in several tissues where Notch signalling is known to control cell-fate decisions, like the vascular system, the nervous system, the gastrointestinal system, and the thymus. Throughout embryonic cardiovascular development, Dll4 expression was seen only on endocardial cells and endothelial cells of the arteries, arterioles, and capillaries, being absent from vascular smooth muscle cells and veins. In the nervous system, expression was detected in the brain, neural tube, retina, and, for the first time, in the olfactory epithelium, vomeronasal organs and para-aortic bodies. Extensive Dll4 expression was also observed in the gut. This detailed expression analysis reveals new clues for both endothelial and non-endothelial Dll4 function in different organs.     

ADAMTS proteinases: a multi-domain,multi-functional family with roles in extracellular matrix turnover and arthritis

Members of the ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) family are known to influence development, angiogenesis, coagulation and progression of arthritis. As proteinases their substrates include the von Willebrand factor precursor and extracellular matrix components such as procollagen, hyalectans (hyaluronan-binding proteoglycans including aggrecan), decorin, fibromodulin and cartilage oligomeric matrix protein. ADAMTS levels and activities are regulated at multiple levels through the control of gene expression, mRNA splicing, protein processing and inhibition by TIMP (tissue inhibitor of metalloproteinases). A recent screen of human cartilage has shown that multiple members of the ADAMTS family may be important in connective tissue homeostasis and pathology.     

Discovery and characterization of a novel, widely expressed metalloprotease, ADAMTS10, and its proteolytic activation

We describe the discovery and characterization of ADAMTS10, a novel metalloprotease encoded by a locus on human chromosome 19 and mouse chromosome 17. ADAMTS10 has the typical modular organization of the ADAMTS family, with five thrombospondin type 1 repeats and a cysteine-rich PLAC (protease and lacunin) domain at the carboxyl terminus. Its domain organization and primary structure is similar to a novel long form of ADAMTS6. In contrast to many ADAMTS proteases, ADAMTS10 is widely expressed in adult tissues and throughout mouse embryo development. In situ hybridization analysis showed widespread expression of Adamts10 in the mouse embryo until 12.5 days of gestation, after which it is then expressed in a more restricted fashion, with especially strong expression in developing lung, bone, and craniofacial region. Mesenchymal, not epithelial, expression in the developing lung, kidney, gonad, salivary gland, and gastrointestinal tract is a consistent feature of Adamts10 regulation. N-terminal sequencing and treatment with decanoyl-Arg-Val-Lys-Arg-chloromethylketone indicate that the ADAMTS10 zymogen is processed by a subtilisin-like proprotein convertase at two sites (Arg64/Gly and Arg233/Ser). The widespread expression of ADAMTS10 suggests that furin, a ubiquitously expressed proprotein convertase, is the likely processing enzyme. ADAMTS10 expressed in HEK293F and COS-1 cells is N-glycosylated and is secreted into the medium, as well as sequestered at the cell surface and extracellular matrix, as demonstrated by cell surface biotinylation and immunolocalization in nonpermeabilized cells. ADAMTS10 is a functional metalloprotease as demonstrated by cleavage of alpha2-macroglobulin, although physiological substrates are presently unknown.     

Expression of the L14 lectin during mouse embryogenesis suggests multiple roles during pre- and post-implantation development.

A cDNA encoding L14, the lactose-binding, soluble lectin of relative molecular mass 14 x 10(3), has been isolated in a differential screen designed to identify genes that are regulated during the differentiation of murine embryonic stem cells in vitro. The expression patterns of the gene and of the encoded protein during mouse embryogenesis are consistent with the lectin playing a role at several stages of development. Firstly, it is initially synthesised in the trophectoderm of expanded blastocysts immediately prior to implantation, suggesting that it may be involved in the attachment of the embryo to the uterine epithelium. Secondly, in the postimplantation embryo, the lectin is abundantly expressed in the myotomes of the somites. This observation, when taken together with data indicating a role for the lectin in myoblast differentiation in culture, suggests that the protein is important in muscle cell differentiation. Finally, within the nervous system expression of this gene is activated early during the differentiation of a particular subset of neurones.     

Expression of versican and ADAMTS1, 4, and 5 during bone development in the rat mandible and hind limb.

Extracellular matrix (ECM) remodeling is achieved by both production and degradation of ECM molecules during bone development. ADAMTS (a disintegrin and metalloprotease with thrombospondin type 1 motifs) constitutes a family of extracellular proteases which are implicated in cleaving the protein versican. The present study was designed to investigate the expression of versican and ADAMTS1, 4, and 5 mRNA during bone development in rat mandibles and hind limbs by RT-PCR and in situ hybridization. Versican was localized by immunohistochemistry. The process of bone development from day 14 postcoitum through week 6 postnatum was divided into the beginning of osteogenesis, woven bone, and lamellar bone stages. Versican protein was abundant in the woven bone matrix, but decreased in the lamellar bone matrix. Versican mRNA was prominent in some osteoblasts with corresponding localization of the cognate protein. The temporal and spatial mRNA expression pattern of ADAMTS1, 4, and 5 was comparable to that of versican. These results suggest that woven bone rich in versican alters into lamellar bone containing little versican during bone development in both mandibles and hind limbs, where some osteoblasts may be involved in production as well as degradation of versican by secreting ADAMTS1, 4, and 5.     

Expression of cysteine proteases in extraembryonic tissues during mouse embryogenesis

The expression of cathepsin B- and L-specific mRNAs as well as active forms of the enzymes was determined in mouse placenta and visceral yolk sac from 7.5 through 17.5 days postconception, a period marked by major anatomic transitions in the mouse conceptus. The level of specific mRNA was determined relative to the 28S ribosomal RNA in a series of multiprobe ribonuclease protection assays using high-specific-activity antisense cathepsin B and L riboprobes. The molecular forms of active cysteine proteases present in the tissues at the time of extraction were detected using a membrane-permeant radiolabeled active site-specific inhibitor, Fmoc-[(125)I(2)]Tyr-Ala-CHN(2). The results of this study show that the expression of active cathepsin L relative to active cathepsin B is significantly higher in visceral yolk sac than in placenta, consistent with a higher proteolytic requirement for the former tissue. Active cathepsin L was highest at Day 9.5 in visceral yolk sac, a stage at which it has been shown that proteolysis in this organ is required for production of amino acids for embryonic protein synthesis. Cathepsin L mRNA was also elevated in the Day 9.5 placenta, but paradoxically this did not result in an increase in cellular active enzyme. An unknown protein, termed p14, highly expressed in placenta, also reacted with the inhibitor. Expression of this protein was highest early during gestation in the ectoplacental cone, suggesting that p14 may be important in the implantation process.     

Processing and localization of ADAMTS-1 and proteolytic cleavage of versican during cumulus matrix expansion and ovulation

ADAMTS-1 (a disintegrin and metalloprotease with thrombospondin motifs-1) is a member of the ADAMTS family of metalloproteases which, together with ADAMTS-4 and ADAMTS-5, has been shown to degrade members of the lectican family of proteoglycans. ADAMTS-1 mRNA is induced in granulosa cells of periovulatory follicles by the luteinizing hormone surge through a progesterone receptor-dependent mechanism. Female progesterone receptor knockout (PRKO) mice are infertile primarily due to ovulatory failure and lack the normal periovulatory induction of ADAMTS-1 mRNA. We therefore investigated the protein localization and function of ADAMTS-1 in ovulating ovaries. Antibodies against two specific peptide regions, the pro-domain and the metalloprotease domain of ADAMTS-1, were generated. Pro-ADAMTS-1 of 110 kDa was identified in mural granulosa cells and appears localized to cytoplasmic secretory vesicles. The mature (85-kDa pro-domain truncated) form accumulated in the extracellular matrix of the cumulus oocyte complex (COC) during the process of matrix expansion. Each form of ADAMTS-1 protein increased >10-fold after the ovulatory luteinizing hormone surge in wild-type but not PRKO mice. Versican is also localized selectively to the ovulating COC matrix and was found to be cleaved yielding a 70-kDa N-terminal fragment immunopositive for the neoepitope DPEAAE generated by ADAMTS-1 and ADAMTS-4 protease activity. This extracellular processing of versican was reduced in ADAMTS-1-deficient PRKO mouse ovaries. These observations suggest that one function of ADAMTS-1 in ovulation is to cleave versican in the expanded COC matrix and that the anovulatory phenotype of PRKO mice is at least partially due to loss of this function.     

小胶质细胞在脑中的生理功能

小胶质细胞是脑中的巨噬细胞,也是脑实质中唯一的一种免疫细胞,因而被看作是中枢神经系统抵御病原入侵的第一道防线。在其他非感染病理状态下,如脑损伤及神经退行性疾病等,小胶质细胞也发挥着保护和毒性损伤的双重作用。相比较其病理功能,人们对小胶质细胞的生理功能长期以来很少关注。然而,近几年关于小胶质细胞生理功能的研究在多个方面都有突破。这些研究结果揭示,小胶质细胞在发育的神经系统中起着调控神经元存活和修饰突触的作用,并且在成熟的健康脑中具有探测和调控神经元活动的功能。将着重对近几年关于小胶质细胞生理功能的相关研究做一综述。     

Post-translational regulation and proteolytic activity of the metalloproteinase ADAMTS8

A disintegrin-like and metalloprotease domain with thrombospondin type 1 motifs (ADAMTS)8 is a secreted protease, which was recently implicated in pathogenesis of pulmonary arterial hypertension (PAH). However, the substrate repertoire of ADAMTS8 and regulation of its activity are incompletely understood. Although considered a proteoglycanase because of high sequence similarity and close phylogenetic relationship to the proteoglycan-degrading proteases ADAMTS1, 4, 5, and 15, as well as tight genetic linkage with ADAMTS15 on human chromosome 11, its aggrecanase activity was reportedly weak. Several post-translational factors are known to regulate ADAMTS proteases such as autolysis, inhibition by endogenous inhibitors, and receptor-mediated endocytosis, but their impacts on ADAMTS8 are unknown. Here, we show that ADAMTS8 undergoes autolysis at six different sites within its spacer domain. We also found that in contrast to ADAMTS4 and 5, ADAMTS8 levels were not regulated through low-density lipoprotein receptor-related protein 1 (LRP1)-mediated endocytosis. Additionally, ADAMTS8 lacked significant activity against the proteoglycans aggrecan, versican, and biglycan. Instead, we found that ADAMTS8 cleaved osteopontin, a phosphoprotein whose expression is upregulated in PAH. Multiple ADAMTS8 cleavage sites were identified using liquid chromatography–tandem mass spectrometry. Osteopontin cleavage by ADAMTS8 was efficiently inhibited by TIMP-3, an endogenous inhibitor of ADAMTS1, 4, and 5, as well as by TIMP-2, which has no previously reported inhibitory activity against other ADAMTS proteases. These differences in post-translational regulation and substrate repertoire differentiate ADAMTS8 from other family members and may help to elucidate its role in PAH.     

The interaction of versican with its binding partners

Versican belongs to the family of the large aggregating chondroitin sulfate proteoglycans located primarily within the extracellular matrix （ECM）. Versican, like other members of its family, has unique N- and C-terminal globular regions, each with multiple motifs. A large glycosaminoglycan-binding region lies between them. This review will begin by outlining these structures, in the context of ECM proteoglycans. The diverse binding partners afforded to versican by virtue of its modular design will then be examined. These include ECM components, such as hyaluronan, type Ⅰ collagen, tenascin-R, fibulin-1, and -2, fibrillin-1, fibronectin, P- and L-selectins, and chemokines. Versican also binds to the cell surface proteins CD44, integrin β1, epidermal growth factor receptor, and P-selectin glycoprotein ligand-1. These multiple interactors play important roles in cell behaviour, and the roles of versican in modulating such processes are discussed.     

Isoaspartate residues dramatically influence substrate recognition and turnover by proteases

Posttranslational modifications influence the structure, stability and biological activity of proteins. Most of the reactions are enzyme-catalyzed, but some, such as asparagine (Asn) and glutamine (Gln) deamidation and the isoaspartate (isoAsp) formation within peptide chains, occur spontaneously. It has been previously shown that certain peptide sequences form isoAsp quite fast if the Asp stretches are exposed to the protein surface, thereby potentially changing susceptibility to proteolysis at these sites. This tempted us to investigate the activity of exo- and endopeptidases against Asp- or isoAsp-containing substrates. Members of the prolyl oligopeptidase family were unable to cleave substrates after proline if isoAsp was placed in the P2-position. Caspases, usually accepting Asp at P1-position of their substrates, did not cleave isoAsp-containing sequences. Similarly, the metal-dependent aminopeptidase amino peptidase N did not turnover N-terminal isoAsp-containing substrates, nor could the endopeptidase matrix metalloproteinase 3 (MMP 3) hydrolyze a serum amyloid A protein-like substrate if the sequence contained isoAsp instead of Asp. Also, the highly specific enterokinase, usually clipping after a stretch of four Asp residues and a lysine in the P1 position, could not turnover substrates if the P2 amino acid was replaced by isoAsp. In contrast, acylamino acid-releasing enzyme and dipeptidyl peptidases 1, 2 and 4 hydrolyzed substrates containing the isoAsp-Ala motif.     

The roles of intramembrane proteases in protozoan parasites

Intramembrane proteolysis is widely conserved throughout different forms of life, with three major types of proteases being known for their ability to cleave peptide bonds directly within the transmembrane domains of their substrates. Although intramembrane proteases have been extensively studied in humans and model organisms, they have only more recently been investigated in protozoan parasites, where they turn out to play important and sometimes unexpected roles. Signal peptide peptidases are involved in endoplasmic reticulum (ER) quality control and signal peptide degradation from exported proteins. Recent studies suggest that repurposing inhibitors developed for blocking presenilins may be useful for inhibiting the growth of Plasmodium, and possibly other protozoan parasites, by blocking signal peptide peptidases. Rhomboid proteases, originally described in the fly, are also widespread in parasites, and are especially expanded in apicomplexans. Their study in parasites has revealed novel roles that expand our understanding of how these proteases function. Within this diverse group of parasites, rhomboid proteases contribute to processing of adhesins involved in attachment, invasion, intracellular replication, phagocytosis, and immune evasion, placing them at the vertex of host–parasite interactions. This article is part of a Special Issue entitled: Intramembrane Proteases.     

The ATP-dependent proteases and proteolytic complexes involved into intracellular protein degradation

The review characterizes the main enzymatic systems of selective proteolysis responsible for maintenance of intracellular proteome in prokaryotes, eukaryotes and archaea. The features of proteolytic components of the ATP-dependent proteases as well as similarity and diversity of their regulatory components belonging to AAA⁺ ATPases are discussed.     

Emerging roles of ADAM and ADAMTS metalloproteinases in cancer

A disintegrin and metalloproteinases (ADAMs) are a recently discovered family of proteins that share the metalloproteinase domain with matrix metalloproteinases (MMPs). Among this family, structural features distinguish the membrane-anchored ADAMs and the secreted ADAMs with thrombospondin motifs referred to as ADAMTSs. By acting on a large panel of membrane-associated and extracellular substrates, they control several cell functions such as adhesion, fusion, migration and proliferation. The current review addresses the contribution of these proteinases in the positive and negative regulation of cancer progression as mainly mediated by the regulation of growth factor activities and integrin functions.     

The characterisation of six ADAMTS proteases in the basal chordate Ciona intestinalis provides new insights into the vertebrate ADAMTS family

ADAMTS, constituting a recently discovered family of secreted zinc-dependent metalloproteases, have been shown to have critical physiological roles through identification of a number of natural animal and human gene mutations. The identification of six ADAMTS genes in the basal chordate Ciona intestinalis provides new insight into how, when and in what order the vertebrate orthologues have evolved. The phylogenetic assignments, based on sequences conserved across all genes, are supported by conserved domain structures within defined sub-families. The phylogeny and the frequent localisation of ADAMTS genes in paralogous regions of the genome are consistent with the vertebrate lineages having arisen by large scale or genome duplication. The high level of conservation in the protease active site of vertebrate orthologues within some sub-families suggests subfunctionalisation, whereas the greater divergence in others would favour the evolution of novel substrate specificities and these observations are borne-out where substrate-specificity is known. The expansion and sub-specialization of the ADAMTS family is a component of the increased complexity of extracellular matrix that is associated with the evolution of vertebrates.     

The roles of versican V1 and V2 isoforms in cell proliferation and apoptosis

Versican is a large chondroitin sulfate proteoglycan belonging to the lectican family. Alternative splicing of versican generates at least four isoforms named V0, V1, V2, and V3. We have shown that the versican V1 isoform not only enhanced cell proliferation, but also modulated cell cycle progression and protected the cells from apoptosis. Futhermore, the V1 isoform was able to not only activate proto-oncogene EGFR expression and modulate its downstream signaling pathway, but also induce p27 degradation and enhance CDK2 kinase activity. As well, the V1 isoform down-regulated the expression of the proapoptotic protein Bad. By contrast, the V2 isoform exhibited opposite biological activities by inhibiting cell proliferation and down-regulated the expression of EGFR and cyclin A. Furthermore, V2 did not contribute apoptotic resistance to the cells. In light of these results, we are reporting opposite functions for the two versican isoforms whose expression is differentially regulated. Our studies suggest that the roles of these two isoforms are associated with the subdomains CSbeta and CSalpha, respectively. These results were confirmed by silencing the expression of versican V1 with small interfering RNA (siRNA), which abolished V1-enhanced cell proliferation and V1-induced reduction of apoptosis.