ADAMs: modulators of cell-cell and cell-matrix interactions

ADAMs contain adhesive and metalloprotease domains. As major ectodomain sheddases, they release a variety of cell-surface proteins, including growth factors, cytokines, cell adhesion molecules and receptors. ADAMs can also cleave and remodel components of the extracellular matrix. Hence, ADAMs are emerging as key modulators of cell-cell and cell-matrix interactions. Important questions, including if and how ADAM adhesive domains promote ADAM protease function, are currently being addressed.     

Induction of new ADAM related proteins from treated human Chang-liver cells

Chang-liver cells is a cell line generated from human liver tissue, which is often used in scientific research. ADAMs are a family of proteins that consist of multi-domains, possess multi-functions and play a central role in normal or abnormal physiological conditions, such as regeneration and tumorigenesis. To investigate the expression and functional alteration of the ADAMs or ADAM related proteins in Chang-liver cells, this cell line was treated with heat stress, modified Hanks solution containing ATP or other buffers. Our results showed that the treatment with Hanks solution containing ATP induces Chang-liver cells to express new ADAM related proteins. To analyze these new ADAM related proteins, a cDNA expression library was constructed for the treated Chang-liver cells. A series of positive clones were obtained through immunoscreening with an ADAMs common antibody. A new ADAM related protein possessing alkaline protease activity was confirmed in these clones.     

Induction of new adam related protein from treated human Chang-liver cells

Chang-liver cells is a cell line generated from human liver tissue, which is often used in scientific research. ADAMs are a family of proteins that consist of multi-domains, possess multi-functions and play a central role in normal or abnormal physiological conditions, such as regeneration and tumorigenesis. To investigate the expression and functional alteration of the ADAMs or ADAM related proteins in Chang-liver cells, this cell line was treated with heat stress, modified Hanks solution containing ATP or other buffers. Our results showed that the treatment with Hanks solution containing ATP induces Chang-liver cells to express new ADAM related proteins. To analyze these new ADAM related proteins, a cDNA expression library was constructed for the treated Chang-liver cells. A series of positive clones were obtained through immunoscreening with an ADAMs common antibody. A new ADAM related protein possessing alkaline protease activity was confirmed in these clones.     

Involvement of ADAMs in tumorigenesis and progression of hepatocellular carcinoma: Is it merely fortuitous or a real pathogenic link?

Hepatocellular carcinoma (HCC) is the most common primary liver cancer and one of the most frequent types of cancer worldwide. It normally develops in patients with chronic liver disease, especially cirrhosis, although some cases without an apparent underlying liver disease have been reported. The pathogenesis of HCC is multi-factorial and complex. Hepatitis viruses are the main factors favoring the development of HCC. In fact, chronic inflammation associated with hepatitis C or B virus infection can lead to progressive liver fibrosis, cirrhosis and ultimately HCC. Chronic inflammation and liver fibrosis cause a continuous remodeling of the extracellular matrix (ECM), a dynamic process that involves several molecules including integrins and matrix processing enzymes. An increasing body of evidence indicates that ADAMs are involved in promoting tumor formation and progression of HCC. A Disintegrin And Metalloproteases (ADAMs) are a group of proteins belonging to the zinc protease superfamily. ADAMs are usually transmembrane proteins that contain disintegrin and metalloprotease domains and are, therefore, able to carry out both cell adhesion and protease activities. Soluble isoforms of ADAMs have also been discovered and characterized. In this review, we focus on the contribution of ADAM proteins to HCC tumorigenesis and cancer progression. The potential role of ADAMs as key modulators of tumor–stroma interactions during tumor progression, by means of the activities of their constituent domains, is also discussed.     

Fungal Proteases and Their Pathophysiological Effects

Proteolytic enzymes play an important role in fungal physiology and development. External digestion of protein substrates by secreted proteases is required for survival and growth of both saprophytic and pathogenic species. Extracellular serine, aspartic, and metalloproteases are considered virulence factors of many pathogenic species. New findings focus on novel membrane-associated proteases such as yapsins and ADAMs and their role in pathology. Proteases from fungi induce inflammatory responses by altering the permeability of epithelial barrier and by induction of proinflammatory cytokines through protease-activated receptors. Many fungal allergens possess proteolytic activity that appears to be essential in eliciting Th2 responses. Allergenic fungal proteases can act as adjuvants, potentiating responses to other allergens. Proteolytic enzymes from fungi contribute to inflammation through interactions with the kinin system as well as the coagulation and fibrinolytic cascades. Their effect on the host protease–antiprotease balance results from activation of endogenous proteases and degradation of protease inhibitors. Recent studies of the role of fungi in human health point to the growing importance of proteases not only as pathogenic agents in fungal infections but also in asthma, allergy, and damp building related illnesses. Proteolytic enzymes from fungi are widely used in biotechnology, mainly in food, leather, and detergent industries, in ecological bioremediation processes and to produce therapeutic peptides. The involvement of fungal proteases in diverse pathological mechanisms makes them potential targets of therapeutic intervention and candidates for biomarkers of disease and exposure.     

Transmembrane collagen XVII,an epithelial adhesion protein,is shed from the cell surface by ADAMs

Collagen XVII, a type II transmembrane protein and epithelial adhesion molecule, can be proteolytically shed from the cell surface to generate a soluble collagen. Here we investigated the release of the ectodomain and identified the enzymes involved. After surface biotinylation of keratinocytes, the ectodomain was detectable in the medium within minutes and remained stable for >48 h. Shedding was enhanced by phorbol esters and inhibited by metalloprotease inhibitors, including hydroxamates and TIMP-3, but not by inhibitors of other protease classes or by TIMP-2. This profile implicated MMPs or ADAMs as candidate sheddases. MMP-2, MMP-9 and MT1-MMP were excluded, but TACE, ADAM-10 and ADAM-9 were shown to be expressed in keratinocytes and to be actively involved. Transfection with cDNAs for the three ADAMs resulted in increased shedding and, vice versa, in TACE-deficient cells shedding was significantly reduced, indicating that transmembrane collagen XVII represents a novel class of substrates for ADAMs. Functionally, release of the ectodomain of collagen XVII from the cell surface was associated with altered keratinocyte motility in vitro.     

Deletion of admB gene encoding a fungal ADAM affects cell wall construction in Aspergillus oryzae

Mammals possess a unique signaling system based on the proteolytic mechanism of a disintegrin and metalloproteinases (ADAMs) on the cell surface. We found two genes encoding ADAMs in Aspergillus oryzae and named them admA and admB. We produced admA and admB deletion strains to elucidate their biological function and clarify whether fungal ADAMs play a similar role as in mammals. The ?admA?admB and ?admB strains were sensitive to cell wall-perturbing agents, congo red, and calcofluor white. Moreover, the two strains showed significantly increased weights of total alkali-soluble fractions from the mycelial cell wall compared to the control strain. Furthermore, ?admB showed MpkA phosphorylation at lower concentration of congo red stimulation than the control strain. However, the MpkA phosphorylation level was not different between ?admB and the control strain without the stimulation. The results indicated that A. oryzae AdmB involved in the cell wall integrity without going through the MpkA pathway.     

ADAM-12 as a Diagnostic Marker for the Proliferation,Migration and Invasion in Patients with Small Cell Lung Cancer

Small cell lung cancer (SCLC) is highly aggressive and is characterized by malignant metastasis. Approximately 90% of patients die due to extensive metastasis. The extracellular matrix (ECM) is a natural barrier that can prevent cellular invasion and metastasis. Therefore, degradation of the ECM must take place in order for extensive metastasis to occur. A disintegrin and metalloprotease (ADAM) is a multi-domain protease that plays an important role in tumorigenesis, as well as tumor development, invasion and metastasis. However, there have been few reports on the expression and role of ADAMs in SCLC. In the current study, the expression and role of ADAMs in SCLC proliferation, invasion and metastasis was investigated. A total of 150 SCLC tissue samples were examined by immunohistochemistry for ADAMs expression. ADAM-12 was found to be abundantly expressed in 72.67% samples and other ADAMs were found to be expressed in 10% to 40% of samples. ADAM-12 levels in serum and urine, from 70 SCLC patients and 40 normal controls, were also measured using ELISA. ADAM-12 expression was significantly higher in SCLC patients than in healthy controls and in patients with extensive disease compared to those with more limited disease. Silencing the expression of ADAM-12 in H1688 cells through the use of specific siRNA significantly reduced cellular proliferation, invasion and metastasis. Supplementing the expression of ADAM-12-L or -S in H345 cells, significantly enhanced cellular proliferation, invasion and metastasis. Animal models with metastatic SCLC also exhibited increased expression of ADAM-12 along with enhanced invasion and metastasis. In brief, ADAM-12 is an independent prognostic factor and diagnostic marker, and is involved in the proliferation, invasion and metastasis of SCLC.     

Translocation of the cytoplasmic domain of ADAM13 to the nucleus is essential for Calpain8-a expression and cranial neural crest cell migration

ADAMs are transmembrane metalloproteases that control cell behavior by cleaving both cell adhesion and signaling molecules. The cytoplasmic domain of ADAMs can regulate the proteolytic activity by controlling the subcellular localization and/or the activation of the protease domain. Here, we show that the cytoplasmic domain of ADAM13 is cleaved and translocates into the nucleus. Preventing this translocation renders the protein incapable of promoting cranial neural crest (CNC) cell migration in?vivo, without affecting its proteolytic activity. In addition, the cytoplasmic domain of ADAM13 regulates the expression of multiple genes in CNC, including the protease Calpain8-a. Restoring the expression of Calpain8-a is sufficient to rescue CNC migration in the absence of the ADAM13 cytoplasmic domain. This study shows that the cytoplasmic domain of ADAM metalloproteases can perform essential functions in the nucleus of cells and may contribute substantially to the overall function of the protein.     

In silico analyses of Fsf1 sequences, a new group of fungal proteins orthologous to the metazoan sideroblastic anemia-related sideroflexin family

Sideroblastic anemias are pathologies observed in metazoan species characterized by accumulation of iron in the mitochondria (sideroblasts), defective erythropoiesis, and iron overload. Some genes have been associated with sideroblastic anemia, e.g. delta-aminolevulinic acid synthase gene (e-ALAS2). Recently, a new sideroblastic-associated protein family was discovered in metazoans and termed sideroflexin (Sfxn). The metazoan Sfxn family comprises five groups of paralogous proteins, present in mitochondria and whose functions are unknown. Using an in silico approach, we have identified and characterized new sideroflexin sequences from the genomes of different fungal species. An in-depth phylogenetic analysis of these new fungal Sfxn sequences (termed Fsf1p) showed that they form a distinct clade within the metazoan Sfxn family. Hydrophobic cluster analysis and transmembrane topological mapping allowed us to compare conserved regions among Fsf1 and Sfxn proteins. The results indicate that Fsf1 probably belongs to an ancient, mitochondrial group of proteins, necessary to maintain the homeostasis of iron within this organelle.     

The cysteine-rich domain regulates ADAM protease function in vivo

ADAMs are membrane-anchored proteases that regulate cell behavior by proteolytically modifying the cell surface and ECM. Like other membrane-anchored proteases, ADAMs contain candidate "adhesive" domains downstream of their metalloprotease domains. The mechanism by which membrane-anchored cell surface proteases utilize these putative adhesive domains to regulate protease function in vivo is not well understood. We address this important question by analyzing the relative contributions of downstream extracellular domains (disintegrin, cysteine rich, and EGF-like repeat) of the ADAM13 metalloprotease during Xenopus laevis development. When expressed in embryos, ADAM13 induces hyperplasia of the cement gland, whereas ADAM10 does not. Using chimeric constructs, we find that the metalloprotease domain of ADAM10 can substitute for that of ADAM13, but that specificity for cement gland expansion requires a downstream extracellular domain of ADAM13. Analysis of finer resolution chimeras indicates an essential role for the cysteine-rich domain and a supporting role for the disintegrin domain. These and other results reveal that the cysteine-rich domain of ADAM13 cooperates intramolecularly with the ADAM13 metalloprotease domain to regulate its function in vivo. Our findings thus provide the first evidence that a downstream extracellular adhesive domain plays an active role in regulating ADAM protease function in vivo. These findings are likely relevant to other membrane-anchored cell surface proteases.     

Cloning, in silico characterization and interaction of cysteine protease and cystatin for establishing their role in early blight disease in tomato

Cysteine protease (CP) and Cysteine protease inhibitor (CPI) or cystatin constitute a critical point in programmed cell death (PCD), a basic biological phenomenon which takes place in the plants, when they are exposed to varying biotic and abiotic stresses. In the present study we isolated and cloned cDNAs encoding cysteine protease and cystatin from early blight infected tomato plants. Using computational biology tools the sequence-structure-function relationships for the tomato cystatin and cysteine protease were elucidated. Interaction between the cystatin and cysteine protease of host and pathogen is higher as compared to interaction shown by cystatin and cysteine protease within the host. The interaction energy of (a)tomato cystatin—tomato cysteine protease, (b)tomato cystatin—fungal cysteine protease and (c)tomato cysteine protease—fungal cystatin are ?319.33 Kcal/mol, ?504.71 Kcal/mol and ?373.731 Kcal/mol respectively. Comparative protein sequence analysis with different plant cystatins and cysteine protease were also done with the sequences of cystatin and cysteine protease isolated from tomato. Structures for all the cystatin and cysteine protease were modeled along with their interactions with fungal cystatin and cysteine protease in order to explore the structural variability and its manifestation at the functional level. This helped to relate the already known functions of these proteins with their sequences as well as the predicted structures. This also served to better understand the CP-CPI interaction operational in developing this protein family and its implication in plant defense during fungal pathogenesis in tomato plants.     

The ADAM family: Insights into Notch proteolysis

Notch signaling is integral to a large number of developmental and homeostasis events, and either gain or loss of Notch signaling results in a wide range of defects. Notch must be processed by several proteases, including a member of the ADAM (a disintegrin and metalloprotease) family to mediate downstream signaling. Until recently, interactions of Notch with specific ADAMs in different contexts were unclear. ADAM10 is now known to be specifically essential for development and homeostasis of mouse epidermis and cardiovascular structures, and ADAM17 may not be able to fully replace ADAM10 in these contexts. However, Notch from T-cell acute lymphoblastic leukemia (T-ALL) patients can be cleaved by both ADAMs 10 and 17. Studies have revealed that ADAM10 is necessary for Notch processing when Notch is activated by a ligand, while ADAM17 is the major protease for processing Notch that is activated independently of ligand in both flies and mammals.     

The ADAM family

Notch signaling is integral to a large number of developmental and homeostasis events, and either gain or loss of Notch signaling results in a wide range of defects. Notch must be processed by several proteases, including a member of the ADAM (a disintegrin and metalloprotease) family to mediate downstream signaling. Until recently, interactions of Notch with specific ADAMs in different contexts were unclear. ADAM10 is now known to be specifically essential for development and homeostasis of mouse epidermis and cardiovascular structures, and ADAM17 may not be able to fully replace ADAM10 in these contexts. However, Notch from T-cell acute lymphoblastic leukemia (T-ALL) patients can be cleaved by both ADAMs 10 and 17. Studies have revealed that ADAM10 is necessary for Notch processing when Notch is activated by a ligand, while ADAM17 is the major protease for processing Notch that is activated independently of ligand in both flies and mammals.     

Heparan sulfate regulates ADAM12 through a molecular switch mechanism

The disintegrin and metalloproteases (ADAMs) are emerging as therapeutic targets in human disease, but specific drug design is hampered by potential redundancy. Unlike other metzincins, ADAM prodomains remain bound to the mature enzyme to regulate activity. Here ADAM12, a protease that promotes tumor progression and chondrocyte proliferation in osteoarthritic cartilage, is shown to possess a prodomain/catalytic domain cationic molecular switch, regulated by exogenous heparan sulfate and heparin but also endogenous cell surface proteoglycans and the polyanion, calcium pentosan polysulfate. Sheddase functions of ADAM12 are regulated by the switch, as are proteolytic functions in placental tissue and sera of pregnant women. Moreover, human heparanase, an enzyme also linked to tumorigenesis, can promote ADAM12 sheddase activity at the cell surface through cleavage of the inhibitory heparan sulfate. These data present a novel concept that might allow targeting of ADAM12 and suggest that other ADAMs may have specific regulatory activity embedded in their prodomain and catalytic domain structures.     

Regulation of human ADAM 12 protease by the prodomain. Evidence for a functional cysteine switch

The ADAMs (a disintegrin and metalloprotease) are a family of multidomain proteins that are believed to play key roles in cell-cell and cell-matrix interactions. We have shown recently that human ADAM 12-S (meltrin alpha) is an active metalloprotease. It is synthesized as a zymogen, with the prodomain maintaining the protease in a latent form. We now provide evidence that the latency mechanism of ADAM 12 can be explained by the cysteine switch model, in which coordination of Zn2+ in the active site of the catalytic domain by a cysteine residue in the prodomain is critical for inhibition of the protease. Replacing Cys179 with other amino acids results in an ADAM 12 proform that is proteolytically active, but latency can be restored by placing cysteine at other positions in the propeptide. None of the amino acids adjacent to the crucial cysteine residue is essential for blocking activity of the protease domain. In addition to its latency function, the prodomain is required for exit of ADAM 12 protease from the endoplasmic reticulum. Tissue inhibitor of metalloprotease-1, -2, and -3 were not found to block proteolytic activity of ADAM 12, hence a physiological inhibitor of ADAM 12 protease in the extracellular environment remains to be identified.