Metalloprotease-disintegrin ADAM 12 binds to the SH3 domain of Src and activates Src tyrosine kinase in C2C12 cells.
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ABSTRACT:
SUBMITTER: Kang Q
PROVIDER: S-EPMC1221530 | biostudies-other | 2000 Dec
REPOSITORIES: biostudies-other
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Project description:ADAM 12, a member of the ADAM family of proteins (containing A Disintegrin And Metalloprotease domain), has been implicated in differentiation and fusion of myoblasts. While the extracellular domain of ADAM 12 contains an active metalloprotease and a region involved in cell adhesion, the function of the cytoplasmic tail of ADAM 12 has been less clear. Here we show that the cytoplasmic domain of ADAM 12 interacts in vitro and in vivo with alpha-actinin-1, an actin-binding and cross-linking protein. Green fluorescent protein fused to ADAM 12 cytoplasmic domain co-localizes with alpha-actinin-1-containing actin stress fibres in C2C12 cells. The interaction between ADAM 12 and alpha-actinin-1 is direct and involves the 58-amino acid C-terminal fragment of ADAM 12 and the 27 kDa N-terminal domain of alpha-actinin-1. Consistently, expression of the 27 kDa fragment of alpha-actinin-1 in C2C12 cells using a mitochondrial targeting system results in recruitment of the co-expressed ADAM 12 cytoplasmic domain to the mitochondrial surface. Moreover, alpha-actinin-1 co-purifies with a transmembrane, His6-tagged form of ADAM 12 expressed in C2C12 myoblasts, indicating that the transmembrane ADAM 12 forms a complex with alpha-actinin-1 in vivo. These results indicate that the actin cytoskeleton may play a critical role in ADAM 12-mediated cell-cell adhesion or cell signalling during myoblast differentiation and fusion.
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Project description:Background and aimsADAM [A Disintegrin And Metalloproteinase] is a family of peptidase proteins which have diverse roles in tissue homeostasis and immunity. Here, we study ADAM-like DECysin-1 [ADAMDEC1] a unique member of the ADAM family. ADAMDEC1 expression is restricted to the macrophage/dendritic cell populations of the gastrointestinal tract and secondary lymphoid tissue. The biological function of ADAMDEC1 is unknown but it has been hypothesised to play a role in immunity. The identification of reduced ADAMDEC1 expression in Crohn's disease patients has provided evidence of a potential role in bowel inflammation.MethodsAdamdec1-/- mice were exposed to dextran sodium sulphate or infected orally with Citrobacter rodentium or Salmonella typhimurium. The clinical response was monitored.ResultsThe loss of Adamdec1 rendered mice more susceptible to the induction of bacterial and chemical induced colitis, as evidenced by increased neutrophil infiltration, greater IL-6 and IL-1? secretion, more weight loss and increased mortality. In the absence of Adamdec1, greater numbers of Citrobacter rodentium were found in the spleen, suggestive of a breakdown in mucosal immunity which resulted in bacteraemia.ConclusionIn summary, ADAMDEC1 protects the bowel from chemical and bacterial insults, failure of which may predispose to Crohn's disease.
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Project description:A disintegrin and metalloproteinases (ADAMs) constitute a protein family essential for extracellular signaling and regulation of cell adhesion. Catalytic activity of ADAMs and their predicted potential for Src-homology 3 (SH3) domain binding show a strong correlation. Here we present a comprehensive characterization of SH3 binding capacity and preferences of the catalytically active ADAMs 8, 9, 10, 12, 15, 17, and 19. Our results revealed several novel interactions, and also confirmed many previously reported ones. Many of the identified SH3 interaction partners were shared by several ADAMs, whereas some were ADAM-specific. Most of the ADAM-interacting SH3 proteins were adapter proteins or kinases, typically associated with sorting and endocytosis. Novel SH3 interactions revealed in this study include TOCA1 and CIP4 as preferred partners of ADAM8, and RIMBP1 as a partner of ADAM19. Our results suggest that common as well as distinct mechanisms are involved in regulation and execution of ADAM signaling, and provide a useful framework for addressing the pathways that connect ADAMs to normal and aberrant cell behavior.
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Project description:A Disintegrin and Metalloprotease (ADAM) are transmembrane proteases displaying multiple functions. ADAM with ThromboSpondin-like motifs (ADAMTS) are secreted proteases characterised by thrombospondin (TS) motifs in their C-terminal domain. The aim of this work was to evaluate the expression pattern of ADAMs and ADAMTS in non small cell lung carcinomas (NSCLC) and to investigate the possible correlation between their expression and cancer progression. Reverse transcriptase-polymerase chain reaction (RT-PCR), Western blot and immunohistochemical analyses were performed on NSCLC samples and corresponding nondiseased tissue fragments. Among the ADAMs evaluated (ADAM-8, -9, -10, -12, -15, -17, ADAMTS-1, TS-2 and TS-12), a modulation of ADAM-12 and ADAMTS-1 mRNA expression was observed. Amounts of ADAM-12 mRNA transcripts were increased in tumour tissues as compared to the corresponding controls. In sharp contrast, ADAMTS-1 mRNA levels were significantly lower in tumour tissues when compared to corresponding nondiseased lung. These results were corroborated at the protein level by Western blot and immunohistochemistry. A positive correlation was observed between the mRNA levels of ADAM-12 and those of two vascular endothelial growth factor (VEGF)-A isoforms (VEGF-A(165) and VEGF-A(121)). Taken together, these results providing evidence for an overexpression of ADAM-12 and a lower expression of ADAMTS-1 in non-small-cell lung cancer suggest that these proteases play different functions in cancer progression.
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Project description:The a disintegrin and metalloproteases (ADAMs) play a pivotal role in the control of development, adhesion, migration, inflammation and cancer. Although numerous substrates of ADAM10 have been identified, the regulation of its surface expression and proteolytic activity is still poorly defined. One current hypothesis is that both processes are in part modulated by protein-protein interactions mediated by the intracellular portion of the protease. For related proteases, especially proline-rich regions serving as docking sites for Src homology domain 3 (SH3) domain-containing proteins proved to be important for mediating regulatory interactions. In order to identify ADAM10-binding SH3 domain proteins, we screened the All SH3 Domain Phager library comprising 305 human SH3 domains using a GST fusion protein with the intracellular region of human ADAM10 as a bait for selection. Of a total of 291 analyzed phage clones, we found 38 SH3 domains that were precipitated with the ADAM10-derived fusion protein but not with GST. We verified the binding to the cytosolic portion of ADAM10 for several candidates by co-immunoprecipitation and/or pull down analyses. Intriguingly, several of the identified proteins have been implicated in regulating surface appearance and/or proteolytic activity of related ADAMs. Thus, it seems likely that they also play a role in ADAM10 biology.
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Project description:A disintegrin and metalloprotease 12 (ADAM12) has been implicated in cell growth, tumor formation, and metastasis. Therefore, we evaluated the role of ADAM12 in colorectal cancer (CRC) progression and prognosis, and elucidated whether targeted downregulation of ADAM12 could lead to therapeutic sensitization. The effect of ADAM12 on tumor cell behavior was assessed in CRC cell lines, CRC tissues, and a mouse xenograft model. ADAM12 overexpression enhanced proliferation, inhibited apoptosis, and acted as positive regulator of cell cycle progression in CRC cells. Phosphorylation of PTEN was decreased and that of Akt was increased by ADAM12 overexpression. These results were reversed upon ADAM12 knockdown. ADAM12 overexpression was significantly associated with the cancer stage, depth of invasion, lymph node metastasis, distant metastasis, and poor survival in CRC patients. In a mouse xenograft model, tumor area, volume, and weight were significantly greater for the ADAM12-pcDNA6-myc-transfected group than for the empty-pcDNA6-myc-transfected group, and significantly lower for the ADAM12-pGFP-C-shLenti-transfected group than for the scrambled pGFP-C-shLenti-transfected group. In conclusion, ADAM12 overexpression is essential for the growth and progression of CRC. Furthermore, ADAM12 knockdown reveals potent anti-tumor activity in a mouse xenograft model. Thus, ADAM12 may serve as a promising biomarker and/or therapeutic target in CRC.
| S-EPMC8073784 | biostudies-literature
Project description:Interferon-gamma (IFN-?) is a pleiotropic cytokine that exerts anti-tumor and anti-osteoclastogenic effects. Although transcriptional and post-transcriptional regulation of IFN-? is well understood, subsequent modifications of secreted IFN-? are not fully elucidated. Previous research indicates that some cancer cells escape immune surveillance and metastasize into bone tissue by inducing osteoclastic bone resorption. Peptidases of the a-disintegrin and metalloproteinase (ADAM) family are implicated in cancer cell proliferation and tumor progression. We hypothesized that the ADAM enzymes expressed by cancer cells degrades IFN-? and attenuates IFN-?-mediated anti-tumorigenic and anti-osteoclastogenic effects. Recombinant ADAM17 degraded IFN-? into small fragments. The addition of ADAM17 to the culture supernatant of stimulated mouse splenocytes decreased IFN-? concentration. However, ADAM17 inhibition in the stimulated mouse T-cells prevented IFN-? degradation. ADAM17-expressing human breast cancer cell lines MCF-7 and MDA-MB-453 also degraded recombinant IFN-?, but this was attenuated by ADAM17 inhibition. Degraded IFN-? lost the functionality including the inhibititory effect on osteoclastogenesis. This is the first study to demonstrate the extracellular proteolytic degradation of IFN-? by ADAM17. These results suggest that ADAM17-mediated degradation of IFN-? may block the anti-tumorigenic and anti-osteoclastogenic effects of IFN-?. ADAM17 inhibition may be useful for the treatment of attenuated cancer immune surveillance and/or bone metastases.
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Project description:The transmembrane protein, ADAM10 (a disintegrin and metalloprotease 10), has key physiologic functions-for example, during embryonic development and in the brain. During transit through the secretory pathway, immature ADAM10 (proADAM10) is converted into its proteolytically active, mature form (mADAM10). Increasing or decreasing the abundance and/or activity of mADAM10 is considered to be a therapeutic approach for the treatment of such diseases as Alzheimer's disease and cancer. Yet biochemical detection and characterization of mADAM10 has been difficult. In contrast, proADAM10 is readily detected-for example, in immunoblots-which suggests that mADAM10 is only a fraction of total cellular ADAM10. Here, we demonstrate that mADAM10, but not proADAM10, unexpectedly undergoes rapid, time-dependent degradation upon biochemical cell lysis in different cell lines and in primary neurons, which prevents the detection of the majority of mADAM10 in immunoblots. This degradation required the catalytic activity of ADAM10, was efficiently prevented by adding active site inhibitors to the lysis buffer, and did not affect proADAM10, which suggests that ADAM10 degradation occurred in an intramolecular and autoproteolytic manner. Inhibition of postlysis autoproteolysis demonstrated efficient cellular ADAM10 maturation with higher levels of mADAM10 than proADAM10. Moreover, a cycloheximide chase experiment revealed that mADAM10 is a long-lived protein with a half-life of approximately 12 h. In summary, our study demonstrates that mADAM10 autoproteolysis must be blocked to allow for the proper detection of mADAM10, which is essential for the correct interpretation of biochemical and cellular studies of ADAM10.-Brummer, T., Pigoni, M., Rossello, A., Wang, H., Noy, P. J., Tomlinson, M. G., Blobel, C. P., Lichtenthaler, S. F. The metalloprotease ADAM10 (a disintegrin and metalloprotease 10) undergoes rapid, postlysis autocatalytic degradation.
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Project description:A Disintegrin and Metalloproteinase (ADAM) is a family of proteolytic enzymes that possess sheddase function and regulate shedding of membrane-bound proteins, growth factors, cytokines, ligands and receptors. Typically, ADAMs have a pro-domain, and a metalloproteinase, disintegrin, cysteine-rich and a characteristic transmembrane domain. Most ADAMs are activated by proprotein convertases, but can also be regulated by G-protein coupled receptor agonists, Ca2+ ionophores and protein kinase C activators. A Disintegrin and Metalloproteinase with Thrombospondin Motifs (ADAMTS) is a family of secreted enzymes closely related to ADAMs. Like ADAMs, ADAMTS members have a pro-domain, and a metalloproteinase, disintegrin, and cysteine-rich domain, but they lack a transmembrane domain and instead have characteristic thrombospondin motifs. Activated ADAMs perform several functions and participate in multiple cardiovascular processes including vascular smooth muscle cell proliferation and migration, angiogenesis, vascular cell apoptosis, cell survival, tissue repair, and wound healing. ADAMs may also be involved in pathological conditions and cardiovascular diseases such as atherosclerosis, hypertension, aneurysm, coronary artery disease, myocardial infarction and heart failure. Like ADAMs, ADAMTS have a wide-spectrum role in vascular biology and cardiovascular pathophysiology. ADAMs and ADAMTS activity is naturally controlled by endogenous inhibitors such as tissue inhibitors of metalloproteinases (TIMPs), and their activity can also be suppressed by synthetic small molecule inhibitors. ADAMs and ADAMTS can serve as important diagnostic biomarkers and potential therapeutic targets for cardiovascular disorders. Natural and synthetic inhibitors of ADAMs and ADAMTS could be potential therapeutic tools for the management of cardiovascular diseases.
| S-EPMC6580420 | biostudies-literature
Project description:The requirement of a metalloprotease, a disintegrin and metalloprotease 17 (ADAM17) for the growth of cultured vascular smooth muscle cells has been demonstrated in vitro. However, whether this metalloprotease is responsible for vascular remodeling in vivo remains unanswered. Rat carotid arteries were analyzed 2 weeks after a balloon angioplasty. The neointimal cells were strongly positive for ADAM17 immunostaining. Marked inhibition of intimal hyperplasia was observed in a dominant-negative ADAM17 adenovirus-treated carotid artery. Proliferating cell nuclear antigen-positive cells and phospho-epidermal growth factor receptor-positive cells in the neointima were reduced by dominant-negative ADAM17 as well. In contrast, the neointima formation, proliferating cell nuclear antigen-positive cells, and phospho-epidermal growth factor receptor-positive cells were markedly enhanced by wild-type ADAM17 adenovirus. In conclusion, ADAM17 activation is involved in epidermal growth factor receptor activation and subsequent neointimal hyperplasia after vascular injury. ADAM17 could be a novel therapeutic target for pathophysiological vascular remodeling.
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