Biochemical and Biophysical Research Communications
Inactivating mutations block the tumor necrosis factor-α-converting enzyme in the early secretory pathway☆
Section snippets
Materials and methods
Antibodies and reagents. Polyclonal antibodies against the extracellular domain of TACE were kindly provided by Dr. Carl Blobel. The polyclonal antibodies directed against the cytoplasmic domain of TACE have been previously described [26]. The monoclonal anti-βAPP antibodies 22C11, mouse IgM anti-actin (Ab-1), and mouse anti-HER2 (CB-11) were from Chemicon International, Oncogene research products, and Biogenex, respectively. Sulfo-NHS-LC-Biotin, immobilized neutravidin, and the SuperSignal
Permanently transfected TACE is processed and transported to the cell surface in M2 cells
Although automatic sequencing of the cDNA encoding TACE from M2 cells did not show the presence of mutations [16], a subsequent systematic scrutiny of the electropherograms showed that M2 mutants express two mutant forms of TACE that result in Cys 225 to Tyr and Cys 600 to Tyr substitutions, respectively (see correction to [16] and Fig. 1). According to the predicted structure of TACE, Cys 225 and Cys 600 form disulfide bonds within the metalloprotease and cysteine-rich domains of TACE,
Discussion
Given the practical importance of protein ectodomain shedding, several years ago we isolated and characterized somatic cell mutants defective in the PMA-activated shedding of the transmembrane growth factor proTGF-α [23], [24]. Subsequently, the ADAM protease TACE was found to be responsible for the shedding of proTGF-α [11]. Previous transient transfection and cell fusion experiments indicated that the component mutated in the shedding-defective cell lines M1 and M2 was different from TACE [25]
Acknowledgements
We thank Dr. Huizhou Fan for helpful discussions on the sequence of TACE from M2 cells. We are also grateful to Dr. Carl Blobel for reagents and constructive suggestions and Drs. Anna Merlos-Suárez and Aldo Borroto Revuelta for critically reading the manuscript. T.V.-T. is the recipient of fellowships from the Instituto de Salud Carlos III. This work was supported by grants from La Marató de TV3, Fondo de Investigación Sanitaria (PI021003), and the EMBO Young Investigator Programme (YIP) to
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2010, Journal of Biological ChemistryCitation Excerpt :24 h after transfection, we observed a significantly lower ADAM10 expression in the presence of the 5′-UTR (Fig. 1A). Consistent with previous findings for ADAM10 and ADAM17, we predominantly detected the immature form of ADAM10 upon overexpression (12, 44–46). Quantification of ADAM10 levels revealed a 3-fold increase of ADAM10 protein in cells transfected with the cDNA construct lacking the 5′-UTR compared with the construct with the 5′-UTR of ADAM10 (Fig. 1B).
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2007, Journal of Biological ChemistryCitation Excerpt :In mammary tumor samples and matched controls, we do not observe a correlation between ADAM17 mRNA and protein levels, leading us to conclude that the levels of active ADAM17 can be post-transcriptionally up-regulated in vivo. Even a modest increase in the levels of ADAM17 mRNA leads to the specific accumulation of the proform of the metalloprotease (see this report and Refs. 27 and 30). Therefore, there seems to exist a mechanism that restricts the levels of active ADAM17, even when the transcription of the gene is up-regulated.
Loss of ectodomain shedding due to mutations in the metalloprotease and cysteine-rich/disintegrin domains of the tumor necrosis factor-α converting enzyme (TACE)
2004, Journal of Biological ChemistryCitation Excerpt :This suggests that the conserved cysteine may be required for the function of ADAM10 as well. After our release of the mutant sequences on September 2, 2004, Villanueva et al. (76) later published a report documenting the existence of the same mutations in M1 and M2 cell lines, while this article was under review. Although their work demonstrated that all the mutant alleles are defective in TGF-α release, there are several important discrepancies between this earlier report and our findings.
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Supplementary data associated with this article can be found, in the online version, at 10.1016/j.bbrc.2003.12.186.