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Inhibitory role of TACE/ADAM17 cytotail in protein ectodomain shedding.

AIM
To determine if the cytotail of the principal sheddase tumor necrosis factor-α converting enzyme (TACE; ADAM17) controls protein ectodomain shedding.


METHODS
Site-directed mutagenesis was performed to derive TACE variants. The resulting TACE expression plasmids with amino acid substitutions in the extracellular, cysteine-rich disintegrin domain (CRD) and/or deleted cytotail, along with an expression vector for the enhanced green fluorescence protein were transfected into shedding-defective M1 mutants stably expressing transmembrane L-selectin or transforming growth factor (TGF)-α. The expression levels of the TACE substrates at the cell surface were determined by flow cytometry.


RESULTS
Consistent with published data, a single point mutation (C600Y) in the CRD led to shedding deficiency. However, removal of the cytotail from the C600Y TACE variant partially restored ectodomain cleavage of TGF-α and L-selectin. Cytotail-deleted mutants with any other substituting amino acid residues in place of Cys600 displayed similar function compared with tail-less C600Y TACE.


CONCLUSION
The cytotail plays an inhibitory role, which becomes evident when it is removed from an enzyme with another mutation that affects the enzyme function.


INTRODUCTION
Protein ectodomain shedding serves as an important means for regulating the function of cell surface proteins required for a variety of physiological processes [1] . For example, generation of freely diffusible epidermal growth factor receptor (EGFR) ligands including transforming growth factor (TGF)-α from their transmembrane precursors is essential for the development of multiple organs [2][3][4] . Ectodomain shedding is also crucial for pathogenesis. Thus, overproduction of soluble EGFR ligands causes cellular transformation [5] ; generation of the cir- Li X et al . Inhibitory TACE/ADAM17 cytotail culating cytokine tumor necrosis factor (TNF)-α from its transmembrane precursor is responsible for cachexia, septic shock and other inflammatory conditions [6][7][8] . TNF-α converting enzyme (TACE) is a principal sheddase that cleaves not only transmembrane TNF-α, but also a large number of other membrane proteins [9] . As a member of the large family of a disintegrin and metalloprotease domains (ADAM), TACE or ADAM17 is a membrane-anchored metalloprotease ( Figure 1) [10,11] . The protease is biosynthesized as a zymogen, in which the catalytic domain is led by an amino-terminal prodomain, and followed in succession by a cysteine-rich disintegrin domain (CRD), a transmembrane segment and a carboxyl terminal cytotail. The TACE zymogen is enzymatically inactive because the prodomain interacts with the active site, causing inaccessibility to protein substrates.

BRIEF ARTICLE
Ectodomain shedding by TACE is a tightly regulated process. Accordingly, a variety of stimuli including growth factors, inflammatory mediators, ionophores, carcinogens and tumor promoters can induce shedding [12][13][14] . However, the cellular and molecular mechanism that controls TACEmediated shedding remains unclear. Although prodomain removal is a prerequisite for TACE to gain catalytic activity [15][16][17] , it does not seem sufficient for shedding activation, because an increase in prodomain removal is not observed following stimulation [11] . Therefore, activation of shedding appears to be through modulation of mature TACE.
It has been established that signaling pathways involving two mitogen-activated protein kinases (MAPKs), Erk and p38, mediate the activation of shedding in response to various stimuli [12,13,18] . The serine-and threonine-rich TACE cytotail is suspected to play a role in the regulation of TACE function. In particular, both MAPKs have been shown to phosphorylate directly the TACE cytotail at Thr-735 [18][19][20] . Erk activity-dependent phosphorylation at Ser-819 has also been demonstrated. Furthermore, Ser-791 is phosphorylated in resting cells, and undergoes dephosphorylation in response to growth factor stimulation [21] . However, mutation of these phosphorylation sites individually or in combination, and even removal of the entire cytotail have no detectable effects on shedding [21][22][23][24] . Thus, the function of the TACE cytotail remains illusive.
There is also evidence suggesting a role for the CRD in TACE-mediated shedding [9,[24][25][26] . We have previously demonstrated that a substitution (C600Y) within the CRD results in enzyme inactivity [9] . Interestingly, in an attempt to examine the function of other cysteines in the CRD, we found that deletion of the cytotail partially restores the shedding activity in the C600Y TACE variant. This finding suggests an inhibitory role for the cytotail in ectodomain shedding and resolves a long mystery with regarding the function of the TACE cytotail, which becomes apparent only when there is another defect in the enzyme.

Cell lines and culture conditions
The shedding-defective M1-L-selectin and M1-TGF-α cells, which overexpress transmembrane L-selectin and TGF-α, respectively, have been described previously [22] . The cell lines were maintained as adherent cultures using DMEM supplemented with 8% FBS and the antibiotics penicillin and streptomycin.

Assays of L-selectin and TGF-α ectodomain shedding
The enzyme activity of wild-type or mutant TACE was determined with cell-based shedding assays using twocolor flow cytometry as described previously [28] . M1-Lselectin and M1-TGF-α cells were transiently cotransfected with a TACE expression vector (or the control RK5 vector) and a mammalian expression plasmid for 247 November 26, 2011|Volume 2|Issue 11| WJBC|www.wjgnet.com enhanced green fluorescence protein (GFP) at a ratio of 3:1. Twenty hours after transfection, cell culture plates were placed on ice and the medium was replaced with cold PBS, supplemented with 10 mmol/L 1, 10-phenanthroline (a metalloproteases inhibitor), 5 mmol/L EDTA, 2% BSA and 0.1% NaN3 (PEB). Cells were scraped off the plates with a Cell Lifter, collected and centrifuged at 1000 r/min at 4 ℃ for 5 min. Following the removal of the supernatant, cells were resuspended in 50 μL PEB containing 500 ng DREG56 (anti-L-

RESULTS
Previously, we have identified a TACE variant carrying a C600Y substitution within the CRD from a sheddingdefective cell line. Cell-based shedding assays confirmed that the variant sheddase lacks the capacity to cleave substrates [9] . On the basis of the tertiary structure of the CRD of ADAM10, which share a high level of sequence homology to that of TACE [9] , Cys-600 forms a disulfide bridge with Cys-593; also, there may be additional 12 disulfide bridges [29] . To determine if other cysteines and/or disulfide bridges are also important for TACE function, we replaced each of the remaining cysteine in the CRD with alanine. For the convenience of vector construction, the C→A mutants were constructed on the ΔC TACE backbone because previous studies have shown that deletion of the cytotail does not affect TACE function [21,23,24] . The enzyme activities of the resulting ΔC/C→A variants were determined in the M1-L-selectin cell line, which overexpresses transmembrane L-selectin but is defective in ectodomain shedding, due to the loss of one TACE allele and a mutation in the other allele resulting in an M435I substitution at the active site [9,28] . Contrary to our prediction, all the ΔC/C→A variants were largely active in L-selectin shedding (Figure 2). Particularly surprising was the L-selectin shedding activity exhibited by the ΔC/C600A variant, because we have previously shown that ΔC/C600A and ΔC/C600Y have no detectable enzyme activity, similar to the full-length C600Y TACE [9] . In that study, the inactivity of C600Y TACE was demonstrated in all three TACE-defective cell lines, with multiple substrates, and using different detec-  Figure. The activity of L-selectin shedding in the TACE constructs was determined as in Figure 2. Transforming growth factor (TGF)-α shedding was determined in a similar manner except the M1-L-selectin cells were replaced with shedding-defective M1-TGF-α cells, which overexpress transmembrane TGF-α, and the anti-Lselectin was substituted with anti-TGF-α. tive methods; however, the ΔC/C600A and ΔC/C600Y constructs were tested only with one TACE substrate using one-color flow cytometry, which cannot discriminate TACE-expressing cells from untransfected cells [9] . Therefore, we believe that the data showing enzymatic inactivity in the ΔC/C600A and ΔC/C600Y constructs were erroneous. In contrast, the shedding-proficiency in the ΔC/C600A mutant, as demonstrated using two-color flow cytometry in which eGFP served as a surrogate marker for TACE expression (Figure 2), ought to be considered reliable.
We next compared the enzyme activities of the fulllength C600Y TACE, the ΔC/C600Y construct, and their respective parental forms (i.e. wild-type TACE and ΔC TACE). Consistent with published data, ΔC TACE demonstrated levels of enzyme activity that were similar to those in wild-type TACE, in the cleavage of transmembrane L-selectin and TGF-α, whereas full-length C600Y TACE completely failed to cleave these substrates ( Figure 3). Evidently, the ΔC/C600Y construct partially regained L-selectin and TGF-α shedding activities, as compared to the C600Y variant ( Figure 3). These results indicate that the cytotail plays an inhibitory role in ectodomain shedding, which became detectable when there was another defect in the enzyme.

DISCUSSION
TACE is responsible for ectodomain shedding of numerous membrane proteins and it is required for a variety of physiological processes, but how its activity is regulated remains unsatisfactorily defined. It was thought, immediately after the sequencing of the TACE cDNA, that the cytotail of the sheddase plays a critical role in the enzyme activity [11] . However, this hypothesis has been questioned because removal of the entire cytotail has demonstrated no detectable effect on TACE-dependent shedding [21][22][23]28] . The lack of an apparent effect of the cytotail deletion (in wild-type TACE) was again reproduced in this work. Nevertheless, removal of the cytotail from the sheddingdefective C600Y TACE variant clearly led to a substantial restoration of enzyme activity. Thus, the cytotail plays an inhibitory role, which becomes noticeable only in the presence of another defect in the enzyme.
How the cytotail exerts an inhibitory activity in shedding has yet to be defined. It is known that the cytotail is phosphorylated in response to activators of ectodomain shedding [11,[18][19][20][21] . We speculate that the phosphorylation alleviates the inhibitory activity of the cytotail, which is also achievable by the deletion of the cytotail.
Cys-600 has been predicted to participate in the formation of a disulfide bridge [29] . Interestingly, recent studies have indicated that agents that affect the redox potential regulate TACE activity [24,25] . Furthermore, protein disulfide isomerase has been shown to inhibit TACE activity through modifying the conformation of the CRD [25] . The fact that the inhibitory role of the cytotail was discovered in the context of Cys-600 mutations sug-gests that the cytotail of TACE regulates disulfide bridging and thus the conformation of the CRD.
In conclusion, our study, aimed at furthering the understanding of how the CRD regulates the function of TACE, has led to the recognition of a new, inhibitory role for the cytotail in ectodomain shedding. However, the original question about which, if any, cysteines other than Cys-600 in the CRD are critical for the enzyme activity of TACE has yet to be answered using full-length constructs.

ACKNOWLEDGMENTS
This work was presented to the Gordon Research Conference on Regulated Proteolysis of Cell Surface Proteins held in Davidson College, NC, United States in July, 2011.

Background
Tumor necrosis factor-α converting enzyme (TACE) is responsible for the cleavage of numerous membrane proteins at the cell surface. The enzyme activity of TACE is tightly regulated by intracellular signaling pathways. The carboxylterminal cytotail has long been thought to function as a signaling transducer in the regulation of TACE function. However, deletion of the cytotail so far has not shown a detectable effect on protein ectodomain shedding.

Research frontiers
In addition to normal development, TACE is required for the pathogenesis of diseases including inflammation and autoimmunity. Identification of new domain function in TACE has implications for molecular cell physiology and pathophysiology, and also may help development of new therapeutic agents.

Innovations and breakthroughs
The present study identifies an inhibitory role for the TACE cytotail. This new function is detectable when the cytotail is removed from a TACE construct with a mutation in the cysteine-rich disintegrin domain, which adversely affects the enzyme function. Previous studies have failed to recognize this important role because the cytotail-truncation mutants used in those studies did not have an additional mutation.

Peer review
Despite a limited number of data presented by the authors, the newly identified function of the cytotail is both interesting and of potential significance. The introduction was somewhat unnecessarily detailed, and a few places could be revised for enhanced clarification.