Two Adjacent Trimeric Fas Ligands Are Required for Fas Signaling and Formation of a Death-Inducing Signaling Complex

Holler, Nils; Tardivel, Aubry; Kovacsovics-Bankowski, Magdalena; Hertig, Sylvie; Gaide, Olivier; Martinon, Fabio; Tinel, Antoine; Deperthes, David; Calderara, Silvio; Schulthess, Therese; Engel, Jürgen; Schneider, Pascal; Tschopp, Jürg

doi:10.1128/mcb.23.4.1428-1440.2003

Cited by 361 publications

(353 citation statements)

References 51 publications

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“…Owing to the lack of the Flag epitope, we were unable to analyze receptor binding of mCD95L(137-279) directly by FACS. Instead, we performed a competition assay, in which the capacity of the non-tagged ligand to protect against cell death induction was determined using a highly active hexameric fusion protein of hCD95L (Fc-hCD95L) and the Fc domain of human IgG1 (see Holler et al 7 ) was determined. While preincubation of KB cells with 50 ng/ml Flag-hCD95L(137-281) was sufficient to rescue about half of the cells from Fc-hCD95L-induced cell death, the non-tagged mCD95L(137-279) failed even at high concentrations (1 mg/ml) to show a protective effect (Figure 3c).…”

Section: Resultsmentioning

confidence: 99%

“…The Next day, cells were treated in triplicates with the indicated concentrations of the various variants of CD95L or TRAIL in the presence and absence of the crosslinking 0.5 mg/ml anti-Flag mAb M2. Holler et al 7 have previously shown that antiFlag-induced multimerization becomes suboptimal at higher ligand concentrations. In our experiments, this becomes evident especially when TRAIL variants were used.…”

Section: Methodsmentioning

confidence: 99%

“…Likewise, hexameric and dodecameric fusion proteins of soluble CD95L and soluble CD40L as well as non-specifically aggregated preparations of TNF ligands produced in E. coli display high activity. [6][7][8] The structural hallmark of the ligands of the TNF family is the carboxy-terminal 'TNF homology domain', which is part of both the transmembrane and soluble forms of TNF ligands. [1][2] The TNF homology domains (THDs) of the various TNF ligands are composed of a framework of aromatic and hydrophobic residues that adopt an almost identical tertiary fold and cause self association into trimers.…”

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confidence: 99%

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Enforced covalent trimerization increases the activity of the TNF ligand family members TRAIL and CD95L

et al. 2007

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Variants of human TRAIL (hTRAIL) and human CD95L (hCD95L), encompassing the TNF homology domain (THD), interact with the corresponding receptors and stimulate CD95 and TRAILR2 signaling after cross-linking. The murine counterparts (mTRAIL, mCD95L) showed no or only low receptor binding and were inactive/poorly active after cross-linking. The stalk region preceding the THD of mCD95L conferred secondary aggregation and restored CD95 activation in the absence of cross-linking. A corresponding variant of mTRAIL, however, was still not able to activate TRAIL death receptors, but gained good activity after cross-linking. Notably, disulfide-bonded fusion proteins of the THD of mTRAIL and mCD95L with a subdomain of the tenascin-C (TNC) oligomerization domain, which still assembled into trimers, efficiently interacted with their cognate cellular receptors and robustly stimulated CD95 and TRAILR2 signaling after secondary cross-linking. Introduction of the TNC domain also further enhanced the activity of THD encompassing variants of hTRAIL and hCD95L. Thus, spatial fixation of the N-terminus of the THD appears necessary in some TNF ligands to ensure proper receptor binding. This points to yet unanticipated functions of the stalk and/or transmembrane region of TNF ligands for the functionality of these molecules and offers a broadly applicable option to generate recombinant soluble ligands of the TNF family with superior activity. Ligands of the tumor necrosis factor (TNF) family fulfill crucial roles in the immune system, but have also been implicated in the development of epithelial and endothelial structures. 1 TNF family ligands are primarily expressed as trimeric type II transmembrane proteins and are often processed into soluble variants that are also organized as trimers. 1,2 Although shedding of some TNF ligands does not interfere with their capability to activate their corresponding receptors and might be even important for their physiological function, other TNF ligands become inactivated by proteolytic processing. 2 Soluble TNF ligands that are not or only poorly active still interact with their cognate receptors. For example, the soluble forms of TNF, CD95L, TRAIL, and CD40L interact with TNFR2, CD95, TRAILR2, and CD40, respectively, but do not or only poorly activate signaling by these receptors. [3][4][5][6] Notably, inactive or poorly active soluble TNF ligands can be converted into highly active molecules by artificially increasing their avidity. For example, soluble flag-tagged variants of TNF, CD95L, TRAIL, and CD40L stimulate robust signaling by TNFR2, CD95, TRAILR2, and CD40, respectively, provided they were cross-linked with the Flag-specific mAb M2. Likewise, hexameric and dodecameric fusion proteins of soluble CD95L and soluble CD40L as well as non-specifically aggregated preparations of TNF ligands produced in E. coli display high activity. [6][7][8] The structural hallmark of the ligands of the TNF family is the carboxy-terminal 'TNF homology domain', which is part of both the transmembrane and s...

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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confidence: 99%

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Enforced covalent trimerization increases the activity of the TNF ligand family members TRAIL and CD95L

et al. 2007

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“…There are 2 models in support of this proposition. The first model is suggested by the observation that the soluble form of Fas ligand has significantly reduced activity compared with its transmembrane form in the induction of apoptosis (42). The second (43) is based on the difference between mechanisms of tolerance induction in B cells in mice transgenic for soluble, as compared with membrane, hen egg lysozyme.…”

Section: Discussionmentioning

confidence: 99%

Transmembrane BAFF from rheumatoid synoviocytes requires interleukin‐6 to induce the expression of recombination‐activating gene in B lymphocytes

Rochas¹,

Hillion²,

Saraux³

et al. 2009

Arthritis & Rheumatism

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“…10,11 For both membrane-bound and sFasL, a spontaneous trimer formation has been suggested, which seems indispensable for triggering the death signal via Fas oligomers. 12 sFasL was found in sera of patients with large granular lymphocytic leukemia and NK cell lymphoma. 13 A number of more recent reports point to a correlation of sFasL serum levels with disease progression, for example, during the asymptomatic stage of HIV infection, in rheumatoid arthritis and osteoarthritis, in ulcerative colitis, and a large number of malignancies.…”

Section: For Review)mentioning

confidence: 98%

ADAM10 regulates FasL cell surface expression and modulates FasL-induced cytotoxicity and activation-induced cell death

et al. 2007

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The apoptosis-inducing Fas ligand (FasL) is a type II transmembrane protein that is involved in the downregulation of immune reactions by activation-induced cell death (AICD) as well as in T cell-mediated cytotoxicity. Proteolytic cleavage leads to the generation of membrane-bound N-terminal fragments and a soluble FasL (sFasL) ectodomain. sFasL can be detected in the serum of patients with dysregulated inflammatory diseases and is discussed to affect Fas-FasL-mediated apoptosis. Using pharmacological approaches in 293T cells, in vitro cleavage assays as well as loss and gain of function studies in murine embryonic fibroblasts (MEFs), we demonstrate that the disintegrin and metalloprotease ADAM10 is critically involved in the shedding of FasL. In primary human T cells, FasL shedding is significantly reduced after inhibition of ADAM10. The resulting elevated FasL surface expression is associated with increased killing capacity and an increase of T cells undergoing AICD. Overall, our findings suggest that ADAM10 represents an important molecular modulator of FasL-mediated cell death.

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Two Adjacent Trimeric Fas Ligands Are Required for Fas Signaling and Formation of a Death-Inducing Signaling Complex

Cited by 361 publications

References 51 publications

Enforced covalent trimerization increases the activity of the TNF ligand family members TRAIL and CD95L

Enforced covalent trimerization increases the activity of the TNF ligand family members TRAIL and CD95L

Transmembrane BAFF from rheumatoid synoviocytes requires interleukin‐6 to induce the expression of recombination‐activating gene in B lymphocytes

ADAM10 regulates FasL cell surface expression and modulates FasL-induced cytotoxicity and activation-induced cell death

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