Structural requirements for signal-induced target binding of FADD determined by functional reconstitution of FADD deficiency
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Updated: Aug 17, 2026
High Precision FRET at Single-molecule Level for Biomolecule Structure Determination
Published on: May 13, 2017
Hongxia Z Imtiyaz1, Yuhang Zhang, Jianke Zhang
1Department of Microbiology and Immunology, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA.
Abstract:
FADD is a key adaptor modulating several signaling pathways such as apoptosis induced by Fas (CD95) and tumor necrosis factor receptor 1, and cell proliferation induced by mitogens. Whereas mutations in Fas disrupt its binding to FADD and cause autoimmune lymphoproliferative (lpr) syndromes, a FADD deficiency blocks embryonic development in mice. To delineate the multifunction of FADD in vivo, we performed functional reconstitution analysis by introducing wild type and mutant FADD into FADD-/- cells or FADD-/- mice lacking the endogenous FADD. An lpr-like FADD mutant, V121N, was reported previously as being defective in Fas binding in vitro. However, we found that in mice V121N can bind to Fas and is functional in signaling apoptosis. Unexpectedly, this lpr-like mutant FADD failed to support mouse development, indicating that the death domain of FADD has an additional function required for embryogenesis, which is independent of that required for receptor-induced apoptosis. Further mutagenesis was targeted at charged residues in the FADD death domain, presumably mediating electrostatic interactions with Fas. We showed that the target binding and apoptosis signaling functions of FADD were not affected when mutations were introduced to a majority of the charged residues. In one exception, replacing arginine 117 with an uncharged residue disrupted target binding and apoptosis signaling, but restoring the positive charge at position 117 failed to reconstitute the FADD function. Therefore, in vivo target binding of FADD involves an additional mechanism distinct from electrostatic interaction.
Insights
Fas-associated death domain (FADD) protein is crucial for embryonic development and apoptosis signaling. A specific FADD mutant supports apoptosis but fails in development, revealing a distinct function for embryogenesis.
Area of Science:
- Molecular and Cellular Biology
- Immunology
- Developmental Biology
Background:
- Fas-associated death domain (FADD) protein is a critical adaptor in Fas and TNF receptor signaling pathways, regulating apoptosis and cell proliferation.
- FADD deficiency in mice leads to embryonic lethality, while Fas mutations causing impaired FADD binding result in autoimmune lymphoproliferative (lpr) syndromes.
Purpose of the Study:
- To investigate the multifaceted roles of FADD in vivo by analyzing its function in FADD-deficient cells and mice.
- To elucidate the specific functions of the FADD death domain in receptor-induced apoptosis and embryonic development.
Main Methods:
- Functional reconstitution analysis using wild-type and mutant FADD introduced into FADD-/- cells and FADD-/- mice.
- Characterization of an lpr-like FADD mutant (V121N) for Fas binding and apoptosis signaling.
- Targeted mutagenesis of charged residues within the FADD death domain to assess their role in target binding and signaling.
Main Results:
- The lpr-like FADD mutant V121N binds to Fas and signals apoptosis in vivo, contrary to previous in vitro findings.
- Despite functional apoptosis signaling, V121N mutant FADD failed to rescue embryonic development, indicating a non-apoptotic role for FADD in embryogenesis.
- Mutations in most charged residues of the FADD death domain did not impair target binding or apoptosis signaling; however, R117 mutations disrupted these functions, and charge restoration failed to rescue activity, suggesting a mechanism beyond simple electrostatic interaction for FADD binding.
Conclusions:
- The FADD death domain possesses essential functions for embryogenesis that are independent of its role in receptor-induced apoptosis.
- In vivo FADD target binding involves a mechanism distinct from simple electrostatic interactions, highlighting novel aspects of FADD's molecular interactions.
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