Precise mapping of the CD95 pre-ligand assembly domain.

International audiencePre-association of CD95 at the plasma membrane is mandatory for efficient death receptor signaling. This homotrimerization occurs through self-association of an extracellular domain called the pre-ligand assembly domain (PLAD). Using novel molecular and cellular tools, we confirmed that CD95-PLAD is necessary to promote CD95 multimerization and plays a pivotal role in the transmission of apoptotic signals. However, while a human CD95 mutant deleted of the previously described PLAD domain (amino acids 1 to 66) fails to interact with its wild-type counterpart and trigger autonomous cell death, deletion of amino acids 1 to 42 does not prevent homo- or hetero (human/mouse)-oligomerization of CD95, and thus does not alter transmission of the apoptotic signal. Overall, these findings indicate that the region between amino acids 43 to 66 corresponds to the minimal motif involved in CD95 homotypic interaction and is necessary to convey an efficient apoptotic signal. Interfering with this PLAD may represent a new therapeutic strategy for altering CD95-induced apoptotic and non-apoptotic signals

Étude de la structure quaternaire du récepteur de mort Fas et développement d'outils moléculaires

Le récepteur Fas (CD95/APO1), un récepteur de mort de la famille des récepteurs au TNF (Tumor Necrosis factor), traduit un signal apoptotique essentiel à l'homéostasie du système immunitaire. Fas est exprimé sous forme préassociée homotrimérique à la membrane plasmique. La transmission du signal de mort serait associée à une augmentation du niveau d'agrégation et à un changement de conformation du récepteur lors de sa liaison avec son ligand naturel, le FasL. L'équipe du Dr Lenardo a mis en évidence que la pré-association du récepteur fait intervenir le domaine PLAD, constitué des 65 premiers acides aminés situés à l'extrémité amino-terminale du récepteur. Afin de définir la séquence Fas impliquée dans la formation de l'homotrimère, différents mutants Fas ont été générés et ces constructions ont été exprimées dans une cellule dépourvue de Fas. Contrairement aux résultats obtenus par le groupe du Dr Lenardo, nos résultats indiquent que les 42 premiers acides aminés ( (1-42)Fas) ne sont pas nécessaires à la formation de l'homotrimère. En revanche, un récepteur tronqué des 66 premiers aminoacides ( (1-66)Fas) ne transmet plus de signal apoptotique. Ces résultats suggèrent que le domaine PLAD serait restreint aux résidus 43 à 66 du récepteur. Ces résultats préliminaires devront être complétés par des études biochimiques de co-immunoprécipitation entre les récepteurs mutants et le récepteur sauvage.CLERMONT FD-BCIU-Santé (631132104) / SudocLYON1-BU Santé (693882101) / SudocSudocFranceF

A chimeric CD95-gp130 construct confirms the presence of a PLAD in the CD95 ectodomain. A.

<p>The intracellular domain of gp130 is pre-associated with JAK kinase. If the ectodomains of CD95 are pre-associated, the intracellular regions of a CD95-gp130 construct will be brought in close proximity, inducing the trans-activation of JAKs and the implementation of a pro-survival signal. <b>B.</b> IL3-addicted BaF3 cells were transfected with ΔExtra-gp130 or CD95-gp130. At 24 hours after transfection, living cells were incubated for 5 days with or without IL3. For each condition, pictures were taken at 10x magnification. Images are representative of five pictures taken in different fields. <b>C.</b> BaF3 cells were transfected with CD95-gp130 or empty vector (numbering indicates different clones). Stable clones were selected and the expression of CD95-gp130 was assessed by flow cytometry. MTT assay was used to assess the cell viability of BaF3 cells in medium deprived of IL3. <b>D.</b> BaF3 cells were treated or untreated with the indicated concentrations of cleaved CD95L in IL3-deprived medium and cell viability was quantified using MTT viability assay.</p

Another cellular model confirms that the PLAD does not encompass residues 1 to 42 of CD95. A

<p>. The anti-mouse CD95 mAb, clone Jo2, induces cell death by aggregating mouse CD95. If human CD95 forms a supernumerary and functional pre-associated hetero-complex with its mouse ortholog, the Jo2-mediated apoptotic signal will be enhanced. <b>B.</b> For each construct, two independent BaF3 clones were selected and the amount of human CD95 at the cell surface was assessed by flow cytometry. <b>C.</b> The BaF3 cells depicted in <b>B</b> were incubated for 24 hours with the indicated concentration of Jo2 and cell death was assessed by MTT assay. Results are given as the means ± SD of three independently performed experiments.</p

The PLAD is instrumental in transmission of the apoptotic signal and does not encompass residues 1 to 42 of CD95. A.

<p>CD95-deficient CEM-IRC cells were transfected with the indicated cDNA and living cells were isolated by Ficoll gradient and lysed. For each condition, 100 µg of protein was loaded per lane and immunoblot analysis was performed using an anti-CD95 mAb (C20). Data are representative of three independent experiments. <b>B.</b> Three independent CEM-IRC clones stably expressing CD95^{wild type}, CD95^(Δ1–42), CD95^(Δ1–66) CD95^(1–210) were selected and the amount of CD95 construct at the plasma membrane was analyzed by flow cytometry. <b>C.</b> CEM-IRC cells shown in <b>F</b> were incubated for 24 hours with the indicated concentrations of Ig-CD95L (left panel) or 7C11 (right panel). Cell death was assessed by MTT assay. Results are given as the means ± SD of three independently performed experiments.</p

The CD95 self-association domain covers the residues 43 to 66. A.

<p>Schematic representation of the different CD95-mCherry constructs. <b>B.</b> Supernatants from HEK cells transfected with the constructs depicted in <b>A</b> were fractionated by gel filtration using a S-300 HR column. Seventeen fractions were harvested and analyzed by immunoblot analysis (anti-DsRed). <b>C.</b> Densitometry analyses were performed on the immunoblots shown in <b>B</b> using Fiji software. For each construct, the estimated quaternary structure is depicted. <b>D.</b> Supernatants from HEK cells transfected with the constructs depicted in <b>A</b> were fractionated by gel filtration using a S-200 HR column. Twenty-one fractions were harvested and analyzed by immunoblot analysis (anti-DsRed). <b>E.</b> Densitometry analyses were performed on the immunoblots shown in <b>D</b> using Fiji software. For each construct, the estimated quaternary structure is depicted.</p