Fas/TNFRSF6/CD95 Overexpression Lysate

HEK293T cells in 10-cm dishes were transiently transfected with a non-lipid polymer transfection reagent specially designed and manufactured for large volume DNA transfection. Transfected cells were cultured for 48hrs before collection. The cells were lysed in modified RIPA buffer (25mM Tris-HCl pH7.6, 150mM NaCl, 1% NP-40, 1mM EDTA, 1xProteinase inhibitor cocktail mix, 1mM PMSF and 1mM Na3VO4, and then centrifuged to clarify the lysate. Protein concentration was measured by BCA protein assay kit.

Tumor Necrosis Family Receptor (TNFR) superfamily member Fas, also known as CD95, APO-1, and TNFRSF6, is a 40-50 kDa type I transmembrane glycoprotein that is traditionally considered a death receptor but also functions in non-apoptotic signaling (1-4). The human Fas/TNFRSF6/CD95 protein is encoded by the FAS gene which contains 9 exons and is located on chromosome 10 (10q23.3-4) (1,2). The mature canonical Fas/TNFRSF6 protein isoform is 335 aa in length, which includes the signal sequence, and has a theoretical molecular weight of 37.7 kDa (1,5). The protein contains an extracellular domain (ECD) consisting of three calcium rich domains (CRDs), a transmembrane domain (TM), and an intracellular domain (ICD) comprised of a calcium-inducing domain (CID) and characteristic dead domain (DD) (1,2,5,6). The Fas protein is expressed on the plasma membrane of activated lymphocytes as a homotrimer formed via CRD1 interactions (1,2,3,6). The DD is crucial for apoptotic signaling which is triggered by the Fas receptor binding its ligand, Fas ligand (FasL) (1,2,6,7). Upon Fas-FasL interaction, the DD recruits an adapter protein Fas-associated DD (FADD) and procaspase-8, generating the death-inducing signaling complex (DISC) (1-4,6-8). Formation of DISC activates caspase-8 and leads to cleavage of caspase-3, initiating a caspase-signaling cascade and cell death (1-4,6-8).

Fas-FasL-mediated apoptosis is important in immune homeostasis and removal of autoreactive T cells, autoreactive B cells, cytotoxic natural killer (NK) cells, and more (1,2,7). Dysfunction and mutations in the Fas receptor and the Fas-FasL signaling axis is associated a loss of apoptotic signaling and removal of autoreactive cells, which correlates with several autoimmune diseases including systemic lupus erythematosus (SLE), autoimmune lymphoproliferative syndrome (ALPS), and multiple sclerosis (MS) (1-4,6,7). In addition to apoptosis and cell death signaling, FasL/TNFRSF6/CD95 mediates other pathways involved in proliferation, survival, and differentiation (3,4,6,8). More specifically, Fas has been shown to activate the NF-kappaB pathway, driving innate immunity which includes IL-1beta production and functioning in host defense (3,4,6,8). Fas is also involved in adaptive immunity playing a role in co-stimulation of CD4+ and CD8+ T cell activation as well as precocious differentiation of naive cells to effector memory T cells (3,4,6). Differentiation into effector memory T cells shows protection against autoimmunity but also limits antitumor response to a form of cancer immunotherapy called adoptive cell transfer (ACT) (3,4). The non-apoptotic roles of the Fas/TNFRSF6/CD95 receptor highlight its potential as a target for both treating autoimmune diseases and in cancer immunotherapy (3,4).

References

1. Singh R, Pradhan V, Patwardhan M, Ghosh K. APO-1/Fas gene: Structural and functional characteristics in systemic lupus erythematosus and other autoimmune diseases. Indian J Hum Genet. 2009;15(3):98-102. https://doi.org/10.4103/0971-6866.60184

2. Magerus A, Bercher-Brayer C, Rieux-Laucat F. The genetic landscape of the FAS pathway deficiencies. Biomed J. 2021;44(4):388-399. https://doi.org/1010.1016/j.bj.2021.06.005

3. Guegan JP, Legembre P. Nonapoptotic functions of Fas/CD95 in the immune response. FEBS J. 2018;285(5):809-827. https://doi.org/10.1111/febs.14292

4. Yi F, Frazzette N, Cruz AC, Klebanoff CA, Siegel RM. Beyond Cell Death: New Functions for TNF Family Cytokines in Autoimmunity and Tumor Immunotherapy. Trends Mol Med. 2018;24(7):642-653. https://doi.org/10.1016/j.molmed.2018.05.004

5. Uniprot (P25445)

6. Guegan JP, Ginestier C, Charafe-Jauffret E, et al. CD95/Fas and metastatic disease: What does not kill you makes you stronger. Semin Cancer Biol. 2020;60:121-131. https://doi.org/10.1016/j.semcancer.2019.06.004

7. Volpe E, Sambucci M, Battistini L, Borsellino G. Fas-Fas Ligand: Checkpoint of T Cell Functions in Multiple Sclerosis. Front Immunol. 2016;7:382. Published 2016 Sep 27. https://doi.org/10.3389/fimmu.2016.00382

8. Cullen SP, Martin SJ. Fas and TRAIL 'death receptors' as initiators of inflammation: Implications for cancer. Semin Cell Dev Biol. 2015;39:26-34. https://doi.org/10.1016/j.semcdb.2015.01.012

This product is for research use only and is not approved for use in humans or in clinical diagnosis. Lysates are guaranteed for 6 months from date of receipt.

Array NBL1-10599

• Fas/TNFRSF6/CD95 Antibodies
• Fas/TNFRSF6/CD95 Antibody Pair
• Fas/TNFRSF6/CD95 ELISA Kits
• Fas/TNFRSF6/CD95 Lysates
• Fas/TNFRSF6/CD95 Proteins
• Fas/TNFRSF6/CD95 Proteins and Enzymes
• Fas/TNFRSF6/CD95 ELISA