Programmed cell death (apoptosis) mediated by the cytokine receptor Fas is critical for the removal of autoreactive T cells, the mechanism of immune privilege, and for maintenance of immune-system homeostasis. Signalling of programmed cell death involves the self-association of a conserved cytoplasmic region of Fas called the death domain and interaction with another death-domain-containing protein, FADD (also known as MORT1). Although death domains are found in several proteins, their three-dimensional structure and the manner in which they interact is unknown. Here we describe the solution structure of the Fas death domain, as determined by NMR spectroscopy. The structure consists of six antiparallel, amphipathic alpha-helices arranged in a novel fold. From the structure and from site-directed mutagenesis, we have identified the region of the death domain involved in self-association and binding to the downstream signalling partner FADD.
When activated, membrane-bound receptors for Fas and tumour-necrosis factor initiate programmed cell death by recruiting the death domain of the adaptor protein FADD to the membrane. FADD then activates caspase 8 (also known as FLICE or MACH) through an interaction between the death-effector domains of FADD and caspase 8. This ultimately leads to the apoptotic response. Death-effector domains and homologous protein modules known as caspase-recruitment domains have been found in several proteins and are important regulators of caspase (FLICE) activity and of apoptosis. Here we describe the solution structure of a soluble, biologically active mutant of the FADD death-effector domain. The structure consists of six antiparallel, amphipathic alpha-helices and resembles the overall fold of the death domains of Fas and p75. Despite this structural similarity, mutations that inhibit protein-protein interactions involving the Fas death domain have no effect when introduced into the FADD death-effector domain. Instead, a hydrophobic region of the FADD death-effector domain that is not present in the death domains is vital for binding to FLICE and for apoptotic activity.
Our previous studies have demonstrated that a generation 5 dendrimer (G5) conjugated with both folic acid (FA) and methotrexate (MTX) has a higher chemotherapeutic index than MTX alone. Despite this, batch-to-batch inconsistencies in the number of FA and MTX molecules linked to each dendrimer led to conjugate batches with varying biological activity, especially when scaleup synthesis was attempted. Since the MTX is conjugated through an ester linkage, there were concerns that biological inconsistency could also result from serum esterase activity and differential bioavailability of the targeted conjugate. In order to resolve these problems, we undertook a novel approach to synthesize a polyvalent G5–MTXn conjugate through click chemistry, attaching the MTX to the dendrimer through an esterase-stable amide linkage. Surface plasmon resonance binding studies show that a G5–MTX10 conjugate synthesized in this manner binds to the FA receptor (FR) through polyvalent interaction showing 4300-fold higher affinity than free MTX. The conjugate inhibits dihydrofolate reductase, and induces cytotoxicity in FR-expressing KB cells through FR-specific cellular internalization. Thus, the polyvalent MTX on the dendrimer serves the dual role as a targeting molecule as well as a chemotherapeutic drug. The newly synthesized G5–MTXn conjugate may serve as a FR-targeted chemotherapeutic with potential for cancer therapy.
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