title: A RANKL G278R mutation causing osteopetrosis identifies a functional amino acid essential for trimer assembly in RANKL and TNF.
abstract: Receptor activator of nuclear factor-κB ligand (RANKL), a trimeric tumor necrosis factor (TNF) superfamily member, is the central mediator of osteoclast formation and bone resorption. Functional mutations in RANKL lead to human autosomal recessive osteopetrosis (ARO), whereas RANKL overexpression has been implicated in the pathogenesis of bone degenerative diseases such as osteoporosis. Following a forward genetics approach using N-ethyl-N-nitrosourea (ENU)-mediated random mutagenesis, we generated a novel mouse model of ARO caused by a new loss-of-function allele of Rankl with a glycine-to-arginine mutation at codon 278 (G278R) at the extracellular inner hydrophobic F β-strand of RANKL. Mutant mice develop severe osteopetrosis similar to Rankl-deficient mice, whereas exogenous administration of recombinant RANKL restores osteoclast formation in vivo. We show that RANKL(G278R) monomers fail to assemble into homotrimers, are unable to bind and activate the RANK receptor and interact with wild-type RANKL exerting a dominant-negative effect on its trimerization and function in vitro. Since G278 is highly conserved within the TNF superfamily, we identified that a similar substitution in TNF, G122R, also abrogated trimerization, binding to TNF receptor and consequently impaired TNF biological activity. Notably, SPD304, a potent small-molecule inhibitor of TNF trimerization that interacts with G122, also inhibited RANKL activity, suggesting analogous inhibitory mechanisms. Our results provide a new disease model for ARO and identify a functional amino acid in the TNF-like core domain essential for trimer formation both in RANKL and in TNF that could be considered a novel potential target for inhibiting their biological activities.
authors: Institute of Immunology, Biomedical Sciences Research Center Alexander Fleming, Vari 16672, Greece. douni@fleming.gr
authors: Eleni
authors: E
authors: Douni
authors: Vagelis
authors: V
authors: Rinotas
authors: Eleni
authors: E
authors: Makrinou
authors: Jochen
authors: J
authors: Zwerina
authors: Josef M
authors: JM
authors: Penninger
authors: Elias
authors: E
authors: Eliopoulos
authors: Georg
authors: G
authors: Schett
authors: George
authors: G
authors: Kollias
mesh_terms: D019943:Amino Acid Substitution / Q000235:genetics*; D000818:Animals; D004195:Disease Models, Animal; D005038:Ethylnitrosourea; D005799:Genes, Dominant / Q000235:genetics; D051379:Mice; D020125:Mutation, Missense / Q000235:genetics; D010010:Osteoclasts / Q000166:cytology / Q000378:metabolism; D010022:Osteopetrosis / Q000139:chemically induced / Q000235:genetics*; D017354:Point Mutation / Q000235:genetics*; D011485:Protein Binding; D055503:Protein Multimerization / Q000235:genetics*; D053245:RANK Ligand / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D053246:Receptor Activator of Nuclear Factor-kappa B / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism*
chemical_list: D053245:RANK Ligand; D053246:Receptor Activator of Nuclear Factor-kappa B; C505687:Tnfrsf11a protein, mouse; C505689:Tnfsf11 protein, mouse; D014409:Tumor Necrosis Factor-alpha; D005038:Ethylnitrosourea