title: Inhibition of osteoclast bone resorption by disrupting vacuolar H+-ATPase a3-B2 subunit interaction.
abstract: Vacuolar H(+)-ATPases (V-ATPases) are highly expressed in ruffled borders of bone-resorbing osteoclasts, where they play a crucial role in skeletal remodeling. To discover protein-protein interactions with the a subunit in mammalian V-ATPases, a GAL4 activation domain fusion library was constructed from an in vitro osteoclast model, receptor activator of NF-κB ligand-differentiated RAW 264.7 cells. This library was screened with a bait construct consisting of a GAL4 binding domain fused to the N-terminal domain of V-ATPase a3 subunit (NTa3), the a subunit isoform that is highly expressed in osteoclasts (a1 and a2 are also expressed, to a lesser degree, whereas a4 is kidney-specific). One of the prey proteins identified was the V-ATPase B2 subunit, which is also highly expressed in osteoclasts (B1 is not expressed). Further characterization, using pulldown and solid-phase binding assays, revealed an interaction between NTa3 and the C-terminal domains of both B1 and B2 subunits. Dual B binding domains of equal affinity were observed in NTa, suggesting a possible model for interaction between these subunits in the V-ATPase complex. Furthermore, the a3-B2 interaction appeared to be moderately favored over a1, a2, and a4 interactions with B2, suggesting a mechanism for the specific subunit assembly of plasma membrane V-ATPase in osteoclasts. Solid-phase binding assays were subsequently used to screen a chemical library for inhibitors of the a3-B2 interaction. A small molecule benzohydrazide derivative was found to inhibit osteoclast resorption with an IC(50) of ∼1.2 μm on both synthetic hydroxyapatite surfaces and dentin slices, without significantly affecting RAW 264.7 cell viability or receptor activator of NF-κB ligand-mediated osteoclast differentiation. Further understanding of these interactions and inhibitors may contribute to the design of novel therapeutics for bone loss disorders, such as osteoporosis and rheumatoid arthritis.
authors.affiliation: Dental Research Institute, Faculty of Dentistry, University of Toronto, Toronto, Ontario M5G 1G6 Canada.
authors.forename: Norbert
authors.initials: N
authors.lastname: Kartner
authors.forename: Yeqi
authors.initials: Y
authors.lastname: Yao
authors.forename: Keying
authors.initials: K
authors.lastname: Li
authors.forename: Gazelle J
authors.initials: GJ
authors.lastname: Crasto
authors.forename: Alessandro
authors.initials: A
authors.lastname: Datti
authors.forename: Morris F
authors.initials: MF
authors.lastname: Manolson
mesh_terms: D000818:Animals; D001862:Bone Resorption / Q000201:enzymology* / Q000235:genetics; D002460:Cell Line; D015536:Down-Regulation; D006801:Humans; D051379:Mice; D010010:Osteoclasts / Q000737:chemistry / Q000201:enzymology*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D020798:Two-Hybrid System Techniques; D025262:Vacuolar Proton-Translocating ATPases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: C484226:Atp6ap1 protein, mouse; D025262:Vacuolar Proton-Translocating ATPases