Inhibition of Osteoclast Bone Resorption by Disrupting Vacuolar H+-ATPase a3-B2 Subunit Interaction

Norbert Kartner,Yeqi Yao,Keying Li,Gazelle J Crasto,Alessandro Datti,Morris F Manolson

doi:10.1074/jbc.m110.123281

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.

Highlights

DAP-02 and Canadian Institutes for Health Research Grant MOP-79322 and PDD-86132. 1 To whom correspondence should be addressed: Faculty of Dentistry, University of Toronto, 124 Edward St., Toronto, Ontario M5G 1G6, Canada
Landolt-Marticorena et al [25] have shown previously, using yeast two-hybrid analysis, that the a subunit of the V0 sector interacts with the A and H subunits of the V1 sector, interactions that are thought to contribute to the stator complex that is required for the function of the V-ATPase molecular motor [26, 27]
Further understanding of the structure and function of V-ATPases will aid the development of targeted therapeutics for the treatment of bone loss diseases, such as osteoporosis and rheumatoid arthritis [13]

Summary

Introduction

DAP-02 and Canadian Institutes for Health Research Grant MOP-79322 and PDD-86132. 1 To whom correspondence should be addressed: Faculty of Dentistry, University of Toronto, 124 Edward St., Toronto, Ontario M5G 1G6, Canada. The initial intention of this work was to determine the set of protein binding partners that interact with the a3 subunit that is highly expressed as part of the plasma membrane V-ATPase of osteoclasts.

Results

Conclusion