Identification of a transporter complex responsible for the cytosolic entry of nitrogen-containing bisphosphonates.

Zhou Yu,Jonathan S Weissman,Chong Yon Park,David M Sabatini,Erin K O'Shea,Timothy R Peterson,Max A Horlbeck,Gregory A Wyant,Lauren E Surface,Monther Abu-Remaileh

doi:10.7554/elife.36620

Abstract

Nitrogen-containing-bisphosphonates (N-BPs) are a class of drugs widely prescribed to treat osteoporosis and other bone-related diseases. Although previous studies have established that N-BPs function by inhibiting the mevalonate pathway in osteoclasts, the mechanism by which N-BPs enter the cytosol from the extracellular space to reach their molecular target is not understood. Here, we implemented a CRISPRi-mediated genome-wide screen and identified SLC37A3 (solute carrier family 37 member A3) as a gene required for the action of N-BPs in mammalian cells. We observed that SLC37A3 forms a complex with ATRAID (all-trans retinoic acid-induced differentiation factor), a previously identified genetic target of N-BPs. SLC37A3 and ATRAID localize to lysosomes and are required for releasing N-BP molecules that have trafficked to lysosomes through fluid-phase endocytosis into the cytosol. Our results elucidate the route by which N-BPs are delivered to their molecular target, addressing a key aspect of the mechanism of action of N-BPs that may have significant clinical relevance.

Highlights

N-BPs are the most commonly prescribed drugs used to treat osteoporosis (Drake et al, 2008)
To gain further insight into the mechanism of action of N-BPs, including the mechanism by which N-BPs are delivered to their molecular target, we implemented an unbiased genome-wide screening approach based on CRISPR-mediated interference (CRISPRi) (Figure 1A) (Gilbert et al, 2014)
We quantified the enrichment/depletion of each single-guide RNA (sgRNA) in the treated population compared to the control population (Figure 1B, Figure 1—figure supplement 1A), and designated the target genes of those sgRNAs enriched in the treated population as resistance hits and those depleted as sensitizing hits (Figure 1B, Figure 1—figure supplement 1B and Supplementary file 1)

Summary

Introduction

N-BPs are the most commonly prescribed drugs used to treat osteoporosis (Drake et al, 2008). Some people have variations of the SLC37A3 and ATRAID genes Testing whether these genetic variations may alter NBPs’ ability to cross the membrane of osteoclasts in mice, might one day help physicians predict which patients with have side effects. N-BPs subsequently inhibit farnesyl diphosphate synthase (FDPS) in the mevalonate pathway and reduce protein prenylation, an essential post-translational lipid modification required for the function of numerous proteins such as Ras, Rab and Rho, thereby inducing apoptosis in osteoclasts and diminishing their bone-resorption activities (Drake et al, 2008; Dunford et al, 2001; Fisher et al, 2000; Hughes et al, 1995; Kavanagh et al, 2006; Luckman et al, 1998a; Luckman et al, 1998b; van Beek et al, 1999) It is not known how highly charged N-BPs exit the endocytic pathway to target FDPS, which is localized to the cytosol and peroxisomes (Martın et al, 2007). An alternative model is that a transporter exists that facilitates the exit of N-BPs from endocytic vesicles

Results and discussion

Materials and methods

Funding Funder