Identification of a transporter complex responsible for the cytosolic entry of nitrogen-containing-bisphosphonates
- Harvard University, United States
- University of California, San Francisco, United States
- Whitehead Institute for Biomedical Research, United States
- Washington University School of Medicine, United States
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.
Data availability
All data generated or analysed during this study are included in the manuscript and supporting files. Source data files have been provided for the two CRISPRi screens shown in Figure 1 and its figure supplement.
-
Data from: Identification and characterization of a transporter complex responsible for the cytosolic entry of nitrogen-containing-bisphosphonatesAvailable at Dryad Digital Repository under a CC0 Public Domain Dedication.
Article and author information
Author details
Jonathan S Weissman
Funding
Howard Hughes Medical Institute
- Zhou Yu
- Lauren E Surface
- Chong Yon Park
- Max A Horlbeck
- Gregory A Wyant
- Monther Abu-Remaileh
- David M Sabatini
- Jonathan S Weissman
- Erin K O'Shea
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Copyright
© 2018, Yu et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
Metrics
-
- 3,734
- views
-
- 631
- downloads
-
- 45
- citations
Views, downloads and citations are aggregated across all versions of this paper published by eLife.
Download links
A two-part list of links to download the article, or parts of the article, in various formats.
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
Identification of a transporter complex responsible for the cytosolic entry of nitrogen-containing-bisphosphonates
eLife 7:e36620.
https://doi.org/10.7554/eLife.36620
Further reading
-
- Biochemistry and Chemical Biology
- Genetics and Genomics
Yerba mate (YM, Ilex paraguariensis) is an economically important crop marketed for the elaboration of mate, the third-most widely consumed caffeine-containing infusion worldwide. Here, we report the first genome assembly of this species, which has a total length of 1.06 Gb and contains 53,390 protein-coding genes. Comparative analyses revealed that the large YM genome size is partly due to a whole-genome duplication (Ip-α) during the early evolutionary history of Ilex, in addition to the hexaploidization event (γ) shared by core eudicots. Characterization of the genome allowed us to clone the genes encoding methyltransferase enzymes that catalyse multiple reactions required for caffeine production. To our surprise, this species has converged upon a different biochemical pathway compared to that of coffee and tea. In order to gain insight into the structural basis for the convergent enzyme activities, we obtained a crystal structure for the terminal enzyme in the pathway that forms caffeine. The structure reveals that convergent solutions have evolved for substrate positioning because different amino acid residues facilitate a different substrate orientation such that efficient methylation occurs in the independently evolved enzymes in YM and coffee. While our results show phylogenomic constraint limits the genes coopted for convergence of caffeine biosynthesis, the X-ray diffraction data suggest structural constraints are minimal for the convergent evolution of individual reactions.
-
- Biochemistry and Chemical Biology
- Structural Biology and Molecular Biophysics
The SARS-CoV-2 main protease (Mpro or Nsp5) is critical for production of viral proteins during infection and, like many viral proteases, also targets host proteins to subvert their cellular functions. Here, we show that the human tRNA methyltransferase TRMT1 is recognized and cleaved by SARS-CoV-2 Mpro. TRMT1 installs the N2,N2-dimethylguanosine (m2,2G) modification on mammalian tRNAs, which promotes cellular protein synthesis and redox homeostasis. We find that Mpro can cleave endogenous TRMT1 in human cell lysate, resulting in removal of the TRMT1 zinc finger domain. Evolutionary analysis shows the TRMT1 cleavage site is highly conserved in mammals, except in Muroidea, where TRMT1 is likely resistant to cleavage. TRMT1 proteolysis results in reduced tRNA binding and elimination of tRNA methyltransferase activity. We also determined the structure of an Mpro-TRMT1 peptide complex that shows how TRMT1 engages the Mpro active site in an uncommon substrate binding conformation. Finally, enzymology and molecular dynamics simulations indicate that kinetic discrimination occurs during a later step of Mpro-mediated proteolysis following substrate binding. Together, these data provide new insights into substrate recognition by SARS-CoV-2 Mpro that could help guide future antiviral therapeutic development and show how proteolysis of TRMT1 during SARS-CoV-2 infection impairs both TRMT1 tRNA binding and tRNA modification activity to disrupt host translation and potentially impact COVID-19 pathogenesis or phenotypes.
