Dual roles of the Sterol Recognition Region in Hedgehog protein modification
Abstract
Nature provides a number of mechanisms to encode dynamic information in biomolecules. In metazoans, there exist rare chemical modifications that occur in entirely unique regimes. One such example occurs in the Hedgehog (Hh) morphogens, proteins singular across all domains of life for the nature of their covalent ligation to cholesterol. The isoform- and context-specific efficiency of this ligation profoundly impacts the activity of Hh morphogens and represents an unexplored facet of Hh ligand-dependent cancers. To elucidate the chemical mechanism of this modification, we have defined roles of the uncharacterized sterol recognition region (SRR) in Hh proteins. We use a combination of sequence conservation, directed mutagenesis, and biochemical assays to specify residues of the SRR participate in cellular and biochemical aspects of Hh cholesterolysis. Our investigations offer a functional portrait of this region, providing opportunities to identify parallel reactivity in nature and a template to design tools in chemical biology.
Additional Information
© The Author(s) 2020. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Received 22 January 2020; Accepted 28 April 2020; Published 21 May 2020. We thank Divya Kolli for preparation of the EGFP-SRR fusion construct, and Gracie Zhang and Colin Lee for performing Western blot control experiments. We are grateful to Prof. Peter Dervan and Prof. James K. Chen for valuable feedback. This work was supported by the Margaret E. Early Medical Research Trust. Data availability: Western blot images for the data in Figs. 2 and 3 are provided in the Supplementary Information, along with all sequences of primers used to generate hSHH mutants. Western blot quantification for Fig. 2a–c, e, and f is available at https://doi.org/10.6084/m9.figshare.12133578.36 Remaining data pertaining to this manuscript is available for corresponding author upon reasonable request. Author Contributions: R.P. and A.E.O. designed experiments. R.P. performed CD spectroscopy, hSHH mutagenesis, cellular hSHH protein analysis, hSHH mutant protein isolation and in vitro cholesterolysis assays, and analyzed data. D.S.P. and E.V. performed hSHH mutagenesis, and E.V. performed Western blot experiments. A.E.O. performed EGFP-SRR mutagenesis, conducted microscopy experiments, and analyzed data. R.P. and A.E.O. wrote the manuscript. The authors declare no competing interests.Errata
In the original published version of the article, an error was introduced into Fig. 1a during the typesetting process. The error has been corrected in the PDF and HTML versions of the paper.Attached Files
Published - s42003-020-0977-2.pdf
Submitted - 2019.12.31.892067v1.full.pdf
Supplemental Material - 42003_2020_977_MOESM1_ESM.pdf
Supplemental Material - 42003_2020_977_MOESM2_ESM.pdf
Supplemental Material - 42003_2020_977_MOESM3_ESM.txt
Supplemental Material - 42003_2020_977_MOESM4_ESM.pdf
Supplemental Material - 42003_2020_977_MOESM5_ESM.pdf
Erratum - s42003-020-1023-0.pdf
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Additional details
- PMCID
- PMC7242414
- Eprint ID
- 100493
- Resolver ID
- CaltechAUTHORS:20200103-101527808
- Margaret E. Early Medical Research Trust
- Created
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2020-01-05Created from EPrint's datestamp field
- Updated
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2022-04-25Created from EPrint's last_modified field