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Hedgehog proteins create a dynamic cholesterol interface.

ABSTRACT: During formation of the Hedgehog (Hh) signaling proteins, cooperative activities of the Hedgehog INTein (Hint) fold and Sterol Recognition Region (SRR) couple autoproteolysis to cholesterol ligation. The cholesteroylated Hh morphogens play essential roles in embryogenesis, tissue regeneration, and tumorigenesis. Despite the centrality of cholesterol in Hh function, the full structure of the Hint-SRR ("Hog") domain that attaches cholesterol to the last residue of the active Hh morphogen remains enigmatic. In this work, we combine molecular dynamics simulations, photoaffinity crosslinking, and mutagenesis assays to model cholesterolysis intermediates in the human Sonic Hedgehog (hSHH) protein. Our results provide evidence for a hydrophobic Hint-SRR interface that forms a dynamic, non-covalent cholesterol-Hog complex. Using these models, we suggest a unified mechanism by which Hh proteins can recruit, sequester, and orient cholesterol, and offer a molecular basis for the effects of disease-causing hSHH mutations.

SUBMITTER: Mafi A

PROVIDER: S-EPMC7906309 | biostudies-literature | 2021

REPOSITORIES: biostudies-literature

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Hedgehog proteins create a dynamic cholesterol interface.

Mafi Amirhossein A Purohit Rahul R Vielmas Erika E Lauinger Alexa R AR Lam Brandon B Cheng Yu-Shiuan YS Zhang Tianyi T Huang Yiran Y Kim Soo-Kyung SK Goddard William A WA Ondrus Alison E AE

PloS one 20210225 2

During formation of the Hedgehog (Hh) signaling proteins, cooperative activities of the Hedgehog INTein (Hint) fold and Sterol Recognition Region (SRR) couple autoproteolysis to cholesterol ligation. The cholesteroylated Hh morphogens play essential roles in embryogenesis, tissue regeneration, and tumorigenesis. Despite the centrality of cholesterol in Hh function, the full structure of the Hint-SRR ("Hog") domain that attaches cholesterol to the last residue of the active Hh morphogen remains e ...[more]

PMID: 33630857

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Project description:BackgroundSonic hedgehog (Shh) signaling plays a crucial role in growth and patterning during embryonic development, and also in stem cell maintenance and tissue regeneration in adults. Aberrant Shh pathway activation is involved in the development of many tumors, and one of the most affected Shh signaling steps found in these tumors is the regulation of the signaling receptor Smoothened by the Shh receptor Patched. In the present work, we investigated Patched activity and the mechanism by which Patched inhibits Smoothened.Methodology/principal findingsUsing the well-known Shh-responding cell line of mouse fibroblasts NIH 3T3, we first observed that enhancement of the intracellular cholesterol concentration induces Smoothened enrichment in the plasma membrane, which is a crucial step for the signaling activation. We found that binding of Shh protein to its receptor Patched, which involves Patched internalization, increases the intracellular concentration of cholesterol and decreases the efflux of a fluorescent cholesterol derivative (BODIPY-cholesterol) from these cells. Treatment of fibroblasts with cyclopamine, an antagonist of Shh signaling, inhibits Patched expression and reduces BODIPY-cholesterol efflux, while treatment with the Shh pathway agonist SAG enhances Patched protein expression and BODIPY-cholesterol efflux. We also show that over-expression of human Patched in the yeast S. cerevisiae results in a significant boost of BODIPY-cholesterol efflux. Furthermore, we demonstrate that purified Patched binds to cholesterol, and that the interaction of Shh with Patched inhibits the binding of Patched to cholesterol.Conclusion/significanceOur results suggest that Patched may contribute to cholesterol efflux from cells, and to modulation of the intracellular cholesterol concentration. This activity is likely responsible for the inhibition of the enrichment of Smoothened in the plasma membrane, which is an important step in Shh pathway activation.

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Hedgehog proteins create a dynamic cholesterol interface.

Publications

Hedgehog proteins create a dynamic cholesterol interface.

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