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1.
Sci Adv ; 9(34): eadh1609, 2023 08 25.
Article in English | MEDLINE | ID: mdl-37611095

ABSTRACT

Patched1 (PTCH1) is a tumor suppressor protein of the mammalian Hedgehog (HH) signaling pathway, implicated in embryogenesis and tissue homeostasis. PTCH1 inhibits the G protein-coupled receptor Smoothened (SMO) via a debated mechanism involving modulating ciliary cholesterol accessibility. Using extensive molecular dynamics simulations and free energy calculations to evaluate cholesterol transport through PTCH1, we find an energetic barrier of ~15 to 20 kilojoule per mole for cholesterol export. In silico data are coupled to in vivo biochemical assays of PTCH1 mutants to probe coupling between cation binding sites, transmembrane motions, and PTCH1 activity. Using complementary simulations of Dispatched1, we find that transition between "inward-open" and solvent "occluded" states is accompanied by Na+-induced pinching of intracellular helical segments. Thus, our findings illuminate the energetics and ion coupling stoichiometries of PTCH1 transport mechanisms, whereby one to three Na+ or two to three K+ couple to cholesterol export, and provide the first molecular description of transitions between distinct transport states.


Subject(s)
Biological Assay , Hedgehog Proteins , Animals , Biological Transport , Binding Sites , Embryonic Development , Mammals
2.
bioRxiv ; 2023 Feb 15.
Article in English | MEDLINE | ID: mdl-36824746

ABSTRACT

Patched1 (PTCH1) is the principal tumour suppressor protein of the mammalian Hedgehog (HH) signalling pathway, implicated in embryogenesis and tissue homeostasis. PTCH1 inhibits the Class F G protein-coupled receptor Smoothened (SMO) via a debated mechanism involving modulating accessible cholesterol levels within ciliary membranes. Using extensive molecular dynamics (MD) simulations and free energy calculations to evaluate cholesterol transport through PTCH1, we find an energetic barrier of ~15-20 kJ mol -1 for cholesterol export. In simulations we identify cation binding sites within the PTCH1 transmembrane domain (TMD) which may provide the energetic impetus for cholesterol transport. In silico data are coupled to in vivo biochemical assays of PTCH1 mutants to probe coupling between transmembrane motions and PTCH1 activity. Using complementary simulations of Dispatched1 (DISP1) we find that transition between 'inward-open' and solvent 'occluded' states is accompanied by Na + induced pinching of intracellular helical segments. Thus, our findings illuminate the energetics and ion-coupling stoichiometries of PTCH1 transport mechanisms, whereby 1-3 Na + or 2-3 K + couple to cholesterol export, and provide the first molecular description of transitions between distinct transport states.

3.
Elife ; 102021 10 26.
Article in English | MEDLINE | ID: mdl-34698632

ABSTRACT

A long-standing mystery in vertebrate Hedgehog signaling is how Patched 1 (PTCH1), the receptor for Hedgehog ligands, inhibits the activity of Smoothened, the protein that transmits the signal across the membrane. We previously proposed (Kinnebrew et al., 2019) that PTCH1 inhibits Smoothened by depleting accessible cholesterol from the ciliary membrane. Using a new imaging-based assay to directly measure the transport activity of PTCH1, we find that PTCH1 depletes accessible cholesterol from the outer leaflet of the plasma membrane. This transport activity is terminated by binding of Hedgehog ligands to PTCH1 or by dissipation of the transmembrane potassium gradient. These results point to the unexpected model that PTCH1 moves cholesterol from the outer to the inner leaflet of the membrane in exchange for potassium ion export in the opposite direction. Our study provides a plausible solution for how PTCH1 inhibits SMO by changing the organization of cholesterol in membranes and establishes a general framework for studying how proteins change cholesterol accessibility to regulate membrane-dependent processes in cells.


Subject(s)
Cell Membrane/metabolism , Cholesterol/metabolism , Patched-1 Receptor/genetics , Smoothened Receptor/genetics , Animals , Escherichia coli , Humans , Mice , Patched-1 Receptor/metabolism , Smoothened Receptor/metabolism
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