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Published on: May 3, 2021
Biochemical Assays to Directly Assess Smoothened Activation by a Conformationally Sensitive Nanobody
Ishan Deshpande1, Aashish Manglik2,3
1Department of Pharmaceutical Chemistry, University of California, San Francisco, San Francisco, CA, USA. Ishan.Deshpande@ucsf.edu.
Abstract:
The Hedgehog signaling pathway coordinates early development and is important in various cancers. Classic approaches to test pathway activation rely on transcriptional readouts or ciliary accumulation of specific pathway components. Although these assays have laid the foundation for studying Hedgehog pathway activation, they integrate the complex molecular actions of the transporter Patched and the seven transmembrane protein Smoothened. Though it is clear that cellular sterols are critical for pathway activity, direct dissection of which sterols drive Smoothened activity is precluded by the complex biosynthetic pathways responsible for cellular sterols. Here we describe a direct method of measuring Smoothened activity in vitro. This assay measures the binding of Smoothened to NbSmo8, a single-domain antibody that is selective for the active-state of Smoothened. Binding of purified Smoothened, reconstituted with specific sterols, to fluorescently labeled NbSmo8 can be rapidly evaluated using a fluorescence-detection size-exclusion chromatography assay. This approach enables a reductionist approach to precisely interrogate the regulatory activities of cellular lipids and sterols during Hedgehog signaling.
Insights
Researchers developed a new in vitro assay to directly measure Smoothened activity, crucial for Hedgehog signaling. This method uses a specific antibody to quantify Smoothened binding, enabling precise study of sterol regulation in this key developmental and cancer pathway.
Area of Science:
- Biochemistry
- Molecular Biology
- Cell Signaling
Background:
- The Hedgehog signaling pathway is vital for embryonic development and implicated in numerous cancers.
- Current methods for assessing pathway activation, such as transcriptional readouts or ciliary protein accumulation, indirectly measure complex molecular events.
- Understanding the precise role of cellular sterols in regulating the seven-transmembrane protein Smoothened (SMO) has been challenging due to intricate sterol biosynthesis.
Purpose of the Study:
- To develop a direct in vitro assay for measuring Smoothened (SMO) activity.
- To enable precise interrogation of the regulatory roles of specific cellular lipids and sterols in Hedgehog signaling.
Main Methods:
- Developed a novel assay measuring the binding of Smoothened to NbSmo8, a single-domain antibody specific for the active state of Smoothened.
- Utilized fluorescence-detection size-exclusion chromatography to rapidly evaluate the binding of purified Smoothened, reconstituted with defined sterols, to fluorescently labeled NbSmo8.
Main Results:
- Successfully established a direct in vitro method to quantify Smoothened activity.
- Demonstrated the ability to rapidly assess Smoothened-NbSmo8 binding in the presence of specific sterols.
Conclusions:
- The developed assay provides a reductionist approach to directly measure Smoothened activity.
- This method facilitates precise investigation into how cellular sterols regulate Hedgehog pathway activation, offering new insights into pathway control in development and disease.
