Xiaochun Li - Hedgehog Signaling: Methods and Protocols

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Cover Illustration Caption: A cryo-EM structure reveals that two Patched receptors engage two distinct sites on one Sonic Hedgehog ligand yielding a signaling-competent complex.

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Hedgehog (HH) signaling has been studied for 40 years. With the dedication of many scientists in the field, it has been shown to play an important role during embryonic development and adult tissue homeostasis in species ranging from insects to mammals. Importantly, aberrant activation of HH signaling has been implicated in various cancers, including basal cell carcinoma, medulloblastoma, and leukemia. Vismodegib, which was developed by Genentech to target the HH pathway, had been approved by U.S. Food and Drug Administration for the treatment of basal cell carcinoma. Therefore, further studies of the HH signal pathway will significantly accelerate our understanding of the molecular mechanisms of animal development and provide additional insights into the treatment of HH-signal linked diseases, particularly cancers.

There are several key protein components in this pathway, including HH, the signaling ligand; Dispatched, a HH secretion regulator; Patched, the receptor of HH; Smoothened, the signal transducer used to transduce the HH signal across the plasma membrane; and GLI, the transcription factor to turn on HH target genes. Recent technical breakthroughs with biophysical and cell biological approaches considerably advanced our understanding of the molecular mechanism underlying the generation and transduction of HH signal, indicating the important role of sterols in this pathway. In this book, the methods of recent interesting studies are described. I wish to express my deep appreciation to all the contributors for their time and efforts to share their expertise and experience.

During development, HH morphogen gradients ensure reproducible and accurate pattern formation. In this book, Roelink and his colleagues describe how to employ genetically modified murine stem cell lines to study morphogen gradients. The full-length HH protein can be cleaved by itself prior to its maturation. In 1996, the Philip Beachy group discovered that the C-terminus of HH catalyzes the autocleavage reaction to covalently add a cholesterol on the carboxyl terminal end of the N-terminus of HH (HH-N). Later studies showed that this modification is essential for proper HH dispersal. After cholesterol modification, a palmitate moiety can be added to the amino terminus of HH-N by Hedgehog Acyltransferase (HHAT). The palmitoylation plays an indispensable role in signal transduction since mutations in HHAT produce a phenotype similar to that of HH knockout in animals. Therefore, HHAT inhibitors potentially can be used to block HH signaling for treating several types of cancers. In this book, Andrei and his colleagues provide a method to monitor HHAT activity and inhibition. Song and his colleagues present a method to identify the cholesterol modification on Smoothened, another key component of HH signal pathway.

In 2003, the Kathryn Anderson laboratory at Sloan Kettering Institute discovered that vertebrate HH signal occurs at the primary cilium, a special organelle present in most vertebrate cells except red blood cells. The dependence on primary cilium makes HH signaling pathway unique among all signaling pathways. During the preparation of this book, Dr. Anderson passed away. Here, I would like to express my highest respect and admiration for her. Cho, Miyamoto, Mukhopadhyay, Rohatgi, Radhakrishnan, Zheng, and their colleagues present the methods to study how the key protein components of this pathway traffic in cilia and how the lipids on the cilia membrane regulate the HH signal.

Patched and Dispatched are structural homologs and responsible for releasing and receiving the HH ligands. In this book, Ogden, Qi, and their colleagues describe methods to analyze the cleavage of Dispatched for HH secretion and assemble the protein complex of Patched1-Hedgehog. Smoothened, a class-F GPCR, plays a role as a signal regulator to transduce the HH signal from the extracellular space to the cytosol. The regulation and modification of the Smoothened protein have been shown to play important role in HH signal transduction. Jia, Jiang, Manglik, Myers, Ikeuchi, Schulte, Zhang, and their colleagues provide the methods to detect the modification of Smoothened and analyze its activity by small molecule or nanobody and assemble the complex of Smoothened with the heterotrimeric Gi protein. Jiang and his colleagues provide an approach to study the phosphorylation of GLI, which contributes to GLI activation. Finally, Kim and his colleagues present a method to study HH-related lung cancer by an orthotopic mouse model.