Smoothened Hedgehog Pathway

Cyclopamine compound leads to cycloptic lambs In 1954, sheep grazing in certain ranges within Idaho were giving birth to lambs with a variety of different deformities like the ones that you see above. These include a single eye and a trunk-like nasal structure above the head. This was happening to as many as 1 out of every 4 lambs in the area. Some thought it was genetic but despite intense investigation, they were unable to find the cause of this sudden rise in deformities. Eventually one of the farmers found that the sheep were becoming sick when they were grazing on a plant called the corn lily (Verratum californicum). Experiments were conducted and found that if the ewe ate this plant on the 14th day of gestation it would cause the cyclops birth defect along with the proboscis structure above the eye. If the plant was consumed at later times, it would lead to gestation, limb, foregut, and tracheal deformities. They later isolated the compound responsible and named it cyclopamine. 30 years later, they learned that cyclopamine interacts with the hedgehog pathway and more specifically acts as a smoothened receptor inhibitor. Later on, a modified version of this compound will be used in the treatment of cancer as I will discuss later. Lee

Inhibition of the Hedgehog Pathway leads to midline, neural tube, and limb deformities In 1978, Wieschause and Volhard discovered the hedgehog pathway in fruit flies. They noted that when this gene was inactivated, it caused the larvae to be covered in spiky looking dendricles that looked a hedgehog, which led to the name. Experiments conducted with animals and cyclopamine at this point have already provided insight into what happens when SHH is inhibited. I was unable to find a proper knockout experiment that involved the SMO gene outside of these cyclopamine cases. However, Chiang et al. conducted an experiment in which they knocked out through truncation the ligand that activates this pathway, the sonic Hedgehog ligand which effectively turned the pathway off. Most of these mutants died at or before birth. As you can see from this picture, when the hedgehog ligand is not present during development, the midline doesn’t develop properly (AB), there is abnormal development of the ventral and dorsal neural tube ©, improper proliferation leading to a decreased size, and improper limb development (M, N). So you can see how critical this pathway is in early development and later on it will become more important for proliferation in the body. Going back to the cyclopamine example, you can see how SHH is responsible for symmetry along with saggital plane. Chiang

Simplified Overview of the Hedgehog Pathway SHH PTCH-1 SMO SUFU/PKA GLI This is a very simplified view of the pathway and obviously other proteins are in play but when SHH ligand is not present, PTCH-1 is able to inhibit SMO and SUFU/PKA are able to inhibit GLI transcription . When SHH is present, PTCH-1 is inhibited and SMO can inhibit SUFU/PKA and GLI is able to serve as a transcription factor.

SMO inhibits SUFU and PKA in order to activate transcription factor, GLI So now that we know about its role in development and a little about its history we can talk about the pathway. The Hedgehog pathway is fascinating because it is one of the five key signal transduction pathways yet so many details of this pathway are still unknown. When the pathway is turned on, sonic hedgehog ligand (SHH) binds to a complex that consists of the transmembrane protein PTCH and one of its co-receptors in this case CDO. The binding causes PTCH to inactive and therefore cease its inhibitory function towards Smoothened which will be elaborated further. Smoothened is then activated which allows further transduction of the signal. SMO inhibits the activity of the Suppressor of Fused (SUFU) and protein kinase A, along with a few other kinases. If they are not inhibited, these kinases phosphorylate serine residues on GLI which then leads to further modification by the proteasome. GLI becomes a repressor protein, translocates to the nucleus and then binds to the DNA, preventing transcription of the target genes. However, when these kinases are inhibited by SMO, GLI avoids this modification and is able to translocate to the nucleus and act as a transcription factor which ultimately leads to transcription of the target genes SMO inhibits these kinases by activating proteins that cause the accumulation and compartmentalization of the kinases in the ciliary tip. One particularly interesting thing about this pathway is that many of the crucial components lie within the cilium. The cilium in this case does not act as a source of movement but rather as a sort of antenna to receive signals like SHH. When PTCH is inhibited by this ligand, it moves away from the cilia and is replaced by SMO. Sharpe

PTCH oxysterol pumping activity inhibits SMO SAG- Known strong SMO activator CPN- SMO inhibitor Oxysterols stimulate Sonic hedgehog signal transduction and proliferation of medulloblastoma cells There is still a great deal of debate as to how PTCH acts to inhibit SMO. Most people do not believe that there is direct binding between PTCH and SMO. The leading theory is quite interesting because its mechanism is unlike the pathways that we have learned previously. When PTCH is active, it acts as a pump that prevents sterols from entering the cell. The sterols when present will bind to SMO. This will induce a confirmation change within SMO that activates the transmembrane receptor. ZGA prevents sterol formation from isoprenoids. OHC (Oxygen Hydrocarbon [number indicates site of hydroxylation] Corcoran

SMO is an oncogene within the Hedgehog Pathway SMO is an oncogene within the Hedgehog Pathway. SMO is a 24 kb gene with 12 exons located on the distal arm of Chromosome 7 Now that we have talked about the pathway, lets look at SMO specifically. As you can see this is a fairly large gene with 12 exons on the distal arm of chromosome 7. Exons 1 and 2 have the 5’ untranslated sequences, the initiation codon, and the signal peptide. The other exons will encode other various parts of the transmembrane receptor. Smoothened is an oncogene. A single activating mutation is enough to elicit density-independent growth within a cell. Xie

SMO is a seven-span transmembrane G-protein coupled receptor Activating Smoothened Mutation in sporadic basal-cell carcinoma Structure of the human smoothened receptor bound to an antitumor agent The smoothened protein is a seven-transmembrane receptor that shares its overall tertiary shape with the frizzled receptor in the wnt pathway. They both have the seven-helical transmembrane domain. They also both share an extracellular cysteine-rich domain. While this receptor shares its base shape with frizzled, it is unique in that it has long extracellular loops that are stabilized by di-sulfide bonds. While we know about SMO general structure, it is still largely unknown how this structure interacts with the pathway and with drugs used to treat it. Wang

Activating mutations interfere with formation of the polar pocket or PTCH-SMO interaction Now we will discuss how this oncogene ties into cancer. In this experiment, Xie examined 47 BCC cell lines in order to look for activating mutations of smoothened. He performed heteroduplex analysis in order to find these mutations. When the sample DNA does not match with the control DNA, you will see two bands present. This can be seen clearly for C BCC 9. They found two cases of one heterozygous missense mutations in Exon 9 (C BCC 9) and one case of a mutation Exon 10 (B326). In Exon 9, a tryptophan was changed to a leucine on the seventh transmembrane domain. This interferes with polar pocket that keeps the receptor in the latent state. They also observed the effect of the SMO mutants in a cell culture and within mice. They found that in vitro, the SMO mutants lost density-dependent inhibition. Within the mice, the SMO mutants affected the basal cells causing the surface epithelium to be non-keratinized and the distinct karato-hyalin granules were absent from the stratum granulosum. While PTCH mutations are the most common source of BCC, you can see how causing SMO to continually inhibit SUFU and PKA can lead to cancer . Xie

Basal Cell Carcinoma is one of the most common types of cancer, although rarely fatal Three out of 10 Caucasians will develop basal-cell cancer within their lifetime, usually around the head or neck. It usually presents as a shiny, pearly skin nodule. Surgical excision is the most common form of treatment but targeted chemotherapies are being developed in order to combat advanced BCCs.

A direct SMO inhibitor, Vismodegib, has been approved by the FDA for use First direct target to the Hedge hog pathway. Acts as a synthetic cyclopamine-like antagonist that targets SMO transmembrane helices. Binds to the heptahelical transmembrane and inactivates the protein. 76% of patients with BCC treated with this drug experienced clinical benefit Efficacy and Safety of Vismodegib in Advanced Basal-Cell Carcinoma

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