Sonic Hedgehog and Cholesterol

1. PHM142 Fall 2012 Coordinator: Dr. Jeffrey Henderson Instructor: Dr. David Hampson Victoria Romanyszyn, GorazdTasev, Alex Bacopoulos, Brittany Rimes Sonic Hedgehog and Cholesterol

2. History of Hedgehog • Christiane Nusselein-Volharddiscovered the hedgehog gene in 1980, winning a Nobel Prize for it • Edward B. Lewis also helped in identifying these genes that control the segmentation pattern of Drosophila melanogaster embryos

3. History of Hedgehog • The mutant phenotype of the hedgehog gene resulted in embryos to be covered with denticles, making it resemble a hedgehog

4. Introduction: Hedgehog • These hedgehog proteins are morphogens—they govern the pattern of tissue development and the positions of the cell types within a tissue. • Similar proteins are also found in vertebrates. • Unlike the fruit fly and other invertebrates, vertebrates have multiple homologs of the Hedgehog gene: the Indian Hedgehog, the Desert Hedgehog, and the Sonic Hedgehog

5. Introduction: Sonic Hedgehog • Sonic hedgehog is a signaling protein crucial in determining the placement of limbs and organs in the developing embryo • It is found on Chromosome 7. • Like all hedgehog proteins, Shh protein undergoes molecular processing in the endoplasmic reticulum

6. Shh Signaling Pathway • Sonic Hedgehog (SHH) is a protein that acts in very large degrees during the developmental phase of the embryo, and to a smaller extent in grown animals • It is made as a ~45kDa precursor, and autocleavesto make a ~20kDa N-terminal signalling protein and a ~25kDa C-terminal piece that has no known signalling function

7. Shh Signaling Pathway • A cholesterol molecule attaches to the carboxylic acid end of the 20kDa piece to make SHH-N • This addition makes the compound able to cross the membrane and enter the interstitial space and subsequently circulation • SHH-N can act in autocrine or paracrine fashion • SHH-N binds to a protein called Patched-1 (PTCH1) on the target cell membrane

8. Shh Signaling Pathway • PTCH1 normally inhibits Smoothened (SMO), but the presence of ligand stops this inhibition • SMO is then allowed to activate certain GLI transcription factors (GLI 1, 2, and 3, in mammals) • GLI 1 and 2 activate and GLI 3 represses transcription of the target genes in the SHH pathway by entering the nucleus and acting on the DNA of the target cell • Mammals have another protein PTCH2 that is similar to PTCH1 and this protein is a weaker inhibitor of SMO, though it acts in a very similar fashion

9. Shh Signaling Pathway

10. The Addition of Cholesterol • The Cholesterol molecule is added at the C-terminus of N-Hhwhich is considered the Signaling section • C-Hhis found to be dispensable and is only used for its catalyzing properties and is otherwise know as the catalyzing section

12. Secretion of N-Hh bound to cholesterol requires the Disp transporter to exit the cell • It is responsible for the release of N-Hhchol that is bound to the membrane • Upon release N-Hhchol forms hexamers • These hexamers then move from cell to cell in a process dependent on HSPG whose synthesis requires Ttv. • Ptcand Hip limit Hh diffusion by sequestering the ligand

13. Developmental Outcomes of Cholesterol-mediated Shh diffusion • Morphogen gradient initiated from Zone of Polarizing Action (ZPA) • Development of digits along anteroposterior axis • Posterior: High Shh concentration • Anterior: Low Shh concentration

14. Dysfunctional Signalling • In absence/dysfunction of cholesterol: • No long-range signalling • No Ptc1-sequestration of Shh

15. Dysfunctional Signalling

16. Summary Page • Sonic hedgehog is a signaling protein crucial in determining the placement of limbs and organs in the developing embryo • N-Shh is the signaling section of Shh that cholesterol binds to • A Disp transporter protein is required to remove N-Hhchol from the cell • C-Shh is the catalytic section of Shh that is dispensable after the cleavage process • N-Shhacts on the Patched protein (Ptc1), which stops inhibition of Smoothened, and allows GLI to enter the nucleus and activate or repress certain parts of the DNA, which allows for a certain specialization of the cell. • Hexamers then form and move from cell to cell in a process dependent on HSPG • A concentration gradient of N-Shh signaling mediates the development of mammalian digits along the antero-posterior axis • High concentrations = posterior digits • Low concentrations = anterior digits • Long-term signaling and Ptc1-mediated negative feedback of signaling is dependent on cholesterol • In the absence of cholesterol, anterior digits do not develop as a result on impaired long distance signaling.

17. References • Heussler, H.S., Sun, M. (2003). Sonic Hedgehog. MolPathol 56(3):129-131. • Jeong, J., McMahon, A.P. (2002). Cholesterol modification of Hedgehog family proteins. J Clin Invest 110(5):591-596. • Kelley, R.I., Roessler, E., Hennekam, R.C.M., Feldman, G.L., Kosaki, K., Jones, M.C., Palumbos, C., Muenke, M. (1996). Holoprosencephaly in RSH/Smith-Lemli-Opitz syndrome: Does abnormal cholesterol metabolism affect function of sonic hedgehog?. American Journal of Medical Genetics. 66(4):478-484. • Lewis, P.M., Dunn, M.P., McMahon, J.A., Logan, M., Martin, J.F., St-Jacques, B., McMahon, A.P. (2001). Cholesterol Modification of Sonic Hedgehog Is Required for Long-Range Signaling Activity and Effective Modulation of Signaling by Ptc1. Cell 105:599-612.