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PDB ID 3m1n

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Contents

Background

Sonic hedgehog is a protein critical to embryonic development first discovered in Drosophila Melanogaster in 1980 by Christiane Nusslein-Volhard and Eric Wieschauns. Loss of function of the HH gene leads to small spiky projections on Drosophila larvae leading to the chosen name. In most vertebrates, there are three HH proteins: sonic hedgehog (SHH), indian hedgehog (IHH), and desert hedgehog (DHH). SHH is the most well studied and understood of the three. Most notably SHH plays a role in embryonic development and differentiation of limbs and the central nervous system. SHH is a morphogen, meaning the concentrations of this protein induce different gene activation and cell fate during development. This process is also known as graded signaling. SHH is responsible for the variation of cell types within the neural tube and is produced by the notochord. SHH is the most studied protein of the Hedgehog Signaling pathway, an integral development pathway in both vertebrates and invertebrates. SHH is 462 amino acids in length and 45 kD. The business end of the protein is a signal domain near the N-terminus that activates the protein when cleaved. The protein is cleaved into two separate regions, each with specific roles in the Hedgehog Signaling Pathway. Nonfunctioning/defective SHH can lead to a plethora of protopathic developmental ailments, including microphthalmia, holoprosencephaly, and triphalangeal thumb-polysyndactyly syndrome. SHH is particularly interesting due to its role in cell division, specifically stem cells (in a fully developed human). As a result, SHH has been directly tied to the cause of some cancers and the target of some anticancer drugs.[1]

Function

Neurulation and Dorsoventral Patterning

Sonic Hedgehog protein (SHH) is a morphogen produced by the notochord active during the major developmental event of neurulation. SHH plays a critical morphological role in progenitor cell fate along the ventral axis, also called ventral patterning, of the newly formed neural tube that will later be differentiated into the central nervous system. Neurulation is the second major embryonic event following gastrulation, which occurs four weeks after human gestation and involves the folding of the neural floor plate located within the ectoderm. This process is highly regulated by a multitude of complicated pathways, genes, and signal transduction molecules [2]. In the absence of SHH, a specific transmembrane receptor, Patched (Ptc1), inhibits the activity of the G protein-coupled receptor Smoothened (Smo). When SHH is present, it binds to Ptc1 and initiates a cascade of events known as the Hedgehog signaling mechanism. Uninhibited Smo produces activated Gli transcription factors that induce gene transcription, thus cell differentiation and proliferation.

Morphogenic Activity

SHH belongs to a class of proteins in which concentration thresholds of that protein affect the biological response of the target cell. These types of proteins are called morphogens, and in the case of SHH, determine ventral patterning along the neural tube. Produced in the notochord and to some extent the neural floor plate, SHH is transported via several transport molecules toward the dorsal end of the neural tube. High concentrations of SHH induces ventral neuron formation, while low concentrations induce motor neuron cell differentiation[3]. This leads to specific progenitor domains along the ventral axis of the neural tube. The gradient is formed due to a lipid modification of the membrane-bound SHH protein. The precursory 45kD SHH protein cleaves via autoproteolysis into two smaller peptide domains: the 19kD and the 26kD C terminus. This autoproteolysis adds a cholesterol moiety to the C terminus of SHH-N, which becomes significant in the long-range activity and regulation of SHH. The SHH-C domain catalytically mediates the autoproteolysis of SHH and diffuses away, while SHH-N undergoes further modification involving a palmitoyl group added to the N terminus catalyzed by the acetyltransferase: Skinny hedgehog (SKI). Once SHH-N has been modified into this dual lipid activated form, it is soluble and diffusible, further increasing its long-range signaling capabilities. The 12 pass transmembrane transporter Dispatched (Dsp) interacts with the activated SHH-N dual lipid moieties dissociating it with the plasma membrane of its secretory cells. The Heparin sulfate proteoglycans Dally and Dally-like transport SHH-N extracellularly to Ptc where it acts like a ligand initiating the Hedgehog Signaling Mechanism. The enzyme "tout-velu" indirectly regulates the movement of SHH-N by regulating the amount of heparin sulfate proteoglycans that are manufactured. The morphogenic process of forming protein gradients was thought to be invariant or rather static once steady state was reached. However, more recent studies have illustrated the importance of the dynamic nature of the protein gradients in ventral patterning.[4] Each of the transport mechanisms and modifications previously discussed has an effect on the rate of SHH transport, thus effecting ventral patterning beyond concentration gradients alone. This model suggests that slowing the transport of SHH-N, such as by dual lipid modification, increases the signal range of the morphogen as a result of concentrating it near the secretory source. The concentration gradient is interpreted by the transcription factors Gli2 and Gli3, leading to the various phenotypic differences in arising cell types.

Structural highlights

The initial SHH precursory protein is composed of two major domains and is 45KD in size. This 462 amino acid protein is then autolytically cleaved into its 19kD SHH-N and 26kD SHH-C domains. During the process of autolytic cleavage, a cholesterol is covalently attached to the C terminal glycine residue of SHH-C. This causes SHH-C to behave like a cholesterol transferase covalently adding the cholesterol to the C terminus of SHH-N. This active SHH-N monomer is 169 amino acids composed of 3 alpha and 8 . SHH-N is associated with both divalent zinc and calcium ions captured in different active sites. The zinc ion is located in the noncatalytic, or pseudo-active site, while the calcium ion is shuttled into the neighboring site. It is worth noting the zinc active site is analogous to zinc hydrolases; however, as previously stated, studies have shown no catalytic activity occurring in this site.[5]These ions are essential for proper protein folding and play a role in SHH-N binding to Ptc1 and hedgehog interacting protein (HHIP).

Binding of SHH-N to HHIP occurs in the noncatalytic active site through three contact points located on each protein. The main contact point of HHIP is brought into the pseudo-active site of SHH-N and forms hydrogen bonds with five histidine residues. Additionally, the adjacent calcium ion within SHH-N stabilizes this event. Within the noncatalytic site, the zinc ion is coordinated by When HHIP interacts with SHH-N, another aspartate residue from HHIP zinc ion to tether the proteins together through the stable of residues around the zinc ion.6

The same noncatalytic site on SHH-N that holds the zinc ion interacts with ptc1; this is quite significant because it suggests competitive binding between ptc1 and HHIP. Interestingly enough, the same aspartate and histidine residues coordinate tetrahedrally around the zinc ion to stabilize the event. However, instead of the adjacent calcium ion stabilizing the reaction, hydrophobic interactions take its place.(1)

Disease

SHH is crucial for differentiation of the ventral prosencephalon as well as midbrain neurons, disruption of SHH function can lead to neural crest cell apoptosis, as well as undifferentiated progenitor cells that cause craniofacial anomalies in humans including microphthalmia and holoprosencephaly.[6]Holoprosencephaly is caused by a missense mutation in the HH gene that results in mutations to the SHH-N domain and is characterized by failure of the embryonic prosencephalon to separate into bilateral hemispheres. This mutation affects the processing of SHH at several steps and decreases the overall level of protein expression and biogenesis.[7] These missense mutations and various stages of SHH processing interference explain the range of phenotypic discrepancies between cases of holoprosencephaly. Aberrant reactivation of the hedgehog signaling pathway in a fully developed human can be the cause of some types of cancer as well as various protopathic developmental diseases. Notably, overexpression of SHH can lead to lung cancer, gastrointestinal cancer, prostate cancer, and pancreatic cancer. [8]The mechanism of SHH in these cancers is not entirely understood; however, there is strong evidence supported by in vitro data that illustrates tumor growth acceleration when SHH-N concentrations are increased and tumor growth inhibition when SHH antagonist cyclopamine is introduced. This data supports the theory that SHH increases the aberrant proliferation of metastatic cells producing tumors via increased intercellular signaling.[8] It has also been suggested that overactivation of the Hedgehog signaling pathway during normal tissue repair can induce tumorigenesis.

Relevance

The process of neurogenesis continues to occur in a fully developed brain in various specific locations. Neurogenesis is regulated by pathophysiological conditions, most notably seizures, and physiology stimuli such as genetic makeup, lifestyle, and age. The process ensures the renewal of various neural tissues, therefore, attributing to overall homeostasis of CNS function. SHH plays a major role in cell proliferation during development as well as adult neurogenesis. The role of SHH in neurogenesis has made it a primary target for anti-stroke drugs as well as neurogenesis enhancement compounds. Some of these include Savlonic Acid, an antioxidant, Resveratrol, polyphenol derivative found in grapes, as well as Epigallocatechin-3-gallate, derived from green tea. This suggests therapeutic applications of SHH and its corresponding pathway that may benefit individuals with neurological disorders such as down syndrome, major depressive disorder, or ischemic stroke, dementia and Alzheimer's disease. [7]SHH has several other subsequent functions in the CNS, including reducing the stress of reactive oxygen species and inflammation, as well as regulation of autophagic related mechanisms to maintain homeostasis under environmental stresses. All of these attributes contribute to the desire to research and target SHH for neurological enhancement and repair drugs.[9]

References

  1. ↑ Rimkus, T.K.; Carpenter, R.L.; Qasem, S.; Chan, M.; Lo, H.-W. Targeting the Sonic Hedgehog Signaling Pathway: Review of Smoothened and GLI Inhibitors. Cancers 2016, 8,22.https://www.mdpi.com/2072-6694/8/2/22
  2. ↑ https://www.sciencedirect.com/topics/neuroscience/neurulation A. Matthew K. Lee, ... Parviz Minoo, in Fetal and Neonatal Physiology (Fifth Edition), 2017 Christopher J. Yuskaitis, Scott L. Pomeroy, in Fetal and Neonatal Physiology (Fifth Edition), 2017
  3. ↑ Choudhry Z, Rikani AA, Choudhry AM, Tariq S, Zakaria F, Asghar MW, Sarfraz MK, Haider K, Shafiq AA, Mobassarah NJ. Sonic hedgehog signalling pathway: a complex network. Ann Neurosci. 2014 Jan;21(1):28-31. doi: 10.5214/ans.0972.7531.210109. PMID:25206052 doi:http://dx.doi.org/10.5214/ans.0972.7531.210109
  4. ↑ doi: https://dx.doi.org/10.1016/b978-0-12-801238-3
  5. ↑ Hitzenberger M, Schuster D, Hofer TS. The Binding Mode of the Sonic Hedgehog Inhibitor Robotnikinin, a Combined Docking and QM/MM MD Study. Front Chem. 2017 Oct 23;5:76. doi: 10.3389/fchem.2017.00076. eCollection 2017. PMID:29109946 doi:http://dx.doi.org/10.3389/fchem.2017.00076
  6. ↑ Chen SD, Yang JL, Hwang WC, Yang DI. Emerging Roles of Sonic Hedgehog in Adult Neurological Diseases: Neurogenesis and Beyond. Int J Mol Sci. 2018 Aug 16;19(8). pii: ijms19082423. doi: 10.3390/ijms19082423. PMID:30115884 doi:http://dx.doi.org/10.3390/ijms19082423
  7. ↑ 7.0 7.1 Singh S, Tokhunts R, Baubet V, Goetz JA, Huang ZJ, Schilling NS, Black KE, MacKenzie TA, Dahmane N, Robbins DJ. Sonic hedgehog mutations identified in holoprosencephaly patients can act in a dominant negative manner. Hum Genet. 2009 Feb;125(1):95-103. doi: 10.1007/s00439-008-0599-0. Epub 2008 Dec , 5. PMID:19057928 doi:http://dx.doi.org/10.1007/s00439-008-0599-0
  8. ↑ 8.0 8.1 Evangelista M, Tian H, de Sauvage FJ. The hedgehog signaling pathway in cancer. Clin Cancer Res. 2006 Oct 15;12(20 Pt 1):5924-8. doi:, 10.1158/1078-0432.CCR-06-1736. PMID:17062662 doi:http://dx.doi.org/10.1158/1078-0432.CCR-06-1736
  9. ↑ 1. Adler, Keith, and Morgan Miller. “Sonic Hedgehog Protein (SHH).” Sonic Hedgehog Protein, Kenyon College, July 2014, biology.kenyon.edu/BMB/jsmol2015/MM_KA_2015/index4.html. http://biology.kenyon.edu/BMB/jsmol2015/MM_KA_2015/index4.html

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