Understanding how posttranslational modifications of the Shh ligand affect signalling pathway activity
PhD student: Salma Ahmed Zeidan
Supervisor at TUD : Ünal Coskun
Supervisor at KCL: Fiona M. Watt
Start date: 01.10.2017
The Hedgehog (Hh) pathway is used throughout development and adult life to regulate disparate cellular activities in an astonishing array of tissue types. Brain, bone, and blood vessel development all require the action of Hh despite their fundamental differences. While other major signalling pathways such as Wnt or BMP employ several ligands to encode different instructions, only three Hedgehog ligands exist in mammals, of which Sonic Hedgehog (Shh) is the most commonly deployed in vertebrate development and cancer. Although ligand concentration and duration of exposure is known to influence signal output, recent data implicate posttranslational modifications of the Shh ligand in pathway regulation.
To generate a functional ligand, Shh is processed in an intein-like manner releasing a 19 kDa N-terminal signalling fragment and a 25 kDa C-terminal fragment that orchestrates the catalytic process between Gly197 and Cys198 of the pre-protein. During processing, Shh is covalently bound to cholesterol at its C-terminal (Shh-Nc) and/or palmitoylated by Hedgehog acyltransferase (HHAT) at its N-terminal cysteine (Shh-Np), forming a stable amide linkage, essential for embryonic development. The mature Shh ligand can be secreted as a sterol free monomer (Shh N*) or as a lipoprotein-associated form when lipid modified in a dual lapidated form (ShhN). All four modified Shh forms are found in vivo in mammalian tissues, where the prevalence of a particular modification is context dependent.
Shh lipid modifications are proposed to alter the amplitude of response a cell will mount upon exposure to the ligand. One possible explanation for this signalling effect might be the interaction between different modified Shh ligands with the direct receptor Patched (Ptch1). Interestingly, X ray-crystallography and mass spectrometry reveals that the N- and C-terminal modifications of ShhN have two spatially distinct binding sites on Ptch1. The palmitoylated N-terminus fits into the cavity between extracellular domain ECD I and ECD II, and the globular part binds to extracellular domain ECD II of Ptch1. While binding of ShhN drives Ptch1 degradation and releases inhibition of the pathway, truncated and palmitoylated Shh, including only the first 22 amino acids (Shh22Np), does not degrade Ptch1, but instead drives its accumulation in the cilium. In addition, Shh22Np also affects trafficking of the downstream effector Smo, and processing of Gli transcription factors which ultimately execute pathway specific gene expression. The idea then emerged that distinct Shh ligand modifications can interact with Ptch1 differently, affecting dynamics of signalling molecules throughout transduction. How this truncated ligand may recapitulate signalling dynamics caused by modified full length proteins in vivo is not known. Here, we test the role of N- and/or C-terminal Shh modifications on the dynamics of signalling components and signal output. These data are essential for understanding how Shh signalling can elicit such distinct cellular responses, in varied biological contexts.
To test whether different Shh modified forms induce variable signalling, I used a Gli1-dependent transcriptional reporter assay. In this assay, cells stably expressing a luminescent reporter for Gli1 promoter activity (Light2 cells) are used to identify how much Gli1 is expressed following treatment with different modified forms of Shh. First, we analysed whether C-terminal cholesterol can alter Gli1 expression. Interestingly, the presence of C-terminal cholesterol in ShhNc drove higher Gli1 expression than sterol-free ShhN*. Similarly, the addition of C-terminal cholesterol to palmitoylated ShhNp (making ShhN), again, increased Gli1 expression in Light2 cells. These data indicate that C-terminal cholesterol controls the amplitude of signal activation. We then studied how the N-terminal palmitoyl group on full-length Shh affects signalling output. We found that N-terminal palmitoylation decreased the effective concentration for 50% of maximal activity (EC50) compared to non-palmitoylated forms with or without cholesteryl. Therefore, ShhNp can elicit the same transcriptional response as ShhN* and ShhNc, but at lower concentrations. These data indicate that N-terminal Shh modification influences the efficiency of pathway activation, which is consistent with concentration dependent effects previously observed in in vitro assays.
In line with our previous data, we hypothesize that different terminus modifications will affect skin regeneration differently. We expect the ShhN to support maximal HF regeneration compared to other forms. This is based on ShhN producing the highest signalling amplitude in Gli1 expression assays. To test our hypothesis, I will use Gli1 – LacZ mice to look at the ability of N- and/or C- terminal modified Shh to activate Shh signalling, thus activating skin regeneration (In collaboration with Prof. Fiona Watt, KCL). Using P21 wounded Gli1-LacZ mice, I plan to treat with different Shh forms in a scarring small wound and quantify 1) the number of activated dermal papilla cells (marked by Sox2), 2) HF formation in wound area, and 3) Gli1 expression in wound area.
Publications:
Glycolysis regulates Hedgehog signalling via the plasma membrane potential. S. Spannl, T. Buhl, I. Nellas, S.A. Zeidan, K.V. Iyer, H. Khaliullina, C. Schultz, A. Nadler, N.A. Dye, S. Eaton. EMBO J. 2020;39:e101767.
ROS Dynamics Delineate Functional States of Hippocampal Neural Stem Cells and Link to Their Activity-Dependent Exit from Quiescence. V.S. Adusumilli, T.L. Walker, R.W. Overall, G.M. Klatt, S.A. Zeidan, S. Zocher, D.G. Kirova, K. Ntitsias, T.J. Fischer, A.M. Sykes, S. Reinhardt, A. Dahl, J. Mansfeld, A.E. Rünker, G. Kempermann. Cell Stem Cell. 2021;28:300-314.
Apico-basal cell compression regulates Lamin A/C levels in epithelial tissues. K.V. Iyer, A. Taubenberger, S.A. Zeidan, N.A. Dye, S. Eaton, F. Jülicher. Nat Commun. 2021;12:1756.