Abstract
The evolutionarily conserved Hedgehog (Hh) signaling pathway is crucial for programmed cell differentiation and proliferation. Dispatched (Disp) is a 12-transmembrane protein that plays a critical role in the Hedgehog (Hh) signaling pathway by releasing the dually lipidated ligand HhN from the membrane, a prerequisite step to the downstream signaling cascade. In this study, we focus on the Disp from water bear, a primitive animal known as the most indestructible on Earth. Using a zebrafish model, we show that the water bear homolog possesses the function of Disp. We have solved its structure to a 6.5-Å resolution using single-particle cryogenic electron microscopy. Consistent with the evolutional conservation of the pathway, the water bear Disp structure is overall similar to the previously reported structures of the fruit fly and human homologs. Although not revealing much detail at this resolution, the water bear Disp shows a different conformation compared to published structures, suggesting that they represent different functional snapshots.
Highlights
Multi-cellular organisms rely on proper cell-cell communications mediated by controlled signaling transduction throughout the life cycle
We report the characterization, expression, purification, and cryogenic electron microscopy (cryo-EM) structure determination of Disp from Hypsibius dujardini, a type of water bear
To elucidate the -unknown architecture of Disp, we first set to overexpress, purify, and determine the cryo-EM structure of the murine homolog as it is one of the most studied in the literature. mDisp was expressed in Expi293 cells with a thermostable green fluorescence protein (TGP) fused to its C-terminal (Cai et al, 2020)
Summary
Multi-cellular organisms rely on proper cell-cell communications mediated by controlled signaling transduction throughout the life cycle. The Hh ligand released by producing cells binds to Ptc, inhibiting the sterol-transport function and activating the downstream signaling cascade that leads to the transcription of several genes in the pathway (Hui and Angers, 2011). To reach and affect receiving cells in long-range signaling events such as tissue patterning (Etheridge et al, 2010), Hh must be firstly released from the membrane of the producing cells This process is facilitated by the transmembrane protein Dispatched (Disp) (Burke et al, 1999). The structural studies revealed its architecture with two sized ECDs connected to the 12-TMH domain Both groups reported the low-resolution map of Disp in complex with the ligand Hh, providing direct evidence for the interaction and revealing the interface information for the binding. The observations raise the possibility of the structural differences being related to Disp’s function and the structures provide a preliminary framework to design experiments to test this hypothesis
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