SHH E176/E177-Zn2+ conformation is required for signaling at endogenous sites

Abstract

Summary Sonic hedgehog (SHH) is a master developmental regulator. In 1995, the SHH crystal structure predicted that SHH-E176 (human)/E177 (mouse) regulates signaling through a Zn2+-dependent mechanism. While Zn2+ is known to be required for SHH protein stability, a regulatory role for SHH-E176 or Zn2+ has not been described. Here, we show that SHH-E176/177 modulates Zn2+-dependent cross-linking in vitro and is required for endogenous signaling, in vivo. While ectopically expressed SHH-E176A is highly active, mice expressing SHH-E177A at endogenous sites (ShhE177/−) are morphologically indistinguishable from mice lacking SHH (Shh−/−), with patterning defects in both embryonic spinal cord and forebrain. SHH-E177A distribution along the embryonic spinal cord ventricle is unaltered, suggesting that E177 does not control long-range transport. While SHH-E177A association with cilia basal bodies increases in embryonic ventral spinal cord, diffusely distributed SHH-E177A is not detected. Together, these results reveal a novel role for E177-Zn2+ in regulating SHH signaling that may involve critical, cilia basal-body localized changes in cross-linking and/or conformation.

Association of Sonic Hedgehog with the extracellular matrix requires its zinc-coordination fold

“…Although mutations of the central zinc coordinating triad are unstable and thus cannot be easily assessed for loss of peptidase function, mutations of several other catalytically important residues (E177, H135 and H181) are not destabilized and show a loss in the ability of Shh to enter the ECM. Together with the observation by Himmelstein and colleagues (Himmelstein et al, 2017) that ShhE177A cannot signal from the notochord to the overlying neural plate strongly supports the idea that a Shh-associated peptidase activity is required for non-cell autonomous signaling by promoting its distribution away from the source cells.…”

“…Zinc is a potent agonist of this putative peptidase activity as it increases ECM association of Shh but not of putative peptidase mutants. The observations that Shh-E177A is unable to mediate signaling from the notochord to the overlying neural tube (non-cell autonomously), and is more capable than Shh to induce the Hh response when expressed in the developing neural tube (likely cell-autonomously) (Himmelstein et al, 2017), provides in vivo evidence that the putative zinc-peptidase activity of Shh is required for non-cell autonomous signaling, but not for the activation of the Hh response pe se. The initial experiments that demonstrated that E177 is dispensable for the activation of the Hh response is easily explained as this mutant ligand was added to the responding cells as a purified and soluble fraction (Fuse et al, 1999), thus bypassing the requirement for the peptidase function that we propose.…”

“…The notion that Shh signaled through a peptidase activity was quickly rejected by demonstrating that mutation of a critical residue involved in catalysis (E177) did not impair the ability of Shh to activate the Hh response (Fuse et al, 1999), and consequently the zinc coordination domain of Shh is often referred to as its "pseudo active" site (Bosanac et al, 2009;Maun et al, 2010). Still, a role for the putative catalytic activity is supported by the finding that Shh-E177A is unable to mediate non-cell autonomous signaling from the notochord to the overlying neural plate (Himmelstein et al, 2017). The ability of Shh-E177A to induce the Hh response when expressed ectopically in the developing brain could be explained by cell-autonomous signaling.…”

Sonic Hedgehog is a member of the Hh/DD-peptidase family that spans the eukaryotic and bacterial domains of life

“…Analysis of this mutant in has revealed two interesting properties. First, Shh-E177A is unable to mediate signaling from the notochord to the overlying neural tube (in trans), but is more capable than Shh to induce the Hh response when expressed in the developing neural tube (likely in cis) [28]. Second, purified ShhN-E177A is more stable in solution than ShhN, indicating a cannibalistic peptidase activity that is intrinsic to ShhN.…”

Gain-of-function Shh mutants activate Smoin cisindependent of Ptch1/2 function

“…Published analysis of this mutant has revealed two interesting properties. First, Shh-E177A is unable to mediate Shh signaling from the notochord to the overlying neural tube (in trans), but is more capable than wt Shh to induce the Hh response when expressed in the developing neural tube (likely in cis) (Himmelstein et al, 2017). Second, ShhN-E177A is more stable in solution than ShhN (Rebollido-Rios et al, 2014).…”