Subverting Hedgehog Protein Autoprocessing by Chemical Induction of Paracatalysis

Abstract

Hedgehog proteins, a family of vital cell signaling factors, are expressed in precursor form, which requires specialized autoprocessing, called cholesterolysis, for full biological activity. Cholesterolysis occurs in cis through the action of the precursor’s C-terminal enzymatic domain, HhC. In this work, we describe HhC activator compounds (HACs), a novel class of noncovalent modulators that induce autoprocessing infidelity, diminishing native cholesterolysis in favor of precursor autoproteolysis, an otherwise minor and apparently nonphysiological side reaction. HAC-induced autoproteolysis generates hedgehog protein that is cholesterol free and hence signaling deficient. The most effective HAC has an AC50 of 9 μM, accelerates HhC autoproteolytic activity by 225-fold, and functions in the presence and absence of cholesterol, the native substrate. HACs join a rare class of “antagonists” that suppress native enzymatic activity by subverting mechanistic fidelity.

Dual roles of the sterol recognition region in Hedgehog protein modification

“…Accordingly, cholesterol ligation can occur in vitro and in cells when the Hh N-terminus is replaced by fluorescent proteins or simply by a His-tag. [18][19][20][21][22] However, the molecular details of the SRR that facilitate cholesterol transfer remain unknown. The SRR, which spans 50-100 residues in different species, bears no clear homology to known proteins in any domain of life.…”

Hedgehog proteins create a dynamic cholesterol interface

“…With the resulting approximation, we created a full lipid bilayer containing 100 POPC molecules in each leaflet with~20% cholesterol content (24 cholesterol molecules in each leaflet), approximating the composition of cholesterol-rich microdomains in the Golgi and endoplasmic reticulum [41][42][43][44]. Several previous studies have demonstrated that Hedge domain residues N-terminal to C198 are not required for cholesterolysis [45][46][47][48]; therefore, we modeled the N-terminal portion of hSHH (C24-G197) as a G197-C198 thioester preceded by a S195-G196 dipeptide for computational simplicity. To prepare the system for MD simulations, we solvated the protein, neutralized charges with NaCl (0.15 M), and adjusted the system to physiological pH (7.4).…”

Nanomolar, noncovalent antagonism of hedgehog cholesterolysis: exception to the “irreversibility rule” for protein autoprocessing inhibition

“…Library screening for HhC inhibitors: We tested 1187 steroid analogs from ChemBridge as potential inhibitors of Hh cholesterolysis using continuous light-to-dark FRET activity assay. [14][15][16] The FRET reporter construct, C-H-Y, has HhC flanked by cyan fluorescent protein (C) and yellow fluorescent protein (Y), providing the FRET donor and acceptor, respectively. FRET is measured as the ratio of emission of 540 nm / 460 nm after excitation at 400 nm.…”

“…There is a striking resemblance between the inhibitors described here and a family of noncovalent, allosteric activators of HhC, which we described previously (Figure 1, right). 14 The inhibitor/activator duality appears to be mediated by the same allosteric site on HhC, and stem from subtle differences in the structure of a heterocycle substituent on the effector.…”

“…10,12,13 We have begun pursuing selective antagonists of HhC that can phenocopy the effects of deactivating HhC mutations. [14][15][16] Inhibitors of HhC cholesterolysis hold promise as chemical tools to understand the complex biology of Hh signaling. HhC inhibitor can also stabilize HhC's dynamic structure 17 and enable structural and functional characterization of this unusual autoprocessing element.…”