The cholesterol membrane anchor of the Hedgehog protein confers stable membrane association to lipid-modified proteins

Abstract

The Hedgehog proteins are potent organizers of animal development. They carry a cholesterol ester at the C terminus of their signaling domain. The membrane anchoring mediated by this lipophilic modification was studied by means of an approach integrating cell biology, biochemistry, biophysics, and organic chemistry techniques. Sterol-modified and fluorescent-labeled Hedgehog-derived peptides and proteins were synthesized and investigated in biophysical and cell-biological assays. These experiments revealed that cholesterol alone anchors proteins to membranes with significant strength and half-times for spontaneous desorption of several hours. Its membrane anchoring ability is comparable to dual lipidation motifs such as double geranylgeranylation or S-palmitoylation plus S-farnesylation found in other lipidated proteins. The experiments also demonstrate that membrane binding changes dramatically if short lipidated peptides are equipped with a large protein. These data suggest that for Hedgehog release and subsequent signaling an interaction partner such as the Dispatched protein is necessary. In addition to these findings the described approach allows one to correlate biophysical data obtained with model peptides with data determined with fully functional proteins and to combine results from in vitro and in vivo experiments. It should be generally applicable to other membrane anchors and proteins.M any proteins involved in key processes of cell growth and differentiation embody lipid modifications that are essential for their biological activity. These modifications serve in most cases as anchoring groups for targeting the proteins to a certain membrane or submembrane compartment. In addition, they may mediate controlled release of proteins from membrane regions to form stable gradients. This is particularly true for the Hedgehog protein (Hh), which is among the key players in patterning numerous types of tissues. Mutations in Hh and its downstream signaling molecules are also associated with numerous oncogenic and disease states.The Hh family of molecules consists of secreted proteins that undergo several posttranslational modifications to gain full activity. In a maturation process they perform an autocatalytic cleavage, generating an N-terminal polypeptide (Hh-Np) containing all of the signaling functions (1-4). During this cleavage process, a cholesterol moiety is attached covalently by an ester function to the C-terminal glycine of the signaling domain (5). The hydrophobicity of the protein is further increased by the addition of a palmitic acid residue to the N terminus of the cleavage product (6, 7).In Drosophila, forms of Hh in which the C terminus has been deleted have much more potent and seemingly longer-ranging signaling activity than the cholesterol-modified forms. This observation led to the idea that the cholesterol moiety acts as an anchor to the cell membrane and limits the spread of Hh (1-3, 8). However, in vertebrate Sonic Hedgehog (Shh) the cholesterol may be required for correct biological ...

Indian hedgehog mutations causing brachydactyly type A1 impair Hedgehog signal transduction at multiple levels

“…This observation was supported by immunofluorescence staining of transfected cells in which the mutant IhhN proteins were found to Figure S1). The tethered IhhN proteins might have undergone an autocatalytic modification with cholesterol, which is essential for Hh membrane association [33]. Furthermore, we showed that IhhN proteins are degraded through the lysosomal pathway, as incubation with two lysosome inhibitors, chloroquine and bafilomycin, significantly reduced the degradation of IhhN proteins, but treatment with proteasome inhibitor MG132 did not ( Figure 2C).…”

Dispatched mediates Hedgehog basolateral release to form the long-range morphogenetic gradient in the Drosophila wing disk epithelium

“…The mature protein has a palmitic acid covalently attached to the amino terminus and a cholesterol moiety attached to the carboxyl-terminus (8). These lipid adducts confer to Hh high affinity for cell membranes (9). Nevertheless, Hh can signal to cells distant from the source (5), suggesting the involvement of specific release mechanisms of this lipid-modified protein (9).…”

“…These lipid adducts confer to Hh high affinity for cell membranes (9). Nevertheless, Hh can signal to cells distant from the source (5), suggesting the involvement of specific release mechanisms of this lipid-modified protein (9). It has been found that dispatched (disp) gene is required exclusively in Hh-producing cells for the release of a fully functional Hh protein (10,11).…”

Addition of a cholesterol group to an HIV-1 peptide fusion inhibitor dramatically increases its antiviral potency

“…Quasi-irreversible binding requires the presence of two long-chain anchors in the molecule, for example, palmitoyl and farnesyl, or hexadecyl and farnesyl (46). The same quasiirreversible binding is achieved with a single cholesteryl moiety (47). The use of cholesterol for membrane targeting of a peptide has just been described to increase the potency of a transitionstate ␤-secretase inhibitor (48).…”