RESUMO
All Hedgehog (Hh) proteins are released from producing cells despite being synthesized as N- and C-terminally lipidated, membrane-tethered molecules. Thus, a cellular mechanism is needed for Hh solubilization. We previously suggested that a disintegrin and metalloprotease (ADAM)-mediated shedding of Sonic hedgehog (ShhNp) from its lipidated N and C termini results in protein solubilization. This finding, however, seemed at odds with the established role of N-terminal palmitoylation for ShhNp signaling activity. We now resolve this paradox by showing that N-palmitoylation of ShhNp N-terminal peptides is required for their proteolytic removal during solubilization. These peptides otherwise block ShhNp zinc coordination sites required for ShhNp binding to its receptor Patched (Ptc), explaining the essential yet indirect role of N-palmitoylation for ShhNp function. We suggest a functional model in which membrane-tethered multimeric ShhNp is at least partially autoinhibited in trans but is processed into fully active, soluble multimers upon palmitoylation-dependent cleavage of inhibitory N-terminal peptides.
Assuntos
Proteínas Hedgehog/química , Proteínas Hedgehog/metabolismo , Palmitatos/farmacologia , Fragmentos de Peptídeos/metabolismo , Processamento de Proteína Pós-Traducional , Receptores de Superfície Celular/metabolismo , Animais , Anticorpos Monoclonais/farmacologia , Diferenciação Celular , Células Cultivadas , Embrião de Galinha , Condrócitos/citologia , Condrócitos/metabolismo , Cristalografia por Raios X , Proteínas Hedgehog/genética , Humanos , Camundongos , Modelos Moleculares , Células NIH 3T3 , Receptores Patched , Receptor Patched-1 , Fragmentos de Peptídeos/genética , Conformação Proteica , Receptores de Superfície Celular/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transdução de SinaisRESUMO
The ectodomains of numerous proteins are released from cells by matrix metalloproteases to yield soluble intercellular regulators. A disintegrin and metalloprotease (ADAM) family members have often been found to be the responsible "sheddases," ADAM17/tumor necrosis factor-alpha-converting enzyme being its best characterized member. In this work, we show that ShhNp (lipidated and membrane-tethered Sonic hedgehog) is released from Bosc23 cells by metalloprotease-mediated ectodomain shedding, resulting in a soluble and biologically active morphogen. ShhNp shedding is increased by ADAM17 coexpression and cholesterol depletion of cells with methyl-beta-cyclodextrin and is reduced by metalloprotease inhibitors as well as ADAM17 RNA interference. We also show that the amount of shed ShhNp is modulated by extracellular heparan sulfate (HS) and that ShhNp shedding depends on specific HS sulfations. Based on those data, we suggest new roles for metalloproteases, including but not restricted to ADAM17, and for HS-proteoglycans in Hedgehog signaling.
