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.

All morphogens of the Hedgehog (Hh) family are synthesized as dual-lipidated proteins, which results in their firm attachment to the surface of the cell in which they were produced. Thus, Hh release into the extracellular space requires accessory protein activities. We suggested previously that the proteolytic removal of N-and Cterminal lipidated peptides (shedding) could be one such activity. More recently, the secreted glycoprotein Scube2 (signal peptide, cubulin domain, epidermal-growth-factor-like protein 2) was also implicated in the release of Shh from the cell membrane. This activity strictly depended on the CUB domains of Scube2, which derive their name from the complement serine proteases and from bone morphogenetic protein-1/tolloid metalloproteinases (C1r/C1s, Uegf and Bmp1). CUB domains function as regulators of proteolytic activity in these proteins. This suggested that sheddases and Scube2 might cooperate in Shh release. Here, we confirm that sheddases and Scube2 act cooperatively to increase the pool of soluble bioactive Shh, and that Scube2-dependent morphogen release is unequivocally linked to the proteolytic processing of lipidated Shh termini, resulting in truncated soluble Shh. Thus, Scube2 proteins act as protease enhancers in this setting, revealing newly identified Scube2 functions in Hh signaling regulation.

Signaling of the cytokine interleukin-6 (IL-6) via its soluble IL-6 receptor (sIL-6R) is responsible for the proinflammatory properties of IL-6 and constitutes an attractive therapeutic target, but how the sIL-6R is generated in vivo remains largely unclear. Here, we use liquid chromatography–mass spectrometry to identify an sIL-6R form in human serum that originates from proteolytic cleavage, map its cleavage site between Pro-355 and Val-356, and determine the occupancy of all O- and N-glycosylation sites of the human sIL-6R. The metalloprotease a disintegrin and metalloproteinase 17 (ADAM17) uses this cleavage site in vitro, and mutation of Val-356 is sufficient to completely abrogate IL-6R proteolysis. N- and O-glycosylation were dispensable for signaling of the IL-6R, but proteolysis was orchestrated by an N- and O-glycosylated sequon near the cleavage site and an N-glycan exosite in domain D1. Proteolysis of an IL-6R completely devoid of glycans is significantly impaired. Thus, glycosylation is an important regulator for sIL-6R generation.

Soluble Interleukin-6 receptor (sIL-6R) mediated trans-signaling is an important pro-inflammatory stimulus associated with pathological conditions, such as arthritis, neurodegeneration and inflammatory bowel disease. The sIL-6R is generated proteolytically from its membrane bound form and A Disintegrin And Metalloprotease (ADAM) 10 and 17 were shown to perform ectodomain shedding of the receptor in vitro and in vivo. However, under certain conditions not all sIL-6R could be assigned to ADAM10/17 activity. Here, we demonstrate that the IL-6R is a shedding substrate of soluble meprin α and membrane bound meprin β, resulting in bioactive sIL-6R that is capable of inducing IL-6 trans-signaling. We determined cleavage within the N-terminal part of the IL-6R stalk region, distinct from the cleavage site reported for ADAM10/17. Interestingly, meprin β can be shed from the cell surface by ADAM10/17 and the observation that soluble meprin β is not capable of shedding the IL-6R suggests a regulatory mechanism towards trans-signaling. Additionally, we observed a significant negative correlation of meprin β expression and IL-6R levels on human granulocytes, providing evidence for in vivo function of this proteolytic interaction.

The fly morphogen Hedgehog (Hh) and its mammalian orthologs, Sonic, Indian, and Desert hedgehog, are secreted signaling molecules that mediate tissue patterning during embryogenesis and function in tissue homeostasis and regeneration in the adult. The function of all Hh family members is regulated at the levels of morphogen multimerization on the surface of producing cells, multimer release, multimer diffusion to target cells, and signal reception. These mechanisms are all known to depend on interactions of positively charged Hh amino acids (the Cardin-Weintraub (CW) motif) with negatively charged heparan sulfate (HS) glycosaminoglycan chains. However, a precise mechanistic understanding of these interactions is still lacking. The proteins of the Hh family are powerful morphogens that control growth and patterning of developing embryos. Current models for Hh activity suggest that the morphogen disperses from a localized source and forms a gradient that patterns fields of responsive cells expressing the Hh receptor Patched (Ptc).Genetic evidence suggests that this process critically depends on heparan sulfate proteoglycan (HSPG) 2 expression. Upon secretion to the cell surface, Drosophila Hh forms nanoscale oligomers on the cell surface that co-localize with HSPGs (1). HS binds to the Cardin-Weintraub (CW) motif found on all known Hhs and regulates their function in flies (2, 3) and mice (4). In Drosophila (5) as well as in mammalian cell culture (6, 7), Hhs are always released from producing cells in multimeric form, as demonstrated by gel filtration analysis of the soluble morphogen. Release of the multimeric morphogen (the processed Hh N-terminal signaling domain, designated HhNp) from the cell surface depends on the expression of Dispatched (8) and A disintegrin and metalloprotease (ADAM) family members that mediate ectodomain shedding from transfected Bosc23 cells (9). HS is involved in the formation of the HhNp extracellular gradient, which, in the fly, depends on the expression of the Drosophila Exostosin (Ext) family of proteins, encoded by the genes tout velu (ttv), brother of tout velu, and sister of tout velu and the glycosylphosphatidylinositollinked HSPGs Dally and Dally-like, corresponding to vertebrate glypicans (2, 3, 10). HS expression and Dally-like/glypican expression are also essential for signal reception and modulation on Ptc-expressing receiving cells (10 -14) and participate in HhNp-Ihog interaction (15). However, the essential role of direct morphogen-HSPG interactions in embryonic patterning was recently challenged (16). In that report, transgenic mice made deficient in two ShhNp CW amino acid residues implicated in HS binding (17) lacked an Shh-related phenotype, suggesting that direct morphogen-HS interactions were not essential for normal development. However, in that report as well as in others (16 -18), CW-dependent HS interactions were characterized using a recombinant, non-physiological monomeric morphogen termed ShhN, whereas embryogenesis depends entirely on the activity of morp...

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