Processed Sonic hedgehog - an active signaling protease?

发育生物学的一个主要挑战是理解细胞如何协调其邻近细胞的发育行为。细胞通常使用分泌的信号分子，如Hedgehog(HH)形态原来控制发育生长和图案形成。然而，HH是一种不同寻常的信号分子。所有脊椎动物HH家族成员都是金属蛋白(包含一个四面体配位的锌离子)，它们是以双重脂质修饰的形式(N-末端棕榈酰化，C-末端胆固醇连接)合成的。这导致金属蛋白多聚化，并牢固地拴在生产细胞的表面。然而，HHS确实从生产细胞中释放出来，并形成依赖于脂肪作用的浓度梯度。这就提出了一个问题，即HH释放是如何实现的，具体地说，HH释放是如何与形态原梯度形成联系的。在此之前，我们已经证明，在脱氧酶介导的Sonic HH(Shh)从生产细胞中释放的过程中，N-末端的脂化是处理相关的N-末端多肽的先决条件。除了Shh的增溶作用外，N端肽的去除还会导致Shh四面体锌配位部位的暴露，该部位是受体(Patted，PTC)的结合部位。该位点先前被N-末端的多肽阻断。重要的是，Shh的锌配位部位与溶葡萄球菌酶活性部位非常相似，这表明Shh具有功能性的蛋白酶活性部位。因此，在这项拨款提案中，我们建议在释放过程中N-端肽的处理与非活性的、膜拴系的Shh酶原转化为活性的锌金属蛋白酶有关。通过处理细胞外基质成分，并可能通过自身降解，Shh蛋白水解酶的激活可能有助于形成其细胞外梯度。为了测试这一想法，我们的目标是通过现代的系统范围的蛋白酶底物图谱技术来表征体外和体内的Shh靶标和特异性。我们还计划测试功能性HH锌配位位点在果蝇体内系统中的假定作用。从这些实验中，我们期待着对体内和体外依赖蛋白分解的HH梯度形成的有趣见解，对发育和疾病具有重要的意义。

A major challenge in developmental biology is to understand how cells coordinate developmental behaviors with that of their neighbours. Cells often employ secreted signaling molecules such as the Hedgehog (Hh) morphogens to control developmental growth and patterning. Hh is an unusual signaling molecule, however. All vertebrate Hh family members are metalloproteins (containing a tetrahedrally coordinated zinc ion) that are synthesized in dually lipid-modified form (N-terminally palmitoylated, C-terminally cholesterol-linked). This results in metalloprotein multimerization and firm tethering to the surface of producing cells. Yet, Hhs do get released from producing cells and form lipidation-dependent concentration gradients. This raises the question of how Hh release is achieved, and specifically, how Hh release is linked to morphogen gradient formation. Previously, we showed that N-terminal lipidation is the prerequisite for the processing of an associated N-terminal peptide during sheddase-mediated Sonic Hh (Shh) release from producing cells. In addition to Shh solubilization, removal of N-terminal peptides results in the exposure of the Shh tetrahedral zinc-coordination site, which serves as the receptor (Patched, Ptc) binding site. This site was previously blocked by the N-terminal peptide. Importantly, the Shh zinc-coordination site and lysostaphin-type protease active sites are strikingly similar, indicating that Shh bears a functional protease active site. In this grant proposal, we thus suggest that N-terminal peptide processing during release is coupled to the conversion of an inactive, membrane-tethered Shh zymogen into an active zinc-metalloprotease. By processing extracellular matrix constituents, and possibly via autodegradation, Shh proteolytic activation may then help shape its extracellular gradient. To test this idea, we aim to characterize in vitro and in vivo Shh targets and specificity via modern, system-wide protease substrate profiling technology. We also plan to test putative roles of functional Hh zinc-coordination sites in the well-characterized Drosophila system in vivo. From these experiments, we expect fascinating insights into proteolysis-dependent Hh gradient formation in vivo and in vitro, with important implications for development and disease.