Processed Sonic hedgehog - an active signaling protease?

发育生物学的一个主要挑战是了解细胞如何与邻近细胞协调发育行为。细胞通常使用分泌的信号分子，如刺猬（Hh）形态因子来控制发育生长和模式。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（含有四面体配位锌离子），以双脂质修饰形式合成（n端棕榈酰化，c端胆固醇连接）。这导致了金属蛋白的多聚和牢固地附着在产生细胞的表面。然而，Hhs确实从生产细胞中释放出来，并形成脂质依赖的浓度梯度。这就提出了Hh释放是如何实现的问题，特别是Hh释放是如何与形态因子梯度形成相关联的。在此之前，我们发现在产生细胞的脱落酶介导的Sonic Hh （Shh）释放过程中，n端脂化是相关n端肽加工的先决条件。除了Shh的溶解作用外，n端肽的去除还会导致Shh四面体锌配位位点的暴露，该位点作为受体（Patched, Ptc）的结合位点。该位点先前被n端肽阻断。重要的是，Shh锌配位位点和溶葡萄球菌蛋白型蛋白酶活性位点惊人地相似，这表明Shh具有功能性蛋白酶活性位点。因此，我们认为释放过程中的n端肽加工与无活性的膜系住Shh酶原转化为活性锌金属蛋白酶有关。通过处理细胞外基质成分，并可能通过自降解，Shh蛋白水解激活可能有助于形成其细胞外梯度。为了验证这一想法，我们的目标是通过现代的全系统蛋白酶底物分析技术来表征体外和体内Shh靶点和特异性。我们还计划在体内测试功能性Hh锌配位位点在果蝇系统中的推测作用。从这些实验中，我们期望对体内和体外蛋白质水解依赖Hh梯度形成的有趣见解，对发育和疾病具有重要意义。

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.