In vivo function of differentially processed Hedgehog morphogens

发育生物学的一个主要挑战是了解细胞如何与其邻居协调发育行为。细胞通常使用分泌的信号分子如刺猬（Hh）形态发生素来控制发育生长和模式化。然而，Hh是一种不寻常的信号分子。它以双重脂质修饰的形式合成（N-末端棕榈酰化，C-末端胆固醇连接），导致形态发生素多聚化和牢固的多聚体拴系到生产细胞的表面。这就提出了Hh释放和信号传导是如何实现的问题。使用重组表达的脊椎动物Hh家族成员Sonic hedgehog（Shh），我们发现A去整合素和金属蛋白酶（ADAM）家族成员介导转染细胞表面上其脂化的N-和C-末端的加工，导致Shh核心蛋白释放。在这种情况下，N-和C-末端脂质是必不可少的膜近端定位的ADAM切割位点及其后续处理。我们进一步提出，Shh加工和增溶与其同时激活相结合。我们发现，未加工的多聚体Shh结合到其受体Patched上接收细胞的能力是自动抑制的N-末端肽，阻止Patched结合位点的反式。然而，蛋白质被截短成Patched结合主管可溶性多聚体棕榈酰化依赖的切割和去除抑制性N-末端肽。基于这个模型，我们解释了生物活性的可溶性Shh棕榈酰酰基转移酶缺陷突变体中表达的缺乏N-末端加工和由此产生的补丁结合位点的封锁。在这里，我们建议在体内测试这一假设，采用果蝇系统。我们将在缺乏内源性Hh功能的细胞克隆中表达JNK截短的非棕榈酰化Hh蛋白变体，并确定其体内生物学活性。表型拯救将证实N-棕榈酰化在Hh功能中的间接作用以及Hh活化对N-末端肽加工的要求。此外，我们将利用果蝇遗传学的力量来解决Hh加工中可能冗余的蛋白酶的功能。

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. It is synthesized in dually lipid-modified form (N-terminally palmitoylated, C-terminally cholesterol-linked), resulting in morphogen multimerization and firm multimer tethering to the surface of producing cells. This raises the question of how Hh release and signaling is achieved. Using the recombinantly expressed vertebrate Hh family member Sonic hedgehog (Shh), we found that A Disintegrin And Metalloprotease (ADAM) family members mediate processing from its lipidated N-and C-termini on the surface of transfected cells, resulting in Shh core protein release. In this scenario, N- and C-terminal lipids are essential for the membrane-proximal positioning of ADAM cleavage sites and their subsequent processing. We further suggested that Shh processing and solubilization are coupled with its simultaneous activation. We showed that the ability of unprocessed multimeric Shh to bind to its receptor Patched on receiving cells is autoinhibited by N-terminal peptides that block the Patched-binding site in trans. However, proteins are truncated into Patched-binding competent soluble multimers upon palmitoylation-dependent cleavage and removal of the inhibitory N-terminal peptides. Based on this model, we explain biological inactivity of soluble Shh expressed in palmitoyl-acyltransferase-deficient mutants by the lack of N-terminal processing and the resulting blockade of the Patched-binding site. Here we propose to test this hypothesis in vivo, employing the Drosophila system. We will express variably N-truncated, non-palmitoylated Hh protein variants in cell clones lacking endogenous Hh function and determine their biolgical activities in vivo. A phenotypic rescue would confirm the indirect role of N-palmitoylation in Hh function and the requirement for N-terminal peptide processing for Hh activation. Moreover, we will use the power of Drosophila genetics to address the function of the presumably redundant proteases involved in Hh processing.