In vivo function of differentially processed Hedgehog morphogens

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。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)，导致形态原多聚体和牢固的多聚体拴在生产细胞的表面。这就提出了如何实现HH释放和信令的问题。利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)家族成员从其脂化的N-末端和C-末端介导了转基因细胞表面的加工，导致Shh核心蛋白的释放。在这种情况下，N-末端和C-末端脂质对于ADAM裂解位点的膜近端定位及其后续处理是必不可少的。我们进一步认为Shh的加工和增溶与其同时激活是耦合的。我们发现，未处理的多聚体Shh与其在接收细胞上修补的受体结合的能力被封闭反式中的修补结合部位的N-末端多肽所自动抑制。然而，蛋白质在棕榈酰化依赖的裂解和去除抑制性N-末端多肽后被截断成补丁结合的有能力的可溶性多聚体。基于这一模型，我们解释了棕榈酰-酰基转移酶缺陷突变体中表达的可溶性Shh的生物学活性丧失的原因是缺乏N-末端处理和由此导致的修补结合位点的阻断。在这里，我们建议使用果蝇系统在体内测试这一假说。我们将在缺乏内源性HH功能的细胞克隆中表达可变N-截短的、非棕榈酰化的HH蛋白变体，并测定它们在体内的生物活性。表型挽救将证实N-棕榈酰化在HH功能中的间接作用，以及N-末端多肽加工对HH激活的要求。此外，我们将使用果蝇遗传学的力量来解决可能参与HH加工的冗余蛋白水解酶的功能。

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.