Synthetic enzyme-substrate tethering obviates the Tolloid-ECM interaction during Drosophila BMP gradient formation.
Synthetic enzyme-substrate tethering obviates the Tolloid-ECM interaction during Drosophila BMP gradient formation.
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DOI：
10.7554/elife.05508
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发表时间：
2015-02-02
期刊：
影响因子：
7.7
通讯作者：
Ashe HL
中科院分区：
文献类型：
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作者：
Winstanley J;Sawala A;Baldock C;Ashe HL
金属蛋白酶Tolloid家族的成员从抑制复合物中释放BMP，以调节胚胎背腹轴模式形成期间BMP梯度的形成。在这里，我们确定机械如何Tolloid活性调节其非催化CUB结构域在果蝇胚胎。我们表明，Tolloid，通过其N-末端CUB结构域，与IV型胶原蛋白，这增强了Tolloid对其底物Sog的活性相互作用，并促进Tsg依赖的刺激裂解。相比之下，两个最C-末端Tld CUB结构域介导Sog相互作用以促进其加工，因为基于我们的结构数据，Tolloid曲率位置将Sog结合在蛋白酶结构域附近。有归因于功能的Tolloid非催化结构域，我们重演胚胎BMP梯度形成在他们的情况下，通过人工拴系的Tld蛋白酶结构域的Sog。我们的研究强调了Tolloid CUB结构域在底物和ECM相互作用中的双向功能如何微调蛋白酶活性以适应特定的发育环境。DOI：http://dx.doi.org/10.7554/eLife.05508.001动物的身体是一个高度组织化的组织和器官结构，其中包含具有专门功能的细胞。为了达到这种组织水平，重要的是胚胎中的细胞知道它们的位置，并接收关于如何发育、何时分裂或移动的正确指令。许多动物都是关于一条从头部到尾部的假想线大致对称的;发育中的胚胎可以向其细胞提供关于它们沿着这条首尾轴和从前到后的轴的位置信息。在胚胎中建立前后轴涉及一个称为骨形态发生蛋白（或BMP）的蛋白质家族。这些蛋白质可以与其他蛋白质结合，这些蛋白质作为信号向细胞提供指令。然而，许多BMP无法执行这项工作，因为它们被与它们结合的抑制分子捕获。属于Tolloid家族的酶可以分解这些抑制剂以释放BMP。抑制剂和Tolloid酶一起在胚胎中产生BMP活性梯度。具有最高活性BMP水平的胚胎一侧设置身体后部的位置，而具有最低活性BMP水平的相对侧成为前部。然而，目前尚不清楚如何控制Tolloid来创建BMP梯度。Tolloid酶的不同部分具有不同的作用;酶的一部分分解抑制分子，并且还有几个所谓的“非催化结构域”。Winstanley等人使用多种方法来研究如何在果蝇胚胎中控制Tolloid。实验表明，Tolloid一端的两个非催化结构域有助于酶与抑制分子结合。在Tolloid酶的另一端，另一个非催化结构域可以与称为胶原蛋白IV的结构蛋白结合。这增强了酶分解抑制分子并释放BMP的能力。这些发现揭示了Tolloid的非催化结构域如何微调这种酶的活性，以产生BMP活性的梯度，这是在动物胚胎中设置前后方向所需的。未来的研究将集中在识别其他与Tolloid的非催化结构域结合的蛋白质，以进一步控制其在发育过程中的活性。DOI：http://dx.doi.org/10.7554/eLife.05508.002网站
Members of the Tolloid family of metalloproteinases liberate BMPs from inhibitory complexes to regulate BMP gradient formation during embryonic dorsal-ventral axis patterning. Here, we determine mechanistically how Tolloid activity is regulated by its non-catalytic CUB domains in the Drosophila embryo. We show that Tolloid, via its N-terminal CUB domains, interacts with Collagen IV, which enhances Tolloid activity towards its substrate Sog, and facilitates Tsg-dependent stimulation of cleavage. In contrast, the two most C-terminal Tld CUB domains mediate Sog interaction to facilitate its processing as, based on our structural data, Tolloid curvature positions bound Sog in proximity to the protease domain. Having ascribed functions to the Tolloid non-catalytic domains, we recapitulate embryonic BMP gradient formation in their absence, by artificially tethering the Tld protease domain to Sog. Our studies highlight how the bipartite function of Tolloid CUB domains, in substrate and ECM interactions, fine-tune protease activity to a particular developmental context. DOI: http://dx.doi.org/10.7554/eLife.05508.001 The body of an animal is a highly organised structure of tissues and organs that contain cells with specialised roles. To achieve this level of organisation, it is important that the cells in the embryo know their location and receive the correct instructions on how to develop, when to divide or move. Many animals are roughly symmetrical about an imaginary line that runs from their head to their tail; a developing embryo can provide its cells with information about their position along this head-to-tail axis and the axis that runs from its front to its back. Setting up the front-to-back axis in the embryo involves a family of proteins called the bone morphogenetic proteins (or BMPs). These proteins can bind to other proteins that act as signals to provide instructions to cells. However, many of the BMPs are unable to perform this job because they are trapped by inhibitory molecules that bind to them instead. Enzymes belonging to the Tolloid family can break down these inhibitors to release the BMPs. Together, the inhibitors and Tolloid enzymes create a gradient of BMP activity across the embryo. The side of the embryo with the highest levels of active BMPs sets the position of the back of the body, while the opposite side—which has the lowest levels of active BMPs—becomes the front. However, it is not clear how Tolloid is controlled to create the BMP gradient. Different parts of the Tolloid enzyme have different roles; one portion of the enzyme breaks down the inhibitory molecules, and there are also several so-called ‘non-catalytic domains’. Winstanley et al. used a combination of approaches to study how Tolloid is controlled in fruit fly embryos. The experiments show that two non-catalytic domains at one end of Tolloid help the enzyme to bind to the inhibitory molecules. At the other end of the Tolloid enzyme, another non-catalytic domain can bind to a structural protein called Collagen IV. This enhances the ability of the enzyme to break down the inhibitory molecules and release the BMPs. These findings reveal how Tolloid's non-catalytic domains can fine-tune the activity of this enzyme to create the gradient of BMP activity that is needed to set the front-to-back direction in animal embryos. Future studies will focus on identifying other proteins that bind to the non-catalytic domains of Tolloid in order to further control its activity during development. DOI: http://dx.doi.org/10.7554/eLife.05508.002