Exosites, ligands and complex formation: novel determinants of protease specificity

蛋白质是生命的三大基石之一，我们体内至少有10万种不同的蛋白质。在它们的生命周期中，所有这些蛋白质都会遇到蛋白质降解酶，称为蛋白酶，这将导致它们的活化，失活，修饰或完全去除。因此，蛋白质水解，即蛋白质的降解，是一个基本过程，必须严格调控。蛋白质的随机降解会导致严重的细胞和组织损伤。蛋白质的选择性降解是由大约700种不同的蛋白酶完成的。所有这些蛋白酶是如何识别它们的蛋白质底物的？我们的研究计划旨在回答这个问题。由于蛋白质可能非常大，蛋白酶确实可能识别其靶底物上的某些表面特征，这些表面特征可能远离实际切割位点。我们已经鉴定出弹性蛋白降解蛋白酶上所谓的外位点，它们专门与该蛋白质相互作用。如果没有弹性蛋白与胞外位点的结合，就不会发生降解。我们还假设，这些外部位点蛋白相互作用将底物定位到正确的方向以允许切割，甚至调节底物的结构以使切割更容易。弹性蛋白是我们体内最丰富但也是最耐蛋白酶的蛋白质之一，负责我们器官的完整性和弹性，包括动脉，肺和皮肤。有趣的是，相同的外切位点也被另一种蛋白酶用于形成蛋白酶复合物，该蛋白酶复合物特异性地降解胶原蛋白，另一种主要的结构蛋白。这告诉我们，相同或相似的外切位点可以利用不同的机制降解不同的蛋白质。使用生物化学，结构和细胞的方法，我们将表征这些外位点的性质和架构，并探索选择性蛋白质降解的新机制。我们还将表征与外切位点相互作用并调节这些蛋白酶的底物特异性的某些配体。

Proteins are one of the three building blocks of life and there are at least 100,000 different of them in our body. During their life-time, all of these proteins will encounter protein-degrading enzymes, called proteases, which will either lead to their activation, inactivation, modification or complete removal. Proteolysis, the degradation of proteins, is thus a fundamental process and must be tightly regulated. A random degradation of proteins would lead to severe cell and tissue damage. The selective protein degradation is accomplished by an army of about 700 different proteases. How do all these proteases recognize their protein substrates? Our research proposal aims to answer this question. As proteins can be very large, proteases do likely recognize certain surface features on their target substrates which can be remote from the actual cleavage site. We have identified so-called exosites on elastin-degrading proteases, which specifically interact with this protein. Without the binding of elastin to the exosites, no degradation occurs. We also hypothesize that these exosite protein interactions situate the substrate into the right orientation to allow cleavage or even modulate the structure of the substrate to make cleavage easier. Elastin is one of the most abundant but also most protease-resistant proteins in our bodies and is responsible for the integrity and elasticity of our organs including arteries, lung and skin. Interestingly, the same exosites are also used by another protease to form a protease complex which specifically degrades collagen, another major structural protein. This tells us that the same or similar exosites can be exploited for the degradation of different proteins using different mechanisms. Using biochemical, structural, and cellular approaches, we will characterize the nature and architectures of those exosites and explore novel mechanisms of selective protein degradation. We will also characterize certain ligands which interact with exosites and regulate the substrate specificity of these proteases.