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

蛋白质是生命的三大组成部分之一，我们体内至少有10万种不同的蛋白质。在它们的一生中，所有这些蛋白质都会遇到蛋白质降解酶，称为蛋白酶，这将导致它们的激活、失活、修饰或完全去除。因此，蛋白质分解，即蛋白质的降解，是一个基本的过程，必须严格控制。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种不同的蛋白酶组成的大军完成的。所有这些酶是如何识别它们的蛋白质底物的？我们的研究提案旨在回答这个问题。由于蛋白质可能非常大，所以蛋白水解酶很可能识别其目标底物上的某些表面特征，这些表面特征可能远离实际的切割位点。我们已经确定了弹性蛋白降解酶上的所谓外切酶，它与这种蛋白特异地相互作用。如果没有弹性蛋白与胞外的结合，就不会发生降解。我们还假设，这些胞外蛋白相互作用使底物处于正确的方向，以允许切割，甚至调节底物的结构，使切割更容易。弹性蛋白是我们身体中含量最丰富但也是最耐蛋白酶的蛋白质之一，它负责我们包括动脉、肺和皮肤在内的器官的完整性和弹性。有趣的是，同样的外切酶也被另一种酶利用来形成一种蛋白酶复合体，它专门降解另一种主要结构蛋白-胶原蛋白。这告诉我们，相同或相似的外生体可以利用不同的机制来降解不同的蛋白质。使用生化、结构和细胞方法，我们将描述这些外显体的性质和结构，并探索选择性蛋白质降解的新机制。我们还将表征某些与外切酶相互作用的配体，并调节这些蛋白酶的底物专一性。

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.