Metalloprotease-Substrate Interactions in Endothelial Cells

在脊椎动物中，白色血细胞通过保护免受细菌、真菌和病毒感染而在免疫系统中发挥重要作用。白色血细胞在体内巡逻，寻找感染的迹象。这种免疫监视涉及白色细胞和血管内壁细胞之间的相互作用。白色细胞通过各种信号，特别是称为趋化因子的分子，被招募到损伤区域。在趋化因子家族中，一个成员CX3CL 1锚定在血管内衬细胞的表面，CX3CL 1既吸引白色细胞，又将它们物理附着在血管上。CX3CL1在被ADAM 17切割后从血管细胞表面释放，ADAM 17是一种充当分子剪刀的酶。然而，ADAM 17与CX3CL1接触和切割CX3CL1的方式还不清楚。 我们的初步工作表明，CX3CL 1通常被限制在沿着血管细胞表面沿着的栅栏细胞内，并且它与ADAM 17分离。 使用最先进的显微镜技术，我们将研究细胞表面的CX3CL1是如何被限制的。 具体来说，我们将确定CX3CL1是否被束缚在维持细胞形状的内部骨架上，或者细胞外分泌的分子是否将CX3CL1捕获在它们的网格内。 我们还将研究ADAM 17是否在细胞表面或细胞内的单独隔室中切割CX3CL1，以及ADAM 17如何进入CX3CL1。 最后，我们将确定CX3CL 1的释放如何影响血管细胞的屏障完整性，以及它们招募白色血细胞的能力。通过了解分子细节，我们的研究将揭示脊椎动物中保守的免疫细胞巡逻的基本过程。我们的工作将进一步阐明ADAM 17分子剪刀在不同物理条件下选择性切割不同类型分子的方式。

In vertebrate animals, white blood cells play an important role in the immune system by protecting against bacterial, fungal, and viral infections. White blood cells patrol the body looking for signs of infection. This immune surveillance involves interactions between white cells and the cells lining blood vessels. White cells are recruited to areas of injury by various signals, especially molecules called chemokines. Among the family of chemokines, one member, CX3CL1, is anchored to the surface of cells lining blood vessels, and CX3CL1 both attracts white cells and physically attaches them to blood vessels.*******CX3CL1 is released from the surface of blood vessel cells after being cut by ADAM17, an enzyme that acts as molecular scissors. However, the way in which ADAM17 makes contact with and cuts CX3CL1 is not well understood. Our preliminary work suggests that CX3CL1 is normally restricted within fenced corrals along the surface of blood vessel cells, and that it is segregated from ADAM17. Using state-of-the-art microscopy techniques, we will examine how CX3CL1 at the surface of the cell is confined. Specifically, we will determine whether CX3CL1 is tethered to the internal skeleton that maintains the shape of the cell, or whether molecules secreted outside the cell trap CX3CL1 within their mesh. We will also examine whether ADAM17 cuts CX3CL1 at the cell surface or in a separate compartment inside the cell, and how ADAM17 gains access to CX3CL1. Lastly, we will determine how release of CX3CL1 affects the barrier integrity of blood vessel cells, and their ability to recruit white blood cells. By understanding the molecular details, our studies will shed light on a fundamental process of immune cell patrolling conserved among vertebrate animals. Our work will further clarify the way in which the ADAM17 molecular scissors can selectively cut different types of molecules under different physical conditions.************