Structure and function of ADAMTSL proteins and their role in tissue organisation

眼韧带、皮肤、血管和肺等弹性组织含有弹性纤维，这些纤维由弹性蛋白和纤维蛋白微原纤维组成。弹性纤维的突变破坏了这些组织的整体功能，导致了一系列疾病。总而言之，它们被称为纤维蛋白病，因为它们通常是由纤维蛋白突变引起的。然而，最近发现了一种名为A去整合素和带有凝血酶反应蛋白1型重复序列的金属蛋白酶(ADAMTS)和ADAMTS-样(ADAMTS-L)蛋白家族的突变可导致相同的疾病。例如，ADAMTSL4基因突变导致异位晶状体(EL)或晶状体脱位，ADAMTSL-2基因突变导致晶状体发育不良，ADAMTS10基因突变导致Weill-Marchesani综合征。ADAMTS家族由多结构域锌金属蛋白酶组成，带有多个非催化辅助结构域。ADAMTSL蛋白由辅助区组成，但缺乏催化结构域和活性。ADAMTS-L4共同定位于包括睫状带在内的眼前房中的纤维蛋白微纤维，这与其将晶状体与睫状体捆绑在一起的作用一致。ADAMTSL-4突变小鼠具有紊乱的纤维蛋白微纤维，这表明ADAMTS-L4在微纤维沉积中发挥了作用(Collin等人，2014年)。然而，对ADAMTSL蛋白的结构、它们与弹性纤维蛋白的相互作用以及它们在基质中的功能知之甚少。因此，本项目的目的是：1)利用表面等离子体共振和微等温滴定量热法研究ADAMTSL蛋白、纤维蛋白和其他弹性纤维蛋白之间的相互作用。将使用生化和基于细胞的方法来研究导致ADAMTSL疾病的突变对蛋白质相互作用和信号传递的影响。2)使用冷冻EM和单颗粒分析来确定ADAMTSL家族成员和ADAMTSL-纤维青素复合体的结构。3)使用免疫组织化学和序列块面扫描电子显微镜成像来确定ADAMTSL-4在眼组织中的组织结构，并与ADAMTSL-4突变小鼠中纤毛带的组织进行比较。结合这些从分子结构到整个组织功能的跨学科方法，将使我们能够了解ADAMTSL蛋白在纤维蛋白组装中的作用以及ADAMTSL-4突变对眼病的影响。

Elastic tissues such as eye ligaments, skin, blood vessels and lung contain elastic fibres which are composed of elastin and fibrillin microfibrils. Mutations in elastic fibres disrupt the overall function of these tissues and result in a wide spectrum of diseases. Collectively they are termed fibrillinopathies as they are usually caused by mutations in fibrillin. However recently, mutations in a family of matrix metalloproteases termed A Disintegrin And Metalloprotease with Thrombospondin type 1 repeats (ADAMTS) and ADAMTS-like (ADAMTS-L) proteins have been found to give rise to the same diseases. For instance, mutations in ADAMTSL4 give rise to ectopia lentis (EL) or dislocated lenses, mutations in ADAMTSL-2 to Geleophysic Dysplasia and mutations in ADAMTS10 to Weill-Marchesani Syndrome.The ADAMTS family is composed of multi-domain zinc metalloproteases with a number of non-catalytic ancillary domains. The ADAMTSL proteins are composed of the ancillary domains but lack the catalytic domain and activity. ADAMTS-L4 co-localises to fibrillin microfibrils in the anterior chamber of the eye including the ciliary zonules, which is consistent with its role in tethering the lens to the ciliary body. An ADAMTSL-4 mutant mouse has disordered fibrillin microfibrils indicating a role for ADAMTS-L4 in microfibril deposition (Collin et al., 2014). However, little is known about the structure of ADAMTSL proteins, the interactions they makes with elastic fibre proteins or their function in the matrix.Therefore the aims of this project are:1) To investigate the interactions between ADAMTSL proteins, fibrillin and other elastic fibre proteins using binding assays such as Surface Plasmon Resonance and micro-isothermal titration calorimetry. The consequence of ADAMTSL disease-causing mutations on protein interactions and signalling will be investigated using biochemical and cell-based approaches.2) To determine the structure of a member of the ADAMTSL family and an ADAMTSL-fibrillin complex using cryoEM and single particle analysis.3) To define the organisation of ADAMTSL-4 in ocular tissue such as the ciliary zonules using immunohistochemistry and serial blockface SEM imaging and to compare with the organization of the ciliary zonules in the ADAMTSL-4 mutant mouse.Underpinning these approaches are advanced imaging techniques including cryo-EM and 3D reconstruction, and serial blockface SEM imaging. Combining these interdisciplinary approaches from molecular structure to whole tissue function will allow us to understand the role of ADAMTSL proteins in fibrillin assembly and the consequence of ADAMTSL-4 mutations on eye disease.