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

ADAMTSL 蛋白的结构和功能及其在组织组织中的作用

• 批准号: 1908761
• 金额: --
• 依托单位: University of Manchester
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2017
• 资助国家: 英国
• 起止时间: 2017 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-1908761
• 关键词: Structure; function; ADAMTSL; proteins; their

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.

弹性组织如眼韧带、皮肤、血管和肺含有弹性纤维，其由弹性蛋白和微纤维组成。弹性纤维的突变破坏了这些组织的整体功能，并导致广泛的疾病。它们统称为原纤维蛋白病，因为它们通常是由突变引起的。然而，最近，已发现基质金属蛋白酶家族中称为具有血小板反应蛋白1型重复的A去整合素和金属蛋白酶（ADAMTS）和ADAMTS样（ADAMTS-L）蛋白的突变引起相同的疾病。例如，ADAMTSL 4中的突变引起晶状体异位（EL）或晶状体脱位，ADAMTSL-2中的突变引起胶状体发育不良，ADAMTS 10中的突变引起Weill-Marchesani综合征ADAMTS家族由具有许多非催化辅助结构域的多结构域锌金属蛋白酶组成。ADAMTSL蛋白由辅助结构域组成，但缺乏催化结构域和活性。ADAMTS-L4共定位于包括睫状小带在内的眼前房中的微纤维中，这与其将透镜束缚于睫状体的作用一致。ADAMTSL-4突变小鼠在微纤维中具有紊乱的微纤维，表明ADAMTS-L4在微纤维沉积中的作用（柯林等人，2014年）。然而，目前对ADAMTSL蛋白的结构、与弹性纤维蛋白的相互作用以及在基质中的功能知之甚少，因此本课题的主要目的是：1）利用表面等离子体共振（SPR）和微量等温滴定量热法等结合分析技术，研究ADAMTSL蛋白、弹性纤维蛋白和其他弹性纤维蛋白之间的相互作用。将使用生物化学和基于细胞的方法研究ADAMTSL致病突变对蛋白质相互作用和信号传导的影响。2）使用cryoEM和单颗粒分析确定ADAMTSL家族成员和复合物中ADAMTSL-β的结构。3）确定ADAMTSL-β的组织结构。4在眼组织如睫状小带中的表达，并与ADAMTSL中睫状小带的组织结构进行比较。这些方法的基础是先进的成像技术，包括冷冻EM和3D重建，以及连续块面SEM成像。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ADAMTSL组装中的作用以及ADAMTSL-4突变对眼部疾病的影响。

项目成果

Fibrillin microfibrils and elastic fibre proteins: Functional interactions and extracellular regulation of growth factors.

• DOI: 10.1016/j.semcdb.2018.07.016
• 发表时间: 2019-05
• 期刊: Seminars in cell & developmental biology
• 影响因子: 7.3
• 作者: Thomson J;Singh M;Eckersley A;Cain SA;Sherratt MJ;Baldock C
• 通讯作者: Baldock C

Molecular Cloning, Lentiviral Transduction, and Expression of Recombinant ADAMTSL2 and ADAMTSL4.

重组 ADAMTSL2 和 ADAMTSL4 的分子克隆、慢病毒转导和表达。

• DOI: 10.1007/978-1-4939-9698-8_12
• 发表时间: 2020
• 期刊: Methods in molecular biology (Clifton, N.J.)
• 作者: Singh M

ADAMTS10-mediated tissue disruption in Weill-Marchesani syndrome.

• DOI: 10.1093/hmg/ddy276
• 发表时间: 2018-11-01
• 期刊: Human molecular genetics
• 影响因子: 3.5
• 作者: Mularczyk EJ;Singh M;Godwin ARF;Galli F;Humphreys N;Adamson AD;Mironov A;Cain SA;Sengle G;Boot-Handford RP;Cossu G;Kielty CM;Baldock C
• 通讯作者: Baldock C

Purification of Recombinant ADAMTSL2.

重组 ADAMTSL2 的纯化。

• DOI: 10.1007/978-1-4939-9698-8_13
• 发表时间: 2020
• 期刊: Methods in molecular biology (Clifton, N.J.)
• 作者: Singh M