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Identification of novel intracellular functions of the Thrombospondin protein family and characterization of the involvement in the endoplasmic reticulum (ER) protein shuttling, secretion and ER stress activation using in vivo and in vitro models.

使用体内和体外模型鉴定血小板反应蛋白家族的新细胞内功能，并表征内质网 (ER) 蛋白穿梭、分泌和 ER 应激激活的参与。

基本信息

批准号：
253341488
负责人：
Dr. Tobias Schips
金额：
--
依托单位：
Cincinnati Childrens Hospital Medical Center
依托单位国家：
德国
项目类别：
Research Fellowships
财政年份：
2013
资助国家：
德国
起止时间：
2012-12-31 至 2014-12-31
项目状态：
已结题

来源：
https://gepris.dfg.de/gepris/projekt/253341488?language=en
关键词：
Identification novel intracellular functions Thrombospondin

项目摘要

Thrombospondin (TSP) proteins are matricellular proteins typically associated with various extracellular functions. After injury, the heart primarily depends on new extracellular matrix (ECM) deposition and remodeling to maintain its structural and functional integrity. This study will attempt to elucidate the novel functional aspects of the TSP proteins in the mouse heart. However, in contrast to the obvious hypothesis that TSPs function within the ECM to affect cardiac remodeling, our recent studies have suggested that the TSP family of proteins functions in a completely novel manner to affect the ECM protein generation, modification and shuttling and secretion from within the cell. Hence our novel hypothesis is that the TSP family of proteins has a critical intracellular function associated with protein quality control, secretion and organization of ECM components. As an added layer of complexity, we will also investigate the potential differences between the 4 primary TSP family members which have both shared but also unique functional domains. This proposal is organized in two aims: i) investigate the specific role of the 2 primary TSP subfamily members during cardiac disease and ii) determine whether each TSP protein regulates the endoplasmic reticulum (ER) function through the activating transcription factor 6 (ATF6). i) To functionally compare the two TSP subgroups (A: TSP1/2; B: TSP3/4) and elucidate their involvement in cardiac homeostasis and disease, we will focus on TSP1 for group A and TSP3 for group B. The use of cardiac specific TSP overexpressing mice and knockout mice will be used to dissect the functional roles of these 2 proteins in vivo. ii) In contrast to previous findings, the sponsors laboratory has recently shown that TSP proteins have a primary function within the cell, which includes protein shuttling, -quality control and mediation of a protective ER stress response via interaction with ATF6. However, whether the role of ER stress in heart diseases is maladaptive or beneficial is still a controversial topic. In this study, gain and loss of function models of TSP and ATF6 will be used to understand their interplay and functional role in the heart. To study the mechanisms of how TSPs modify protein quality control and intracellular transport and secretion, knockout mouse embryonic fibroblasts (MEF) will serve as an in vitro model. Furthermore, in order to define the specific protein domains responsible for the interplay between TSPs and ATF6 to induce a protective ER stress response, overexpression of single protein domain constructs of TSPs and ATF6 will be used in TSP or ATF6 knockout MEFs.

血小板反应蛋白 (TSP) 蛋白是基质细胞蛋白，通常与各种细胞外功能相关。损伤后，心脏主要依靠新的细胞外基质（ECM）沉积和重塑来维持其结构和功能的完整性。本研究将尝试阐明小鼠心脏中 TSP 蛋白的新功能。然而，与 TSP 在 ECM 内发挥作用以影响心脏重塑的明显假设相反，我们最近的研究表明，TSP 蛋白家族以一种全新的方式发挥作用，影响 ECM 蛋白的生成、修饰以及细胞内的穿梭和分泌。因此，我们的新假设是 TSP 蛋白质家族具有与蛋白质质量控制、ECM 成分的分泌和组织相关的关键细胞内功能。作为复杂性的额外一层，我们还将研究 4 个主要 TSP 家族成员之间的潜在差异，这些成员既有共享的功能域，又有独特的功能域。该提案有两个目的：i) 研究 2 个主要 TSP 亚家族成员在心脏病期间的具体作用，ii) 确定每种 TSP 蛋白是否通过激活转录因子 6 (ATF6) 调节内质网 (ER) 功能。 i) 为了在功能上比较两个 TSP 亚组（A：TSP1/2；B：TSP3/4）并阐明它们在心脏稳态和疾病中的参与，我们将重点关注 A 组的 TSP1 和 B 组的 TSP3。将使用心脏特异性 TSP 过表达小鼠和基因敲除小鼠来剖析这 2 种蛋白质在体内的功能作用。 ii) 与之前的研究结果相反，申办者实验室最近表明 TSP 蛋白在细胞内具有主要功能，其中包括蛋白质穿梭、质量控制以及通过与 ATF6 相互作用介导保护性 ER 应激反应。然而，内质网应激在心脏病中的作用是适应不良还是有益仍然是一个有争议的话题。在这项研究中，TSP 和 ATF6 的功能获得和丧失模型将用于了解它们在心脏中的相互作用和功能作用。为了研究 TSP 如何改变蛋白质质量控制以及细胞内运输和分泌的机制，敲除小鼠胚胎成纤维细胞 (MEF) 将作为体外模型。此外，为了定义负责 TSP 和 ATF6 之间相互作用以诱导保护性 ER 应激反应的特定蛋白结构域，TSP 和 ATF6 的单蛋白结构域构建体的过表达将用于 TSP 或 ATF6 敲除 MEF。