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.

凝血酶原蛋白(TSP)是一种典型的与多种细胞外功能相关的基质蛋白。心脏损伤后，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。本研究试图阐明TSP蛋白在小鼠心脏中的新功能。然而，与TSP在ECM中发挥作用影响心脏重构的明显假设相反，我们最近的研究表明，TSP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蛋白家族具有与蛋白质质量控制、细胞外基质成分的分泌和组织相关的关键细胞内功能。作为一个额外的复杂性层，我们还将调查4个主要TSP家族成员之间的潜在差异，这些成员既有共同的功能域，也有独特的功能域。这项研究有两个目的：一是研究两个主要的TSP亚家族成员在心脏疾病中的具体作用；二是确定每个TSP蛋白是否通过激活转录因子6(ATF6)来调节内质网(ER)的功能。1)为了从功能上比较两个TSP亚群(A：TSP1/2；B：TSP3/4)，阐明它们在心脏稳态和疾病中的作用，我们将重点关注A组的TSP1和B组的TSP3。我们将利用心脏特异的TSP过表达小鼠和基因敲除小鼠来剖析这两种蛋白在体内的功能作用。Ii)与以前的发现不同，赞助商的实验室最近表明，TSP蛋白在细胞内具有主要功能，包括蛋白质穿梭、质量控制以及通过与ATF6的相互作用调节保护性内质网应激反应。然而，内质网应激在心脏病中的作用是不适应的还是有益的，仍然是一个有争议的话题。在本研究中，将利用TSP和ATF6的得失功能模型来了解它们在心脏中的相互作用和功能作用。为了研究TSPs如何改变蛋白质质量控制和细胞内运输和分泌的机制，将以敲除小鼠胚胎成纤维细胞(MEF)作为体外模型。此外，为了确定TSP和ATF6之间相互作用的特定蛋白结构域，以诱导保护性的内质网应激反应，将在TSP或ATF6基因敲除的MEF中使用TSP和ATF6单一蛋白结构域的过表达。

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.