Lack of type VII collagen (C7) disrupts cellular proteostasis yet the mechanism remains undescribed. By studying the relationship between C7 and the extracellular matrix (ECM)-associated proteins thrombospondin-1 (TSP1), type XII collagen (C12) and tissue transglutaminase (TGM2) in primary human dermal fibroblasts from multiple donors with or without the genetic disease recessive dystrophic epidermolysis bullosa (RDEB) (n=31), we demonstrate that secretion of each of these proteins is increased in the presence of C7. In dermal fibroblasts isolated from patients with RDEB, where C7 is absent or defective, association with the COPII outer coat protein SEC31 and ultimately secretion of each of these ECM-associated proteins is reduced and intracellular levels are increased. In RDEB fibroblasts, overall collagen secretion (as determined by the levels of hydroxyproline in the media) is unchanged while traffic from the ER to Golgi of TSP1, C12 and TGM2 occurs in a type I collagen (C1) dependent manner. In normal fibroblasts association of TSP1, C12 and TGM2 with the ER exit site transmembrane protein Transport ANd Golgi Organization-1 (TANGO1) as determined by proximity ligation assays, requires C7. In the absence of wild-type C7, or when ECM-associated proteins are overexpressed, C1 proximity and intracellular levels increase resulting in elevated cellular stress responses and elevated TGFβ signaling. Collectively, these data demonstrate a role for C7 in loading COPII vesicle cargo and provides a mechanism for disrupted proteostasis, elevated cellular stress and increased TGFβ signaling in patients with RDEB. Furthermore, our data point to a threshold of cargo loading that can be exceeded with increased protein levels leading to pathological outcomes in otherwise normal cells.

缺乏 VII 型胶原蛋白 (C7) 会破坏细胞蛋白稳态，但其机制仍未得到描述。通过研究 C7 与细胞外基质 (ECM) 相关蛋白血小板反应蛋白 - 1 (TSP1)、XII 型胶原蛋白 (C12) 和组织转谷氨酰胺酶 (TGM2) 之间的关系，这些原代人皮肤成纤维细胞来自多个供体，有或没有隐性遗传病营养不良性大疱性表皮松解症 (RDEB) (n=31)，我们证明在 C7 存在的情况下，这些蛋白质中的每一种的分泌都会增加。在从 RDEB 患者分离的真皮成纤维细胞中，C7 缺失或有缺陷，与 COPII 外层蛋白 SEC31 的结合以及这些 ECM 相关蛋白的最终分泌减少，细胞内水平增加。在 RDEB 成纤维细胞中，总体胶原蛋白分泌（由培养基中的羟脯氨酸水平决定）没有变化，而 TSP1、C12 和 TGM2 从 ER 到高尔基体的流量以 I 型胶原蛋白 (C1) 依赖性方式发生。在正常成纤维细胞中，TSP1、C12 和 TGM2 与 ER 出口位点跨膜蛋白的关联Transport AN d Golgi Organization- 1 ( TANGO1 )由邻近连接分析确定，需要 C7 。在没有野生型 C7 的情况下，或当 ECM 相关蛋白过表达时，C1 接近度和细胞内水平增加，导致细胞应激反应升高和 TGFβ 信号传导升高。总的来说，这些数据证明了 C7 在装载 COPII 囊泡货物中的作用，并提供了 RDEB 患者蛋白质稳态破坏、细胞应激升高和 TGFβ 信号增加的机制。此外，我们的数据指出了货物装载的阈值，该阈值可以随着蛋白质水平的增加而超过，从而导致正常细胞出现病理结果。