Novel mechanism underlying fibrinogen biogenesis in the endoplasmic reticulum

大量的疾病现在被认为是由蛋白质错误折叠和随后的聚集引起的“构象疾病”。一个例子是肝纤维蛋白原储存疾病(HFSD)，其根本原因是内质网(ER)滞留和纤维蛋白原突变体聚集，导致肝脏损伤、低纤维蛋白原血症和大量出血。然而，内质网中野生型和突变型纤维蛋白原的生物发生和质量控制的分子事件仍不清楚。在这一应用的初步数据中，我们偶然发现纤维蛋白原的生物发生受到主要ER质量控制机制-ER相关降解(ERAD)的Sel1L-Hrd1蛋白复合体的调控。Sel1L-Hrd1 ERAD代表ERAD在进化上最保守的分支，以错误折叠的ER蛋白为胞浆蛋白酶体降解的靶点。肝细胞特异性Sel1L缺陷小鼠表现出肝细胞内质网中含有纤维蛋白原的包涵体，肝损伤和低纤维蛋白原血症，类似于人类HFSD患者。事实上，纤维蛋白原的野生型和致病突变体都能被Sel1L-Hrd1 ERAD降解。这些数据表明，肝细胞Sel1L-Hrd1 ERAD在纤维蛋白原生物发生和肝内稳态中起着关键作用。因此，这一应用的首要假设是Sel1L-Hrd1 ERAD是纤维蛋白原生物发生、凝血和肝脏动态平衡的关键调控机制，它通过靶向错误折叠的纤维蛋白原蛋白来降解蛋白酶体。ERAD对纤维蛋白原的这种作用可防止在肝脏中形成有毒的纤维蛋白原包涵体和聚集体。我们将完成两个目标：(1)阐明Sel1L-Hrd1 ERAD在凝血和纤维蛋白原生物发生中的意义和分子机制；(2)阐明Sel1L-Hrd1 ERAD在HFSD中的病理意义。这些研究的完成不仅将阐明ERAD在纤维蛋白原早期生物发生中的意义和分子机制，而且将对与蛋白质错误折叠相关的其他构象疾病的机制和治疗策略产生更广泛的影响。

A large number of diseases are now recognized as ‘conformational diseases’, caused by protein misfolding and subsequent aggregation. One example is hepatic fibrinogen storage disease (HFSD), where the underlying cause is the endoplasmic reticulum (ER) retention and aggregation of fibrinogen mutants, leading to liver damage, hypofibrinogenemia, and excessive bleeding. However, the molecular events underlying the biogenesis and quality control of, both wildtype and mutant fibrinogen, in the ER remain unknown. In the preliminary data of this application, we serendipitously found that the biogenesis of fibrinogen is regulated by the Sel1L-Hrd1 protein complex of the principal ER quality-control machinery, ER-associated degradation (ERAD). Sel1L-Hrd1 ERAD represents the most evolutionarily conserved branch of ERAD and targets misfolded ER proteins for cytosolic proteasomal degradation. Hepatocyte-specific Sel1L-deficient mice exhibit fibrinogen-containing inclusions in the ER of hepatocytes, hepatic damage and hypofibrinogenemia, resembling human patients with HFSD. Indeed, both wildtype and disease-causing mutants of fibrinogens are degraded by Sel1L-Hrd1 ERAD. These data point to a critical role of hepatocyte Sel1L-Hrd1 ERAD in fibrinogen biogenesis and live homeostasis. Hence, the overarching hypothesis of this application is that Sel1L-Hrd1 ERAD is a critical regulatory mechanism for fibrinogen biogenesis, coagulation and liver homeostasis by targeting misfolded, either wildtype or mutant, fibrinogen proteins for proteasomal degradation. This action of ERAD towards fibrinogen prevents the formation of toxic fibrinogen inclusions and aggregates in the liver. We will accomplish two Aims: (1) Demonstrate the significance and molecular mechanism of hepatic Sel1L-Hrd1 ERAD in coagulation and fibrinogen biogenesis; and (2) Delineate the pathological importance of Sel1L-Hrd1 ERAD in HFSD. Completion of these studies will not only delineate the significance and molecular mechanism underlying the ERAD function in early biogenesis of fibrinogen, but also have broader impact on the mechanism and therapeutical strategy for other conformational diseases associated with protein misfolding.