Biosynthesis and Trafficking of Surfactant Protein C In Health and Disease

说明(申请人提供)：肺表面活性蛋白C(SP-C)是一种肺特异性疏水肽，可增强表面活性磷脂的生物物理活性，它被合成为21kD的前肽(prosp-C21)，并经翻译后加工得到3.7kD的表面活性肺泡形式。SP-C基因(SFTPC)杂合性表达与间质性肺疾病(ILD)相关的研究结果，突显了SP-C对肺部健康和疾病的重要性。我们实验室最近的研究：(I)在prosp-C NH2末端及其配体E3连接酶Nedd4-2中发现了一个新的PPDY基序，作为靶向prosp-C远端分泌途径的关键元件；(Ii)从功能上表征了在伴发ILD的患者中发现的易于聚集的SFTPC突变(“Brichos”结构域)导致的上皮细胞功能障碍的后果；(Iii)发现了第二类表现明显的与ILD相关的SFTPC突变(“非Brichos”)，虽然没有保留ER，但错误地与非天然细胞器相关。我们已发表的和新的初步数据也表明，非Brichos SP-C突变体通过内质网应激非依赖性途径破坏内体/溶酶体功能以及产生细胞毒性的宏观自噬。这个项目寻求建立在我们长期研究SP-C生物合成代谢的细胞和分子机制的基础上，进一步关注对肺细胞生物学至关重要的三个新兴主题：蛋白质运输、细胞器动态平衡和细胞保护。具体目标1将集中于确定4个关键因素，即SP-C NH2 PPDY基序、Nedd4-2 WW结构域、prosp-C单泛素化以及适配蛋白高尔基相关、？-Adaptin同源物、Arf结合蛋白3(GGA3)在prosp-C顺行运输中的作用。此外，这一靶向机制的破坏引发的prosp-C的近端滞留的后果将使用两个良好的体外模型系统和新一代可诱导的Cre重组酶驱动的Nedd4-2缺乏症小鼠模型来研究，以评估体内的质量控制反应。在特定的目标2中，将评估由于表达错误的非Brichos SP-C突变异构体而导致AT2细胞功能障碍和细胞毒性的机制，并与易于聚集的COOH折叠突变体的反应进行比较。这项研究的结果将加深我们对ILD中伴随纤维化肺重塑的AT2细胞功能障碍的分子机制的理解，也将为与肺实质疾病和上皮功能障碍相关的其他疾病的蛋白平衡和细胞信号的“个性化”操作提供一个框架和模型系统。

DESCRIPTION (provided by applicant): Surfactant protein C (SP-C), a lung-specific hydrophobic peptide that enhances the biophysical activity of surfactant phospholipid, is synthesized as a 21 kD propeptide (proSP-C21) and post-translationally processed to yield a 3.7 kD surface active alveolar form. The importance of SP-C to lung health and disease has been underscored by observations that heterozygous expression of mutations in the SP-C gene (SFTPC) in humans is associated with interstitial lung disease (ILD). Recent studies from our laboratory have: (i) identified a novel PPDY motif in the proSP-C NH2 terminus and its ligand the E3 ligase Nedd4-2 as crucial elements in the targeting of proSP-C to the distal secretory pathway; (ii) functionally characterized the consequences of improper folding of the proSP-C COOH terminus for epithelial cell dysfunction seen with aggregation prone SFTPC mutations ("BRICHOS" domain) found in patients with accompanying ILD; (iii) uncovered a second class of phenotypically distinct ILD-associated SFTPC mutations ("Non-BRICHOS") that, while not ER retained, are instead mistrafficked to non-native organelles. Our published and new preliminary data also indicate that non-BRICHOS SP-C mutants disrupt both endosomal/ lysosomal function as well as macroautophagy producing cytotoxicity by ER stress independent pathways. This project seeks to build upon our long-standing discovery program studying cellular and molecular mechanisms underlying the biosynthetic metabolism of SP-C by further focusing on 3 emerging themes critical to lung cell biology: protein trafficking, organelle homeostasis, and cytoprotection. Specific Aim 1 will focus on defining the role of 4 key factors, the SP-C NH2 PPDY motif, Nedd4-2 WW domains, proSP-C monoubiquitination, and the adapter protein Golgi-associated, ?-adaptin homologues, Arf-binding protein 3 (GGA3) in anterograde trafficking of proSP-C. In addition, the consequences of proximal retention of proSP-C initiated by disruption of this targeting machinery will be studied using both well-characterized in vitro model systems and a new generation inducible, Cre-recombinase-driven mouse model of Nedd4-2 deficiency to evaluate quality control responses in vivo. In Specific Aim 2, mechanisms underlying AT2 cell dysfunction and cytotoxicity resulting from expression of mistrafficked non-BRICHOS SP-C mutant isoforms will be evaluated and compared with responses to aggregation prone COOH folding mutants. Results from the proposed studies will enhance our understanding of the molecular mechanisms underlying not only the pathophysiology of AT2 cell dysfunction that accompanies fibrotic lung remodeling in ILD but will provide a framework and model systems for "personalized" manipulation of proteostasis and cellular signaling for other disorders associated with parenchymal lung disease and epithelial dysfunction.