Biosynthesis and Trafficking of Surfactant Protein C In Health and Disease

描述（由申请人提供）：表面活性剂蛋白C （SP-C）是一种肺特异性疏水肽，可增强表面活性剂磷脂的生物物理活性，合成为21 kD的前肽（pro - c21），翻译后加工产生3.7 kD的表面活性肺泡形式。人类SP-C基因（SFTPC）突变的杂合表达与间质性肺病（ILD）相关，这一观察结果强调了SP-C对肺部健康和疾病的重要性。我们实验室最近的研究发现：(i)在普洛斯- c NH2末端发现了一个新的PPDY基序及其配体E3连接酶Nedd4-2是将普洛斯- c靶向到远端分泌途径的关键元件；（ii）从功能上描述了在伴有ILD的患者中发现的易聚集的SFTPC突变（“BRICHOS”结构域）导致的上皮细胞功能障碍中，pro - c COOH末端折叠不当的后果；（iii）发现了第二类表型不同的与ild相关的SFTPC突变（“Non-BRICHOS”），这些突变虽然没有保留ER，但却被误载到非天然细胞器上。我们已发表的和新的初步数据还表明，非brichos SP-C突变体通过内质网应激独立途径破坏内体/溶酶体功能以及产生细胞毒性的巨噬。该项目旨在建立我们长期的发现计划，研究SP-C生物合成代谢的细胞和分子机制，进一步关注对肺细胞生物学至关重要的3个新兴主题：蛋白质运输，细胞器稳态和细胞保护。特异性目标1将侧重于确定4个关键因子的作用，即SP-C NH2 PPDY基序，Nedd4-2 WW结构域，普洛斯- c单泛素化以及适配蛋白高尔基相关，γ-适配蛋白同源物，arf结合蛋白3 （GGA3）在普洛斯- c顺行运输中的作用。此外，研究人员将利用体外模型系统和新一代可诱导的、由cred -重组酶驱动的Nedd4-2缺失小鼠模型来评估体内质量控制反应，研究这种靶向机制破坏引发的近端pro -c滞留的后果。在Specific Aim 2中，将评估AT2细胞功能障碍和细胞毒性的机制，并将其与COOH折叠突变体对聚集倾向的反应进行比较。拟议研究的结果将增强我们对ILD中伴随纤维化肺重塑的AT2细胞功能障碍的病理生理学基础的分子机制的理解，而且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质停滞和细胞信号的“个性化”操作提供框架和模型系统。

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, γ-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.