Biosynthesis and Trafficking of Surfactant Protein C In Health and Disease

描述（由申请人提供）：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，合成为21 kD前肽（proSP-C21），并进行后处理以产生3.7 kD表面活性肺泡形式。SP-C对肺部健康和疾病的重要性已经通过观察而得到强调，即人类SP-C基因（SFTPC）中突变的杂合表达与间质性肺病（ILD）相关。本实验室最近的研究发现：（1）在proSP-C的NH_2端发现了一个新的PPDY基序，其配体E_3连接酶Nedd_4 -2是proSP-C靶向远端分泌途径的关键元件;（ii）在功能上表征了proSP-C COOH末端的不适当折叠对易聚集SFTPC突变所见的上皮细胞功能障碍的后果（iii）揭示了第二类表型上不同的ILD相关的SFTPC突变（“非BRICHOS”），其虽然不保留ER，但反而错配至非天然细胞器。我们发表的和新的初步数据还表明，非BRICHOS SP-C突变体破坏了内体/溶酶体功能以及通过ER应激非依赖性途径产生细胞毒性的大自噬。该项目旨在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3个新兴主题：蛋白质运输，细胞器稳态和细胞保护。具体目标1将集中于定义4个关键因子的作用，SP-C NH 2 PPDY基序，Nedd 4 -2 WW结构域，proSP-C单泛素化，以及高尔基体相关的衔接蛋白，？adaptin同系物，Arf结合蛋白3（GGA 3）在proSP-C的顺行运输。此外，将使用充分表征的体外模型系统和新一代诱导型Cre重组酶驱动的Nedd 4 -2缺陷小鼠模型研究通过破坏该靶向机制启动的proSP-C近端保留的后果，以评估体内质量控制响应。在特定目标2中，将评价由错误表达的非BRICHOS SP-C突变体亚型表达引起的AT 2细胞功能障碍和细胞毒性的潜在机制，并与对易聚集COOH折叠突变体的反应进行比较。从拟议的研究结果将提高我们的理解，不仅AT 2细胞功能障碍的病理生理学，伴随着纤维化肺重塑ILD的分子机制，但将提供一个框架和模型系统的“个性化”的蛋白质稳态和细胞信号转导的其他疾病相关的实质性肺疾病和上皮功能障碍的操纵。

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.