Rab38在肺泡II型细胞中对板层小体生物生成的调控机制研究

Rab38 is a lung epithelium-restricted master regulator of intracellular membrane trafficking. Rab38 has a null mutation in a rat model [i.e., Fawn-hooded hypertension (FHH) rat] of Hermansky-Pudlak syndrome (HPS), but coordinates the molecular functions of three (AP3, BLOC-2 & -3) out of four multiprotein complexes mutated in human HPS patients. We have shown that the Rab38-null mutation in the FHH rat produces the giant lamellar body (GLB) pathology found in fibrotic lungs of HPS patients. Rab38 targets to 30-40% LBs at any moment; yet, 1) reexpressing EGFP-Rab38 alone rescues the GLB pathology in cultured FHH alveolar type II (ATII) pneumocytes. Our preliminary results further show that 2) Rab38 specifically targeted to a subgroup of larger and older LBs produced during a late stage of the LB biogenesis process, and the selective targeting resulted in inhibition of growth in size of the targeted LBs. Importantly, 3) trafficking of a lysosomal lipid-metabolizing enzyme, peroxiredoxin-6 (Prdx6), to LBs was abolished in the Rab38-null FHH ATII cells. These results support our hypothesis that Rab38 regulates Prdx6 trafficking to a subgroup of larger and older LBs for proper control of local lipid metabolism; disruption of this process leads to the GLB pathology. To test this hypothesis, we will establish 1）Prdx6 is a cargo of the Rab38-regulated trafficking in the ATII pneumocyte, 2）the causal relationship between the Rab38-null mutation and the GLB pathology, 3) Rab38 preferentially regulates a subgroup of larger, older LBs and BLOC-3's role in identifying these LBs, and 4) the role of molecular machine Rab38-Varp-VAMP7 during the membrane fusion process between the Rab38-labeled trafficking vesicles and the targeted LBs. To accomplish these goals, we have developed innovative confocal and multiphoton microscopy that allows quantitative analysis of molecular localization, concentration, dynamics and interaction on individual LBs in single cultured ATII cells. We have also developed the lipophilic dye DPH as a sensitive indicator of surfactant lipid contents in individual LBs, thus allowing quantitative analysis of causal factors contributing to the GLB pathology in cultured ATII cells. Moreover, we have established stable primary ATII cells (i.e., Matrigel-cultured rat ATII cells) allowing RNAi knockdown experiments to specifically perturb individual components of the molecular machines involved in trafficking Prdx6 to LBs. Alternatively, rescue experiments will be carried out by reexpressing components of these molecular machines in cultured ATII cells using plasmid transfection or virus transduction methods. Together, these approaches will enable us to investigate a molecular mechanism underlying the GLB pathology proposed to be a root cause of the HPS-associated pulmonary fibrosis. In addition, we will establish Rab38 as the first molecular marker for a subgroup of LBs produced during a phased LB biogenesis process.

因肺泡II型细胞中板层小体的巨大化（GLB）病变而引起的肺纤维化是类溶酶体功能障碍（HPS）病人的致死原因。Rab38是肺上皮细胞对其囊泡运输进行调控的关键信号分子，并且与在HPS病人中发现的四种蛋白复合体中的三种（即AP3，BLOC-2和-3）发生直接分子相互作用。FHH大鼠中的基因突变导致Rab38蛋白表达缺失，其肺泡II型细胞也发生GLB病变。现有证据表明：Rab38与其上、下游分子伙伴协作向一类体积较大、生成时间较长的板层小体亚群输送脂类分子降解酶Prdx6；作用于这一过程的分子机器的损伤造成GLB病变。为了验证以上机制，我们将证明：1）Rab38调控Prdx6的囊泡运输，2）Prdx6运输缺陷与GLB病变之间的因果关系，3）Rab38和BLOC-3选择性调控针对一类较成熟板层小体的囊泡运输，4）Rab38-Varp-VAMP7分子机器作用于运输囊泡与目标板层小体的膜融合过程。

细胞器的生物生成机制是细胞生物学研究的基本问题，其缺损也与人类疾病发生息息相关。其中，与溶酶体或类溶酶体（lysosome-related organelle）功能障碍有关的人类遗传疾病已被发现超过50多种。与溶酶体相似, 类溶酶体包含一个酸性的内腔和部分溶酶体酶以作用于生物物质降解。与类溶酶体缺陷有关的疾病主要为白化病、出血难止、免疫缺陷、和因肺泡II型细胞中板层小体（Lamellar Body）病变而引起的肺纤维化疾病。Rab38属于Rab-family small GTPases；Rab GTPase是细胞内对囊泡运输进行调控的关键信号分子，而Rab38在经典类溶酶体（即黑素体）的生物生成过程中发挥至关重要的作用。在Rab38缺失的大鼠中，其肺泡II细胞中的类溶酶体，即板层小体，发生巨大化（giant lamellar body）病变。对于Hermansky-Pudlak syndrome病人，因板层小体巨大化病变而导致的肺纤维化是至死疾病。本项目1）建立了一种从小鼠动物模型分离并体外培养肺泡II型原代细胞的方法，2）开发了一种定量分析共聚焦-多光子荧光显微镜分析技术；3）应用定量分析显微镜技术发我们发现了Rab38在大鼠肺泡II型细胞中特异性地标记并调控一种直径更大、发育更完全的板层小体亚群；4）应用荧光显微镜技术在大鼠肺泡II型细胞中发现了Rab38-Varp-VAMP7分子相互作用，并应用免疫共沉淀（Co-immunoprecipitation；Co-IP）技术在人源肺癌A594细胞中验证了Rab38-Varp-VAMP7分子机器的组装。根据实验结果，我们提出了一种由磷脂分子转运体ABCA3和膜转运调控分子Rab38共同调控LB细胞器生物生成的分子机制假说：即ABCA3促进LB的新生和成长，Rab38直接调控其中体型较大、发育更成熟的LB亚群，并可能提供抑制ABCA3功能来控制LB的大小，从而防止LB的巨大化病变。本项目研究了细胞生物学中的一个根本问题，即类溶酶体生物生成过程中的囊泡运输调控机制。我们研究的分子对象，Rab38，是对板层小体囊泡运输进行上游调控的关键信号分子。我们的研究结果表明Rab38是防止板层小体巨大化病变的关键调控分子，从而为治疗相关肺纤维化疾病奠定理论基础。