通过与循环内体互作影响大致密核心颗粒生物发生的机制研究

Large dense core vesicle (LDCV) in Paneth cell is a type of lysosome-related oragnelles (LRO). Its biogenesis is complicated through interactions with various organelles. Hermansky-Pudlak syndrome (HPS) is characterized by multiple LRO defects. Many HPS1 patients present inflammatory bowel disease, but the pathogenesis is unknown. It has been reported that the melanosomes (a type of LRO) are enlarged in HPS1 mouse mutants, in which recycling endosomes (REs) are involved. Our unpublished data has shown that another type of LRO, LDCV in Paneth cell, exhibited enlargement in HPS1 mouse mutant, implicating a conserved mechanism in the maturation of LDCVs. That is, LDCV may interact with RE, which has no report yet. Our organelle distributional assays showed that HPS1 deficiency led to abnormal subcellular distribution of Rab38 and VAMP7. We proposed that the deficiency of HPS1, as a GEF component of Rab38, affects the activity and subcellular localization of Rab38. Abnormal Rab38 distribution affects the recruitment of v-SNARE proteins (such as VAMP7) and the interaction between LDCV and REs in late maturation stage of LDCVs, resulting in defective maturation and secretion of LDCVs. In this project, we will determine the interaction between LDCVs and REs from the perspective of physical contacts and functional relevance, and explore the molecular machinery that regulates the interaction between LDCVs and REs, to provide new insights into the pathogenesis and treatment of inflammatory bowel diseases.

潘氏细胞大致密核心颗粒（LDCV）是一种溶酶体相关细胞器（LRO），其发生过程复杂，涉及到与多种细胞器互作。HPS综合征的病理特征是多种LRO的缺陷。HPS1患者表现有炎性肠病的发病机制不明。有报道HPS1小鼠突变体的黑素小体变大，其成熟过程需循环内体RE参与。我们前期工作表明，HPS1突变体小鼠潘氏细胞LDCV直径变大，其表型与黑素小体类似，暗示不同LRO的发生可能共享某些机制，即LDCV的成熟可能与RE有关，但尚未见报道。小肠隐窝蔗糖密度梯度离心结果显示，HPS1缺陷引起Rab38和VAMP7亚细胞分布改变。HPS1作为Rab38的GEF因子组分，其缺陷可能影响Rab38活性和亚细胞定位，进而影响LDCV成熟后期v-SNARE蛋白招募和通过RE的回收，导致LDCV成熟和分泌障碍。本项目将从物理接触和生理功能角度揭示LDCV与RE互作，并阐明互作分子机器，为炎性肠病发生和防治提供新思路。

Hermansky-Pudlak综合征（HPS）是一类表现为眼皮肤白化、出血倾向、慢性肠炎等一系列组织器官损伤的疾病。其致病原因与溶酶体相关细胞器（LRO）的生物学发生过程缺陷相关。其中HPS1型患者表现为炎性肠病，由小肠潘氏细胞中的大致密核心颗粒功能异常导致。潘氏细胞的大致密核心颗粒（LDCVs）是一种溶酶体相关细胞器（LRO），其发生过程涉及到与多种细胞器互作。我们的研究发现，HPS1患者及HPS1缺陷的ep小鼠肠道菌群微生物组发生变化，证明HPS1与炎性肠病的发生存在相关性；ep小鼠隐窝内的潘氏细胞数目减少，潘氏细胞内的LDCV数目增多，直径变大，说明HPS1在潘氏细胞LDCV的发生中发挥了重要作用；同时发现ep小鼠存在溶菌酶分泌缺陷，提示HPS1 影响潘氏细胞LDCV的功能行使，并特异性地参与了其中溶菌酶的分泌调控从而影响肠道微生物组成。进一步机制研究证明LDCV的成熟与循环内体（Recycling endosome, RE）相关。通过密度梯度离心发现，循环内体标记蛋白Rab11与LDCV特异性的抗菌肽Lysozyme有相同的分布，与Rab2a、VAMP7部分共定位，提示LDCV与RE存在相互作用和物质交换的可能；HPS1作为Rab38的GEF因子组分，其缺陷影响Rab38/Rab32活性和亚细胞定位，导致VAMP7和Rab2a分布改变，进而影响LDCV成熟过程中Rab2a，VAMP7的招募和通过RE的回收，导致LDCV成熟和分泌障碍。本项目从物理接触和生理功能角度阐述了LDCV与RE互作，为炎性肠病发生和防治提供新思路。