Revealing potential Rab proteins participate in regulation of secretory autophagy machinery.

The Kaohsiung journal of medical sciences Pub Date : 2024-07-01 Epub Date: 2024-05-28 DOI:10.1002/kjm2.12848
Pei-Wen Lin, Man-Ling Chu, Yu-Wen Liu, Yu-Cing Chen, Yao-Hsiang Shih, Sheng-Hui Lan, Shang-Ying Wu, I-Ying Kuo, Hong-Yi Chang, Hsiao-Sheng Liu, Ying-Ray Lee
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Abstract

Autophagy can be classified as degradative and secretory based on distinct functions. The small GTPase proteins Rab8a and Rab37 are responsible for secretory autophagy-mediated exocytosis of IL-1β, insulin, and TIMP1 (tissue inhibitor of 54 metalloproteinase 1). Other Rab family members participating in secretory autophagy are poorly understood. Herein, we identified 26 overlapped Rab proteins in purified autophagosomes of mouse pancreatic β-cell "Min-6" and human lung cancer cell "CL1-5-Q89L" with high secretory autophagy tendency by LC-MS/MS proteomics analysis. Six Rab proteins (Rab8a, Rab11b, Rab27a, Rab35, Rab37, and Rab7a) were detected in autophagosomes of four cell lines, associating them with autophagy-related vesicle trafficking. We used CL1-5-Q89L cell line model to evaluate the levels of Rab proteins colocalization with autophagy LC3 proteins and presence in purified autophagosomes. We found five Rab proteins (Rab8a, Rab11b, Rab27a, Rab35, and Rab37) are highly expressed in the autophagosome compared to the normal control by immunoblotting under active secretion conditions. However, only Rab8a, Rab35, and Rab37 showing high colocalization with LC3 protein by cofocal microscopy. Despite the discrepancy between the image and immunoblotting analysis, our data sustains the speculation that Rab8a, Rab11b, Rab27a, Rab35, and Rab37 are possibly associated with the secretory autophagy machinery. In contrast, Rab7a shows low colocalization with LC3 puncta and low level in the autophagosome, suggesting it regulates different vesicle trafficking machineries. Our findings open a new direction toward exploring the role of Rab proteins in secretory autophagy-related cargo exocytosis and identifying the cargoes and effectors regulated by specific Rab proteins.

揭示 Rab 蛋白参与调节分泌型自噬机制的潜力
根据不同的功能,自噬可分为降解性自噬和分泌性自噬。小 GTPase 蛋白 Rab8a 和 Rab37 负责分泌性自噬介导的 IL-1β、胰岛素和 TIMP1(54 金属蛋白酶 1 的组织抑制剂)的外渗。对参与分泌性自噬的其他 Rab 家族成员还知之甚少。在此,我们通过LC-MS/MS蛋白质组学分析,在具有高分泌性自噬倾向的小鼠胰腺β细胞 "Min-6 "和人肺癌细胞 "CL1-5-Q89L "纯化的自噬体中发现了26个重叠的Rab蛋白。在四种细胞系的自噬体中检测到了六种Rab蛋白(Rab8a、Rab11b、Rab27a、Rab35、Rab37和Rab7a),它们与自噬相关的囊泡运输有关。我们利用CL1-5-Q89L细胞系模型评估了Rab蛋白与自噬LC3蛋白的共定位水平以及在纯化的自噬体中的存在情况。通过免疫印迹法,我们发现在主动分泌条件下,与正常对照组相比,5种Rab蛋白(Rab8a、Rab11b、Rab27a、Rab35和Rab37)在自噬体中高表达。然而,在共焦显微镜下,只有 Rab8a、Rab35 和 Rab37 与 LC3 蛋白高度共焦。尽管图像与免疫印迹分析之间存在差异,但我们的数据证实了 Rab8a、Rab11b、Rab27a、Rab35 和 Rab37 可能与分泌型自噬机制有关的推测。相比之下,Rab7a与LC3点的共定位程度较低,在自噬体中的水平也较低,这表明它调控着不同的囊泡转运机制。我们的研究结果为探索Rab蛋白在分泌型自噬相关货物外排中的作用以及鉴定受特定Rab蛋白调控的货物和效应物开辟了一个新方向。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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