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J or H mtDNA haplogroups in retinal pigment epithelial cells: Effects on cell physiology, cargo in extracellular vesicles, and differential uptake of such vesicles by naïve recipient cells
Biochimica et Biophysica Acta (BBA) - General Subjects ( IF 3 ) Pub Date : 2020-11-18 , DOI: 10.1016/j.bbagen.2020.129798
Crystal Nicholson 1 , Masaaki Ishii 1 , Balasubramaniam Annamalai 1 , Kyrie Chandler 1 , Marilyn Chwa 2 , M Cristina Kenney 2 , Navjot Shah 1 , Bärbel Rohrer 3
Affiliation  

Purpose

Extracellular vesicles (EVs) are predicted to represent the internal state of cells. In polarized RPE monolayers, EVs can mediate long-distance communication, requiring endocytosis via protein-protein interactions. EV uptake from oxidatively stressed donor cells triggers loss in transepithelial resistance (TER) in recipient monolayers mediated by HDAC6. Here, we examine EVs released from RPE cells with identical nuclear genes but different mitochondrial (mt)DNA haplogroups (H, J). J-cybrids produce less ATP, and the J-haplogroup is associated with a higher risk for age-related macular degeneration.

Methods

Cells were grown as mature monolayers to either collect EVs from apical surfaces or to serve as naïve recipient cells. Transfer assays, transferring EVs to a recipient monolayer were performed, monitoring TER and EV-uptake. The presence of known EV surface proteins was quantified by protein chemistry.

Results

H- and J-cybrids were confirmed to exhibit different levels of TER and energy metabolism. EVs from J-cybrids reduced TER in recipient ARPE-19 cells, whereas EVs from H-cybrids were ineffective. TER reduction was mediated by HDAC6 activity, and EV uptake required interaction between integrin and its ligands and surface proteoglycans. Protein quantifications confirmed elevated levels of fibronectin and annexin A2 on J-cybrid EVs.

Conclusions

We speculate that RPE EVs have a finite set of ligands (membrane proteoglycans and integrins and/or annexin A2) that are elevated in EVs from stressed cells; and that if EVs released by the RPE could be captured from serum, that they might provide a disease biomarker of RPE-dependent diseases.



中文翻译:

视网膜色素上皮细胞中的 J 或 H mtDNA 单倍群:对细胞生理学、细胞外囊泡中的货物以及幼稚受体细胞对此类囊泡的差异摄取的影响

目的

细胞外囊泡(EV)预计代表细胞的内部状态。在极化的 RPE 单层中,EV 可以介导长距离通讯,需要通过蛋白质-蛋白质相互作用进行内吞作用。从氧化应激的供体细胞摄取 EV 会触发 HDAC6 介导的受体单层细胞跨上皮抵抗 (TER) 的丧失。在这里,我们检查了 RPE 细胞释放的 EV,其核基因相同,但线粒体 (mt)DNA 单倍群不同 (H、J)。J-cybrids 产生较少的 ATP,并且 J-单倍群与年龄相关性黄斑变性的较高风险相关。

方法

细胞生长为成熟的单层细胞,以从顶端表面收集 EV 或作为初始受体细胞。进行了转移测定,将 EV 转移至受体单层,监测 TER 和 EV 的摄取。通过蛋白质化学对已知 EV 表面蛋白的存在进行定量。

结果

H-和J-cybrids被证实表现出不同水平的TER和能量代谢。来自 J-cybrids 的 EV 减少了受体 ARPE-19 细胞中的 TER,而来自 H-cybrids 的 EV 则无效。TER 的减少是由 HDAC6 活性介导的,EV 的摄取需要整合素及其配体和表面蛋白聚糖之间的相互作用。蛋白质定量证实 J-cybrid EV 上的纤连蛋白和膜联蛋白 A2 水平升高。

结论

我们推测 RPE EV 具有一组有限的配体(膜蛋白聚糖和整合素和/或膜联蛋白 A2),这些配体在来自应激细胞的 EV 中升高;如果可以从血清中捕获 RPE 释放的 EV,那么它们可能会提供 RPE 依赖性疾病的疾病生物标志物。

更新日期:2020-11-18
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