Ascorbate peroxidase-mediated in situ labelling of proteins in secreted exosomes.
Ascorbate peroxidase-mediated in situ labelling of proteins in secreted exosomes.
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DOI：
10.1002/jev2.12239
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发表时间：
2022-06
影响因子：
16
通讯作者：
中科院分区：
文献类型：
--
作者：
胞外囊泡外泌体通过转运大分子如蛋白质和核糖核酸（RNA）来介导细胞间的通讯。以高精度确定货物内容将有助于破译外泌体在各种情况下介导的生物过程。用于探测外泌体货物分子的现有方法依赖于先前的外泌体分离程序。在这里，我们报告了一种用于外泌体货物识别的原位标记方法，该方法绕过了外泌体分离步骤。在该方法中，工程化抗坏血酸过氧化物酶APEX的变体与外泌体货物蛋白如CD63融合，在活细胞中的外泌体产生囊泡中或在条件培养基中的分泌外泌体中特异性表达，以在短时间内诱导APEX变体附近的蛋白质的生物素化。对肾近端小管衍生细胞分泌的外泌体中通过该方法生物素化的蛋白质进行的质谱分析表明，氧化应激可导致核糖体蛋白在含有CD63融合APEX变体的外泌体亚群中积累。
The extracellular vesicle exosome mediates intercellular communication by transporting macromolecules such as proteins and ribonucleic acids (RNAs). Determining cargo contents with high accuracy will help decipher the biological processes that exosomes mediate in various contexts. Existing methods for probing exosome cargo molecules rely on a prior exosome isolation procedure. Here we report an in situ labelling approach for exosome cargo identification, which bypasses the exosome isolation steps. In this methodology, a variant of the engineered ascorbate peroxidase APEX, fused to an exosome cargo protein such as CD63, is expressed specifically in exosome‐generating vesicles in live cells or in secreted exosomes in the conditioned medium, to induce biotinylation of the proteins in the vicinity of the APEX variant for a short period of time. Mass spectrometry analysis of the proteins biotinylated by this approach in exosomes secreted by kidney proximal tubule‐derived cells reveals that oxidative stress can cause ribosomal proteins to accumulate in an exosome subpopulation that contains the CD63‐fused APEX variant.
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影响因子：
4
作者：
Momen-Heravi F;Balaj L;Alian S;Trachtenberg AJ;Hochberg FH;Skog J;Kuo WP
通讯作者：
Kuo WP
影响因子：
64.5
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Ting, Alice Y.
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通讯作者：
Jean, Steve
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3.3
作者：
Cijsouw T;Ramsey AM;Lam TT;Carbone BE;Blanpied TA;Biederer T
通讯作者：
Biederer T
影响因子：
48
作者：
Lam, Stephanie S.;Martell, Jeffrey D.;Ting, Alice Y.
通讯作者：
Ting, Alice Y.