Proteomic Profiling Reveals the Transglutaminase-2 Externalization Pathway in Kidneys after Unilateral Ureteric Obstruction
Proteomic Profiling Reveals the Transglutaminase-2 Externalization Pathway in Kidneys after Unilateral Ureteric Obstruction
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DOI：
10.1681/asn.2017050479
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发表时间：
2018-03-01
影响因子：
13.6
通讯作者：
Verderio, Elisabetta A. M.
中科院分区：
文献类型：
--
作者：
Furini, Giulia;Schroeder, Nina;Verderio, Elisabetta A. M.
小管上皮细胞（tec）向周围间质输出谷氨酰胺转酶2 （TG2）的增加改变了细胞外稳态平衡，导致纤维化膜扩张。尽管在CKD动物模型中，细胞外TG2的沉默改善了进行性肾瘢痕形成，但TG2从tec分泌并促进疾病进展的途径尚未阐明。在这项研究中，我们开发了一种全球蛋白质组学方法，以TG2敲除肾脏作为阴性对照，确定TG2在单侧输尿管梗阻（UUO）肾脏中负责TG2外化的结合伙伴。我们报告了一项稳健且无偏倚的分析，分析了纤维化肾脏中TG2的膜相互作用组相对于UUO后的整个蛋白质组，并通过SWATH质谱检测。数据已存入ProteomeX-change，标识符为PXD008173。TG2相互作用组中的外泌体蛋白簇支持了TG2在纤维化过程中由胞外膜囊泡分泌的假设。在已建立的TEC细胞系中，我们发现TG2存在于内体（外泌体）和质膜起源（微囊泡/外泌体）的囊泡中，TGF-b1刺激TG2的分泌。敲除syndecan-4 （SDC4）会严重损害TG2外泌体的分泌。TG2可与SDC4共沉淀，但不能与SDC4共沉淀。在体外，egfp标记的TG2在原发皮质tec的质膜上突起/收缩的球状元件（泡）中积累，而SDC4敲除会破坏泡的形成，影响TG2的释放。通过这种体内和体外结合的方法，我们已经解剖了CKD中TECs分泌TG2的途径。
Increased export of transglutaminase-2 (TG2) by tubular epithelial cells (TECs) into the surrounding interstitium modifies the extracellular homeostatic balance, leading to fibrotic membrane expansion. Although silencing of extracellular TG2 ameliorates progressive kidney scarring in animal models of CKD, the pathway through which TG2 is secreted from TECs and contributes to disease progression has not been elucidated. In this study, we developed a global proteomic approach to identify binding partners of TG2 responsible for TG2 externalization in kidneys subjected to unilateral ureteric obstruction (UUO) using TG2 knockout kidneys as negative controls. We report a robust and unbiased analysis of the membrane interactome of TG2 in fibrotic kidneys relative to the entire proteome after UUO, detected by SWATH mass spectrometry. The data have been deposited to the ProteomeX-change with identifier PXD008173. Clusters of exosomal proteins in the TG2 interactome supported the hypothesis that TG2 is secreted by extracellular membrane vesicles during fibrosis progression. In established TEC lines, we found TG2 in vesicles of both endosomal (exosomes) and plasmamembrane origin (microvesicles/ectosomes), and TGF-b1 stimulated TG2 secretion. Knockout of syndecan-4 (SDC4) greatly impaired TG2 exosomal secretion. TG2 coprecipitated with SDC4 from exosome lysate but not ectosome lysate. Ex vivo, EGFP-tagged TG2 accumulated in globular elements (blebs) protruding/retracting from the plasma membrane of primary cortical TECs, and SDC4 knockout impaired bleb formation, affecting TG2 release. Through this combined in vivo and in vitro approach, we have dissected the pathway through which TG2 is secreted from TECs in CKD.