Propiverine, a frequently-prescribed pharmaceutical for the treatment of symptoms associated with overactive bladder syndrome, provoked massive intranuclear and cytosolic protein inclusions in rat proximal tubule epithelium, primarily consisting of the peroxisomal targeting signal 1 (PTS1) containing protein d-amino acid oxidase (DAAO). As this type of nephropathy was also observed for other drugs, the aim was to determine whether propiverine interferes with trafficking and/or import of peroxisomal proteins. To elucidate this, DAAO- and propiverine-specific interaction partners from human HEK293 and rat WKPT cell lines and rat kidney and liver homogenate were determined using co-immunoprecipitation with subsequent nano-ESI-LC-MS/MS analyses. Corroboration of the role of DAAO- and/or propiverine-specific interaction partners in the drug-induced DAAO accumulation was sought via specific immunofluorescence staining of rat kidney sections from control and propiverine-treated rats. Above analyses demonstrated the interaction of propiverine with several protein classes, foremost peroxisomal proteins (DAAO, MFE2, HAOX2) and proteins of the protein quality control system, i.e. chaperones (HSP70 and DnaJ co-chaperones), proteases and proteasomal proteins (regulatory subunits of the 26S proteasome; Rpn1/2). The immunofluorescence analysis revealed mislocalization of many PTS1-proteins (DAAO, CAT, MFE2, ACOX1, EHHADH) in rat renal sections, strongly suggesting that propiverine primarily binds to PTS1 proteins resulting in the formation of PTS1 but not PTS2 or peroxisomal membrane protein (PMP) accumulations. Moreover, chaperones involved in peroxisomal trafficking (HSC70, DnaJB1) and peroxisomal biogenesis factor proteins (PEX3, PEX5, PEX7), also presented with distinct mislocalization patterns. Concomitantly, an increased number of peroxisomes was observed, suggestive of a compensatory mechanism for the presumably suboptimally functioning peroxisomes.

Overall, the data presented suggested that propiverine interacts exclusively with DAAO or with a selected number of PTS1 proteins. The consequence of this interaction is the abrogated trafficking and peroxisomal import of PTS1 proteins concomitant with their nuclear and cytosolic accumulation due to inhibited degradation and imbalanced protein homeostasis.

Propiverine是治疗与膀胱过度活动症相关的症状的常用药物，在大鼠近端小管上皮中引起大量的核内和胞浆蛋白包裹体，主要由过氧化物酶体靶向信号1（PTS1）组成，蛋白d-氨基酸氧化酶（DAAO）。由于在其他药物中也观察到了这种类型的肾病，目的是确定普罗维林是否干扰过氧化物酶体蛋白的运输和/或进口。为了阐明这一点，使用共免疫沉淀法和随后的纳米ESI-LC-MS / MS分析确定了来自人HEK293和大鼠WKPT细胞系以及大鼠肾脏和肝脏匀浆的DAAO和丙脯氨酸特异性相互作用伴侣。通过对来自对照和经异丙肾上腺素治疗的大鼠的大鼠肾脏切片进行特异性免疫荧光染色，寻求在药物诱导的DAAO积累中证实DAAO和/或丙肝素特异性相互作用伙伴的作用。上面的分析证明了普罗维林与几种蛋白质类型的相互作用，其中最主要的是过氧化物酶体蛋白（DAAO，MFE2，HAOX2）和蛋白质质量控​​制系统的蛋白质，即伴侣蛋白（HSP70和DnaJ伴侣蛋白），蛋白酶和蛋白酶体蛋白（26S蛋白酶体的调节亚基； Rpn1 / 2）。免疫荧光分析揭示了大鼠肾切片中许多PTS1蛋白（DAAO，CAT，MFE2，ACOX1，EHHADH）的定位错误，强烈表明丙肝素主要与PTS1蛋白结合导致PTS1的形成但不与PTS2或过氧化物酶体膜蛋白（PMP）结合）积累。此外，参与过氧化物酶体运输（HSC70，DnaJB1）和过氧化物酶体生物发生因子蛋白（PEX3，PEX5，PEX7）的伴侣蛋白也呈现出明显的错位模式。伴随地，观察到过氧化物酶体的数量增加，这暗示了过氧化物酶体的亚最佳功能的补偿机制。

总体而言，所提供的数据表明，丙脯氨酸只能与DAAO或与选定数量的PTS1蛋白相互作用。这种相互作用的结果是，由于抑制了降解和蛋白质稳态失衡，PTS1蛋白的运输被废止和过氧化物酶体的导入及其核和胞质的积累。