Understanding renal nuclear protein accumulation: an in vitro approach to explain an in vivo phenomenon

Abstract

Proper subcellular trafficking is essential to prevent protein mislocalization and aggregation. Transport of the peroxisomal enzyme D-amino acid oxidase (DAAO) appears dysregulated by specific pharmaceuticals, e.g., the anti-overactive bladder drug propiverine or a norepinephrine/serotonin reuptake inhibitor (NSRI), resulting in massive cytosolic and nuclear accumulations in rat kidney. To assess the underlying molecular mechanism of the latter, we aimed to characterize the nature of peroxisomal and cyto-nuclear shuttling of human and rat DAAO overexpressed in three cell lines using confocal microscopy. Indeed, interference with peroxisomal transport via deletion of the PTS1 signal or PEX5 knockdown resulted in induced nuclear DAAO localization. Having demonstrated the absence of active nuclear import and employing variably sized mCherry- and/or EYFP-fusion proteins of DAAO and catalase, we showed that peroxisomal proteins ≤134 kDa can passively diffuse into mammalian cell nuclei-thereby contradicting the often-cited 40 kDa diffusion limit. Moreover, their inherent nuclear presence and nuclear accumulation subsequent to proteasome inhibition or abrogated peroxisomal transport suggests that nuclear localization is a characteristic in the lifecycle of peroxisomal proteins. Based on this molecular trafficking analysis, we suggest that pharmaceuticals like propiverine or an NSRI may interfere with peroxisomal protein targeting and import, consequently resulting in massive nuclear protein accumulation in vivo.

Identification of d -amino acid oxidase and propiverine interaction partners and their potential role in the propiverine-mediated nephropathy

“…Since none of the tested, established human renal cell lines (e.g. RPTEC/TERT1) expressed endogenous DAAO, HEK293 cells were transfected with DAAO and were proven to be highly suitable to study peroxisomal trafficking and protein homeostasis as shown earlier [12]. Indeed, recent data confirmed that peroxisomes of HEK293 cells reacted in a comparable manner to rat proximal tubule epithelial cell peroxisomes in vivo (Maier et al, 2017, unpublished data).…”

“…EYFP-tagged rat and human DAAO (hereafter referred to as EYFP-rDAAO and EYFP-hDAAO, respectively), and mutants lacking the PTS1 sequence (DAAO-ΔPTS) thereof were used to identify DAAO-specific interaction partners in HEK293 and WKPT cells. As described earlier [12], EYFP-rDAAO and EYFP-hDAAO were predominantly localized in peroxisomes due to the C-terminal PTS1 sequence and are subsequently referred to as DAAO perox , while the DAAO-ΔPTS mutants lacking the Cterminal PTS1 sequence were localized simultaneously in the cytosol and the nucleus [12] and are subsequently referred to as DAAO cyt . GFP-Trap ® coupled magnetic beads allowed efficient immobilization and purification of EYFP-DAAO ( Fig.…”

“…With the exception of EYFPr/hDAAO cyt (ΔPTS mutants), only stably transfected, monoclonal cell lines were used for all ensuing experiments. To produce ΔPTS1 mutants of rDAAO and hDAAO (DAAO cyt ), site-directed mutagenesis was performed as previously described [12] and cells were subsequently transiently transfected with FuGENE [12]. HEK293 (human embryonic kidney) cells were purchased from the Leibniz Institute DSMZ -German Collection of Microorganisms and Cell Cultures and cultured in Dulbecco's modified Eagle's medium (DMEM) containing low glucose (1 g/ L) supplemented with 10% (v/v) fetal bovine serum (FBS) gold, 100 U/ mL penicillin and 0.1 mg/mL streptomycin.…”

“…Indeed, a very low and transient level of PEX5-DAAO interaction is expected when considering the model system used, i.e. stably expressing EYFP-DAAO HEK293 cells where DAAO is primarily localized to peroxisomes [12]. To demonstrate the rather transient and brief interaction of DAAO with PEX5, HEK293 cells were transiently transfected with EYFP-hDAAO and a Co-IP was initiated directly after transfection.…”

“…Indeed, recent in vitro analyses of cyto-nuclear shuttling of human and rat DAAO demonstrated that interference with the peroxisomal transport, i.e. deletion of the PTS1 sequence or PEX5 knockdown, resulted in cytosolic DAAO localization as well as passive diffusion into the nucleus [12]. The latter supports the hypothesis that propiverine and/or its metabolite(s) may interfere at the level of proteins intricately involved in DAAO trafficking from its cytosolic synthesis to the correct integration into the peroxisome [12].…”