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

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.

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

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.

Trophic state and geographic gradients influence planktonic cyanobacterial diversity and distribution in New Zealand lakes.

Cyanobacteria are commonly associated with eutrophic lakes, where they often form blooms and produce toxins. However, they are a ubiquitous component of phytoplankton in lakes of widely varying trophic status. We hypothesised that cyanobacterial diversity would vary among lakes of differing trophic status, but that the relative importance of geographical and hydromorphological characteristics driving these patterns would differ across trophic groups. DNA from 143 New Zealand lakes that spanned a range of geographic, hydromorphological and trophic gradients was analysed using automated rRNA intergenic spacer analysis and screened for genes involved in cyanotoxin production. Statistical analysis revealed significant delineation among cyanobacterial communities from different trophic classes. Multivariate regression indicated that geographical features (latitude, longitude and altitude) were significant in driving cyanobacterial community structure; however, partitioning of their effects varied among trophic categories. High-throughput sequencing was undertaken on selected samples to investigate their taxonomic composition. The most abundant and diverse (71 operational taxonomic units) taxon across all lake types was the picocyanobacteria genus Synechococcus Cyanotoxins (microcystins n = 23, anatoxins n = 1) were only detected in eutrophic lowland lakes. Collectively, these data infer that increasing eutrophication of lakes will have broad-scale impacts on planktonic cyanobacteria diversity and the prevalence of cyanotoxins.