Abstract
BackgroundPromyelocytic Leukemia (PML) protein can interact with a multitude of cellular factors and has been implicated in the regulation of various processes, including protein sequestration, cell cycle regulation and DNA damage responses. Previous studies reported that misfolded proteins or proteins containing polyglutamine tracts form aggregates with PML, chaperones, and components of the proteasome, supporting a role for PML in misfolded protein degradation.ResultsIn the current study, we have identified a reactive oxygen species (ROS) dependent aggregation of PML, small ubiquitin-like modifier 1 (SUMO-1), heat shock protein 70 (HSP70) and 20S proteasomes in human cell lines that have been transiently transfected with vectors expressing the puromycin resistance gene, puromycin n-acetyl transferase (pac). Immunofluorescent studies demonstrated that PML, SUMO-1, HSP70 and 20S proteasomes aggregated to form nuclear inclusions in multiple cell lines transfected with vectors expressing puromycin (puro) resistance in regions distinct from nucleoli. This effect does not occur in cells transfected with identical vectors expressing other antibiotic resistance genes or with vectors from which the pac sequence has been deleted. Furthermore, ROS scavengers were shown to ablate the effect of puro vectors on protein aggregation in transfected cells demonstrating a dependency of this effect on the redox state of transfected cells.ConclusionTaken together we propose that puromycin vectors may elicit an unexpected misfolded protein response, associated with the formation of nuclear aggresome like structures in human cell lines. This effect has broad implications for cellular behavior and experimental design.
Highlights
Promyelocytic Leukemia (PML) protein can interact with a multitude of cellular factors and has been implicated in the regulation of various processes, including protein sequestration, cell cycle regulation and DNA damage responses
During the course of our studies we observed an apparent relocalization of PML nuclear bodies (NBs) in cell lines transfected with shRNA vectors targeting specific E3 ligases (MDM2 and CHIP)
Nuclear inclusions containing heat shock protein 70 (HSP70), proteasomes and PML have been well characterized in cells overexpressing non-PolyQ aggregation prone proteins, viral proteins and in cells treated with proteasomal inhibitors [16,17,25,26,27]. These studies suggest that PML bodies can represent nuclear aggresomes and potential sites of nuclear misfolded protein degradation. Based on these previous studies, we propose that a protein aggregation/misfolded protein response may account for nuclear aggregation of PML, HSP70, SUMO1 and proteasomes into nuclear inclusions in this study
Summary
Promyelocytic Leukemia (PML) protein can interact with a multitude of cellular factors and has been implicated in the regulation of various processes, including protein sequestration, cell cycle regulation and DNA damage responses. The PML protein localizes to punctate nuclear structures called PML nuclear bodies (NBs) which are discrete foci (0.1–0.2 μm diameter) that are present in most mammalian cells. PML is essential for the formation of PML NBs, the bodies themselves are composed of many other proteins such as SUMO, SP100, CBP and Daxx. SUMO-1 (Small Ubiquitin like Modifier 1) belongs to a family of ubiquitin like proteins that may be covalently attached to protein substrates and are typically associated with protein localization. SUMO modification of PML is required for NB formation [1,2]. Far over 50 proteins have been (page number not for citation purposes)
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