Puromycin-based vectors promote a ROS-dependent recruitment of PML to nuclear inclusions enriched with HSP70 and Proteasomes.
BACKGROUND
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. 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.


RESULTS
In 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.


CONCLUSIONS
Taken 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.
Moran
Diarmuid M
DM
Department of Radiation and Cellular Oncology, University of Chicago, Chicago, Illinois, USA. diarmuidmoran@uchicago.edu
Shen
Hong
H
Maki
Carl G
CG
D002460:Cell Line; D005822:Genetic Vectors / Q000235:genetics*; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D045586:Intranuclear Inclusion Bodies / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D000071156:Promyelocytic Leukemia Protein; D046988:Proteasome Endopeptidase Complex / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D017510:Protein Folding / Q000187:drug effects; D011691:Puromycin / Q000494:pharmacology*; D017382:Reactive Oxygen Species / Q000378:metabolism*; D025842:SUMO-1 Protein / Q000235:genetics / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D025521:Tumor Suppressor Proteins / Q000235:genetics / Q000378:metabolism*
D018840:HSP70 Heat-Shock Proteins; D009687:Nuclear Proteins; D000071156:Promyelocytic Leukemia Protein; D017382:Reactive Oxygen Species; D025842:SUMO-1 Protein; C495501:SUMO1 protein, human; D014157:Transcription Factors; D025521:Tumor Suppressor Proteins; C070576:PML protein, human; D011691:Puromycin; D046988:Proteasome Endopeptidase Complex