Imaging of radiation effects on cellular 26S proteasome function in situ.
OBJECTIVE
The classical radiobiological paradigm is that DNA is the target for cell damage caused by ionising radiation. However, evidence is accumulating that other constituents, such as the membrane, organelles, and proteins, are also important targets. We have shown that the isolated 26S proteasome is one such target and here we wish to substantiate it within the cell, in situ.


METHODS
We used confocal microscopy to quantitatively detect and subcellularly localise radiation-induced 26S proteasome inhibition in cells expressing an ornithine decarboxylase degron that targets a fused Zoanthus species green (ZsGreen) fluorescent protein reporter specifically to the 26S proteasome.


RESULTS
Exposure of cells to a range of radiation doses, even as low as 0.05 Gy inhibited 26S activity within minutes. Initially, punctate nuclear ZsGreen fluorescence was observed that became cytoplasmic after seven hours -- a pattern distinct from the diffuse homogeneous fluorescence of cells incubated in the conventional proteasome inhibitor MG-132.


CONCLUSIONS
Our study clearly indicates that the 26S proteasome is a radiation target with physiological consequences and introduces a new perspective in mechanistic investigations of cellular responses to stresses.
Brush
James M
JM
Department of Radiation Oncology, David Geffen School of Medicine at UCLA, Los Angeles, California 90095-1714, USA.
Kim
Kwanghee
K
Sayre
James W
JW
McBride
William H
WH
Iwamoto
Keisuke S
KS
D002453:Cell Cycle / Q000528:radiation effects; D016923:Cell Death / Q000528:radiation effects; D045744:Cell Line, Tumor; D004307:Dose-Response Relationship, Radiation; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics; D006801:Humans; D009955:Ornithine Decarboxylase / Q000235:genetics; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*; D011839:Radiation, Ionizing; D013312:Stress, Physiological / Q000528:radiation effects; D025801:Ubiquitin / Q000378:metabolism
D025801:Ubiquitin; D049452:Green Fluorescent Proteins; D046988:Proteasome Endopeptidase Complex; C061553:ATP dependent 26S protease; D009955:Ornithine Decarboxylase