Excess protein in nuclei isolated from heat-shocked cells results from a reduced extractability of nuclear proteins.
An excellent correlation has been established between the quantity of protein associated with nuclei isolated from heat-shocked cells and the level of hyperthermic cell killing. However, controversy remains about whether increases in nuclear-associated protein result from a heat-induced migration of cytoplasmic proteins into the nucleus or because hyperthermia reduces the solubility of nuclear proteins in the detergent buffers commonly used to isolate nuclei. To address this controversy, the nuclear protein content was measured in whole and detergent-extracted cells before and following hyperthermia. It was found that hyperthermia caused no significant change in the nuclear protein content of whole, unextracted cells, and when fluorescently labeled proteins were microinjected into the cytoplasm no gross change in the selective permeability of the nuclear membrane to soluble proteins was observed during or following hyperthermia. Measurements in extracted cells showed that the detergent buffers removed protein from both the nucleus and cytoplasm of control, nonheated cells and that hyperthermia reduced the extractability of both nuclear and cytoplasmic proteins. The amount of protein found in nuclei isolated from heated cells approached that observed in nuclei within nonheated whole cells as the hyperthermic exposure was increased. Thus, the dose-dependent, two- to threefold increase in the protein content of nuclei isolated from heated cells represents a heat-induced reduction in the extractability of proteins normally present within cell nuclei and does not result from a mass migration of cytoplasmic proteins into the nucleus, although some specific proteins (e.g., the 70 KDa heat shock protein) do migrate to the nucleus following heat shock. Differential scanning calorimetry (DSC) measurements of whole cells, isolated nuclei, cytoplasts, and karyoplasts supported these conclusions and suggested that most of the detergent-insoluble proteins remaining in the nuclei and cytoplasm of heated cells are in their native state. Thus, a relatively small amount of denatured protein may be sufficient to initiate and sustain insoluble protein aggregates comprised of mostly native proteins. Analyses of the DSC data also implied that the previously identified critical target proteins, predicted to have a Tm of 46.0 degrees C, are present in both the nucleus and cytoplasm.
Borrelli
M J
MJ
Department of Radiation Oncology, William Beaumont Hospital, Royal Oak, Michigan 48073, USA.
Lepock
J R
JR
Frey
H E
HE
Lee
Y J
YJ
Corry
P M
PM
D016475:3T3 Cells; D000818:Animals; D016466:CHO Cells; D002152:Calorimetry, Differential Scanning; D002467:Cell Nucleus / Q000737:chemistry* / Q000378:metabolism; D006224:Cricetinae; D003902:Detergents / Q000494:pharmacology; D005434:Flow Cytometry; D016650:Fluorescein-5-isothiocyanate / Q000031:analogs & derivatives / Q000378:metabolism; D005557:Formaldehyde / Q000494:pharmacology; D006360:Heat-Shock Proteins / Q000032:analysis*; D006358:Hot Temperature; D051379:Mice; D008845:Microinjections; D008856:Microscopy, Fluorescence; D009685:Nuclear Envelope / Q000378:metabolism; D009687:Nuclear Proteins / Q000032:analysis*; D010539:Permeability; D011108:Polymers / Q000494:pharmacology; D011506:Proteins / Q000378:metabolism*; D012710:Serum Albumin, Bovine / Q000378:metabolism; D012995:Solubility
D003902:Detergents; D006360:Heat-Shock Proteins; D009687:Nuclear Proteins; D011108:Polymers; D011506:Proteins; C039217:fluorescein isothiocyanate bovine serum albumin; D005557:Formaldehyde; D012710:Serum Albumin, Bovine; D016650:Fluorescein-5-isothiocyanate; C003043:paraform