Effect of mutation of amino acids 246-251 (KRKHKK) in HSP72 on protein synthesis and recovery from hypoxic injury.
Heat shock protein (HSP)72, the inducible form of HSP70, protects cells against a variety of injuries, but underlying mechanisms are poorly defined. To investigate the protective effects of HSP72, multiple clones expressing wild-type (WT) HSP72 and two mutants with defective nucleolar and nuclear localization (M45 and 985A, respectively) were made with the tet-off system in C2C12 cells. Four different parameters of cell function/injury were examined after simulated ischemia: protein synthesis, polysome formation, DNA synthesis, and lactate dehydrogenase (LDH release). Overexpression of WT HSP72 was also compared to nontransfected C2C12 cells. As expected, overexpression of HSP72 protected against simulated ischemia and reoxygenation for all parameters. In contrast, both M45 and 985A showed abnormal protein synthesis and polysome formation, both after simulated ischemia and under control conditions. Total RNA was slightly reduced in M45 and 985A at baseline, but 1 h after hypoxia, RNA levels were protected in all clones but significantly decreased in nontransfected C2C12 cells. Clones expressing 985A had nuclear retention of mRNA, suggesting that HSP72 is needed for nuclear export of RNA. All clones, both WT and mutant, had protection of DNA synthesis compared to C2C12 cells, but 985A had greater release of LDH after injury than any other group. These results support a multifactoral protective effect of HSP72, some aspects dependent on nuclear localization with stress and some not. The protection of protein synthesis and polysome formation, and abnormalities in these with the mutants, support a role for HSP72 in these processes both in the normal cell and in injury.
Voss
M R
MR
Molecular and Cellular Cardiology, Genomics and Biomedical Sciences Facility, Rm. 6317, Univ. of California, Davis, 451 East Health Sciences Way, Davis, CA 95616, USA.
Gupta
S
S
Stice
J P
JP
Baumgarten
G
G
Lu
L
L
Tristan
J M
JM
Knowlton
A A
AA
D000818:Animals; D015687:Cell Hypoxia / Q000502:physiology; D002460:Cell Line; D050884:HSP72 Heat-Shock Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018869:Heat-Shock Response / Q000502:physiology*; D051379:Mice; D018482:Muscle, Skeletal / Q000502:physiology*; D016297:Mutagenesis, Site-Directed; D032446:Myoblasts / Q000502:physiology*; D011132:Polyribosomes / Q000502:physiology; D014176:Protein Biosynthesis / Q000502:physiology*; D012313:RNA / Q000378:metabolism*; D013329:Structure-Activity Relationship
D050884:HSP72 Heat-Shock Proteins; D012313:RNA