High hydrostatic pressure inhibits the biosynthesis of eukaryotic elongation factor-2.
High continuous hydrostatic pressure is known to inhibit the total cellular protein synthesis. In this study, our goal was to identify pressure-regulated proteins by using two dimensional gel electrophoresis and mass spectrometry. This analysis showed that under 30 MPa continuous hydrostatic pressure the biosynthesis of eukaryotic elongation factor-2 (eEF-2) was inhibited both in HeLa carcinoma and T/C28a4 chondrocytic cell lines. Western blot analysis of HeLa cells revealed that the cellular protein level of eEF-2 decreased by 40%-50% within 12 h of the pressure treatment. However, the steady-state mRNA level of eEF-2 was not affected by the pressure. Cycloheximide addition after 4 h-pressure treatment suggested that the half-life of eEF-2 protein was shorter in pressurized cells. eEF-2 is responsible for the translocation of ribosome along the specific mRNA during translation, and its phosphorylation prevents the ribosomal translocation. Therefore, increased phosphorylation of eEF-2 was considered as one mechanism that could explain the reduced level of protein synthesis in pressurized HeLa cell cultures. However, Western blot analysis with an antibody recognizing the Thr56-phosphorylated form of eEF-2 showed that phosphorylation of eEF-2 was not elevated in pressurized samples. In conclusion, the inhibition of protein synthesis under high pressure occurs independent of the phosphorylation of eEF-2. However, this inhibition may result from the decrease of cellular eEF-2 protein.
Elo
Mika A
MA
Department of Anatomy, University of Kuopio, 70211 Kuopio, Finland.
Karjalainen
Hannu M
HM
Sironen
Reijo K
RK
Valmu
Leena
L
Redpath
Nicholas T
NT
Browne
Gareth J
GJ
Kalkkinen
Nisse
N
Helminen
Heikki J
HJ
Lammi
Mikko J
MJ
D000595:Amino Acid Sequence; D001483:Base Sequence; D015152:Blotting, Northern; D015153:Blotting, Western; D017931:DNA Primers; D015180:Electrophoresis, Gel, Two-Dimensional; D005057:Eukaryotic Cells / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D020652:Peptide Elongation Factor 2 / Q000096:biosynthesis* / Q000737:chemistry / Q000378:metabolism; D010766:Phosphorylation; D011312:Pressure; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D017931:DNA Primers; D020652:Peptide Elongation Factor 2