Heat shock impairs the interaction of cap-binding protein complex with 5' mRNA cap

Area of Science:

• Molecular Biology
• Cell Biology
• Biochemistry

Background:

• Heat shock triggers a global inhibition of protein synthesis in eukaryotic cells.
• The cap-binding protein complex, a component of eukaryotic initiation factor 4F, is crucial for restoring protein synthesis.
• Previous work demonstrated that this complex can restore translation in cell-free systems from heat-shocked cells.

Purpose of the Study:

• To investigate the effect of heat shock on the amount and activity of the cap-binding protein complex.
• To elucidate the molecular mechanisms by which heat shock inhibits translation initiation.
• To examine the phosphorylation state of cap-binding complex subunits during heat shock.

Main Methods:

• Preparation of cell-free protein synthesizing systems from heat-shocked Ehrlich cells.
• Purification of the cap-binding protein complex using 7-methyl-guanosine triphosphate Sepharose affinity chromatography.
• Analysis of protein phosphorylation states using biochemical assays.

Main Results:

• Cell-free systems from heat-shocked cells showed reduced protein synthesis, mirroring cellular inhibition.
• Heat shock reduced the amount of functional cap-binding complex in cell extracts.
• Purification revealed less cap-binding complex from heat-shocked cells, indicating impaired binding to the mRNA 5' cap.
• Reduced phosphorylation was observed in both p220·p28 complex and free p28 during heat shock.

Conclusions:

• Heat shock impairs the binding of the cap-binding complex to the mRNA 5' cap.
• Proteolysis and competitive inhibitors were ruled out as causes for this impairment.
• Potential mechanisms include reduced association between p220 and p28 subunits or a non-competitive inhibitor.
• Altered phosphorylation of p28 and p220 may play a role in heat shock-induced translational inhibition.