Hypusine formation in protein by a two-step process in cell lysates

Area of Science:

• Biochemistry
• Molecular Biology
• Cell Biology

Background:

• Eukaryotic initiation factor 4D (eIF-4D) is a protein involved in translation initiation.
• eIF-4D undergoes post-translational modification by spermidine, forming the rare amino acid hypusine from lysine.
• Understanding this modification is crucial for comprehending protein synthesis regulation.

Purpose of the Study:

• To investigate the biochemical pathway of hypusine formation in eIF-4D.
• To identify the enzyme responsible for deoxyhypusine formation and its properties.
• To explore the role of polyamines in this post-translational modification.

Main Methods:

• Analysis of protein modification in intact cells and cell lysates.
• Isoelectric focusing and molecular weight determination to confirm protein identity.
• Enzyme assays using cell lysates, polyamine depletion (alpha-difluoromethylornithine), and enzyme inhibitors (guazatine).

Main Results:

• Deoxyhypusine formation occurred in cell lysates at pH 9.5 and converted to hypusine at pH 7.1.
• Unmodified eIF-4D accumulated in spermidine-deficient cells.
• Guazatine significantly inhibited deoxyhypusine formation, indicating a specific enzyme's involvement.
• The enzyme showed a basic pH optimum and sensitivity to guazatine, but not other amine oxidase inhibitors.

Conclusions:

• Deoxyhypusine formation in eIF-4D is catalyzed by a novel guazatine-sensitive enzyme.
• This enzyme functions optimally at basic pH and is crucial for hypusine biosynthesis.
• Polyamines, specifically spermidine, are essential for the post-translational modification of eIF-4D.