Deoxyhypusine Modification of Eukaryotic Translation Initiation Factor 5A (eIF5A) Is Essential for Trypanosoma brucei Growth and for Expression of Polyprolyl-containing Proteins

J Biol Chem. 2015 Aug 7;290(32):19987-98. doi: 10.1074/jbc.M115.656785. Epub 2015 Jun 16.

Abstract

The eukaryotic protozoan parasite Trypanosoma brucei is the causative agent of human African trypanosomiasis. Polyamine biosynthesis is essential in T. brucei, and the polyamine spermidine is required for synthesis of a novel cofactor called trypanothione and for deoxyhypusine modification of eukaryotic translation initiation factor 5A (eIF5A). eIF5A promotes translation of proteins containing polyprolyl tracts in mammals and yeast. To evaluate the function of eIF5A in T. brucei, we used RNA interference (RNAi) to knock down eIF5A levels and found that it is essential for T. brucei growth. The RNAi-induced growth defect was complemented by expression of wild-type human eIF5A but not by a Lys-50 mutant that blocks modification by deoxyhypusine. Bioinformatics analysis showed that 15% of the T. brucei proteome contains 3 or more consecutive prolines and that actin-related proteins and cysteine proteases were highly enriched in the group. Steady-state protein levels of representative proteins containing 9 consecutive prolines that are involved in actin assembly (formin and CAP/Srv2p) were significantly reduced by knockdown of eIF5A. Several T. brucei polyprolyl proteins are involved in flagellar assembly. Knockdown of TbeIF5A led to abnormal cell morphologies and detached flagella, suggesting that eIF5A is important for translation of proteins needed for these processes. Potential specialized functions for eIF5A in T. brucei in translation of variable surface glycoproteins were also uncovered. Inhibitors of deoxyhypusination would be expected to cause a pleomorphic effect on multiple cell processes, suggesting that deoxyhypusine/hypusine biosynthesis could be a promising drug target in not just T. brucei but in other eukaryotic pathogens.

Keywords: Trypanosoma brucei; eukaryotic initiation factor 5A (eIF5A); hypusine; parasite metabolism; polyamine; spermidine.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Escherichia coli / genetics
  • Escherichia coli / metabolism
  • Flagella / genetics
  • Flagella / metabolism
  • Flagella / ultrastructure
  • Gene Knockdown Techniques
  • Humans
  • Lysine / analogs & derivatives*
  • Lysine / metabolism
  • Microfilament Proteins / genetics
  • Microfilament Proteins / metabolism
  • Molecular Sequence Data
  • Peptide Initiation Factors / antagonists & inhibitors
  • Peptide Initiation Factors / genetics
  • Peptide Initiation Factors / metabolism*
  • Peptides / metabolism
  • Protein Processing, Post-Translational*
  • Proteome / genetics
  • Proteome / metabolism
  • Protozoan Proteins / antagonists & inhibitors
  • Protozoan Proteins / genetics
  • Protozoan Proteins / metabolism*
  • RNA, Messenger / antagonists & inhibitors
  • RNA, Messenger / genetics
  • RNA, Messenger / metabolism*
  • RNA, Protozoan / antagonists & inhibitors
  • RNA, Protozoan / genetics
  • RNA, Protozoan / metabolism*
  • RNA, Small Interfering / genetics
  • RNA, Small Interfering / metabolism
  • RNA-Binding Proteins / antagonists & inhibitors
  • RNA-Binding Proteins / genetics
  • RNA-Binding Proteins / metabolism*
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Sequence Alignment
  • Sequence Homology, Amino Acid
  • Trypanosoma brucei brucei / genetics
  • Trypanosoma brucei brucei / metabolism*
  • Trypanosoma brucei brucei / ultrastructure

Substances

  • Microfilament Proteins
  • Peptide Initiation Factors
  • Peptides
  • Proteome
  • Protozoan Proteins
  • RNA, Messenger
  • RNA, Protozoan
  • RNA, Small Interfering
  • RNA-Binding Proteins
  • Recombinant Proteins
  • eukaryotic translation initiation factor 5A
  • polyproline
  • deoxyhypusine
  • Lysine