The Ribosome is the ultimate receptor for Trypsin Modulating Oostatic Factor (TMOF)

Authors: BOROVSKY, DOV - University Of Colorado; ROUGE, PIERRE - University Of Toulouse; Shatters, Robert - Bob

Submitted to: Biomolecules EISSN 2218-273X
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/12/2022
Publication Date: 4/14/2022
Citation: Borovsky, D., Rouge, P., Shatters, R.G. 2022. The Ribosome is the ultimate receptor for Trypsin Modulating Oostatic Factor (TMOF). Biomolecules 12:577. https://doi.org/10.3390/biom12040577.
DOI: https://doi.org/10.3390/biom12040577

Interpretive Summary: Over 30 years ago, a peptide hormone (called TMOF) was identified in mosquitoes that controlled the biosynthesis of digestive enzymes in the gut of females that had taken a blood meal. It was further shown that this peptide was toxic to mosquito larvae and a number of other insects when fed in their diet. These results led to the use of this peptide in mosquito control strategies; however, it was never understood how this peptide blocked the production of the digestive enzymes. This paper provides strong evidence that the peptide exherts its activity by binding the protein synthesis machinery of cells and that through this binding, it inhibits protein synthesis. Furthermore, it was shown that this peptide has a bifunctional activity of not only blocking insect gut cell synthesis of digestive enzymes and killing bacteria through total shutdown of protein synthesis in these microbes. This bifunctional protein shows promise for its use in controlling pest insects and bacterial pathogens in important crop plants and our understanding of the molecular function of TMOF aids in our ability to adapt this peptide for use in agricultural crop production.

Technical Abstract: AeaTMOF a mosquito decapeptide that controls trypsin biosynthesis in female and larval mosquitoes enters the gut epithelial cells of female mosquitos using ABC-tmfA receptor/importer. To study the ultimate targeted receptor after AeaTMOF enters the cell, AeaTMOF was incubated in vitro with either E. coli or Spodoptera frugiperda protein expressing extracts containing 70S and 80S ribosomes, respectively. The effect of AeaTMOF on luciferase biosynthesis in vitro using 70S ribosomes was compared with that of oncocin112, an antimicrobial protein synthesis peptide (1-13). IC50 of 1 'M and 2 'M, respectively for both peptides were determined. Incubation with protein expressing system and S. frugiperda 80S ribosomes determined an IC50 of 1.8 'M for Aedes aegypti larval late trypsin biosynthesis. Incubation of purified E. coli ribosome with increasing concentration of AeaTMOF shows that the binding of AeaTMOF to the bacterial ribosome exhibits high affinity (KD = 23 + 3.4 nM, Bmax = 0.553 + 0.023 pmol/'g ribosome and Kassoc = 4.3 x107 M-1). Molecular modeling and docking experiments show that AeaTMOF binds bacterial and Drosophila ribosome (50S and 60S, respectively) at the entrance of the ribosome exit tunnel blocking tRNA entrance and preventing protein biosynthesis. Recombinant E. coli cells that express only ABC-tmfA importer are inhibited by AeaTMOF but not by oncocin112 (1-13). These result suggest that the ribosome is the ultimate targeted receptor of AeaTMOF.