Insect Receptors: Biochemical, Physiological and Molecular Studies

A Biomolecules Special Issue on insect receptors was a great opportunity to invite colleagues from all over the world to contribute original articles and timely reviews on the subject [...

The Ribosome Is the Ultimate Receptor for Trypsin Modulating Oostatic Factor (TMOF)

International audienceAedes aegypti Trypsin Modulating Oostatic Factor (AeaTMOF). a mosquito decapeptide that controls trypsin biosynthesis in female and larval mosquitoes. enters the gut epithelial cells of female mosquitoes using ABC-tmfA receptor/importer. To study the ultimate targeted receptor after AeaTMOF enters the cell, AeaTMOF was incubated in vitro with either Escherichia 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 (1–13), a ribosome-binding antibacterial peptide. The IC50 of 1 μM and 2 μM, respectively, for both peptides was determined. Incubation with a protein-expressing system and S. frugiperda 80S ribosomes determined an IC50 of 1.8 μM 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 a high affinity (KD = 23 ± 3.4 nM, Bmax = 0.553 ± 0.023 pmol/μg ribosome and Kassoc = 4.3 × 107 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 the 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 results suggest that the ribosome is the ultimate targeted receptor of AeaTMOF

Bactericidal Properties of Proline-Rich <i>Aedes aegypti</i> Trypsin Modulating Oostatic Factor (<i>Aea</i>TMOF)

The antimicrobial properties of proline-rich Aedes aegypti decapeptide TMOF (AeaTMOF) and oncocin112 (1–13) were compared. Incubations with multidrug-resistant Escherichia coli cells showed that AeaTMOF (5 mM) was able to completely inhibit bacterial cell growth, whereas oncocin112 (1–13) (20 mM) partially inhibited bacterial growth as compared with bacterial cells that were not multidrug-resistant cells. AeaTMOF (5 mM) was very effective against Acinetobacter baumannii and Pseudomonas aeruginosa, completely inhibiting cell growth during 15 h incubations. AeaTMOF (5 mM) completely inhibited the Gram-positive bacteria Staphylococcus aureus and Bacillus thurengiensis sups. Israelensis cell growth, whereas oncocin112 (1–13) (10 and 20 mM) failed to affect bacterial cell growth. E. coli cells that lack the SbmA transporter were inhibited by AeaTMOF (5 mM) and not by oncocin112 (1–13) (10 to 20 mM), indicating that AeaTMOF can use other bacterial transporters than SbmA that is mainly used by proline-rich antimicrobial peptides. Incubation of E. coli cells with NaAzide showed that AeaTMOF does not use ABC-like transporters that use ATP hydrolysis to import molecules into bacterial cells. Three-dimensional modeling and docking of AeaTMOF to SbmA and MdtM transporters showed that AeaTMOF can bind these proteins, and the binding location of AeaTMOF inside these protein transporters allows AeaTMOF to be transported into the bacterial cytosol. These results show that AeaTMOF can be used as a future antibacterial agent against both multidrug-resistant Gram-positive and -negative bacteria

On The Mechanism of Amylose Branching by Potato Q‐Enzyme

1 When potato Q‐enzyme converts amylose into an amylopectin‐like molecule, the action is by a random, endo‐type transglycosylation of the substrate chains. 2 Inter‐chain transfer takes place during the formation of the amylopectin branch linkage. This is seen in experiments in which radioactive label was transferred between substrates of disparate molecular weight. Intra‐chain transfer, leading to the formation of a branch linkage, is not excluded by these experiments. 3 The minimum length of amylose chain that can act as an acceptor in the transglycosylation reaction, under the experimental conditions described, is greater than 40 glucose units. 4 The requirement of Q‐enzyme for substrate chains at least 40 glucose units in length is interpreted as meaning that a stabilized secondary and tertiary structure must be established in the substrate before it can be utilized by Q‐enzyme, and that the forces that provide such conformation are sufficiently strong only when the chains are longer than the minimum. Inter‐chain transfer is seen as taking place by one of two mechanisms. The first involves the reaction of the enzyme with a chain that has a stabilized (helical?) conformation. An enzyme‐donor chain intermediate is formed, that then reacts with an acceptor chain to complete the transglycosylation. The second mechanism envisages the substrate for the enzyme as being a complex formed between two chains (a double helix?). The enzyme encounters the complex and carries out an inter‐chain transglycosylation reaction

Effect of ACE inhibitors and TMOF on growth, development and trypsin activity of larval Spodoptera littoralis

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Culex quinquefasciatus Late Trypsin Biosynthesis Is Translationally Regulated by Trypsin Modulating Oostatic Factor

International audienceTrypsin is a serine protease that is synthesized by the gut epithelial cells of female mosquitoes; it is the enzyme that digests the blood meal. To study its molecular regulation, Culex quinquefasciatus late trypsin was purified by diethylaminoethyl (DEAE), affinity, and C 18 reverse-phase high performance liquid chromatography (HPLC) steps, and the N-terminal amino acid sequence was determined for molecular cloning. Five overlapping segments of the late trypsin cDNA were amplified by PCR, cloned, and the full sequence (855 bp) was characterized. Three-dimensional models of the pro-trypsin and activated trypsin were built and compared with other trypsin models. Trypsin modulating oostatic factor (TMOF) concentrations in the hemolymph were determined by ELISA and compared with trypsin activity in the gut after the blood meal. The results showed that there was an increase in TMOF concentrations circulating in the hemolymph which has correlated to the reduction of trypsin activity in the mosquito gut. Northern blot analysis of the trypsin transcripts after the blood meal indicated that trypsin activity also followed the increase and decrease of the trypsin transcript. Injections of different amounts of TMOF (0.025 to 50 μg) decreased the amounts of trypsin in the gut. However, Northern blot analysis showed that TMOF injections did not cause a decrease in trypsin transcript abundance, indicating that TMOF probably affected trypsin translation