A New Family of Receptor Tyrosine Kinases with a Venus Flytrap Binding Domain in Insects and Other Invertebrates Activated by Aminoacids

Background: Tyrosine kinase receptors (RTKs) comprise a large family of membrane receptors that regulate various cellular processes in cell biology of diverse organisms. We previously described an atypical RTK in the platyhelminth parasite Schistosoma mansoni, composed of an extracellular Venus flytrap module (VFT) linked through a single transmembrane domain to an intracellular tyrosine kinase domain similar to that of the insulin receptor. Methods and Findings: Here we show that this receptor is a member of a new family of RTKs found in invertebrates, and particularly in insects. Sixteen new members of this family, named Venus Kinase Receptor (VKR), were identified in many insects. Structural and phylogenetic studies performed on VFT and TK domains showed that VKR sequences formed monophyletic groups, the VFT group being close to that of GABA receptors and the TK one being close to that of insulin receptors. We show that a recombinant VKR is able to autophosphorylate on tyrosine residues, and report that it can be activated by L-arginine. This is in agreement with the high degree of conservation of the alpha amino acid binding residues found in many amino acid binding VFTs. The presence of high levels of vkr transcripts in larval forms and in female gonads indicates a putative function of VKR in reproduction and/or development. Conclusion: The identification of RTKs specific for parasites and insect vectors raises new perspectives for the control of human parasitic and infectious diseases

<i>Schistosoma mansoni</i> Sirtuins: Characterization and Potential as Chemotherapeutic Targets

<div><p>Background</p><p>The chemotherapy of schistosomiasis currently depends on the use of a single drug, praziquantel. In order to develop novel chemotherapeutic agents we are investigating enzymes involved in the epigenetic modification of chromatin. Sirtuins are NAD+ dependent lysine deacetylases that are involved in a wide variety of cellular processes including histone deacetylation, and have been demonstrated to be therapeutic targets in various pathologies, including cancer.</p><p>Methodology, Principal Findings</p><p>In order to determine whether <i>Schistosoma mansoni</i> sirtuins are potential therapeutic targets we first identified and characterized their protein sequences. Five sirtuins (SmSirt) are encoded in the <i>S. mansoni</i> genome and phylogenetic analysis showed that they are orthologues of mammalian Sirt1, Sirt2, Sirt5, Sirt6 and Sirt7. Both SmSirt1 and SmSirt7 have large insertion in the catalytic domain compared to their mammalian orthologues. SmSirt5 is the only mitochondrial sirtuin encoded in the parasite genome (orthologues of Sirt3 and Sirt4 are absent) and transcripts corresponding to at least five splicing isoforms were identified. All five sirtuins are expressed throughout the parasite life-cycle, but with distinct patterns of expression. Sirtuin inhibitors were used to treat both schistosomula and adult worms maintained in culture. Three inhibitors in particular, Sirtinol, Salermide and MS3 induced apoptosis and death of schistosomula, the separation of adult worm pairs, and a reduction in egg laying. Moreover, Salermide treatment led to a marked disruption of the morphology of ovaries and testes. Transcriptional knockdown of <i>SmSirt1</i> by RNA interference in adult worms led to morphological changes in the ovaries characterized by a marked increase in mature oocytes, reiterating the effects of sirtuin inhibitors and suggesting that SmSirt1 is their principal target.</p><p>Conclusion, Significance</p><p>Our data demonstrate the potential of schistosome sirtuins as therapeutic targets and validate screening for selective sirtuin inhibitors as a strategy for developing new drugs against schistosomiasis.</p></div

Freshly perfused adult couples were maintained in culture for 6 days and incubated with 10 µM or 20 µM of Salermide (A), Sirtinol (B), MS3 (C), HR103 (D), MS13 (E) and CS13 (F).

<p>DMSO solvent was use as the control. Percentages of paired worm were determined daily. The results are expressed as mean % paired worms (± SEM, three independent experiments).</p

Effects of <i>SmSirt1</i> transcript knockdown in adult worms.

<p>RNA interference was carried out by the electroporation of adult worms with dsRNA for <i>SmSirt1</i> or <i>luciferase</i> (negative control) as described in the Methods section. (A) RNAi led to a reduction of about 80% in the level of transcripts for <i>SmSirt1</i> compared to controls knocked down for the irrelevant <i>luciferase</i> gene. (B) Microscopic examination was carried out 5 days after RNAi treatment as previously. This treatment caused no obvious morphological changes to male worm testes (B) compared to the control (A). Testes are indicated by red arrows. In contrast, RNAi treatment led to a marked increase in mature oocytes in the ovaries (D) compared to the control (C) particularly in the posterior part (MO). A mature oocyte in the anterior part of the ovary (IO), normally containing only immature oocytes (D) is arrowed. Results shown are representative of four independent experiments.</p

Alignment of the protein sequences of <i>S.</i><i>mansoni</i> sirtuins catalytic domains with orthologues from other species.

<p>Sequences used in the alignment of sirtuin class I (Figure A) in addition to SmSirt1 and SmSirt2 are <i>Clonorchis sinensis</i> Sirt1 (CsSirt1 Accession No: GAA56043.1) Sirt2 (CsSirt2 GAA29763.1), <i>Homo sapiens</i> Sirt1 (HsSirt1: AAH12499.1) Sirt2 (HsSirt2: NP_036369.2), <i>Drosophila melanogaster</i> Sirt1 (DmSirt1: NP_477351.1) Sirt2 (DmSirt2: NP_650880.2), <i>Caenorhabditis elegans</i> Sirt1 (CeSirt1: NP_001255484.1) Sirt2 (CeSirt2: NP_001024673.1). Sequences used in the alignment of sirtuin class III (Figure B) in addition to SmSirt5 are <i>C. sinensis</i> Sirt5 (CsSirt5: GAA47482.1), <i>H. sapiens</i> Sirt5 (HsSirt5: NP_036373.1), <i>Aplysia californica</i> Sirt5 (AcSirt5: NP_001191422.1), <i>Camponotus floridanus</i> Sirt5 (CfSirt5: EFN62888.1). Sequences used in the alignment of sirtuin class IV (Figure C) in addition to SmSirt6 and SmSirt7 are <i>C. sinensis</i> Sirt7 (CsSirt7: GAA293.65.2), <i>H. sapiens</i> Sirt6 (HsSirt6: CAG33481.1) Sirt7 (HsSirt7: NP_057622.1), <i>Xenopus laevis</i> Sirt6 (XlSirt6: NP_001085592.1), <i>C. floridanus</i> Sirt6 (CfSirt6: EFN65179.1) Sirt7 (CfSirt7: EFN70308.1), <i>D. melanogaster</i> Sirt6 (DmSirt6: NP_649990.2) Sirt7 (NP_651664.2). Solid lines above the sequence alignment indicate which regions of the proteins compose the Rossmann-fold domain (red), cofactor binding loop (black), small domain (blue) and loop regions (green). Highlighted residues in the sequence alignment indicate which amino acids are involved in NAD+ binding (green), zinc binding (blue) and acetyl-lysine peptide binding (red).</p

Total number of eggs laid during 5 days treatment with sirtuin inhibitors at 10 µM and 20 µM.

<p>Results obtained with (A) Salermide, (B) Sirtinol, (C) MS3, (D) HR103, (E) MS13 and (F) CS13 were expressed as mean % of eggs laid by treated worms compared to controls (± SD, three independent experiments). Statistical analyses were performed using student's t-test, and significance is displayed as follows: ** p<0.01, ***p<0.001.</p