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The mosquito-larvicidal binary toxin from Bacillus sphaericus is composed of two polypeptides called BinA and BinB with molecular masses of approximately 42 and 51 kDa. Both components are required for full activity, with BinB acting as a specificity determinant and BinA being responsible for toxic action. To investigate the role of the selected charged residues in BinA, four mutants were generated by replacing charged amino acids with alanine (R97A, E98A, R101A, and E114A). All mutant proteins were produced at high levels and formed inclusion bodies similar to that of the wild type. Mosquito-larvicidal assays against Culex quinquefasciatus larvae revealed that the mutant R97A completely lost its activity and mutants E98A, R101A, and E114A showed significantly reduced toxicity. Intrinsic fluorescence spectroscopy analysis indicated that alanine substitutions at these positions did not alter the overall structure of the toxin. Binding of the mutants to BinB was not different from that of the wild type, suggesting that these mutations did not affect BinA-BinB interaction. Results demonstrated that R97, E98, R101, and E114 neither play a direct role in maintenance of BinA structure nor are involved in BinA-BinB interaction. Since these residues are required for full activity, they may play an important role during toxin internalization and/or toxic action of BinA inside the target cells.

Advanced prostate cancer is typically treated with anti-androgens to reduce cancer growth, but patients almost inevitably develop treatment resistance and castration-resistant disease. Recently, extracellular vesicles known as exosomes, which are secreted from the endosomal compartments in which they are formed, have been implicated in drug resistance mechanisms. Here we investigate whether growth regulation by the amino acid-dependent kinase complex, mechanistic Target of Rapamycin Complex 1 (mTORC1), and associated extracellular vesicle secretion might be involved in the adaptive responses to anti-androgens. We show that expression and intracellular localisation of the glutamine-sensing PAT4 (SLC36A4) amino acid transporter is increased in malignant versus benign prostatic tissue, mirroring earlier in vivo fly studies suggesting that these transporters are more effective at promoting growth from internal versus cell surface membranes. Furthermore, androgens induce PAT4 expression in prostate cancer cell lines and PAT4 is required for a proportion of androgen-stimulated mTORC1 activation and growth. Consistent with previous studies in other cancer cell lines, we find that glutamine depletion, PAT4 knockdown and mTORC1 inhibition all independently increase the production of a specific exosome subtype, Rab11a-exosomes, which has recently been implicated in pro-tumorigenic signalling responses to mTORC1 inhibition. Furthermore, we show that these exosomes are also induced by anti-androgens. We hypothesise that the uptake of Rab11a-exosomes by cells with higher PAT4 levels could provide a growth-promoting boost, enabling them to out-compete others with lower PAT4 expression, resulting in tumours that are more resistant to nutrient-deprivation and anti-androgen treatment.

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