title: Azaspiracid-1 inhibits the maturation of cathepsin D in mammalian cells.
abstract: Azaspiracid-1 (AZA-1) inhibits endocytosis, but the consequences of this alteration on cellular processes are unknown. We hypothesized that the inhibition of endocytosis is a key step of the mode of action of AZA-1, leading to perturbation of cellular processes dependent on proper functioning of endocytic machinery. We tested this working hypothesis by probing whether AZA-1 can alter the maturation of cathepsin D in MCF-7 epithelial cells, as a model system. We found that cell treatment with AZA-1 inhibited the conversion of 52 kDa procathepsin D into the mature 30 kDa protein. The effects induced by AZA-1 were similar to those elicited by chlorpromazine and other agents preventing proper maturation of lysosomal enzymes, indicating that the inhibition of endocytic transfer of proforms to late endosomes/lysosomess is responsible for the effect induced by the toxin. By immunofluorescence microscopy, we found no colocalization of cathepsin D and the early endosomal marker EEA-1 in control cells, where most of cathepsin D resides in late endosomes/lysosomes. Co-localization of cathepsin D and EEA-1 immunoreactivity, in turn, was found in cells exposed to AZA-1, indicating that the toxin blocks protein maturation at the early steps of endocytosis, causing accumulation of procathepsin D in early endosomes. The molecular alteration induced by AZA-1 involved both secreted and intracellular pools of procathepsin D, showing that the toxin effect does not result from a general impairment of vesicular trafficking but is the outcome of a perturbed centripetal process. Furthermore, AZA-1 was found to inhibit procathepsin D maturation also in normal fibroblasts, showing that this molecular response is induced by this toxin in different cell types. The data we obtained corroborated our hypothesis and provide a unified molecular frame for the mode of action of AZAs in animal models, involving a primary alteration of endocytic processes.
authors: Dipartimento di Scienze della Vita, Università di Modena e Reggio Emilia, Via Campi 287, Modena, Italy.
authors: Gian Luca
authors: GL
authors: Sala
authors: Mirella
authors: M
authors: Bellocci
authors: Federica
authors: F
authors: Callegari
authors: Gian Paolo
authors: GP
authors: Rossini
mesh_terms: D000818:Animals; D049872:Bivalvia / Q000737:chemistry; D002402:Cathepsin D / Q000032:analysis / Q000378:metabolism*; D045744:Cell Line, Tumor; D002478:Cells, Cultured; D004705:Endocytosis / Q000187:drug effects; D004792:Enzyme Precursors / Q000032:analysis / Q000378:metabolism*; D004847:Epithelial Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D008247:Lysosomes / Q000187:drug effects / Q000378:metabolism; D008387:Marine Toxins / Q000633:toxicity*; D051379:Mice; D013141:Spiro Compounds / Q000633:toxicity*; D000095702:Polyether Toxins
chemical_list: D002402:Cathepsin D; D004792:Enzyme Precursors; D008387:Marine Toxins; D013141:Spiro Compounds; C406592:azaspiracid; D000095702:Polyether Toxins; C058345:procathepsin D