Dissecting the process of activation of cancer-promoting zinc-requiring ectoenzymes by zinc metalation mediated by ZnT transporters [Membrane Biology]

Zinc-requiring ectoenzymes, including both secreted and membrane-bound enzymes, are considered to capture zinc in their active site for their activation in the early secretory pathway. This idea has been confirmed by our studies conducted using tissue nonspecific alkaline phosphatase (TNAP), which is elaborately activated by means of a two-step mechanism by Zn transporter 5 (ZnT5)-ZnT6 heterodimers and ZnT7 homodimers, through protein stabilization followed by enzyme activation with zinc in the early secretory pathway. However, the molecular basis of the activation process in other zinc-requiring ectoenzymes remains largely unknown. In this study, we investigated this activation process by using three cancer-promoting zinc-requiring ectoenzymes, autotaxin (ATX), matrix metalloproteinase 9 (MMP9), and carbonic anhydrase IX (CAIX), and the chicken DT40 cell mutants that we generated; we specifically focused on clarifying whether the same or similar activation mechanism operates in these ectoenzymes. ATX activation required ZnT5-ZnT6 heterodimers and ZnT7 homodimers in a manner similar to TNAP activation, although the protein stability of ATX was differently regulated from that of TNAP. MMP9 required ZnT5-ZnT6 heterodimers and ZnT7 homodimers for its activation as well as secretion: MMP9 was not secreted into the spent medium unless both ZnT zinc-transport complexes were present. Lastly, CAIX activation by zinc was mediated not only by ZnT5-ZnT6 heterodimers and ZnT7 homodimers, but also by ZnT4 homodimers; thus, these three ZnT zinc-transport complexes redundantly contribute to CAIX activation. Our results provide pivotal insights into the activation processes of zinc-requiring ectoenzymes, and, furthermore, offer novel insights for potential cancer-therapy applications given the cancer-promoting potencies of ATX, MMP9, and CAIX.

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