Metalation and activation of Zn²⁺enzymes via early secretory pathway-resident ZNT proteins

Zinc (Zn²⁺), an essential trace element, binds to various proteins, including enzymes, transcription factors, channels, and signaling molecules and their receptors, to regulate their activities in a wide range of physiological functions. Zn²⁺ proteome analyses have indicated that approximately 10% of the proteins encoded by the human genome have potential Zn²⁺ binding sites. Zn²⁺binding to the functional site of a protein (for enzymes, the active site) is termed Zn²⁺metalation. In eukaryotic cells, approximately one-third of proteins are targeted to the endoplasmic reticulum; therefore, a considerable number of proteins mature by Zn²⁺metalation in the early secretory pathway compartments. Failure to capture Zn²⁺ in these compartments results in not only the inactivation of enzymes (apo-Zn²⁺ enzymes), but also their elimination via degradation. This process deserves attention because many Zn²⁺ enzymes that mature during the secretory process are associated with disease pathogenesis. However, how Zn²⁺is mobilized via Zn²⁺ transporters, particularly ZNTs, and incorporated in enzymes has not been fully elucidated from the cellular perspective and much less from the biophysical perspective. This review focuses on Zn²⁺ enzymes that are activated by Zn²⁺ metalation via Zn²⁺ transporters during the secretory process. Further, we describe the importance of Zn²⁺ metalation from the physiopathological perspective, helping to reveal the importance of understanding Zn²⁺ enzymes from a biophysical perspective

Metalation and Maturation of Zinc Ectoenzymes: A Perspective

Numerous zinc ectoenzymes are folded and activated in the compartments of the early secretory pathway, such as the ER and the Golgi apparatus, before reaching their final destination. During this process, zinc must be incorporated into the active site; therefore, metalation of the nascent protein is indispensable for the expression of the active enzyme. However, to date, the molecular mechanism underlying this process has been poorly investigated. This is in sharp contrast to the physiological and pathophysiological roles of zinc ectoenzymes, which have been extensively investigated over the past decades. This manuscript concisely outlines the present understanding of zinc ectoenzyme activation through metalation by zinc and compares this with copper ectoenzyme activation, in which elaborate copper metalation mechanisms are known. Moreover, based on the comparison, several hypotheses are discussed. Approximately 80 years have passed since the first zinc enzyme was identified; therefore, it is necessary to improve our understanding of zinc ectoenzymes from a biochemical perspective, which will further our understanding of their biological roles

A Postsynaptic Mechanism of Zinc Transport Driving Inhibition of NMDA Receptors

Zinc is an essential element with diverse signaling functions in the central nervous system. Extracellular zinc acts on a variety of receptors to modulate neurotransmission. Notably, zinc binds and inhibits the GluN2A subunit of NMDA receptors (NMDARs) with high affinity. Inside the cell, zinc also triggers diverse signaling cascades, ranging from zinc-induced gene expression to cell death triggered by high concentrations of zinc. To maintain sufficient signaling without tipping the scales towards cell death, a complex system of transporters, metalloproteins, and ion channels regulate the localization and concentration of zinc. The zinc transporter, ZnT3, concentrates the majority of loosely bound ‘labile’ zinc into synaptic vesicles from where it then is released into the cleft in an activity-dependent manner. Current modeling of vesicular zinc assumes that ZnT3-dependent zinc is released and subsequently diffuses across the cleft and this is sufficient to account for its actions on postsynaptic targets, including NMDARs. Interestingly, the transporter ZnT1 is located in the postsynaptic density and binds directly to the GluN2A subunit of NMDARs, suggesting that ZnT1’s transport of zinc out of the cytoplasm into the extracellular space may contribute to NMDAR inhibition. This suggests that ZnT1 and intracellular zinc may critically regulate zinc inhibition of NMDARs through ZnT1’s interaction with GluN2A. To explore this question, we developed a novel peptide that specifically disrupts the interaction between GluN2A and ZnT1. We found that either disrupting ZnT1’s association with GluN2A or chelating intracellular zinc is sufficient to block endogenous inhibition of NMDARs, even in the presence of presynaptic zinc release. ZnT1, in addition to transporting cytosolic zinc, is also upregulated by intracellular zinc through the metal regulatory transcription factor 1. We found that increasing intracellular zinc is sufficient to drive upregulation of ZnT1-GluN2A interactions and subsequent inhibition of NMDARs. Together these data reveal a novel mechanism in which presynaptic release, intracellular zinc, and ZnT1 cooperatively drive inhibition of NMDARs. These findings add complexity to our current understanding of zinc dynamics at the synapses and provide a novel mechanism for modulating zinc and NMDAR signaling