PHD2 enzyme is an intracellular manganese sensor that initiates the homeostatic response against elevated manganese

Abstract

Intracellular sensors detect changes in levels of essential metals to initiate homeostatic responses. But, a mammalian manganese (Mn) sensor is unknown, representing a major gap in understanding of Mn homeostasis. Using human-relevant models, we recently reported that: 1) the primary homeostatic response to elevated Mn is upregulation of hypoxia-inducible factors (HIFs), which increases expression of the Mn efflux transporter SLC30A10; and 2) elevated Mn blocks the prolyl hydroxylation of HIFs by prolyl hydroxylase domain (PHD) enzymes, which otherwise targets HIFs for degradation. Thus, the mammalian mechanism for sensing elevated Mn likely relates to PHD inhibition. Moreover, 1) Mn substitutes for a catalytic iron (Fe) in PHD structures; and 2) exchangeable cellular levels of Fe and Mn are comparable. Therefore, we hypothesized that elevated Mn directly inhibits PHD by replacing its catalytic Fe. In vitro assays using catalytically active PHD2, the primary PHD isoform, revealed that Mn inhibited, and Fe supplementation rescued, PHD2 activity. However, a mutation in PHD2 (D315E) that selectively reduced Mn binding without substantially impacting Fe binding or enzymatic activity resulted in complete insensitivity of PHD2 to Mn in vitro. Additionally, hepatic cells expressing full-length PHD2 D315E were less sensitive to Mn-induced HIF activation and SLC30A10 upregulation than PHD2 wild-type . These results: 1) define a fundamental Mn sensing mechanism for controlling Mn homeostasis—elevated Mn inhibits PHD2, which functions as a Mn sensor, by outcompeting its catalytic Fe, and PHD2 inhibition activates HIF signaling to up-regulate SLC30A10; and 2) identify a unique mode of metal sensing that may have wide applicability.