Six-Transmembrane Epithelial Antigen of Prostate 1 (STEAP1) Has a Single b Heme and Is Capable of Reducing Metal Ion Complexes and Oxygen

Abstract

STEAP1, six-transmembrane epithelial antigen of prostate member 1, is strongly expressed in several types of cancer cells, particularly in prostate cancer, and inhibition of its expression reduces the rate of tumor cell proliferation. However, the physiological function of STEAP1 remains unknown. Here for the first time, we purified a mammalian (rabbit) STEAP1 at a milligram level, permitting its high-quality biochemical and biophysical characterizations. We found that STEAP1 likely assembles as a homotrimer and forms a heterotrimer when co-expressed with STEAP2. Each STEAP1 protomer binds one heme prosthetic group that is mainly low-spin with a pair of histidine axial ligands, with small portions of high-spin and P450-type heme. In its ferrous state, STEAP1 is capable of reducing transition metal ion complexes of Fe and Cu. Ferrous STEAP1 also reacts readily with O through an outer sphere redox mechanism. Kinetics with all three substrates are biphasic with ∼80 and ∼20% for the fast and slow phases, respectively, in line with its heme heterogeneity. STEAP1 retained a low level of bound FAD during purification, and the binding equilibrium constant, K, was ∼30 μM. These results highlight STEAP as a novel metal reductase and superoxide synthase and establish a solid basis for further research into understanding how STEAP1 activities may affect cancer progression.

Structural basis for targeting human cancer antigen STEAP1 with antibodies

“…S3D), which is missing in STEAP1. In line with this, STEAP3 and 4 exhibit a low micromolar affinity for FAD (Kd = ~1 µM) (26,29), whereas the affinity of STEAP1 for FAD is much weaker (Kd = 34 µM) (30). The cryo-EM density in this region could therefore correspond to a loosely bound FAD cofactor, although the weak density does not allow for modelling the complete cofactor.…”

“…A previous pioneering study reported the biophysical and electrochemical characterization of N-terminally truncated rabbit STEAP1, purified from insect cells in lauryl maltose neopentyl glycol (LMNG) detergent (30). Our initial attempts to purify full-length, human STEAP1 from mammalian HEK-cells using a similar protocol were hampered by the loss of the noncovalently bound heme-b cofactor during the purification, suggesting that STEAP1 was not natively folded.…”

“…The reductase-like architecture of STEAP1 ( Fig. 3) and its heme-redox potential of -114 to -118 mV (30), indicate that the protein could be functional in reducing metal-ion complexes in vivo. However, to the best of our knowledge there is currently no experimental data available that shows that STEAP1 is capable of reducing ferric iron in a physiological setting.…”

“…It has been proposed that STEAP1 may have a functional role in heterooligomeric complexes with other STEAP paralogs (19,30). In support, its expression often correlates with the expression of STEAP2 in cancers (17) and both proteins co-purify in detergent (30), suggesting they could form a functional complex. Further indications for a functional hetero-trimeric STEAP complex emerged from the recent cryo-EM structures of homo-trimeric human STEAP4 (29), which revealed a domain-swapped architecture, with the intracellular OxRD positioned beneath the TMD of the adjacent protomer.…”

“…However, a recent study revealed that dithionite-reduced, purified STEAP1 retains heme and is capable of reducing metal-ion complexes and oxygen (30), indicating that the absence of a binding site for an electron-donating substrate like NADPH could explain the lack of reductase activity for STEAP1. It has been proposed that STEAP1 may have a functional role in heterooligomeric complexes with other STEAP paralogs (19,30). In support, its expression often correlates with the expression of STEAP2 in cancers (17) and both proteins co-purify in detergent (30), suggesting they could form a functional complex.…”