Electron relay via diffusible and protein-bound flavin cofactor in human six-transmembrane epithelial antigen of the prostate (STEAP)

Six transmembrane epithelial antigen of the prostate (STEAP) is a family of membrane-embedded metal ion reductases that transfer electrons across the cell membranes. STEAPs are unique to mammals and implicated in metabolic and inflammatory responses and are significantly upregulated in many types of cancer cells. There are four members in the family, STEAP1 - 4, and all STEAPs have a transmembrane domain (TMD) that has a conserved heme binding site, and STEAP2 - 4, but not STEAP1, have an intracellular reductase domain (RED) that binds to NADPH and FAD. NADPH, FAD, and heme form an electron transfer chain that allows electron flow across the cell membranes, however, the mechanism of the stepwise cross-membrane electron transfer remains unclear. It is also unclear how STEAP1, which does not have a RED, acquires and transfers electrons. We expressed and purified human STEAP2 (hSTEAP2), and constructed the electron transfer chain in vitro. Purified hSTEAP2 mediates electron transfer from NADPH to FAD and to heme, with a NADPH oxidation rate of 0.0026 per second. The time course for reduction of heme is more complex with an initial rate of ~ 0.00016 per second. We also found that the heme in hSTEAP2 has a low-spin electron structure and thus a rigid coordination, which is consistent with its high occupancy in the purified protein and its role as part of the electron transfer chain. We then determined the structure of hSTEAP2 in complex with NADP, FAD, and heme by cryo-electron microscopy to 3.2 Å. Human STEAP2 forms a homotrimer and its structure is similar to that of hSTEAP4. NADP+, FAD, and heme are well-resolved in the structure, and while the current conformation would allow electron transfer from FAD to heme, the FAD isoalloxazine ring is ~ 19 Å away from NADPH and does not support hydride transfer. Significant structural changes are required to accommodate dissociation of the FAD isoalloxazine ring from the TMD such that the FAD may become diffusible after its reduction. To test this hypothesis and also to find out how STEAP1 may transfer electrons, we reconstructed an electron transfer chain for STEAP1 and found that the heme in STEAP1 can be reduced by FAD produced either by the full-length STEAP2 or by the soluble RED domain from STEAP4. These results support a diffusible FAD mechanism and demonstrate that STEAP1 is capable of mediating electron transfer across the cell membranes. In summary, our study established a structural and functional framework for further analyses for resolving the mechanism of electron transfer in STEAPs.

Molecular Hydrogen Attenuates High Hydrostatic Pressure-Induced Neuronal Cell Damage by Reversing Dysfunction of Mitochondrial Electron Transfer Chain

Cellular hydrostatic pressure beyond its normal range can induce the accumulation of reactive oxidative species (ROS) generated by mitochondria and lead to pathological conditions such as glaucomatous optic neuropathy. However, little is known about how the mitochondrial electron transfer chain (ETC) is affected by elevated pressure. Moreover, the protective effects of hydrogen on various pathological conditions have been observed by reductions in ROS, yet the role of hydrogen in high hydrostatic pressure (HHP)-induced cell damage remains obscure. The goal of this study was to investigate the effect of HHP on ETC activity and whether hydrogen exerts protective effects against HHP-induced damage in cultured neuronal cells. Cultured SH-SY5Y human neuroblastoma cells were exposed to an elevated ambient hydrostatic pressure of 50 mmHg for a period of 2 to 6 h. HHP impaired the activities of ETC complexes, and these effects were reversed by hydrogen. Significant increases in apoptotic rates and intracellular ROS levels were observed in HHP-treated SH-SY5Y cells. Hydrogen significantly inhibited the apoptotic rates and reduced the levels of ROS. These findings suggest that HHP induces cell damage by causing ETC dysfunction to increase oxidative stress and that hydrogen may act as a protective agent to alleviate HHP-induced neuronal injury.

Disrupting Mitochondrial Electron Transfer Chain Complex I Decreases Immune Checkpoints in Murine and Human Acute Myeloid Leukemic Cells

Oxidative metabolism is crucial for leukemic stem cell (LSC) function and drug resistance in acute myeloid leukemia (AML). Mitochondrial metabolism also affects the immune system and therefore the anti-tumor response. The modulation of oxidative phosphorylation (OxPHOS) has emerged as a promising approach to improve the therapy outcome for AML patients. However, the effect of mitochondrial inhibitors on the immune compartment in the context of AML is yet to be explored. Immune checkpoints such as ectonucleotidase CD39 and programmed dead ligand 1 (PD-L1) have been reported to be expressed in AML and linked to chemo-resistance and a poor prognosis. In the present study, we first demonstrated that a novel selective electron transfer chain complex (ETC) I inhibitor, EVT-701, decreased the OxPHOS metabolism of murine and human cytarabine (AraC)-resistant leukemic cell lines. Furthermore, we showed that while AraC induced an immune response regulation by increasing CD39 expression and by reinforcing the interferon-γ/PD-L1 axis, EVT-701 reduced CD39 and PD-L1 expression in vitro in a panel of both murine and human AML cell lines, especially upon AraC treatment. Altogether, this work uncovers a non-canonical function of ETCI in controlling CD39 and PD-L1 immune checkpoints, thereby improving the anti-tumor response in AML.

Disrupting Mitochondrial Electron Transfer Chain Complex I Decreases Immune Checkpoints in Murine and Human Acute Myeloid Leukemic Cells

Oxidative metabolism is crucial for leukemic stem cell (LSC) function and drug resistance in acute myeloid leukemia (AML). Mitochondrial metabolism also affects the immune system and therefore the antitumor response. Modulation of oxidative phosphorylation (OxPHOS) has emerged as a promising approach to improve therapy outcome for AML patients. However, the effect of mitochondrial inhibitors on the immune compartment in the context of AML is yet to be explored. Immune checkpoints such as the ecto-nucleotidase CD39 and programmed dead ligand 1 (PD-L1) have been reported to be expressed in AML and linked to chemoresistance and poor prognosis. In the present study, we first demonstrated that a novel selective electron transfer chain complex (ETC) I inhibitor, EVT-701, decreased OxPHOS metabolism of murine and human cytarabine (AraC)-resistant leukemic cell lines. Furthermore, we showed that, while AraC induced immune response regulation by increasing CD39 expression and by reinforcing interferon-γ/PD-L1 axis, EVT-701 reduced CD39 and PD-L1 expression in vitro in a panel of both murine and human AML cell lines, especially upon AraC treatment. Altogether, this work uncovers a non-canonical function of ETCI in controlling CD39 and PD-L1 immune checkpoints, thereby improving the anti-tumor response in AML.

The Significance of Chloroplast NAD(P)H Dehydrogenase Complex and Its Dependent Cyclic Electron Transport in Photosynthesis

Chloroplast NAD(P)H dehydrogenase (NDH) complex, a multiple-subunit complex in the thylakoid membranes mediating cyclic electron transport, is one of the most important alternative electron transport pathways. It was identified to be essential for plant growth and development during stress periods in recent years. The NDH-mediated cyclic electron transport can restore the over-reduction in stroma, maintaining the balance of the redox system in the electron transfer chain and providing the extra ATP needed for the other biochemical reactions. In this review, we discuss the research history and the subunit composition of NDH. Specifically, the formation and significance of NDH-mediated cyclic electron transport are discussed from the perspective of plant evolution and physiological functionality of NDH facilitating plants’ adaptation to environmental stress. A better understanding of the NDH-mediated cyclic electron transport during photosynthesis may offer new approaches to improving crop yield.