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.

Differential Regulation of Degradation and Immune Pathways Underlies Adaptation of the Ectosymbiotic Nematode Laxus Oneistus to Oxic-Anoxic Interfaces

Eukaryotes may experience oxygen deprivation under both physiological and pathological conditions. Because oxygen shortage leads to a reduction in cellular energy production, all eukaryotes studied so far conserve energy by suppressing their metabolism. However, the molecular physiology of animals that naturally and repeatedly experience anoxia is underexplored. One such animal is the marine nematode Laxus oneistus. It thrives, invariably coated by its sulfur-oxidizing symbiont Candidatus Thiosymbion oneisti, in anoxic sulfidic or hypoxic sand. Here, transcriptomics and proteomics showed that, whether in anoxia or not, L. oneistus mostly expressed genes involved in ubiquitination, energy generation, oxidative stress response, immune response, development, and translation. Importantly, ubiquitination genes were also highly expressed when the nematode was subjected to anoxic sulfidic conditions, together with genes involved in autophagy, detoxification and ribosome biogenesis. We hypothesize that these degradation pathways were induced to recycle damaged cellular components (mitochondria) and misfolded proteins into nutrients. Remarkably, when L. oneistus was subjected to anoxic sulfidic conditions, lectin and mucin genes were also upregulated, potentially to promote the attachment of its thiotrophic symbiont. Furthermore, the nematode appeared to survive oxygen deprivation by using an alternative electron carrier (rhodoquinone) and acceptor (fumarate), to rewire the electron transfer chain. On the other hand, under hypoxia, genes involved in costly processes (e.g., amino acid biosynthesis, development, feeding, mating) were upregulated, together with the worm’s Toll-like innate immunity pathway and several immune effectors (e.g., Bacterial Permeability Increasing proteins, fungicides). In conclusion, we hypothesize that, in anoxic sulfidic sand, L. oneistus upregulates degradation processes, rewires oxidative phosphorylation and by reinforces its coat of bacterial sulfur-oxidizers. In upper sand layers, instead, it appears to produce broad-range antimicrobials and to exploit oxygen for biosynthesis and development.

Differential regulation of degradation and immune pathways underlies adaptation of the symbiotic nematode Laxus oneistus to oxic-anoxic interfaces

Eukaryotes may experience oxygen deprivation under both physiological and pathological conditions. Because oxygen shortage leads to a reduction in cellular energy production, all eukaryotes studied so far conserve energy by suppressing their metabolism. However, the molecular physiology of animals that naturally and repeatedly experience anoxia underexplored. One such animal is the symbiotic marine nematode Laxus oneistus. It thrives, invariably coated by its sulfur-oxidizing bacterium Candidatus Thiosymbion oneisti, in anoxic sulfidic or hypoxic sand. Here, transcriptomics and proteomics showed that, whether in anoxia or not, L. oneistus mostly expressed genes involved in ubiquitination, energy generation, oxidative stress response, immune response, development, and translation. Importantly, ubiquitination genes were also highly expressed when the nematode was subjected to anoxic sulfidic conditions, together with genes involved in autophagy, detoxification, chaperone-encoding genes, and ribosome biogenesis. We hypothesize that these degradation pathways were induced to recycle damaged cellular components (mitochondria) and misfolded proteins into nutrients. Remarkably, when L. oneistus was subjected to anoxic sulfidic conditions, lectin genes were also upregulated, potentially to promote the attachment of its thiotrophic anaerobic symbiont. Furthermore, L. oneistus appeared to survive oxygen deprivation by using an alternative electron carrier (rhodoquinone) and acceptor (fumarate), to rewire the electron transfer chain. On the other hand, under hypoxia, genes involved in costly processes (e.g., amino acid biosynthesis, development, feeding, mating) were upregulated, together with the worm's Toll-like innate immunity pathway and several immune effectors (e.g., Bacterial Permeability Increasing proteins, fungicides). In conclusion, we hypothesize that, in anoxic sulfidic sand, L. oneistus survives by overexpressing degradation processes, rewiring oxidative phosphorylation and by reinforcing its coat of bacterial sulfur-oxidizers. In upper sand layers, instead, it appears to produce broad-range antimicrobials and to exploit oxygen for biosynthesis and development.

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.

Physiological And Proteomic Analyses Reveals That Brassinosteroids Application Improves The Chilling Stress Tolerance of Pepper Seedlings

Brassinosteroids (BRs) are important in plant resistance to chilling stress. However, limited information is available regarding the specific mechanisms involved at proteomic level. We utilized iTRAQ proteomic approach, physiological assays and information obtained from cellular ultrastructure to clarify the underlying molecular mechanism of BRs to alleviate chilling stress in pepper (Capsicum annuum L.). Foliar application of 24-epibrassinolide (EBR) improved photosynthesis and improved cell structure by presenting a distinct mesophyll cell and chloroplast with well-developed thylakoid membranes in the leaves of pepper seedlings. We identified 346 differentially expressed proteins (DEPs), including 217 up-regulated proteins and 129 down-regulated proteins in plants under chilling (Chill) and Chill + EBR treated plants. Most of the DEPs were related to multiple pathways, including photosynthesis, carbohydrates metabolism, energy metabolism, protein biosynthesis, amino acids synthesis, redox and stress defence (ascorbate peroxidase, glutathione peroxidase, and superoxide dismutase). Up-regulated DEPs were associated with photosynthetic electron transfer chain, oxidative phosphorylation, GSH metabolism pathway, Calvin cycle and signaling pathway. The physiochemical analysis showed that EBR treatment improved the tolerance of pepper seedlings to chilling stress.

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.

Lactate dehydrogenases amplify reactive oxygen species in cancer cells in response to oxidative stimuli

Previous studies demonstrated that superoxide could initiate and amplify LDH-catalyzed hydrogen peroxide production in aqueous phase, but its physiological relevance is unknown. Here we showed that LDHA and LDHB both exhibited hydrogen peroxide-producing activity, which was significantly enhanced by the superoxide generated from the isolated mitochondria from HeLa cells and patients’ cholangiocarcinoma specimen. After LDHA or LDHB were knocked out, hydrogen peroxide produced by Hela or 4T1 cancer cells were significantly reduced. Re-expression of LDHA in LDHA-knockout HeLa cells partially restored hydrogen peroxide production. In HeLa and 4T1 cells, LDHA or LDHB knockout or LDH inhibitor FX11 significantly decreased ROS induction by modulators of the mitochondrial electron transfer chain (antimycin, oligomycin, rotenone), hypoxia, and pharmacological ROS inducers piperlogumine (PL) and phenethyl isothiocyanate (PEITC). Moreover, the tumors formed by LDHA or LDHB knockout HeLa or 4T1 cells exhibited a significantly less oxidative state than those formed by control cells. Collectively, we provide a mechanistic understanding of a link between LDH and cellular hydrogen peroxide production or oxidative stress in cancer cells in vitro and in vivo.

Recent advances in the structural diversity of reaction centers

Photosynthetic reaction centers (RC) catalyze the conversion of light to chemical energy that supports life on Earth, but they exhibit substantial diversity among different phyla. This is exemplified in a recent structure of the RC from an anoxygenic green sulfur bacterium (GsbRC) which has characteristics that may challenge the canonical view of RC classification. The GsbRC structure is analyzed and compared with other RCs, and the observations reveal important but unstudied research directions that are vital for disentangling RC evolution and diversity. Namely, (1) common themes of electron donation implicate a Ca2+ site whose role is unknown; (2) a previously unidentified lipid molecule with unclear functional significance is involved in the axial ligation of a cofactor in the electron transfer chain; (3) the GsbRC features surprising structural similarities with the distantly-related photosystem II; and (4) a structural basis for energy quenching in the GsbRC can be gleaned that exemplifies the importance of how exposure to oxygen has shaped the evolution of RCs. The analysis highlights these novel avenues of research that are critical for revealing evolutionary relationships that underpin the great diversity observed in extant RCs.

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.