Bi-directional electron transfer between H2 and NADPH mitigates light fluctuation responses in green algae

The metabolism of green algae has been the focus of much research over the last century. These photosynthetic organisms can thrive under various conditions and adapt quickly to changing environments by concomitant usage of several metabolic apparatuses. The main electron coordinator in their chloroplasts, nicotinamide adenine dinucleotide phosphate (NADPH), participates in many enzymatic activities and is also responsible for inter-organellar communication. Under anaerobic conditions, green algae also accumulate molecular hydrogen (H2), a promising alternative for fossil fuels. However, to scale-up its accumulation, a firm understanding of its integration in the photosynthetic apparatus is still required. While it is generally accepted that NADPH metabolism correlates to H2 accumulation, the mechanism of this collaboration is still vague and relies on indirect measurements. Here, we investigated this connection in Chlamydomonas reinhardtii using simultaneous measurements of both dissolved gases concentration, NADPH fluorescence and electrochromic shifts at 520-546 nm. Our results indicate that energy transfer between H2 and NADPH is bi-directional and crucial for the maintenance of redox balance under light fluctuations. At light onset, NADPH consumption initially eventuates in H2 evolution, which initiates the photosynthetic electron flow. Later on, as illumination continues the majority of NADPH is diverted to the Calvin-Benson-Bassham cycle. Dark onset triggers re-assimilation of H2, which produces NADPH and so, enables initiation of dark fermentative metabolism.

Aberrant ROS Served as an Acquired Vulnerability of Cisplatin-resistant Lung Cancer

BackgroundLung cancer has become a global health issue in recent decades. Despite of high rate of resistance, cisplatin-base chemotherapy is still the main treatment of lung cancer patients who are not suitable for TKI-based targeted therapy and immunotherapy. Thus, overcoming cisplatin resistance is urgently needed.MethodsA small panel is established to screen chemicals and compounds overcoming cisplatin resistance. Survival fractions as well as proliferation are determined by MTT assay. Colony formation assay, JC-1 assay, EdU assay, ROS assay as well as apoptosis and cell cycle assay are performed to verify vitalities of different groups. Quantifications of NADP+/NADPH and GSH/GSSG are carried out according to standard protocols. Xenograft model is generated to evaluate the in vivo role of APR-246.ResultsIn this study, we identify NADPH metabolism and reactive oxygen species (ROS) levels as the main cause accounting for cisplatin resistance of H460. Based on a small panel consisting common chemotherapy drugs and compounds, APR-246 is proved an effective compound specifically inhibiting proliferation and colony formation of cisplatin resistant H460 (H460-Cis) cells. APR-246 significantly causes G0/G1 accumulation and S phase inhibition of H460-Cis cells. Besides, APR-246 can obviously lead to severe mitochondria dysfunction as well as elevated apoptosis by altering apoptosis-related protein expressions in H460-Cis cells. Further study proves that it is the aberrant ROS levels as well as NRF2/SLC7A11/GSH axis dysfunction accounting for the specific anti-tumor effects of APR-246. Mechanistically, NRF2 is specifically ubiquitylated degraded in APR-246 treated H460-Cis cells, which in turn decrease NRF2/SLC7A11/GSH axis activity.ConclusionOur study uncovered new insights into the biology driving cisplatin resistance of lung cancer and highlights potentials of APR-246 as future therapeutic agents again cisplatin resistance.

NADPH homeostasis in cancer: functions, mechanisms and therapeutic implications

Nicotinamide adenine dinucleotide phosphate (NADPH) is an essential electron donor in all organisms, and provides the reducing power for anabolic reactions and redox balance. NADPH homeostasis is regulated by varied signaling pathways and several metabolic enzymes that undergo adaptive alteration in cancer cells. The metabolic reprogramming of NADPH renders cancer cells both highly dependent on this metabolic network for antioxidant capacity and more susceptible to oxidative stress. Modulating the unique NADPH homeostasis of cancer cells might be an effective strategy to eliminate these cells. In this review, we summarize the current existing literatures on NADPH homeostasis, including its biological functions, regulatory mechanisms and the corresponding therapeutic interventions in human cancers, providing insights into therapeutic implications of targeting NADPH metabolism and the associated mechanism for cancer therapy.

Limitations of Deuterium-Labelled Substrates for Quantifying NADPH Metabolism in Heterotrophic Arabidopsis Cell Cultures

NADPH is the primary source of cellular reductant for biosynthesis, and strategies for increasing productivity via metabolic engineering need to take account of the requirement for reducing power. In plants, while the oxidative pentose phosphate pathway is the most direct route for NADPH production in heterotrophic tissues, there is increasing evidence that other pathways make significant contributions to redox balance. Deuterium-based isotopic labelling strategies have recently been developed to quantify the relative production of NADPH from different pathways in mammalian cells, but the application of these methods to plants has not been critically evaluated. In this study, LC-MS was used to measure deuterium incorporation into metabolites extracted from heterotrophic Arabidopsis cell cultures grown on [1-2H]glucose or D2O. The results show that a high rate of flavin-enzyme-catalysed water exchange obscures labelling of NADPH from deuterated substrates and that this exchange cannot be accurately accounted for due to exchange between triose- and hexose-phosphates. In addition, the duplication of NADPH generating reactions between subcellular compartments can confound analysis based on whole cell extracts. Understanding how the structure of the metabolic network affects the applicability of deuterium labelling methods is a prerequisite for development of more effective flux determination strategies, ensuring data are both quantitative and representative of endogenous biological processes.

2H/1H variation in microbial lipids is controlled by NADPH metabolism

The hydrogen-isotopic compositions (2H/1H ratios) of lipids in microbial heterotrophs are known to vary enormously, by at least 40% (400‰) relative. This is particularly surprising, given that most C-bound H in their lipids appear to derive from the growth medium water, rather than from organic substrates, implying that the isotopic fractionation between lipids and water is itself highly variable. Changes in the lipid/water fractionation are also strongly correlated with the type of energy metabolism operating in the host. Because lipids are well preserved in the geologic record, there is thus significant potential for using lipid 2H/1H ratios to decipher the metabolism of uncultured microorganisms in both modern and ancient ecosystems. But despite over a decade of research, the precise mechanisms underlying this isotopic variability remain unclear. Differences in the kinetic isotope effects (KIEs) accompanying NADP+ reduction by dehydrogenases and transhydrogenases have been hypothesized as a plausible mechanism. However, this relationship has been difficult to prove because multiple oxidoreductases affect the NADPH pool simultaneously. Here, we cultured five diverse aerobic heterotrophs, plus five Escherichia coli mutants, and used metabolic flux analysis to show that 2H/1H fractionations are highly correlated with fluxes through NADP+-reducing and NADPH-balancing reactions. Mass-balance calculations indicate that the full range of 2H/1H variability in the investigated organisms can be quantitatively explained by varying fluxes, i.e., with constant KIEs for each involved oxidoreductase across all species. This proves that lipid 2H/1H ratios of heterotrophic microbes are quantitatively related to central metabolism and provides a foundation for interpreting 2H/1H ratios of environmental lipids and sedimentary hydrocarbons.