Production of fully assembled and active Aquifex aeolicus F1FO ATP synthase in Escherichia coli

Background

F₁F_O ATP synthases catalyze the synthesis of ATP from ADP and inorganic phosphate driven by ion motive forces across the membrane. A number of ATP synthases have been characterized to date. The one from the hyperthermophilic bacterium Aquifex aeolicus presents unique features, i.e. a putative heterodimeric stalk. To complement previous work on the native form of this enzyme, we produced it heterologously in Escherichia coli.

Methods

We designed an artificial operon combining the nine genes of A. aeolicus ATP synthase, which are split into four clusters in the A. aeolicus genome. We expressed the genes and purified the enzyme complex by affinity and size-exclusion chromatography. We characterized the complex by native gel electrophoresis, Western blot, and mass spectrometry. We studied its activity by enzymatic assays and we visualized its structure by single-particle electron microscopy.

Results

We show that the heterologously produced complex has the same enzymatic activity and the same structure as the native ATP synthase complex extracted from A. aeolicus cells. We used our expression system to confirm that A. aeolicus ATP synthase possesses a heterodimeric peripheral stalk unique among non-photosynthetic bacterial F₁F_O ATP synthases.

Conclusions

Our system now allows performing previously impossible structural and functional studies on A. aeolicus F₁F_O ATP synthase.

General significance

More broadly, our work provides a valuable platform to characterize many other membrane protein complexes with complicated stoichiometry, i.e. other respiratory complexes, the nuclear pore complex, or transporter systems.     

The driver and passenger effects of isocitrate dehydrogenase 1 and 2 mutations in oncogenesis and survival prolongation

Mutations in isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2) are key events in the development of glioma, acute myeloid leukemia (AML), chondrosarcoma, intrahepatic cholangiocarcinoma (ICC), and angioimmunoblastic T-cell lymphoma. They also cause D-2-hydroxyglutaric aciduria and Ollier and Maffucci syndromes. IDH1/2 mutations are associated with prolonged survival in glioma and in ICC, but not in AML. The reason for this is unknown. In their wild-type forms, IDH1 and IDH2 convert isocitrate and NADP⁺ to α-ketoglutarate (αKG) and NADPH. Missense mutations in the active sites of these enzymes induce a neo-enzymatic reaction wherein NADPH reduces αKG to D-2-hydroxyglutarate (D-2HG). The resulting D-2HG accumulation leads to hypoxia-inducible factor 1α degradation, and changes in epigenetics and extracellular matrix homeostasis. Such mutations also imply less NADPH production capacity. Each of these effects could play a role in cancer formation. Here, we provide an overview of the literature and discuss which downstream molecular effects are likely to be the drivers of the oncogenic and survival-prolonging properties of IDH1/2 mutations. We discuss interactions between mutant IDH1/2 inhibitors and conventional therapies. Understanding of the biochemical consequences of IDH1/2 mutations in oncogenesis and survival prolongation will yield valuable information for rational therapy design: it will tell us which oncogenic processes should be blocked and which “survivalogenic” effects should be retained.     

SOX1 links the function of neural patterning and Notch signalling in the ventral spinal cord during the neuron-glial fate switch

During neural development the transition from neurogenesis to gliogenesis, known as the neuron-glial (Ν/G) fate switch, requires the coordinated function of patterning factors, pro-glial factors and Notch signalling. How this process is coordinated in the embryonic spinal cord is poorly understood. Here, we demonstrate that during the N/G fate switch in the ventral spinal cord (vSC) SOX1 links the function of neural patterning and Notch signalling. We show that, SOX1 expression in the vSC is regulated by PAX6, NKX2.2 and Notch signalling in a domain-specific manner. We further show that SOX1 regulates the expression of Hes1 and that loss of Sox1 leads to enhanced production of oligodendrocyte precursors from the pMN. Finally, we show that Notch signalling functions upstream of SOX1 during this fate switch and is independently required for the acquisition of the glial fate perse by regulating Nuclear Factor I A expression in a PAX6/SOX1/HES1/HES5-independent manner. These data integrate functional roles of neural patterning factors, Notch signalling and SOX1 during gliogenesis.     

Progress and perspective on cyanobacterial glycogen metabolism engineering

As important oxygenic photoautotrophs, cyanobacteria are also generally considered as one of the most promising microbial chassis for photosynthetic biomanufacturing. Diverse synthetic biology and metabolic engineering approaches have been developed to enable the efficient harnessing of carbon and energy flow toward the synthesis of desired metabolites in cyanobacterial cell factories. Glycogen metabolism works as the most important natural carbon sink mechanism and reserve carbon source, storing a large portion of carbon and energy from the Calvin-Benson-Bassham (CBB) cycle, and thus is traditionally recognized as a promising engineering target to optimize the efficacy of cyanobacterial cell factories. Multiple strategies and approaches have been designed and adopted to engineer glycogen metabolism in cyanobacteria, leading to the successful regulation of glycogen synthesis and storage contents in cyanobacteria cells. However, disturbed glycogen metabolism results in weakened cellular physiological functionalities, thereby diminishing the robustness of metabolism. In addition, the effects of glycogen removal as a metabolic engineering strategy to enhance photosynthetic biosynthesis are still controversial. This review focuses on the efforts and effects of glycogen metabolism engineering on the physiology and metabolism of cyanobacterial chassis strains and cell factories. The perspectives and prospects provided herein are expected to inspire novel strategies and tools to achieve ideal control over carbon and energy flow for biomanufacturing.     

Kinetic analysis of inhibition of α-glucosidase by leaf powder from Morus australis and its component iminosugars

ABSTRACT

Mulberry leaves contain iminosugars, such as 1-deoxynojirimycin (1-DNJ), fagomine, and 2-O-α-D-galactopyranosyl deoxynojirimycin (GAL-DNJ) that inhibit α-glucosidase. In this study, we quantified iminosugars in Morus australis leaves and made the kinetic analysis in the hydrolysis of maltose by α-glucosidase. By LC-MS/MS, the concentrations of 1-DNJ, fagomine, and GAL-DNJ in the powdered leaves were 4.0, 0.46, and 2.5 mg/g, respectively, and those in the roasted ones were 1.0, 0.24, and 0.73 mg/g, respectively, suggesting that the roasting process degraded iminosugars. Steady-state kinetic analysis revealed that the powdered and roasted leaves exhibited competitive inhibition. At pH 6.0 at 37ºC, the IC₅₀ values of the extracts from the boiled powdered or roasted leaves were 0.36 and 1.1 mg/mL, respectively. At the same condition, the IC₅₀ values of 1-DNJ, fagomine, and GAL-DNJ were 0.70 μg/mL, 0.18 mg/mL, and 2.9 mg/mL, respectively. These results suggested that in M. australis, 1-DNJ is a major inhibitor of α-glucosidase.