Autonomous oscillations found in gene expression and metabolic, cardiac and neuronal systems have attracted significant attention both because of their obvious biological roles and their intriguing dynamics. In addition, de novo designed oscillators have been demonstrated, using components that are not part of the natural oscillators. Such oscillators are useful in testing the design principles and in exploring potential applications not limited by natural cellular behaviour. To achieve transcriptional and metabolic integration characteristic of natural oscillators, here we designed and constructed a synthetic circuit in Escherichia coli K12, using glycolytic flux to generate oscillation through the signalling metabolite acetyl phosphate. If two metabolite pools are interconverted by two enzymes that are placed under the transcriptional control of acetyl phosphate, the system oscillates when the glycolytic rate exceeds a critical value. We used bifurcation analysis to identify the boundaries of oscillation, and verified these experimentally. This work demonstrates the possibility of using metabolic flux as a control factor in system-wide oscillation, as well as the predictability of a de novo gene-metabolic circuit designed using nonlinear dynamic analysis.
SUMMARY Ferroportin exports iron into plasma from absorptive enterocytes, erythrophagocytosing macrophages, and hepatic stores. The hormone hepcidin controls cellular iron export and plasma iron concentrations by binding to ferroportin and causing its internalization and degradation. We explored the mechanism of hepcidin-induced endocytosis of ferroportin, the key molecular event in systemic iron homeostasis. Hepcidin binding caused rapid ubiquitination of ferroportin in cell lines overexpressing ferroportin and in murine bone marrow-derived macrophages. No hepcidin-dependent ubiquitination was observed in C326S ferroportin mutant which does not bind hepcidin. Substitutions of lysines between residues 229 and 269 in the third cytoplasmic loop of ferroportin prevented hepcidin-dependent ubiquitination and endocytosis of ferroportin, and promoted cellular iron export even in the presence of hepcidin. The human ferroportin mutation K240E, previously associated with clinical iron overload, caused hepcidin resistance in vitro by interfering with ferroportin ubiquitination. Our study demonstrates that ubiquitination is the functionally-relevant signal for hepcidin-induced ferroportin endocytosis.
• An injection of heat-killedBrucella abortus in mice causes prolonged anemia with features similar to human anemia of inflammation.• Ablation of hepcidin ameliorates anemia of inflammation in this model and allows faster recovery.Anemia is a common complication of infections and inflammatory diseases, but the few mouse models of this condition are not well characterized. We analyzed in detail the pathogenesis of anemia induced by an injection of heat-killed Brucella abortus and examined the contribution of hepcidin by comparing wild-type (WT) to iron-depleted hepcidin-1 knockout (Hamp-KO) mice. B abortus-treated WT mice developed severe anemia with a hemoglobin nadir at 14 days and partial recovery by 28 days. After an early increase in inflammatory markers and hepcidin, WT mice manifested hypoferremia, despite iron accumulation in the liver. Erythropoiesis was suppressed between days 1 and 7, and erythrocyte destruction was increased as evidenced by schistocytes on blood smears and shortened red blood cell lifespan. Erythropoietic recovery began after 14 days but was iron restricted. In B abortus-treated Hamp-KO compared with WT mice, anemia was milder, not iron restricted, and had a faster recovery. Similarly to severe human anemia of inflammation, the B abortus model shows multifactorial pathogenesis of inflammatory anemia including iron restriction from increased hepcidin, transient suppression of erythropoiesis, and shortened erythrocyte lifespan. Ablation of hepcidin relieves iron restriction and improves the anemia. (Blood. 2014;123(8):1129-1136 Introduction Anemia of inflammation (AI) is a feature of a wide spectrum of inflammatory disorders, including connective tissue disease, infections, certain malignancies, and chronic kidney disease.1 AI is typically a normocytic normochromic anemia with a shortened erythrocyte lifespan and suppressed erythropoiesis, despite adequate levels of circulating erythropoietin.2 Perhaps the most consistent feature of AI is a derangement of systemic iron homeostasis characterized by hypoferremia with intact iron stores 1 and decreased availability of iron for erythrocyte production.Hepcidin, a 25-amino acid peptide hormone produced primarily by hepatocytes, 3 is the principal regulator of iron homeostasis in health and during inflammation. 4 Excessive production of this hormone causes iron sequestration in macrophages and hypoferremia, as was shown in transgenic mice with hepcidin overexpression 5 and in the human genetic syndrome of hepcidin excess, iron-refractory iron-deficiency anemia due to mutations in matriptase-2/TMPRSS6. 6 Hepcidin acts by binding to ferroportin, the sole known cellular iron exporter, displayed on the surface of macrophages, hepatocytes, and the basolateral membranes of enterocytes. Hepcidin binding to ferroportin causes ferroportin endocytosis and degradation.7 During inflammation or infection, hepcidin is strongly induced, largely by interleukin 6 (IL-6) 8 via the Janus kinase-signal transducer and activator of transcription pathway.9-11 The ex...
Highlights d Alveolar epithelial progenitor cells are transcriptionally distinct upon KRAS expression d Alveolar epithelial organoids recapitulate early-stage lung adenocarcinoma d Oncogenic KRAS leads to loss of lineage identity in AT2 cells d Bulk, scRNA-seq, and proteomics data from murine and human KRAS mutant AT2 cells
Artificial transcriptional networks have been used to achieve novel, nonnative behavior in bacteria. Typically, these artificial circuits are isolated from cellular metabolism and are designed to function without intercellular communication. To attain concerted biological behavior in a population, synchronization through intercellular communication is highly desirable. Here we demonstrate the design and construction of a gene-metabolic circuit that uses a common metabolite to achieve tunable artificial cell-cell communication. This circuit uses a threshold concentration of acetate to induce gene expression by acetate kinase and part of the nitrogen-regulation two-component system. As one application of the cell-cell communication circuit we created an artificial quorum sensor. Engineering of carbon metabolism in Escherichia coli made acetate secretion proportional to cell density and independent of oxygen availability. In these cells the circuit induced gene expression in response to a threshold cell density. This threshold can be tuned effectively by controlling ⌬pH over the cell membrane, which determines the partition of acetate between medium and cells. Mutagenesis of the enhancer sequence of the glnAp 2 promoter produced variants of the circuit with changed sensitivity demonstrating tunability of the circuit by engineering of its components. The behavior of the circuit shows remarkable predictability based on a mathematical design model.
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