Cellular Requirements Research Articles

Natural deep eutectic solvents: cytotoxic profile.

The purpose of this study was to investigate the cytotoxic profiles of different ternary natural deep eutectic solvents (NADESs) containing water. For this purpose, five different NADESs were prepared using choline chloride as a salt, alongside five hydrogen bond donors (HBD) namely glucose, fructose, sucrose, glycerol, and malonic acid. Water was added as a tertiary component during the eutectics preparation, except for the malonic acid-based mixture. Coincidentally, the latter was found to be more toxic than any of the water-based NADESs. A trend was observed between the cellular requirements of cancer cells, the viscosity of the NADESs, and their cytotoxicity. This study also highlights the first time application of the conductor-like screening model for real solvent (COSMO-RS) software for the analysis of the cytotoxic mechanism of NADESs. COSMO-RS simulation of the interactions between NADESs and cellular membranes’ phospholipids suggested that NADESs strongly interacted with cell surfaces and that their accumulation and aggregation possibly defined their cytotoxicity. This reinforced the idea that careful selection of NADESs components is necessary, as it becomes evident that organic acids as HBD highly contribute to the increasing toxicity of these neoteric mixtures. Nevertheless, NADESs in general seem to possess relatively less acute toxicity profiles than their DESs parents. This opens the door for future large scale utilization of these mixtures.

Role for the αV Integrin Subunit in Varicella-Zoster Virus-Mediated Fusion and Infection

Varicella-zoster virus (VZV) is an alphaherpesvirus that causes varicella and herpes zoster. Membrane fusion is essential for VZV entry and the distinctive syncytium formation in VZV-infected skin and neuronal tissue. Herpesvirus fusion is mediated by a complex of glycoproteins gB and gH-gL, which are necessary and sufficient for VZV to induce membrane fusion. However, the cellular requirements of fusion are poorly understood. Integrins have been implicated to facilitate entry of several human herpesviruses, but their role in VZV entry has not yet been explored. To determine the involvement of integrins in VZV fusion, a quantitative cell-cell fusion assay was developed using a VZV-permissive melanoma cell line. The cells constitutively expressed a reporter protein and short hairpin RNAs (shRNAs) to knock down the expression of integrin subunits shown to be expressed in these cells by RNA sequencing. The αV integrin subunit was identified as mediating VZV gB/gH-gL fusion, as its knockdown by shRNAs reduced fusion levels to 60% of that of control cells. A comparable reduction in fusion levels was observed when an anti-αV antibody specific to its extracellular domain was tested in the fusion assay, confirming that the domain was important for VZV fusion. In addition, reduced spread was observed in αV knockdown cells infected with the VZV pOka strain relative to that of the control cells. This was demonstrated by reductions in plaque size, replication kinetics, and virion entry in the αV subunit knockdown cells. Thus, the αV integrin subunit is important for VZV gB/gH-gL fusion and infection. Varicella-zoster virus (VZV) is a highly infectious pathogen that causes chickenpox and shingles. A common complication of shingles is the excruciating condition called postherpetic neuralgia, which has proven difficult to treat. While a vaccine is now available, it is not recommended for immunocompromised individuals and its efficacy decreases with the recipient's age. These limitations highlight the need for new therapies. This study examines the role of integrins in membrane fusion mediated by VZV glycoproteins gB and gH-gL, a required process for VZV infection. This knowledge will further the understanding of VZV entry and provide insight into the development of better therapies.

Long‐Term Intra‐Aortic Balloon Pump Support as Bridge to Left Ventricular Assist Device Implantation

The intra-aortic balloon pump (IABP) can be used to bridge critically ill end-stage heart failure patients to left ventricular assist device (LVAD) implantation. However, the IABP's potential association with hemorrhagic complications raises concerns regarding its utilization in these patients. We investigated whether preoperative long-term IABP support increases hemorrhagic complications post-LVAD implantation. Ten patients undergoing IABP support prior to LVAD implantation (IABP-LVAD group) were compared with 16 who did not require IABP support (LVAD group). Mean duration of IABP support was 25.8 days. Preoperatively, both groups were comparable in all measured parameters and indices of end-organ function. Perioperative (defined as three weeks post-LVAD implantation, including the procedure) cellular and noncellular blood transfusion requirements were similar between IABP-LVAD and LVAD groups (19.8 ± 9.95 vs. 19.76 ± 29.69 RBC units, p = 0.96; 3.8 ± 4.14 vs. 2.0 ± 6.44 plateletpheresis units p = 0.84; 23 ± 16.04 vs. 25.14 ± 37.8 fresh frozen plasma units, p = 0.45). Perioperative minimum hematocrit (33.6 ± 5.6 vs. 36.59 ± 4.8, p = 0.38) and minimum platelet count (199 ± 153 vs. 144 ± 65, p = 0.52) were similar in the two groups. Two patients in the IABP-LVAD group and three patients in the LVAD group underwent reoperation post-LVAD implantation for bleeding. Length of ICU stay was longer in the LVAD group but did not reach statistical significance (6.2 ± 6.22 days in the IABP-LVAD group versus 13.45 ± 10.95 days in the LVAD group, p = 0.06). Long-term IABP support as a bridge to LVAD implantation is not associated with increased hemorrhagic complications post-LVAD implantation. doi: 10.1111/jocs.12759 (J Card Surg 2016;31:467-471).

Multi-step Th17 differentiation in response to segmented filamentous bacteria in the mouse intestine

Abstract Th17 cells have significant roles in maintaining homeostasis and regulating host defense against various pathogens in our bodies. Our laboratory initially identified segmented filamentous bacteria (SFB) as a unique commensal that is sufficient for Th17 cell differentiation and promotion of Th17-dependent autoimmune diseases such as a mouse model of spontaneous arthritis. The molecular and cellular requirements of SFB-induced Th17 cell differentiation are still unclear. To understand the whole process of Th17 cells differentiation in vivo, we developed SFB-specific T cell receptor transgenic (7B8) mice. We can trace SFB-specific Th17 cell differentiation and response by transferring fluorescently-labeled 7B8 naïve T cells into SFB-gavaged host mice. Using this approach, we have elucidated the requirements for cytokines and antigen presenting cells (APCs) to understand the process of Th17 cell differentiation. Here, we describe that initial induction and expansion of Th17 cells occurs in mesenteric lymph nodes (MLN), and their subsequent migration to intestine is integrin β7-dependent. Although RORγt expression in Th17 cells is mainly dependent on IL-6 signaling in the MLN, IL-23R signaling also contributes to RORγt expression in Th17 cells in the ileum in the absence of IL-6. CD103+ CD11b+ APCs are not important for induction and initial expansion of SFB-specific Th17 cells in MLN, but play important roles in maintenance of SFB-specific Th17 cells in the ileum. Taken together, these results indicate that Th17 cell differentiation proceeds in multiple discrete stages.

Costimulatory pathways mediating T-dependent germinal center responses: distinct cellular requirements for CD40 and B7 costimulation

Abstract T cell-dependent (TD) germinal center (GC) and antibody responses require coordinated interactions of T cells with B cells and antigen-presenting dendritic cells (DCs). Although B7 and CD40 costimulatory pathways are critical for these responses, a comprehensive analysis of cell type-specific pathways has been hindered by the absence of models for conditional expression of B7 or CD40. Here we report generation of conditional knockout mice for both B7 and CD40, and the use of these lines together with conditional MHC class II (MHCII) knockouts and bone marrow chimeric strategies, to analyze pathways involved in TD GC and antibody responses. Our results indicate that MHCII-restricted recognition of both DCs and B cells is necessary for generation of antigen-specific TFH cells, GC B cells, and antibody response. Notably, the requirements for expression of B7 and CD40 were distinct, with B7 expression required on DCs but not on B cells, while CD40 was required on B cells but not DCs for generation of TFH cells, antigen-specific GC B cells, and antigen-specific class-switched antibody responses. These findings identify requirements for cell and costimulatory interactions in TD GC and antibody responses and support a unique model in which major costimulatory pathways function through interaction of T cells with distinct cell populations.

Onset of Inflammation With Ischemia: Implications for Donor Lung Preservation and Transplant Survival.

Lungs stored ahead of transplant surgery experience ischemia. Pulmonary ischemia differs from ischemia in the systemic organs in that stop of blood flow in the lung leads to loss of shear alone because the lung parenchyma does not rely on blood flow for its cellular oxygen requirements. Our earlier studies on the ischemia-induced mechanosignaling cascade showed that the pulmonary endothelium responds to stop of flow by production of reactive oxygen species (ROS). We hypothesized that ROS produced in this way led to induction of proinflammatory mediators. In this study, we used lungs or cells subjected to various periods of storage and evaluated the induction of several proinflammatory mediators. Isolated murine, porcine and human lungs in situ showed increased expression of cellular adhesion molecules; the damage-associated molecular pattern protein high-mobility group box 1 and the corresponding pattern recognition receptor, called the receptor for advanced glycation end products; and induction stabilization and translocation of hypoxia-inducible factor 1α and its downstream effector VEGFA, all of which are participants in inflammation. We concluded that signaling with lung preservation drives expression of inflammatory mediators that potentially predispose the donor lung to an inflammatory response after transplant.

B cells establish, but do not maintain, long-lived murine anti-peanut IgE(a).

Peanut allergy (PNA) has been reported to be transferred to tolerant recipients through organ and bone marrow (BM) transplantation. The roles T and B cells play in establishing, and the roles B cell subsets play in maintaining lifelong anti-peanut IgE levels are unknown. To determine the cellular requirements for the transfer of murine PNA and to determine the role CD20(+) cells play in maintaining long-lived anti-peanut IgE levels. We developed a novel adoptive transfer model to investigate the cellular requirements for transferring murine PNA. We also treated peanut-allergic (PA) mice with anti-CD20 antibody and measured IgE levels throughout treatment. Purified B220(+) cells from PA splenocytes and purified CD4(+) cells from naïve (NA) splenocytes are the minimal requirements for the adoptive transfer of PNA. Prolonged treatment of allergic mice with anti-CD20 antibody results in significant depletion of B cell subsets but does not affect anti-peanut IgE levels, symptoms, or numbers of IgE antibody secreting cells (ASCs) in the BM. Adoptive transfer of BM and spleen cells from allergic donors treated with anti-CD20 antibody does not result in the transfer of PNA in NA recipients, demonstrating that anti-CD20 antibody treatment depletes B cells capable of differentiating into peanut-specific IgE ASCs. Peanut allergy can be established in a NA hosts with B220(+) cells from PA donors and CD4(+) cells from peanut-NA donors. However, long-term depletion of B220(+) cells with anti-CD20 antibody does not affect anti-peanut IgE levels. These results highlight a novel role for B cells in the development of PNA and provide evidence that long-lived anti-peanut IgE levels may be maintained by long-lived ASCs.

Steady-State Growth under Inorganic Carbon Limitation Conditions Increases Energy Consumption for Maintenance and Enhances Nitrous Oxide Production in Nitrosomonas europaea.

Nitrosomonas europaea is a chemolithoautotrophic bacterium that oxidizes ammonia (NH3) to obtain energy for growth on carbon dioxide (CO2) and can also produce nitrous oxide (N2O), a greenhouse gas. We interrogated the growth, physiological, and transcriptome responses of N. europaea to conditions of replete (>5.2 mM) and limited inorganic carbon (IC) provided by either 1.0 mM or 0.2 mM sodium carbonate (Na2CO3) supplemented with atmospheric CO2 IC-limited cultures oxidized 25 to 58% of available NH3 to nitrite, depending on the dilution rate and Na2CO3 concentration. IC limitation resulted in a 2.3-fold increase in cellular maintenance energy requirements compared to those for NH3-limited cultures. Rates of N2O production increased 2.5- and 6.3-fold under the two IC-limited conditions, increasing the percentage of oxidized NH3-N that was transformed to N2O-N from 0.5% (replete) up to 4.4% (0.2 mM Na2CO3). Transcriptome analysis showed differential expression (P ≤ 0.05) of 488 genes (20% of inventory) between replete and IC-limited conditions, but few differences were detected between the two IC-limiting treatments. IC-limited conditions resulted in a decreased expression of ammonium/ammonia transporter and ammonia monooxygenase subunits and increased the expression of genes involved in C1 metabolism, including the genes for RuBisCO (cbb gene cluster), carbonic anhydrase, folate-linked metabolism of C1 moieties, and putative C salvage due to oxygenase activity of RuBisCO. Increased expression of nitrite reductase (gene cluster NE0924 to NE0927) correlated with increased production of N2O. Together, these data suggest that N. europaea adapts physiologically during IC-limited steady-state growth, which leads to the uncoupling of NH3 oxidation from growth and increased N2O production. Nitrification, the aerobic oxidation of ammonia to nitrate via nitrite, is an important process in the global nitrogen cycle. This process is generally dependent on ammonia-oxidizing microorganisms and nitrite-oxidizing bacteria. Most nitrifiers are chemolithoautotrophs that fix inorganic carbon (CO2) for growth. Here, we investigate how inorganic carbon limitation modifies the physiology and transcriptome of Nitrosomonas europaea, a model ammonia-oxidizing bacterium, and report on increased production of N2O, a potent greenhouse gas. This study, along with previous work, suggests that inorganic carbon limitation may be an important factor in controlling N2O emissions from nitrification in soils and wastewater treatment.

Silica, Silicosis, and Autoimmunity.

Inhalation of dust containing crystalline silica is associated with a number of acute and chronic diseases including systemic autoimmune diseases. Evidence for the link with autoimmune disease comes from epidemiological studies linking occupational exposure to crystalline silica dust with the systemic autoimmune diseases systemic lupus erythematosus, systemic sclerosis, and rheumatoid arthritis. Although little is known regarding the mechanism by which silica exposure leads to systemic autoimmune disease, there is a voluminous literature on silica exposure and silicosis that may help identify immune processes that precede development of autoimmunity. The pathophysiology of silicosis consists of deposition of silica particles in the alveoli of the lung. Ingestion of these particles by macrophages initiates an inflammatory response, which stimulates fibroblasts to proliferate and produce collagen. Silica particles are encased by collagen leading to fibrosis and the nodular lesions characteristic of the disease. The steps in the development of silicosis, including acute and chronic inflammation and fibrosis, have different molecular and cellular requirements, suggesting that silica-induced inflammation and fibrosis may be mechanistically separate. Significantly, it is unclear whether silica-induced inflammation and fibrosis contribute similarly to the development of autoimmunity. Nonetheless, the findings from human and animal model studies are consistent with an autoimmune pathogenesis that begins with activation of the innate immune system leading to proinflammatory cytokine production, pulmonary inflammation leading to activation of adaptive immunity, breaking of tolerance, and autoantibodies and tissue damage. The variable frequency of these immunological features following silica exposure suggests substantial genetic involvement and gene/environment interaction in silica-induced autoimmunity. However, numerous questions remain unanswered.

Touch and go: nuclear proteolysis in the regulation of metabolic genes and cancer.

The recruitment of transcription factors to promoters and enhancers is a critical step in gene regulation. Many of these proteins are quickly removed from DNA after they completed their function. Metabolic genes in particular are dynamically regulated and continuously adjusted to cellular requirements. Transcription factors controlling metabolism are therefore under constant surveillance by the ubiquitin–proteasome system, which can degrade DNA‐bound proteins in a site‐specific manner. Several of these metabolic transcription factors are critical to cancer cells, as they promote uncontrolled growth and proliferation. This review highlights recent findings in the emerging field of nuclear proteolysis and outlines novel paradigms for cancer treatment, with an emphasis on multiple myeloma.

Direct Visualization of Ebola Virus Fusion Triggering in the Endocytic Pathway

ABSTRACTEbola virus (EBOV) makes extensive and intricate use of host factors in the cellular endosomal/lysosomal pathway to release its genome into the cytoplasm and initiate infection. Following viral internalization into endosomes, host cysteine proteases cleave the EBOV fusion glycoprotein (GP) to unmask the binding site for its intracellular receptor, the cholesterol transporter Niemann-Pick C1 (NPC1). GP-NPC1 interaction is required for viral entry. Despite these and other recent discoveries, late events in EBOV entry following GP-NPC1 binding and culminating in GP-catalyzed fusion between viral and cellular lipid bilayers remain enigmatic. A mechanistic understanding of EBOV membrane fusion has been hampered by the failure of previous efforts to reconstitute fusion in vitro or at the cell surface. This report describes an assay to monitor initial steps directly in EBOV membrane fusion—triggering of GP and virus-cell lipid mixing—by single virions in live cells. Fusogenic triggering of GP occurs predominantly in Rab7-positive (Rab7+) endosomes, absolutely requires interaction between proteolytically primed GP and NPC1, and is blocked by key GP-specific neutralizing antibodies with therapeutic potential. Unexpectedly, cysteine protease inhibitors do not inhibit lipid mixing by virions bearing precleaved GP, even though they completely block cytoplasmic entry by these viruses, as shown previously. These results point to distinct cellular requirements for different steps in EBOV membrane fusion and suggest a model in which host cysteine proteases are dispensable for GP fusion triggering after NPC1 binding but are required for the formation of fusion pores that permit genome delivery.

Regulation of Mitochondrial Signaling and Quality Control by the 18KDA Translocator Protein (TSPO)

The 18-kDa translocator protein (TSPO) is localized to the outer mitochondrial membrane (OMM) where it is found in complex with the voltage dependent anion channel (VDAC). TSPO expression is elevated in cancer and inflammatory conditions and it is well known for its ligand binding properties and application in PET imaging as a marker of neuroinflammation. TSPO is implicated in multiple mitochondrial functions including steroidogenesis, apoptosis and ATP production although precise molecular mechanisms remain to be elucidated. We recently demonstrated that increased TSPO expression limits mitochondrial ubiquitination and mitophagy through a reactive oxygen species (ROS) -dependent process, which leads to changes in mitochondrial morphology and biomass.Here we have corroborated this pathway by establishing an interplay with the protein kinase A (PKA) signaling complex, which is recruited to mitochondria by TSPO via acyl-CoA binding domain containing 3 (ACBD3). Using targeted luminescent protein chimeras, we show that the combined accumulation of TSPO and PKA alters subcellular Ca2+ dynamics and metabolism, which we propose arises from increased VDAC phosphorylation and subsequent changes in channel activity. Of note, this pathway is activated under glutamate-induced cell stress in the neuronal cell line, SH-SY5Y.This work highlights an important role for the OMM in fine-tuning mitochondrial signaling and mitophagy. As an inducible factor, TSPO represents a critical physiological element that facilitates mitochondrial adaptation to altered cellular metabolic requirements under stress conditions.

Systemic lupus erythematosus in 2015: Cellular and metabolic requirements of effector T cells.

Key advances in lupus research in 2015 highlight the contribution of T cells to the pathogenesis of the disease. The findings not only shed light on the regulation and activity of these cells, but also suggest several novel therapeutic targets.

Regulation of Carbohydrate Metabolism, Lipid Metabolism, and Protein Metabolism by AMPK.

This chapter summarizes AMPK function in the regulation of substrate and energy metabolism with the main emphasis on carbohydrate and lipid metabolism, protein turnover, mitochondrial biogenesis, and whole-body energy homeostasis. AMPK acts as whole-body energy sensor and integrates different signaling pathway to meet both cellular and body energy requirements while inhibiting energy-consuming processes but also activating energy-producing ones. AMPK mainly promotes glucose and fatty acid catabolism, whereas it prevents protein, glycogen, and fatty acid synthesis.

Control of YAP/TAZ Activity by Metabolic and Nutrient-Sensing Pathways.

Metabolism is a fundamental cellular function that can be reprogrammed by signaling pathways and oncogenes to meet cellular requirements. An emerging paradigm is that signaling and transcriptional networks can be in turn regulated by metabolism, allowing cells to coordinate their metabolism and behavior in an integrated manner. The activity of the YAP/TAZ transcriptional coactivators, downstream transducers of the Hippo cascade and powerful pro-oncogenic factors, was recently found to be regulated by metabolic pathways, such as aerobic glycolysis and mevalonate synthesis, and by the nutrient-sensing LKB1-AMPK and TSC-mTOR pathways. We discuss here current data linking YAP/TAZ to metabolism and suggest how this coupling might coordinate nutrient availability with genetic programs that sustain tissue growth, neoplastic cell proliferation, and tumor malignancy.

Limiting Cholesterol Biosynthetic Flux Spontaneously Engages Type I IFN Signaling

Cellular lipid requirements are achieved through a combination of biosynthesis and import programs. Using isotope tracer analysis, we show that type I interferon (IFN) signaling shifts the balance of these programs by decreasing synthesis and increasing import of cholesterol and long chain fatty acids. Genetically enforcing this metabolic shift in macrophages is sufficient to render mice resistant to viral challenge, demonstrating the importance of reprogramming the balance of these two metabolic pathways invivo. Unexpectedly, mechanistic studies reveal that limiting flux through the cholesterol biosynthetic pathway spontaneously engages a type I IFN response in a STING-dependent manner. The upregulation of type I IFNs was traced to a decrease in the pool size of synthesized cholesterol and could be inhibited by replenishing cells with free cholesterol. Taken together, these studies delineate a metabolic-inflammatory circuit that links perturbations in cholesterol biosynthesis with activation of innate immunity.

Comparative proteomic analysis by iTRAQ-2DLC-MS/MS provides insight into the key proteins involved in Cronobacter sp. biofilm formation

Cronobacter sp., an opportunistic foodborne pathogen, can cause high mortality in neonatal and premature infants because this pathogen is associated with powdered infant formula (PIF) contamination. Biofilm formation is recognized as the most effective means to help Cronobacter survive throughout the long shelf-life of PIF, thus promoting resistance to cleaning agents and disinfectants. Crystal violet (CV) staining and fluorescence microscopy analyses were used to compare the biofilm formation ability among different Cronobacter strains, and then the weak biofilm former Cronobacter sakazakii ATCC 29544 and the strong biofilm former Cronobacter dubliniensis DSM 18707 were selected for further proteomic analysis. Two-dimensional liquid chromatography–tandem mass spectrometry, which was coupled with isobaric tags for relative and absolute quantification (iTRAQ) labeling, was employed to quantitatively identify the proteins that were differentially expressed in C. sakazakii ATCC 29544 compared to C. dubliniensis DSM 18707. In total, 1190 proteins were detected. Gene Ontology (GO) analysis indicated that these differentially expressed proteins are related to biological binding, cell structure, signal transduction, cell adhesion, and cellular interaction. Among these differential proteins, the expression levels of 177 non-redundant proteins were altered significantly by more than 5-fold, with 96 up-regulated proteins and 81 down-regulated. This study provides important information regarding the cellular requirements for the underlying mechanism of Cronobacter biofilm formation and its survival in harsh environments.

Deranged iron status in psoriasis: the impact of low body mass.

BackgroundIron deficiency (ID) frequently complicates inflammatory-mediated chronic disorders, irrespective of anaemia. Psoriasis is a chronic, immune-mediated skin disease with systemic pro-inflammatory activation; thus, these patients may be prone to develop ID. ID adversely affects immune cells function, which can further contribute to disease progression. This study investigates iron status in psoriasis.MethodsSerum concentrations of ferritin, transferrin saturation (Tsat), soluble transferrin receptor (sTfR), and hepcidin were assessed as the biomarkers of iron status in 39 patients with psoriasis (17 men, age: 47 ± 10 years) and 44 healthy subjects (30 men, age: 53 ± 6 years).ResultsCompared with healthy controls, patients with psoriasis demonstrated similar haematologic status but deranged iron status as evidenced by decreased Tsat and elevated sTfR (negative tissue iron balance) and low levels of hepcidin (depleted iron stores) (all P < 0.05 vs. controls). In patients, the levels of interleukin-6 (level of pro-inflammatory activation) significantly correlated with hepcidin (R = 0.54), but not with ferritin, Tsat, and sTfR. Biomarkers reflecting ID were not associated with the severity of the disease (assessed with the Psoriasis Area and Severity Index) but significantly correlated low body mass index (BMI). Patients with BMI < 24 kg/m2 compared with those with BMI ≥ 24 kg/m2 demonstrated lower levels of ferritin (40 ± 30 vs. 186 ± 128 ng/mL, P < 0.001) and hepcidin (4.9 ± 2.3 vs. 10.7 ± 6.7 ng/mL, P = 0.03).ConclusionPsoriasis is associated with deranged iron status characterized by depleted iron stores with concomitant unmet cellular iron requirements. The magnitude of these abnormalities is particularly strong in patients with low body mass index. Whether iron deficiency may become a therapeutic target in psoriasis needs to be investigated.

Growth-rate dependency of de novo resveratrol production in chemostat cultures of an engineered Saccharomyces cerevisiae strain.

IntroductionSaccharomyces cerevisiae has become a popular host for production of non-native compounds. The metabolic pathways involved generally require a net input of energy. To maximize the ATP yield on sugar in S. cerevisiae, industrial cultivation is typically performed in aerobic, sugar-limited fed-batch reactors which, due to constraints in oxygen transfer and cooling capacities, have to be operated at low specific growth rates. Because intracellular levels of key metabolites are growth-rate dependent, slow growth can significantly affect biomass-specific productivity. Using an engineered Saccharomyces cerevisiae strain expressing a heterologous pathway for resveratrol production as a model energy-requiring product, the impact of specific growth rate on yeast physiology and productivity was investigated in aerobic, glucose-limited chemostat cultures.ResultsStoichiometric analysis revealed that de novo resveratrol production from glucose requires 13 moles of ATP per mole of produced resveratrol. The biomass-specific production rate of resveratrol showed a strong positive correlation with the specific growth rate. At low growth rates a substantial fraction of the carbon source was invested in cellular maintenance-energy requirements (e.g. 27 % at 0.03 h−1). This distribution of resources was unaffected by resveratrol production. Formation of the by-products coumaric, phloretic and cinnamic acid had no detectable effect on maintenance energy requirement and yeast physiology in chemostat. Expression of the heterologous pathway led to marked differences in transcript levels in the resveratrol-producing strain, including increased expression levels of genes involved in pathways for precursor supply (e.g. ARO7 and ARO9 involved in phenylalanine biosynthesis). The observed strong differential expression of many glucose-responsive genes in the resveratrol producer as compared to a congenic reference strain could be explained from higher residual glucose concentrations and higher relative growth rates in cultures of the resveratrol producer.ConclusionsDe novo resveratrol production by engineered S. cerevisiae is an energy demanding process. Resveratrol production by an engineered strain exhibited a strong correlation with specific growth rate. Since industrial production in fed-batch reactors typically involves low specific growth rates, this study emphasizes the need for uncoupling growth and product formation via energy-requiring pathways.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-015-0321-6) contains supplementary material, which is available to authorized users.

PKA Phosphorylates the ATPase Inhibitory Factor 1 and Inactivates Its Capacity to Bind and Inhibit the Mitochondrial H(+)-ATP Synthase.

The mitochondrial H(+)-ATP synthase synthesizes most of cellular ATP requirements by oxidative phosphorylation (OXPHOS). The ATPase Inhibitory Factor 1 (IF1) is known to inhibit the hydrolase activity of the H(+)-ATP synthase insituations that compromise OXPHOS. Herein, we demonstrate that phosphorylation of S39 in IF1 by mitochondrial protein kinase A abolishes its capacity to bind the H(+)-ATP synthase. Only dephosphorylated IF1 binds and inhibits both the hydrolase and synthase activities of the enzyme. The phosphorylation status of IF1 regulates the flux of aerobic glycolysis and ATP production through OXPHOS in hypoxia and during the cell cycle. Dephosphorylated IF1 is present in human carcinomas. Remarkably, mouse heart contains a large fraction of dephosphorylated IF1 that becomes phosphorylated and inactivated upon invivo β-adrenergic stimulation. Overall, we demonstrate the essential function of the phosphorylation of IF1 in regulating energy metabolism and speculate that dephosho-IF1 might play a role in signaling mitohormesis.