Nitrogen sourcing during viral infection of marine cyanobacteria

The building blocks of a virus derived from de novo biosynthesis during infection and/or catabolism of preexisting host cell biomass, and the relative contribution of these 2 sources has important consequences for understanding viral biogeochemistry. We determined the uptake of extracellular nitrogen (N) and its biosynthetic incorporation into both virus and host proteins using an isotope-labeling proteomics approach in a model marine cyanobacterium Synechococcus WH8102 infected by a lytic cyanophage S-SM1. By supplying dissolved N as 15N postinfection, we found that proteins in progeny phage particles were composed of up to 41% extracellularly derived N, while proteins of the infected host cell showed almost no isotope incorporation, demonstrating that de novo amino acid synthesis continues during infection and contributes specifically and substantially to phage replication. The source of N for phage protein synthesis shifted over the course of infection from mostly host derived in the early stages to more medium derived later on. We show that the photosystem II reaction center proteins D1 and D2, which are auxiliary metabolic genes (AMGs) in the S-SM1 genome, are made de novo during infection in an apparently light-dependent manner. We also identified a small set of host proteins that continue to be produced during infection; the majority are homologs of AMGs in S-SM1 or other viruses, suggesting selective continuation of host protein production during infection. The continued acquisition of nutrients by the infected cell and their utilization for phage replication are significant for both evolution and biogeochemical impact of viruses.

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Availability of intestinal microbial lysine for whole body lysine homeostasis in human subjects

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.1999.277.4.e597 ◽

1999 ◽

Vol 277 (4) ◽

pp. E597-E607 ◽

Cited By ~ 15

Author(s):

Cornelia C. Metges ◽

Antoine E. El-Khoury ◽

Lidewij Henneman ◽

Klaus J. Petzke ◽

Ian Grant ◽

...

Keyword(s):

De Novo ◽

Human Subjects ◽

Isotope Ratio Mass Spectrometry ◽

Acid Synthesis ◽

Whole Body ◽

Microbial Protein ◽

Amino Acid Synthesis ◽

Adequate Diet ◽

Appearance Rate ◽

Intravenous And Oral Administration

We have investigated whether there is a net contribution of lysine synthesized de novo by the gastrointestinal microflora to lysine homeostasis in six adults. On two separate occasions an adequate diet was given for a total of 11 days, and a 24-h (12-h fast, 12-h fed) tracer protocol was performed on the last day, in which lysine turnover, oxidation, and splanchnic uptake were measured on the basis of intravenous and oral administration ofl-[1-13C]lysine andl-[6,6-2H2]lysine, respectively. [15N2]urea or15NH4Cl was ingested daily over the last 6 days to label microbial protein. In addition, seven ileostomates were studied with15NH4Cl. [15N]lysine enrichment in fecal and ileal microbial protein, as precursor for microbial lysine absorption, and in plasma free lysine was measured by gas chromatography-combustion-isotope ratio mass spectrometry. Differences in plasma [13C]- and [2H2]lysine enrichments during the 12-h fed period were observed between the two15N tracer studies, although the reason is unclear, and possibly unrelated to the tracer form per se. In the normal adults, after15NH4Cl and [15N2]urea intake, respectively, lysine derived from fecal microbial protein accounted for 5 and 9% of the appearance rate of plasma lysine. With ileal microbial lysine enrichment, the contribution of microbial lysine to plasma lysine appearance was 44%. This amounts to a gross microbial lysine contribution to whole body plasma lysine turnover of between 11 and 130 mg ⋅ kg−1 ⋅ day−1, depending on the [15N]lysine precursor used. However, insofar as microbial amino acid synthesis is accompanied by microbial breakdown of endogenous amino acids or their oxidation by intestinal tissues, this may not reflect a net increase in lysine absorption. Thus we cannot reliably estimate the quantitative contribution of microbial lysine to host lysine homeostasis with the present paradigm. However, the results confirm the significant presence of lysine of microbial origin in the plasma free lysine pool.

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Proteasome Inhibition in Brassica napus Roots Increases Amino Acid Synthesis to Offset Reduced Proteolysis

Plant and Cell Physiology ◽

10.1093/pcp/pcaa047 ◽

2020 ◽

Vol 61 (6) ◽

pp. 1028-1040

Author(s):

Dan Pereksta ◽

Dillon King ◽

Fahmida Saki ◽

Amith Maroli ◽

Elizabeth Leonard ◽

...

Keyword(s):

Amino Acid ◽

Brassica Napus ◽

De Novo ◽

Metabolic Response ◽

Sugar Metabolism ◽

Proteasome Inhibition ◽

Acid Synthesis ◽

Amino Acid Synthesis ◽

Amino Acid Depletion ◽

Metabolic Activities

Abstract Cellular homeostasis is maintained by the proteasomal degradation of regulatory and misfolded proteins, which sustains the amino acid pool. Although proteasomes alleviate stress by removing damaged proteins, mounting evidence indicates that severe stress caused by salt, metal(oids), and some pathogens can impair the proteasome. However, the consequences of proteasome inhibition in plants are not well understood and even less is known about how its malfunctioning alters metabolic activities. Lethality causes by proteasome inhibition in non-photosynthetic organisms stem from amino acid depletion, and we hypothesized that plants respond to proteasome inhibition by increasing amino acid biosynthesis. To address these questions, the short-term effects of proteasome inhibition were monitored for 3, 8 and 48 h in the roots of Brassica napus treated with the proteasome inhibitor MG132. Proteasome inhibition did not affect the pool of free amino acids after 48 h, which was attributed to elevated de novo amino acid synthesis; these observations coincided with increased levels of sulfite reductase and nitrate reductase activities at earlier time points. However, elevated amino acid synthesis failed to fully restore protein synthesis. In addition, transcriptome analysis points to perturbed abscisic acid signaling and decreased sugar metabolism after 8 h of proteasome inhibition. Proteasome inhibition increased the levels of alternative oxidase but decreased aconitase activity, most sugars and tricarboxylic acid metabolites in root tissue after 48 h. These metabolic responses occurred before we observed an accumulation of reactive oxygen species. We discuss how the metabolic response to proteasome inhibition and abiotic stress partially overlap in plants.

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Temporal Viral Genome-Protein Interactions Define Distinct Stages of Productive Herpesviral Infection

mBio ◽

10.1128/mbio.01182-18 ◽

2018 ◽

Vol 9 (4) ◽

Cited By ~ 22

Author(s):

Jill A. Dembowski ◽

Neal A. DeLuca

Keyword(s):

Life Cycle ◽

Protein Interactions ◽

Viral Genome ◽

Isotope Labeling ◽

De Novo ◽

Affinity Purification ◽

Human Pathogens ◽

Host Proteins ◽

Viral Dna ◽

Hsv 1

ABSTRACTHerpesviruses utilize multiple mechanisms to redirect host proteins for use in viral processes and to avoid recognition and repression by the host. To investigate dynamic interactions between herpes simplex virus type 1 (HSV-1) DNA and viral and host proteins throughout infection, we developed an approach to identify proteins that associate with the infecting viral genome from nuclear entry through packaging. To accomplish this, virus stocks were prepared in the presence of ethynyl-modified nucleotides to enable covalent tagging of viral genomes after infection for analysis of viral genome-protein interactions by imaging or affinity purification. Affinity purification was combined with stable isotope labeling of amino acids in cell culture (SILAC) mass spectrometry to enable the distinction between proteins that were brought into the cell by the virus or expressed within the infected cell before or during infection. We found that input viral DNA progressed within 6 h through four temporal stages where the genomes sequentially (i) interacted with intrinsic antiviral and DNA damage response proteins, (ii) underwent a robust transcriptional switch mediated largely by ICP4, (iii) engaged in replication, repair, and continued transcription, and then (iv) transitioned to a more transcriptionally inert state engagingde novo-synthesized viral structural components while maintaining interactions with replication proteins. Using a combination of genetic, imaging, and proteomic approaches, we provide a new and temporally compressed view of the HSV-1 life cycle based on input genome-proteome dynamics.IMPORTANCEHerpesviruses are highly prevalent and ubiquitous human pathogens. Studies of herpesviruses and other viruses have previously been limited by the ability to directly study events that occur on the viral DNA throughout infection. We present a new powerful approach, which allows for the temporal investigation of viral genome-protein interactions at all phases of infection. This work has integrated many results from previous studies with the discovery of novel factors potentially involved in viral infection that may represent new antiviral targets. In addition, the study provides a new view of the HSV-1 life cycle based on genome-proteome dynamics.

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Target of Rapamycin Inhibition in Chlamydomonas reinhardtii Triggers de Novo Amino Acid Synthesis by Enhancing Nitrogen Assimilation

The Plant Cell ◽

10.1105/tpc.18.00159 ◽

2018 ◽

Vol 30 (10) ◽

pp. 2240.1-2254 ◽

Cited By ~ 19

Author(s):

Umarah Mubeen ◽

Jessica Jüppner ◽

Jessica Alpers ◽

Dirk K. Hincha ◽

Patrick Giavalisco

Keyword(s):

Amino Acid ◽

Chlamydomonas Reinhardtii ◽

Nitrogen Assimilation ◽

De Novo ◽

Acid Synthesis ◽

Target Of Rapamycin ◽

Amino Acid Synthesis

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Mechanism of De Novo Branched-Chain Amino Acid Synthesis as an Alternative Electron Sink in Hypoxic Aspergillus nidulans Cells

Applied and Environmental Microbiology ◽

10.1128/aem.02135-09 ◽

2010 ◽

Vol 76 (5) ◽

pp. 1507-1515 ◽

Cited By ~ 36

Author(s):

Motoyuki Shimizu ◽

Tatsuya Fujii ◽

Shunsuke Masuo ◽

Naoki Takaya

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Aspergillus Nidulans ◽

De Novo ◽

Acid Synthesis ◽

Branched Chain Amino Acids ◽

Amino Acid Synthesis ◽

Lactate Dehydrogenase Activity ◽

Branched Chain Amino Acid ◽

Branched Chain

ABSTRACT Although branched-chain amino acids are synthesized as building blocks of proteins, we found that the fungus Aspergillus nidulans excretes them into the culture medium under hypoxia. The transcription of predicted genes for synthesizing branched-chain amino acids was upregulated by hypoxia. A knockout strain of the gene encoding the large subunit of acetohydroxy acid synthase (AHAS), which catalyzes the initial reaction of the synthesis, required branched-chain amino acids for growth and excreted very little of them. Pyruvate, a substrate for AHAS, increased the amount of hypoxic excretion in the wild-type strain. These results indicated that the fungus responds to hypoxia by synthesizing branched-chain amino acids via a de novo mechanism. We also found that the small subunit of AHAS regulated hypoxic branched-chain amino acid production as well as cellular AHAS activity. The AHAS knockout resulted in higher ratios of NADH/NAD+ and NADPH/NADP+ under hypoxia, indicating that the branched-chain amino acid synthesis contributed to NAD+ and NADP+ regeneration. The production of branched-chain amino acids and the hypoxic induction of involved genes were partly repressed in the presence of glucose, where cells produced ethanol and lactate and increased levels of lactate dehydrogenase activity. These indicated that hypoxic branched-chain amino acid synthesis is a unique alternative mechanism that functions in the absence of glucose-to-ethanol/lactate fermentation and oxygen respiration.

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LIF and CNTF, which share the gp130 transduction system, stimulate hepatic lipid metabolism in rats

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.1996.271.3.e521 ◽

1996 ◽

Vol 271 (3) ◽

pp. E521-E528 ◽

Cited By ~ 4

Author(s):

K. Nonogaki ◽

X. M. Pan ◽

A. H. Moser ◽

J. Shigenaga ◽

I. Staprans ◽

...

Keyword(s):

Fatty Acid ◽

Lipid Metabolism ◽

Fatty Acid Synthesis ◽

De Novo ◽

Acid Synthesis ◽

Interleukin 4 ◽

Dependent Manner ◽

Hepatic Triglyceride ◽

Serum Triglycerides ◽

Triglyceride Secretion

We determined the effects of leukemia inhibitory factor (LIF) and ciliary neurotrophic factor (CNTF) on lipid metabolism in intact rats. Administration of LIF and CNTF increased serum triglycerides in a dose-dependent manner with peak values at 2 h. The effects of LIF and CNTF on serum cholesterol were very small, and serum glucose was unaffected. Both LIF and CNTF stimulated hepatic triglyceride secretion, hepatic de novo fatty acid synthesis, and lipolysis. Pretreatment with phenylisopropyl adenosine, which inhibits lipolysis, partially inhibited LIF- and CNTF-induced hypertriglyceridemia. Interleukin-4, which inhibits cytokine-induced hepatic fatty acid synthesis, also partially inhibited LIF- and CNTF-induced hypertriglyceridemia. These results indicate that both lipolysis and de novo fatty acid synthesis play a role in providing fatty acids for the increase in hepatic triglyceride secretion. Neither indomethacin nor adrenergic receptor antagonists affected the hypertriglyceridemia. The combination of LIF plus CNTF showed no additive effects consistent with the action of both cytokines through the gp130 transduction system. Thus LIF and CNTF have similar effects on lipid metabolism; they join a growing list of cytokines that stimulate hepatic triglyceride secretion and may mediate the changes in lipid metabolism that accompany the acute phase response.

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Division and adaptation to host nutritional environment of apicomplexan parasites depend on apicoplast lipid metabolic plasticity and host organelles remodelling

10.1101/585737 ◽

2019 ◽

Cited By ~ 2

Author(s):

Souad Amiar ◽

Nicholas J. Katris ◽

Laurence Berry ◽

Sheena Dass ◽

Melanie J. Shears ◽

...

Keyword(s):

Host Cell ◽

De Novo ◽

Acid Synthesis ◽

Apicomplexan Parasites ◽

Metabolic Plasticity ◽

Lipid Source ◽

Nutritional Conditions ◽

Membrane Bound ◽

Nutritional Environment

AbstractApicomplexan parasites are unicellular eukaryotes responsible for major human diseases including malaria and toxoplasmosis. Apicomplexan parasites must obtain and combine lipids both from host cell scavenging andde novosynthesis to maintain parasite propagation and survival within their human host. Major questions on the actual role for each lipid source or how these are regulated upon fluctuating host nutritional conditions remain unanswered. Characterization of an apicoplast acyltransferase TgATS2, shows that the apicoplast provides local (lyso)phosphatidic acid balance, which is required for the recruitment of a novel dynamin (TgDrpC) critical during parasite cytokinesis. Disruption of TgATS2 led parasites to shift metabolic lipid acquisition fromde novosynthesis towards host scavenging. Importantly, both lipid scavenging andde novosynthesis pathways exhibit major metabolic and cellular plasticity upon sensing host lipid-deprived environments through concomitant (i) up-regulation ofde novofatty acid synthesis capacities in the apicoplast, and (ii) parasite-driven host cell remodelling to generate multi-membrane-bound structures from host organelles that are imported towards the parasite.

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Quantitative Mass Spectrometry Catalogues Salmonella Pathogenicity Island-2 Effectors and Identifies Their Cognate Host Binding Partners

Journal of Biological Chemistry ◽

10.1074/jbc.m111.224600 ◽

2011 ◽

Vol 286 (27) ◽

pp. 24023-24035 ◽

Cited By ~ 44

Author(s):

Sigrid D. Auweter ◽

Amit P. Bhavsar ◽

Carmen L. de Hoog ◽

Yuling Li ◽

Y. Alina Chan ◽

...

Keyword(s):

Host Cell ◽

Isotope Labeling ◽

Bacterial Pathogens ◽

Pathogenicity Island ◽

Secretion System ◽

Host Protein ◽

Host Cells ◽

Bacterial Disease ◽

Secretion Systems ◽

Interaction Partners

Gram-negative bacterial pathogens have developed specialized secretion systems to transfer bacterial proteins directly into host cells. These bacterial effectors are central to virulence and reprogram host cell processes to favor bacterial survival, colonization, and proliferation. Knowing the complete set of effectors encoded by a particular pathogen is the key to understanding bacterial disease. In addition, the identification of the molecular assemblies that these effectors engage once inside the host cell is critical to determining the mechanism of action of each effector. In this work we used stable isotope labeling of amino acids in cell culture (SILAC), a powerful quantitative proteomics technique, to identify the proteins secreted by the Salmonella pathogenicity island-2 type three secretion system (SPI-2 T3SS) and to characterize the host interaction partners of SPI-2 effectors. We confirmed many of the known SPI-2 effectors and were able to identify several novel substrate candidates of this secretion system. We verified previously published host protein-effector binding pairs and obtained 11 novel interactions, three of which were investigated further and confirmed by reciprocal co-immunoprecipitation. The host cell interaction partners identified here suggest that Salmonella SPI-2 effectors target, in a concerted fashion, cellular processes such as cell attachment and cell cycle control that are underappreciated in the context of infection. The technology outlined in this study is specific and sensitive and serves as a robust tool for the identification of effectors and their host targets that is readily amenable to the study of other bacterial pathogens.

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Central Resistin Regulates Hypothalamic and Peripheral Lipid Metabolism in a Nutritional-Dependent Fashion

Endocrinology ◽

10.1210/en.2007-1708 ◽

2008 ◽

Vol 149 (9) ◽

pp. 4534-4543 ◽

Cited By ~ 72

Author(s):

María J. Vázquez ◽

C. Ruth González ◽

Luis Varela ◽

Ricardo Lage ◽

Sulay Tovar ◽

...

Keyword(s):

Fatty Acid ◽

Lipid Metabolism ◽

Mrna Expression ◽

Fatty Acid Synthase ◽

De Novo ◽

Acid Synthesis ◽

Inhibitory Effect ◽

De Novo Lipogenesis ◽

Dependent Manner ◽

Fed State

Evidence suggests that the adipocyte-derived hormone resistin (RSTN) directly regulates both feeding and peripheral metabolism through, so far, undefined hypothalamic-mediated mechanisms. Here, we demonstrate that the anorectic effect of RSTN is associated with inappropriately decreased mRNA expression of orexigenic (agouti-related protein and neuropeptide Y) and increased mRNA expression of anorexigenic (cocaine and amphetamine-regulated transcript) neuropeptides in the arcuate nucleus of the hypothalamus. Of interest, RSTN also exerts a profound nutrition-dependent inhibitory effect on hypothalamic fatty acid metabolism, as indicated by increased phosphorylation levels of both AMP-activated protein kinase and its downstream target acetyl-coenzyme A carboxylase, associated with decreased expression of fatty acid synthase in the ventromedial nucleus of the hypothalamus. In addition, we also demonstrate that chronic central RSTN infusion results in decreased body weight and major changes in peripheral expression of lipogenic enzymes, in a tissue-specific and nutrition-dependent manner. Thus, in the fed state central RSTN is associated with induced expression of fatty acid synthesis enzymes and proinflammatory cytokines in liver, whereas its administration in the fasted state does so in white adipose tissue. Overall, our results indicate that RSTN controls feeding and peripheral lipid metabolism and suggest that hepatic RSTN-induced insulin resistance may be mediated by central activation of de novo lipogenesis in liver.

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