Purpose: Many transformed cells and embryonic stem cells are dependent on the biosynthesis of the universal methyl-donor S-adenosylmethionine (SAM) from methionine by the enzyme MAT2A to maintain their epigenome. We hypothesized that cancer stem cells (CSCs) rely on SAM biosynthesis and that the combination of methionine depletion and MAT2A inhibition would eradicate CSCs.Methods: Human triple (ER/PR/HER2)-negative breast carcinoma (TNBC) cell lines were cultured as CSC-enriched mammospheres in control or methionine-free media. MAT2A was inhibited with siRNAs or cycloleucine. The effects of methionine restriction and/or MAT2A inhibition on the formation of mammospheres, the expression of CSC markers (CD44 hi /C24 low ), MAT2A and CSC transcriptional regulators, apoptosis induction, and histone modifications were determined. A murine model of metastatic TNBC was utilized to evaluate the effects of dietary methionine restriction, MAT2A inhibition and the combination.Results: Methionine restriction inhibited mammosphere formation and reduced the CD44 hi / C24 low CSC population; these effects were partly rescued by SAM. Methionine depletion induced MAT2A expression (mRNA and protein) and sensitized CSCs to inhibition of MAT2A (siRNAs or cycloleucine). Cycloleucine enhanced the effects of methionine depletion on H3K4me3 demethylation and suppression of Sox9 expression. Dietary methionine restriction induced
Zinc is an essential cofactor for many proteins. A key mechanism of zinc homeostasis during deficiency is “zinc sparing” in which specific zinc-binding proteins are repressed to reduce the cellular requirement. In this report, we evaluated zinc sparing across the zinc proteome of Saccharomyces cerevisiae. The yeast zinc proteome of 582 known or potential zinc-binding proteins was identified using a bioinformatics analysis that combined global domain searches with local motif searches. Protein abundance was determined by mass spectrometry. In zinc-replete cells, we detected over 2500 proteins among which 229 were zinc proteins. Based on copy number estimates and binding stoichiometries, a replete cell contains ~9 million zinc-binding sites on proteins. During zinc deficiency, many zinc proteins decreased in abundance and the zinc-binding requirement decreased to ~5 million zinc atoms per cell. Many of these effects were due at least in part to changes in mRNA levels rather than simply protein degradation. Measurements of cellular zinc content showed that the level of zinc atoms per cell dropped from over 20 million in replete cells to only 1.7 million in deficient cells. These results confirmed the ability of replete cells to store excess zinc and suggested that the majority of zinc-binding sites on proteins in deficient cells are either unmetalated or mismetalated. Our analysis of two abundant zinc proteins, Fba1 aldolase and Met6 methionine synthetase, supported that hypothesis. Thus, we have discovered widespread zinc sparing mechanisms and obtained evidence of a high accumulation of zinc proteins that lack their cofactor during deficiency.
Highlights d COQ9 specifically accesses and binds membraneembedded aromatic isoprenes d An exposed tryptophan and an amphipathic helix control lipid and membrane binding d Interactions with the peripheral membrane enzyme COQ7 suggest lipid presentation d In vivo CoQ production relies on COQ9's membrane, lipid, and protein interactions
Each year over 90 million units of blood are transfused worldwide. Our dependence on this blood supply mandates optimized blood management and storage. During storage, red blood cells undergo degenerative processes resulting in altered metabolic characteristics which may make blood less viable for transfusion. However, not all stored blood spoils at the same rate, a difference that has been attributed to variable rates of energy usage and metabolism in red blood cells. Specific metabolite abundances are heritable traits; however, the link between heritability of energy metabolism and red blood cell storage profiles is unclear. Herein we performed a comprehensive metabolomics and proteomics study of red blood cells from 18 mono- and di-zygotic twin pairs to measure heritability and identify correlations with ATP and other molecular indices of energy metabolism. Without using affinity-based hemoglobin depletion, our work afforded the deepest multi-omic characterization of red blood cell membranes to date (1280 membrane proteins and 330 metabolites), with 119 membrane protein and 148 metabolite concentrations found to be over 30% heritable. We demonstrate a high degree of heritability in the concentration of energy metabolism metabolites, especially glycolytic metabolites. In addition to being heritable, proteins and metabolites involved in glycolysis and redox metabolism are highly correlated, suggesting that crucial energy metabolism pathways are inherited en bloc at distinct levels. We conclude that individuals can inherit a phenotype composed of higher or lower concentrations of these proteins together. This can result in vastly different red blood cells storage profiles which may need to be considered to develop precise and individualized storage options. Beyond guiding proper blood storage, this intimate link in heritability between energy and redox metabolism pathways may someday prove useful in determining the predisposition of an individual toward metabolic diseases.
Pathogenicity islands and plasmids bear genes for pathogenesis of various Escherichia coli pathotypes. Although there is a basic understanding of the contribution of these virulence factors to disease, less is known about variation in regulatory networks in determining disease phenotypes. Here, we dissected a regulatory network directed by the conserved iron homeostasis regulator, ferric uptake regulator (Fur), in uropathogenic E. coli (UPEC) strain CFT073. Comparing anaerobic genome-scale Fur DNA binding with Fur-dependent transcript expression and protein levels of the uropathogen to that of commensal E. coli K-12 strain MG1655 showed that the Fur regulon of the core genome is conserved but also includes genes within the pathogenicity/genetic islands. Unexpectedly, regulons indicative of amino acid limitation and the general stress response were also indirectly activated in the uropathogen fur mutant, suggesting that induction of the Fur regulon increases amino acid demand. Using RpoS levels as a proxy, addition of amino acids mitigated the stress. In addition, iron chelation increased RpoS to the same levels as in the fur mutant. The increased amino acid demand of the fur mutant or iron chelated cells was exacerbated by aerobic conditions, which could be partly explained by the O2-dependent synthesis of the siderophore aerobactin, encoded by an operon within a pathogenicity island. Taken together, these data suggest that in the iron-poor environment of the urinary tract, amino acid availability could play a role in the proliferation of this uropathogen, particularly if there is sufficient O2 to produce aerobactin. IMPORTANCE Host iron restriction is a common mechanism for limiting the growth of pathogens. We compared the regulatory network controlled by Fur in uropathogenic E. coli (UPEC) to that of nonpathogenic E. coli K-12 to uncover strategies that pathogenic bacteria use to overcome iron limitation. Although iron homeostasis functions were regulated by Fur in the uropathogen as expected, a surprising finding was the activation of the stringent and general stress responses in the uropathogen fur mutant, which was rescued by amino acid addition. This coordinated global response could be important in controlling growth and survival under nutrient-limiting conditions and during transitions from the nutrient-rich environment of the lower gastrointestinal (GI) tract to the more restrictive environment of the urinary tract. The coupling of the response of iron limitation to increased demand for amino acids could be a critical attribute that sets UPEC apart from other E. coli pathotypes.
Introduction Each year over 90 million units of blood are transfused worldwide. Our dependence on this blood supply requires optimized blood collection and storage. During storage, red blood cells (RBCs) undergo degenerative processes resulting in altered metabolic characteristics. In the past decade numerous studies have implicated longer storage of RBCs in adverse patient outcomes. The post-storage ATP level in blood is the single best predictor of transfused RBC in vivo recovery. Although the rate of ATP decline is highly variable between individuals, post-storage ATP levels are primarily determined by inheritance. Understanding the effect of storage on energy metabolism pathways is thus of vital importance to maintaining a safe and effective blood supply. Methods We performed comprehensive metabolomics and proteomics studies of mono- and di-zygotic twin pairs to measure heritability of molecules and identify correlations with ATP and other markers in energy metabolism. Metabolite levels were measured at six time points from 0-56 days to elucidate changes that occur during storage. An obstacle for RBC proteomics is the massive quantity of hemoglobin, constituting 97% of protein material. This was avoided by preparing RBC membrane fractions, which mitigated the need for hemoglobin depletion. All proteomics data was collected on an Orbitrap Elite hybrid ion trap-orbitrap mass spectrometer (Thermo Fisher Scientific). Metabolomics data was collected by Metabolon Inc. and was collated with proteomics results to give a complete view of RBC metabolism. Preliminary Data Our optimized method for collecting proteomics data in RBCs has yielded the greatest depth of coverage observed without the use of commercial hemoglobin depletion. Purified RBCs were lysed and centrifuged to collect membrane fractions allowing us to identify 1280 proteins and 330 metabolites from mono and di-zygotic twins. Of these, 146 proteins and 148 metabolites were found to be over 30% heritable. We observe a high degree of heritability in metabolites involved in energy metabolism, especially glycolysis. This is supported by the heritability in key regulatory enzymes including phosphofructokinase (PFK) (57%) and bisphosphoglycerate mutase (BPGM) (50%). Additionally we observe high correlations between both glycolytic proteins and metabolites suggesting that this crucial energy metabolism pathway is inherited en blocat various levels. A number of the correlations we observed can be combined to produce a model to predict post-storage ATP levels. Five key parameters in this model include PFK, carbonic anhydrase 1 (CA1), band 3, BPGM, and pH. Strikingly, concentrations of all protein components of this model were at least 45% heritable. Band 3, BPGM, and CA1 correlate negatively with post storage ATP levels and together shuttle flux away from glycolysis and ATP production. We also observe a positive correlation between pH and post-storage ATP. A negative correlation observed between CA1, which is 84% heritable, and post-storage ATP, is especially significant in that it provides a hypothetical model for the heritability of ATP decline during storage. Our model proposes that RBC units, which are stored in gas permeable bags allowing CO2 to diffuse into the bag, are subject to genetically determined, CA1-mediated production of carbonic acid, resulting in inhibition of PFK. This model is further supported by negative correlations between CA1 and pH during storage. We propose that heritable concentrations of CA1 negatively influence pH, which allosterically inhibits PFK and impedes energy metabolism and subsequently ATP production. We conclude that individuals inherit a phenotype composed of higher or lower concentrations of key energy metabolism proteins that regulate flux through glycolysis during RBC storage. Heritability of energy metabolism can result in markedly different RBC storage profiles and knowledge of heritable RBC energy metabolism can be used to improve and individualize RBC storage methods. Disclosures Hess: ASH: Patents & Royalties: 4 US patents related to RBC storage solution AS-7.
1The Zap1 transcription factor of Saccharomyces cerevisiae is a key regulator in the genomic 2 responses to zinc deficiency. Among the genes regulated by Zap1 during zinc deficiency is the 3 autophagy-related gene ATG41. Here, we report that Atg41 is required for growth in zinc-4 deficient conditions but not when zinc is abundant or when other metals are limiting. 5Consistent with a role for Atg41 in macroautophagy, we show that nutritional zinc deficiency 6 induces autophagy and that mutation of ATG41 diminishes that response. Several experiments 7 indicated that the importance of ATG41 function to growth during zinc deficiency is not 8 because of its role in macroautophagy but rather is due to one or more autophagy-independent 9 functions. For example, rapamycin treatment fully induced autophagy in zinc-deficient atg41Δ 10 mutants but failed to improve growth. In addition, atg41Δ mutants showed a far more severe 11 growth defect than any of several other autophagy mutants tested, and atg41Δ mutants 12 showed increased Hsf1 activity, an indicator of protein homeostasis stress, while other 13 autophagy mutants did not. An autophagy-independent function for ATG41 in sulfur 14 metabolism during zinc deficiency was suggested by analyzing the transcriptome of atg41Δ 15 mutants during the transition from zinc-replete to deficient conditions. Analysis of sulfur 16 metabolites confirmed that Atg41 is needed for the normal accumulation of methionine, 17 homocysteine, and cysteine in zinc-deficient cells. Therefore, we conclude that Atg41 plays 18 roles in both macroautophagy and sulfur metabolism during zinc deficiency. 19 20 4
Zinc homeostasis is essential for all organisms. The Zap1 transcriptional activator regulates these processes in the yeast Saccharomyces cerevisiae. During zinc deficiency, Zap1 increases expression of zinc transporters and proteins involved in adapting to the stress of zinc deficiency. Transcriptional activation by Zap1 can also repress expression of some genes, e.g., RTC4. In zinc-replete cells, RTC4 mRNA is produced with a short transcript leader that is efficiently translated. During deficiency, Zap1-dependent expression of an RNA with a longer transcript leader represses the RTC4 promoter. This long leader transcript (LLT) is not translated due to the presence of small open reading frames upstream of the RTC4 coding region. In this study, we show that the RTC4 LLT RNA also plays a second function, i.e., repression of the adjacent GIS2 gene. In generating the LLT transcript, RNA polymerase II transcribes RTC4 through the GIS2 promoter. Production of the LLT RNA correlates with the decreased expression of GIS2 mRNA and mutations that prevent synthesis of the LLT RNA or terminate it before the GIS2 promoter renders GIS2 mRNA expression and Gis2 protein accumulation constitutive. Thus, we have discovered an unusual regulatory mechanism that uses a bicistronic RNA to control two genes simultaneously.
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