The distributions of Fe in mitochondria isolated from respiring, respiro-fermenting, and fermenting yeast cells were determined with an integrative biophysical approach involving Mössbauer and ...electronic absorption spectroscopies, electron paramagnetic resonance, and inductively coupled plasma emission mass spectrometry. Approximately 40% of the Fe in mitochondria from respiring cells was present in respiration-related proteins. The concentration and distribution of Fe in respiro-fermenting mitochondria, where both respiration and fermentation occur concurrently, were similar to those of respiring mitochondria. The concentration of Fe in fermenting mitochondria was also similar, but the distribution differed dramatically. Here, levels of respiration-related Fe-containing proteins were diminished ∼3-fold, while non-heme HS FeII species, non-heme mononuclear HS FeIII, and FeIII nanoparticles dominated. These changes were rationalized by a model in which the pool of non-heme HS FeII ions serves as feedstock for Fe−S cluster and heme biosynthesis. The integrative approach enabled us to estimate the concentration of respiration-related proteins.
Mössbauer spectroscopy was used to detect pools of Fe in mitochondria from fermenting yeast cells, including those consisting of nonheme high-spin (HS) FeII species, FeIII nanoparticles, and ...mononuclear HS FeIII species. At issue was whether these species were located within mitochondria or on their exterior. None could be removed by washing mitochondria extensively with ethylene glycol tetraacetic acid or bathophenanthroline sulfonate (BPS), FeII chelators that do not appear to penetrate mitochondrial membranes. However, when mitochondrial samples were sonicated, BPS coordinated the FeII species, forming a low-spin FeII complex. This treatment also diminished the levels of both FeIII species, suggesting that all of these Fe species are encapsulated by mitochondrial membranes and are protected from chelation until membranes are disrupted. 1,10-Phenanthroline is chemically similar to BPS but is membrane soluble; it coordinated nonheme HS FeII in unsonicated mitochondria. Further, the HS FeIII species and nanoparticles were not reduced by dithionite until the detergent deoxycholate was added to disrupt membranes. There was no correlation between the percentage of nonheme HS FeII species in mitochondrial samples and the level of contaminating proteins. These results collectively indicate that the observed Fe species are contained within mitochondria. Mössbauer spectra of whole cells were dominated by HS FeIII features; the remainder displayed spectral features typical of isolated mitochondria, suggesting that the Fe in fermenting yeast cells can be coarsely divided into two categories: mitochondrial Fe and (mostly) HS FeIII ions in one or more non-mitochondrial locations.
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Despite similar clinical symptoms, peanut-allergic (PA) individuals may respond quite differently to the same therapeutic interventions.
This study aimed to determine whether inherent ...qualities of cell response at baseline could influence response to peanut oral immunotherapy (PnOIT).
We first performed ex vivo T-cell profiling on peanut-reactive CD154+CD137+ T (pTeff) cells from 90 challenge-confirmed PA individuals. We developed a gating strategy for unbiased assessment of the phenotypic distribution of rare pTeff cells across different memory CD4+ T-cell subsets to define patient immunotype. In longitudinal samples of 29 PA participants enrolled onto the IMPACT trial of PnOIT, we determined whether patient immunotype at baseline could influence response to PnOIT.
Our data emphasize the heterogeneity of pTeff cell responses in PA participants with 2 mutually exclusive phenotypic entities (CCR6−CRTH2+ and CCR6+CRTH2−). Our findings lead us to propose that peanut allergy can be classified broadly into at least 2 discrete subtypes, termed immunotypes, with distinct immunologic and clinical characteristics that are based on the proportion of TH2A pTeff cells. PnOIT induced elimination of TH2A pTeff cells in the context of the IMPACT clinical trial. Only 1 PA patient with a low level of TH2A pTeff cells at baseline experienced long-lasting benefit of remission after PnOIT discontinuation.
Dividing PA patients according to their individual peanut-specific T-cell profile may facilitate patient stratification in clinical settings by identifying which immunotypes might respond best to different therapies.
Introduction:
Despite the great diversity in cell of origin and other clinical features, hematological malignancies share a core set of pathways and processes that drive tumorigenesis. Pathways ...critical to the normal growth and development of the hematopoietic compartment, such as MAPK, MYC signaling, NF-kB, PI3K-AKT, B and T cell receptor signaling are frequently mutated and disrupted. Additionally, dysregulation of cytokines, apoptosis, the DNA damage response, and epigenetic factors are frequently seen in gene expression as well as mutational profiles across a variety of these cancers.
To address the need to profile such pathways across multiple classes of biological macromolecules from small amounts of sample, NanoString has developed 3D Biology™ Technology, which enables DNA, RNA, and protein to be simultaneously detected from a single sample utilizing digital molecular barcoding technology on the nCounter® system. For hematology-oncology specifically, the nCounter® Vantage 3D™ DNA:RNA:Protein Heme Assay embodies a collection of probes, molecular barcodes, and workflows to measure 180 mRNA targets, more than 30 protein targets (total and phosphorylated forms including JAK-STAT, Src, Syk, Ik-Ba, PI3K-Akt), and more than 120 DNA mutations for the conserved and crucial pathways underlying hematological malignancies.
Methods and Results:
Development of 3D Biology™ Technology is based on Nanostring's proven digital molecular barcoding technology. Probe-reporter complexes are formed through highly specific and predictable nucleic acid hybridization events allowing for parallel design and development across multiple analytes. The RNA portion is composed of probes designed using a well-established pipeline and are verified against synthetic oligonucleotide targets and Universal Human Reference total RNA (Agilent). The protein portion utilizes a mixture of antibodies each of which has been chemically labeled with a target-specific ‘alien’ synthetic oligonucleotide. The capacity for each antibody within the mix to specifically detect its target is tested against lysates and formalin-fixed, paraffin-embedded (FFPE) tissue samples. Finally, the DNA panel is composed of novel ‘SNV’ probes that can discriminate between targets that differ by a single base. These probes are designed and screened against synthetic mutant and reference (hg19) allele targets for specificity and sensitivity to ensure detection down to 5% allele frequency with >95% sensitivity. Standard genomic DNA samples from NIST (NA12878) and Horizon Discovery are also used to verify assay performance.
Workflows have been established and validated for DNA:RNA:Protein profiling for fresh/frozen or FFPE-preserved samples. For fresh/frozen samples, the assay enables simultaneous DNA:RNA:Protein profiling from as little as 5 ng DNA, 25 ng RNA, and 250 ng protein. This is achieved by extracting genomic DNA and creating a lysate for RNA and protein analysis. The purified DNA is pre-amplified before overnight hybridization with SNV probes and reporters and the lysate is processed to permit a combined overnight hybridization reaction with both RNA- and protein-specific probes and reporters. For FFPE samples, equivalent profiling can be obtained from two 100 mm2, 5-micron thick FFPE sections; one section is incubated with antibodies and the second section is used for extraction of RNA and DNA using a standard kit. After hybridization, processed DNA, RNA, and protein are pooled and co-analyzed using an nCounter system and nSolver™ software (alpha version 4.0). Concordant results were obtained from both workflows for two heme-derived cell lines, CCRF-CEM and Hut78, showing reproducibility between fresh/frozen and FFPE sample types. Additionally, all known DNA mutations were detected and Hut78 showed higher and concomitant RNA and protein expression of NF-kB p65 (RELA), BCL-xL (BCL2L1), Stat3 (STAT3), and IkBa (IKBA ) than occurs in CCRF-CEM cell lines. Results from human archived tissues will also be shown.
Conclusion:
Combined analysis of SNV, mRNA, and protein expression in a 3D Biology experiment promises broad and unique utility for hematology-oncology research, particularly for the evaluation of blood, lymph node, and bone marrow biopsies.
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Yah1p, an Fe 2S 2-containing ferredoxin located in the matrix of Saccharomyces cerevisiae mitochondria, functions in the synthesis of Fe/S clusters and heme a prosthetic groups. EPR, Mossbauer ...spectroscopy, and electron microscopy were used to characterize the Fe that accumulates in Yah1p-depleted isolated intact mitochondria. Gal- YAH1 cells were grown in standard rich media (YPD and YPGal) under O 2 or argon atmospheres. Mitochondria were isolated anaerobically, then prepared in the as-isolated redox state, the dithionite-treated state, and the O 2-treated state. The absence of strong EPR signals from Fe/S clusters when Yah1p was depleted confirms that Yah1p is required in Fe/S cluster assembly. Yah1p-depleted mitochondria, grown with O 2 bubbling through the media, accumulated excess Fe (up to 10 mM) that was present as 2-4 nm diameter ferric nanoparticles, similar to those observed in mitochondria from yfh1Delta cells. These particles yielded a broad isotropic EPR signal centered around g = 2, characteristic of superparamagnetic relaxation. Treatment with dithionite caused Fe (3+) ions of the nanoparticles to become reduced and largely exported from the mitochondria. Fe did not accumulate in mitochondria isolated from cells grown under Ar; a significant portion of the Fe in these organelles was in the high-spin Fe (2+) state. This suggests that the O 2 used during growth of Gal- YAH1 cells is responsible, either directly or indirectly, for Fe accumulation and for oxidizing Fe (2+) --> Fe (3+) prior to aggregation. Models are proposed in which the accumulation of ferric nanoparticles is caused either by the absence of a ligand that prevents such precipitation in wild-type mitochondria or by a more oxidizing environment within the mitochondria of Yah1p-depleted cells exposed to O 2. The efficacy of reducing accumulated Fe along with chelating it should be considered as a strategy for its removal in diseases involving such accumulations.
Mitochondria from respiring cells were isolated under anaerobic conditions. Microscopic images were largely devoid of contaminants, and samples consumed O(2) in an NADH-dependent manner. Protein and ...metal concentrations of packed mitochondria were determined, as was the percentage of external void volume. Samples were similarly packed into electron paramagnetic resonance tubes, either in the as-isolated state or after exposure to various reagents. Analyses revealed two signals originating from species that could be removed by chelation, including rhombic Fe(3+) (g = 4.3) and aqueous Mn(2+) ions (g = 2.00 with Mn-based hyperfine). Three S = 5/2 signals from Fe(3+) hemes were observed, probably arising from cytochrome c peroxidase and the a(3):Cu(b) site of cytochrome c oxidase. Three Fe/S-based signals were observed, with averaged g values of 1.94, 1.90 and 2.01. These probably arise, respectively, from the Fe(2)S(2)(+) cluster of succinate dehydrogenase, the Fe(2)S(2)(+) cluster of the Rieske protein of cytochrome bc (1), and the Fe(3)S(4)(+) cluster of aconitase, homoaconitase or succinate dehydrogenase. Also observed was a low-intensity isotropic g = 2.00 signal arising from organic-based radicals, and a broad signal with g (ave) = 2.02. Mössbauer spectra of intact mitochondria were dominated by signals from Fe(4)S(4) clusters (60-85% of Fe). The major feature in as-isolated samples, and in samples treated with ethylenebis(oxyethylenenitrilo)tetraacetic acid, dithionite or O(2), was a quadrupole doublet with DeltaE (Q) = 1.15 mm/s and delta = 0.45 mm/s, assigned to Fe(4)S(4)(2+) clusters. Substantial high-spin non-heme Fe(2+) (up to 20%) and Fe(3+) (up to 15%) species were observed. The distribution of Fe was qualitatively similar to that suggested by the mitochondrial proteome.
Yah1p, an Fe2S2-containing ferredoxin located in the matrix of Saccharomyces cerevisiae mitochondria, functions in the synthesis of Fe/S clusters and heme a prosthetic groups. EPR, Mössbauer ...spectroscopy, and electron microscopy were used to characterize the Fe that accumulates in Yah1p-depleted isolated intact mitochondria. Gal-YAH1 cells were grown in standard rich media (YPD and YPGal) under O2 or argon atmospheres. Mitochondria were isolated anaerobically, then prepared in the as-isolated redox state, the dithionite-treated state, and the O2-treated state. The absence of strong EPR signals from Fe/S clusters when Yah1p was depleted confirms that Yah1p is required in Fe/S cluster assembly. Yah1p-depleted mitochondria, grown with O2 bubbling through the media, accumulated excess Fe (up to 10 mM) that was present as 2−4 nm diameter ferric nanoparticles, similar to those observed in mitochondria from yfh1Δ cells. These particles yielded a broad isotropic EPR signal centered around g = 2, characteristic of superparamagnetic relaxation. Treatment with dithionite caused Fe3+ ions of the nanoparticles to become reduced and largely exported from the mitochondria. Fe did not accumulate in mitochondria isolated from cells grown under Ar; a significant portion of the Fe in these organelles was in the high-spin Fe2+ state. This suggests that the O2 used during growth of Gal-YAH1 cells is responsible, either directly or indirectly, for Fe accumulation and for oxidizing Fe2+ → Fe3+ prior to aggregation. Models are proposed in which the accumulation of ferric nanoparticles is caused either by the absence of a ligand that prevents such precipitation in wild-type mitochondria or by a more oxidizing environment within the mitochondria of Yah1p-depleted cells exposed to O2. The efficacy of reducing accumulated Fe along with chelating it should be considered as a strategy for its removal in diseases involving such accumulations.
Methods are presented to aid in the study of iron metabolism in isolated mitochondria. The "iron-ome" of mitochondria, including the type and concentration of all Fe-containing species in the ...organelle, is evaluated by integrating the results of four spectroscopic methods, including Mössbauer spectroscopy, electron paramagnetic resonance, electronic absorption spectroscopy, and inductively coupled plasma mass spectrometry. Although this systems biology approach only allows groups of Fe centers to be assessed, rather than individual species, it affords new and useful information. There are many considerations in executing this approach, and this chapter focuses on the practical methods that we have developed for this purpose. First, large quantities of mitochondria are required, and so published isolation methods must be scaled up. Second, mitochondria are isolated under strict anaerobic conditions to allow control of redox state and to protect O(2)-sensitive Fe-containing proteins from degradation. Third, the importance of packing mitochondria for both spectroscopic and analytical characterizations is developed. By measuring the volume of packed samples and the percentage of mitochondria contained within that volume, absolute Fe and protein concentrations within the organelle can be obtained. Packing samples into spectroscopy holders also affords maximal signal intensities, which are critical for these studies. Custom inserts designed for this purpose are described. Also described are the designs of a 25-L glass bioreactor, a mechanical cell homogenizer, a device for inserting short EPR tubes into the standard Oxford Instruments EPR cryostat, and a device for transferring samples from Mössbauer holders to EPR tubes while maintaining samples at liquid N(2) temperatures. A brief summary of what we have learned by use of these methods is included.
An integrative biophysical and bioanalytical approach to studying the Fe
distribution in isolated mitochondria was developed. This procedure involved
large-scale growths, the inclusion of a chelator ...in isolation buffers and an
anaerobic isolation protocol. Electron microscopy confirmed that mitochondrial
membranes were intact and that samples were largely devoid of contaminants.
The Fe-ome-the sum of all Fe species in mitochondria--was studied using a
combination of EPR, Mossbauer Spectroscopy, Electron Absorption, ICP-MS
and Protein analysis.
Isolated mitochondria were packed prior to analysis to improve the S/N
ratio. The residual buffer content of sample pellets was determined by use of a
radio-labeled buffer. There was essentially no difference in the packing
efficiency of mitochondria isolated from respiring and fermenting cells. The
determined packing factor, 0.80, was used to calculate concentrations of
individual species in neat mitochondria.
The Fe-omes of mitochondria isolated from cells grown on respiring,
respirofermenting and fermenting media were determined. Neat mitochondria
contained ~ 750 mM Fe, regardless of whether the cells had been grown on
respiring or fermenting media. The Fe distribution of respirofermenting samples
(which can undergo respiration and fermentation simultaneously) was nearly
identical to that of respiring mitochondria. Fermenting samples had a very
different Fe-distribution.
Nearly 40 % of the iron in respiring mitochondria was present in
respiratory complexes including cytochrome c, cytochrome bc1, succinate
dehydrogenase, and cytochrome c oxidase. Fermenting mitochondria contain
an Fe-ome dominated by non-protein centers. Approximately 80 % of the Fe
was present as a combination of nonheme HS Fe2+, nonheme Fe3+ and Fe3+
nanoparticles. These centers were present in roughly equal amounts. The
remaining 20 % of the Fe was present as respiratory complexes which have
concentrations ~ 1/2 to 1/3 that of respiring mitochondria.
A model is presented in which the nonheme HS Fe2+ species serves as a
feedstock for Fe/S and heme biosynthesis. When the cell is growing on
respiring media, this metabolic reservoir diminishes as respiratory complexes
are constantly synthesized. Under fermentative growth, the metabolic pool
increases due to the reduced demand for respiration-related prosthetic groups.
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