The detrimental effects of ionizing radiation (IR) involve a highly orchestrated series of events that are amplified by endogenous signaling and culminating in oxidative damage to DNA, lipids, ...proteins, and many metabolites. Despite the global impact of IR, the molecular mechanisms underlying tissue damage reveal that many biomolecules are chemoselectively modified by IR.
The development of high-throughput "omics" technologies for mapping DNA and protein modifications have revolutionized the study of IR effects on biological systems. Studies in cells, tissues, and biological fluids are used to identify molecular features or biomarkers of IR exposure and response and the molecular mechanisms that regulate their expression or synthesis.
In this review, chemical mechanisms are described for IR-induced modifications of biomolecules along with methods for their detection. Included with the detection methods are crucial experimental considerations and caveats for their use. Additional factors critical to the cellular response to radiation, including alterations in protein expression, metabolomics, and epigenetic factors, are also discussed.
Throughout the review, the synergy of combined "omics" technologies such as genomics and epigenomics, proteomics, and metabolomics is highlighted. These are anticipated to lead to new hypotheses to understand IR effects on biological systems and improve IR-based therapies.
The activation of mostly quiescent haematopoietic stem cells (HSCs) is a prerequisite for life-long production of blood cells
. This process requires major molecular adaptations to allow HSCs to meet ...the regulatory and metabolic requirements for cell division
. The mechanisms that govern cellular reprograming upon stem-cell activation, and the subsequent return of stem cells to quiescence, have not been fully characterized. Here we show that chaperone-mediated autophagy (CMA)
, a selective form of lysosomal protein degradation, is involved in sustaining HSC function in adult mice. CMA is required for protein quality control in stem cells and for the upregulation of fatty acid metabolism upon HSC activation. We find that CMA activity in HSCs decreases with age and show that genetic or pharmacological activation of CMA can restore the functionality of old mouse and human HSCs. Together, our findings provide mechanistic insights into a role for CMA in sustaining quality control, appropriate energetics and overall long-term HSC function. Our work suggests that CMA may be a promising therapeutic target for enhancing HSC function in conditions such as ageing or stem-cell transplantation.
In this study we interrogated the metabolome of human acute myeloid leukemia (AML) stem cells to elucidate properties relevant to therapeutic intervention. We demonstrate that amino acid uptake, ...steady-state levels, and catabolism are all elevated in the leukemia stem cell (LSC) population. Furthermore, LSCs isolated from de novo AML patients are uniquely reliant on amino acid metabolism for oxidative phosphorylation and survival. Pharmacological inhibition of amino acid metabolism reduces oxidative phosphorylation and induces cell death. In contrast, LSCs obtained from relapsed AML patients are not reliant on amino acid metabolism due to their ability to compensate through increased fatty acid metabolism. These findings indicate that clinically relevant eradication of LSCs can be achieved with drugs that target LSC metabolic vulnerabilities.
•Leukemia stem cells (LSCs) rely on amino acids for survival•LSCs use amino acids to fuel oxidative phosphorylation (OXPHOS)•Venetoclax with azacitidine inhibits OXPHOS in LSCs from leukemia patients•Venetoclax with azacitidine targets OXPHOS by inhibiting amino acid metabolism
By interrogating metabolic properties of human acute myeloid leukemia, Jones et al. reveal increased amino acid metabolism in leukemia stem cells (LSCs), which rely on amino acids for oxidative phosphorylation and survival. Venetoclax with azacitidine induces LSC toxicity by decreasing amino acid uptake.
Metabolomics has emerged in the past decade as a highly attractive and impactful technique for phenotype-level profiling in diverse biological applications. Most recently, the dual developments of ...high-throughput analytical techniques along with dramatically increased sensitivity of high-resolution mass spectrometers have enabled the routine analysis of hundreds of unique samples per day. We have previously reported a robust 3 min isocratic metabolomics platform for the quantification of amino acids and the key pathways of central carbon and nitrogen metabolism. Building on this work, we describe here a 5 min reverse phase gradient followed by global, untargeted profiling of the hydrophilic metabolome. In addition to observing those metabolites measured in the 3 min run, the use of the longer gradient run here also allows for coverage of less polar compounds such as fatty acids and acylcarnitines, both key players in mitochondrial and lipid metabolism, without a significant sacrifice in throughput.
Dendritic cell (DC) activation by Toll-like receptor (TLR) agonists causes rapid glycolytic reprogramming that is required to meet the metabolic demands of their immune activation. Recent efforts in ...the field have identified an important role for extracellular glucose sourcing to support DC activation. However, the contributions of intracellular glucose stores to these processes have not been well characterized. We demonstrate that DCs possess intracellular glycogen stores and that cell-intrinsic glycogen metabolism supports the early effector functions of TLR-activated DCs. Inhibition of glycogenolysis significantly attenuates TLR-mediated DC maturation and impairs their ability to initiate lymphocyte activation. We further report that DCs exhibit functional compartmentalization of glucose- and glycogen-derived carbons, where these substrates preferentially contribute to distinct metabolic pathways. This work provides novel insights into nutrient homeostasis in DCs, demonstrating that differential utilization of glycogen and glucose metabolism regulates their optimal immune function.
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•Dendritic cells (DCs) express machinery for cell-intrinsic glycogen metabolism•Intracellular glycogen stores are catabolized to support DC effector function•Inhibition of glycogen catabolism impairs dendritic cell activation•Glycogen-derived carbons preferentially support DC citrate synthesis
Thwe et al. show that dendritic cells (DCs) possess intracellular glycogen stores that fuel their activation-associated glycolysis induction and immune effector function. They uncover a novel mechanism of metabolic regulation in DCs by which glucose- and glycogen-derived carbons preferentially contribute to distinct metabolic pathways.
Liquid chromatography coupled to mass spectrometry (LC-MS)-based metabolomics and lipidomics offers invaluable tools to qualitatively and quantitatively study biological systems. Historically, ...unbiased (or discovery) analysis has been performed independently of targeted, quantitative analysis such as multiple reaction monitoring (MRM). These practices have been aptly carried out based on technical limitations of each assay. The wide mass scanning ranges typical of discovery approaches limit assay sensitivity, while targeted methods that improve analyte detection do not acquire data on ions not included in the targeted assay design. Recent improvements to quadrupole-Orbitrap technology have improved both scan speed as well as sensitivity, thus making these instruments more robust. By combining the improved robustness and coverage with stable isotope dilution (SID) techniques, advantages of the separate assays can now be realized in a single run, thereby improving the throughput of this type of analysis.
We have previously demonstrated that oxidative phosphorylation is required for the survival of human leukemia stem cells (LSCs) from patients with acute myeloid leukemia (AML). More recently, we ...demonstrated that LSCs in patients with de novo AML rely on amino acid metabolism to drive oxidative phosphorylation. Notably, although overall levels of amino acids contribute to LSC energy metabolism, our current findings suggest that cysteine may be of particular importance for LSC survival. We demonstrate that exogenous cysteine is metabolized exclusively to glutathione. Upon cysteine depletion, glutathione synthesis is impaired, leading to reduced glutathionylation of succinate dehydrogenase A (SDHA), a key component of electron transport chain complex (ETC) II. Loss of SDHA glutathionylation impairs ETC II activity, thereby inhibiting oxidative phosphorylation, reducing production of ATP, and leading to LSC death. Given the role of cysteine in driving LSC energy production, we tested cysteine depletion as a potential therapeutic strategy. Using a novel cysteine-degrading enzyme, we demonstrate selective eradication of LSCs, with no detectable effect on normal hematopoietic stem/progenitor cells. Together, these findings indicate that LSCs are aberrantly reliant on cysteine to sustain energy metabolism, and that targeting this axis may represent a useful therapeutic strategy.
•LSCs rely on cysteine for survival.•Cysteine metabolism is required for ETC II activity in LSCs.
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An emerging metabolic theory of pulmonary hypertension (PH) suggests that cellular and mitochondrial metabolic dysfunction underlies the pathology of this disease. We and others have previously ...demonstrated the existence of hyperproliferative, apoptosis-resistant, proinflammatory adventitial fibroblasts from human and bovine hypertensive pulmonary arterial walls (PH-Fibs) that exhibit constitutive reprogramming of glycolytic and mitochondrial metabolism, accompanied by an increased ratio of glucose catabolism through glycolysis versus the tricarboxylic acid cycle. However, the mechanisms responsible for these metabolic alterations in PH-Fibs remain unknown. We hypothesized that in PH-Fibs microRNA-124 (miR-124) regulates PTBP1 (polypyrimidine tract binding protein 1) expression to control alternative splicing of pyruvate kinase muscle (PKM) isoforms 1 and 2, resulting in an increased PKM2/PKM1 ratio, which promotes glycolysis and proliferation even in aerobic environments.
Pulmonary adventitial fibroblasts were isolated from calves and humans with severe PH (PH-Fibs) and from normal subjects. PTBP1 gene knockdown was achieved via PTBP1-siRNA; restoration of miR-124 was performed with miR-124 mimic. TEPP-46 and shikonin were used to manipulate PKM2 glycolytic function. Histone deacetylase inhibitors were used to treat cells. Metabolic products were determined by mass spectrometry-based metabolomics analyses, and mitochondrial function was analyzed by confocal microscopy and spectrofluorometry.
We detected an increased PKM2/PKM1 ratio in PH-Fibs compared with normal subjects. PKM2 inhibition reversed the glycolytic status of PH-Fibs, decreased their cell proliferation, and attenuated macrophage interleukin-1β expression. Furthermore, normalizing the PKM2/PKM1 ratio in PH-Fibs by miR-124 overexpression or PTBP1 knockdown reversed the glycolytic phenotype (decreased the production of glycolytic intermediates and byproducts, ie, lactate), rescued mitochondrial reprogramming, and decreased cell proliferation. Pharmacological manipulation of PKM2 activity with TEPP-46 and shikonin or treatment with histone deacetylase inhibitors produced similar results.
In PH, miR-124, through the alternative splicing factor PTBP1, regulates the PKM2/PKM1 ratio, the overall metabolic, proliferative, and inflammatory state of cells. This PH phenotype can be rescued with interventions at various levels of the metabolic cascade. These findings suggest a more integrated view of vascular cell metabolism, which may open unique therapeutic prospects in targeting the dynamic glycolytic and mitochondrial interactions and between mesenchymal inflammatory cells in PH.
Red blood cell storage in the blood bank promotes the progressive accumulation of metabolic alterations that may ultimately impact the erythrocyte capacity to cope with oxidant stressors. However, ...the metabolic underpinnings of the capacity of RBCs to resist oxidant stress and the potential impact of donor biology on this phenotype are not known. Within the framework of the REDS-III RBC-Omics study, RBCs from 8,502 healthy blood donors were stored for 42 days and tested for their propensity to hemolyze following oxidant stress. A subset of extreme hemolyzers donated a second unit of blood, which was stored for 10, 23, and 42 days and profiled again for oxidative hemolysis and metabolomics (599 samples). Alterations of RBC energy and redox homeostasis were noted in donors with high oxidative hemolysis. RBCs from females, donors over 60 years old, donors of Asian/South Asian race-ethnicity, and RBCs stored in additive solution-3 were each independently characterized by improved antioxidant metabolism compared to, respectively, males, donors under 30 years old, Hispanic and African American race ethnicity donors, and RBCs stored in additive solution-1. Merging metabolomics data with results from an independent GWAS study on the same cohort, we identified metabolic markers of hemolysis and G6PD-deficiency, which were associated with extremes in oxidative hemolysis and dysregulation in NADPH and glutathione-dependent detoxification pathways of oxidized lipids. Donor sex, age, ethnicity, additive solution and G6PD status impact the metabolism of the stored erythrocyte and its susceptibility to hemolysis following oxidative insults.
The vitreous humor is a highly aqueous eye fluid interfacing with the retina and lens and providing shape. Its molecular composition provides a readout for the eye’s physiological status. Changes in ...cellular metabolism underlie vitreoretinal pathologies, but despite routine surgical collection of vitreous, only limited reports of metabolism in the vitreous of human patients have been described. Vitreous samples from patients with rhegmatogenous retinal detachment (n = 25) and proliferative diabetic retinopathy (n = 9) were profiled along with control human vitreous samples (n = 8) by untargeted mass-spectrometry-based metabolomics. Profound changes were observed in diabetic retinopathy vitreous, including altered glucose metabolism and activation of the pentose phosphate pathway, which provides reducing equivalents to counter oxidative stress. In addition, purine metabolism was altered in diabetic retinopathy, with decreased xanthine and elevated levels of related purines (inosine, hypoxanthine, urate, allantoate) generated in oxidant-producing reactions. In contrast, the vitreous metabolite profiles of retinal detachment patients were similar to controls. In total, our results suggest a rewiring of vitreous metabolism in diabetic retinopathy that underlies disease features such as oxidative stress and furthermore illustrates how the vitreous metabolic profile may be impacted by disease.
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