Changes In ATP Levels Research Articles

Intracellular Mg2+ concentrations are differentially regulated in the sperm head and mid-piece in acrosome reaction inducing conditions.

The sperm ability to fertilize involves the regulation of ATP levels. Because inside cells, ATP is complexed with Mg2+ ions, changes in ATP levels result in changes in intracellular Mg2+ concentration ([Mg2+]i), which can be followed using intracellular Mg2+ sensors such as Mag-520. In this work, we tested conditions known to decrease sperm ATP such as starvation and capacitation. As expected, in these conditions [Mg2+]i increased in all cell compartments. In contrast, when ATP increases, such as adding nutrients to starved sperm, [Mg2+]i significantly decreases in all compartments. On the other hand, when the acrosome reaction was induced, either with progesterone or with ionomycin, [Mg2+]i was differentially regulated in the head and mid-piece. While Mag-520 fluorescence increased in the sperm mid-piece, it decreased in the head. These changes were observed in capacitated as well as in starved sperm but not in sperm incubated in conditions that do not support capacitation. Changes in [Mg2+]i were still observed when the sperm were incubated in high extracellular Mg2+ suggesting that this decrease is not due to Mg2+ efflux. Interestingly, the progesterone and ionomycin effects on [Mg2+]i were abolished on sperm incubated in Ca2+-free media. Altogether, these results indicate that [Mg2+]i is regulated in sperm during capacitation and acrosomal reaction, and suggest that these measurements can serve to evaluate ATP levels in real time.

Optimization of T Cell Activation Protocols to Enrich Stem Cell Memory Phenotypes: Metabolic and Health Assays as Predictive Tools

Abstract The generation of T cells with a stem cell memory phenotype (T_SCM) holds significant promise for immunotherapy due to their longevity and potent antitumor activity. This study aimed to optimize T cell activation and expansion conditions to selectively enrich for the T_SCM population. Utilizing a suite of cell health and metabolism assays, we monitored T cell cultures to identify predictive markers of T_SCM enrichment. We observed that early activation steps correlated with a surge in ATP production and alterations in cell reducing potential, indicative of metabolic reprogramming. Subsequent activation phases were marked by an upregulation in both catabolic and anabolic pathways, as evidenced by the increased turnover of key metabolites including glucose, lactate, glutamine, glutamate, branched-chain amino acids (BCAA), and pyruvate. Notably, the rate of T cell activation influenced the composition of the resultant T cell populations, with the media composition further affecting these dynamics. Our findings suggest that metabolic assays, particularly those measuring changes in ATP levels and primary metabolite flux, can serve as effective early predictors of T_SCM enrichment. These insights may guide the development of more efficacious T cell-based immunotherapies by facilitating the targeted generation of T cells with a stem cell-like memory phenotype.

Cardiolipin-mediated temporal response to hydroquinone toxicity in human retinal pigmented epithelial cell line.

Hydroquinone, a potent toxic agent of cigarette smoke, damages retinal pigmented epithelial cells by triggering oxidative stress and mitochondrial dysfunction, two events causally related to the development and progression of retinal diseases. The inner mitochondrial membrane is enriched in cardiolipin, a phospholipid susceptible of oxidative modifications which determine cell-fate decision. Using ARPE-19 cell line as a model of retinal pigmented epithelium, we analyzed the potential involvement of cardiolipin in hydroquinone toxicity. Hydroquinone exposure caused an early concentration-dependent increase in mitochondrial reactive oxygen species, decrease in mitochondrial membrane potential, and rise in the rate of oxygen consumption not accompanied by changes in ATP levels. Despite mitochondrial impairment, cell viability was preserved. Hydroquinone induced cardiolipin translocation to the outer mitochondrial membrane, and an increase in the colocalization of the autophagosome adapter protein LC3 with mitochondria, indicating the induction of protective mitophagy. A prolonged hydroquinone treatment induced pyroptotic cell death by cardiolipin-mediated caspase-1 and gasdermin-D activation. Cardiolipin-specific antioxidants counteracted hydroquinone effects pointing out that cardiolipin can act as a mitochondrial "eat-me signal" or as a pyroptotic cell death trigger. Our results indicate that cardiolipin may act as a timer for the mitophagy to pyroptosis switch and propose cardiolipin-targeting compounds as promising approaches for the treatment of oxidative stress-related retinal diseases.

Modeling the heterogeneity of sodium and calcium homeostasis between cortical and hippocampal astrocytes and its impact on bioenergetics.

Emerging evidence indicates that neuronal activity-evoked changes in sodium concentration in astrocytes Na a represent a special form of excitability, which is tightly linked to all other major ions in the astrocyte and extracellular space, as well as to bioenergetics, neurotransmitter uptake, and neurovascular coupling. Recently, one of us reported that Na a transients in the neocortex have a significantly higher amplitude than those in the hippocampus. Based on the extensive data from that study, here we develop a detailed biophysical model to further understand the origin of this heterogeneity and how it affects bioenergetics in the astrocytes. In addition to closely fitting the observed experimental Na a changes under different conditions, our model shows that the heterogeneity in Na a signaling leads to substantial differences in the dynamics of astrocytic Ca2+ signals in the two brain regions, and leaves cortical astrocytes more susceptible to Na+ and Ca2+ overload under metabolic stress. The model also predicts that activity-evoked Na a transients result in significantly larger ATP consumption in cortical astrocytes than in the hippocampus. The difference in ATP consumption is mainly due to the different expression levels of NMDA receptors in the two regions. We confirm predictions from our model experimentally by fluorescence-based measurement of glutamate-induced changes in ATP levels in neocortical and hippocampal astrocytes in the absence and presence of the NMDA receptor's antagonist (2R)-amino-5-phosphonovaleric acid.

Dual-site fluorescent probe for bioimaging of upregulated adenosine triphosphate under the stresses of endogenous and exogenous H2S

To provide new insights in the way of the potential interaction between biological small molecules, it is essential to develop molecular tools that are capable of tracking two related biomolecules at the cellular level. Therefore, we successfully designed and synthesized a novel dual-site turn on fluorescent probe that could simultaneously detect fluctuations of H2S and ATP concentrations in two separate fluorescence channels without spectral crosstalk. Taking advantage of fluorescence imaging technology, we visualized the dynamic changes of ATP levels under the stimulation of exogenous hydrogen sulfide and cysteine. We anticipate that NT-RD can be used to further investigate the relationship between H2S and ATP in the processes of inhibiting or stimulating tumor growth.

Metabolic adaptation to the chronic loss of Ca2+ signaling induced by KO of IP3 receptors or the mitochondrial Ca2+ uniporter

Calcium signaling is essential for regulating many biological processes. Endoplasmic reticulum inositol trisphosphate receptors (IP3Rs) and the mitochondrial Ca2+ uniporter (MCU) are key proteins that regulate intracellular Ca2+ concentration. Mitochondrial Ca2+ accumulation activates Ca2+-sensitive dehydrogenases of the tricarboxylic acid (TCA) cycle that maintain the biosynthetic and bioenergetic needs of both normal and cancer cells. However, the interplay between calcium signaling and metabolism is not well understood. In this study, we used human cancer cell lines (HEK293 and HeLa) with stable KOs of all three IP3R isoforms (triple KO [TKO]) or MCU to examine metabolic and bioenergetic responses to the chronic loss of cytosolic and/or mitochondrial Ca2+ signaling. Our results show that TKO cells (exhibiting total loss of Ca2+ signaling) are viable, displaying a lower proliferation and oxygen consumption rate, with no significant changes in ATP levels, even when made to rely solely on the TCA cycle for energy production. MCU KO cells also maintained normal ATP levels but showed increased proliferation, oxygen consumption, and metabolism of both glucose and glutamine. However, MCU KO cells were unable to maintain ATP levels and died when relying solely on the TCA cycle for energy. We conclude that constitutive Ca2+ signaling is dispensable for the bioenergetic needs of both IP3R TKO and MCU KO human cancer cells, likely because of adequate basal glycolytic and TCA cycle flux. However, in MCU KO cells, the higher energy expenditure associated with increased proliferation and oxygen consumption makes these cells more prone to bioenergetic failure under conditions of metabolic stress.

PD06-10 URINARY ATP LEVELS: A POTENTIAL BIOMARKER OF OVERACTIVE BLADDER TREATMENT RESPONSE FOLLOWING ONABOTULINUM TOXIN-A INJECTIONS

You have accessJournal of UrologyUrodynamics/Lower Urinary Tract Dysfunction/Female Pelvic Medicine: Female Incontinence: Therapy I (PD06)1 Sep 2021PD06-10 URINARY ATP LEVELS: A POTENTIAL BIOMARKER OF OVERACTIVE BLADDER TREATMENT RESPONSE FOLLOWING ONABOTULINUM TOXIN-A INJECTIONS Samantha Freeman, Laura Tellechea, Ilir Agalliu, Melissa Laudano, Nitya Abraham, and Sylvia Suadicani Samantha FreemanSamantha Freeman More articles by this author , Laura TellecheaLaura Tellechea More articles by this author , Ilir AgalliuIlir Agalliu More articles by this author , Melissa LaudanoMelissa Laudano More articles by this author , Nitya AbrahamNitya Abraham More articles by this author , and Sylvia SuadicaniSylvia Suadicani More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000001974.10AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Overactive bladder (OAB) is diagnosed clinically based on symptoms. However, a biomarker may help provide objective measurement of disease severity and improvement after treatment. ATP is released from the urothelium and levels tend to be higher in animal models and patients with OAB. Bladder onabotulinum toxin-A (BTX-A) injections decrease stretch-induced ATP release in animal models, yet little is known about the effect of BTX-A on human urine ATP levels. The objective of this study is to quantify changes in voided urine ATP levels and correlate with OAB-V8 symptom improvement in women treated with BTX-A injection. METHODS: Urine samples were collected and frozen on dry ice from women prior to and after 100 units of BTX-A treatment. Urine ATP levels were quantified using the luciferin-luciferase luminescent assay. OAB-V8 questionnaires were administered at time of urine collection. Women with positive urine cultures or neurogenic bladder were excluded. Degree of OAB-V8 score improvement was used to categorize participants: non-responders (change<8), mild responders (change 8-16), moderate responders (change 17-24), and marked responders (change>24). Percent change in ATP level was compared based on OAB-V8 score improvement and statistical significance was determined using the Mann-Whitney test. RESULTS: Urine was collected from 23 female patients (OAB-V8 scores 2 to 40); 7 were excluded. Median age was 53.5 years (49.5-61.5). 8 patients had chronic pain disorders, 2 patients had Type 2 diabetes, and 6 patients had prior pelvic surgery. After BTX-A injections, responders had significantly lower median ATP levels compared to non-responders at 2 weeks (p=0.0268). When patients were classified according to OAB-V8 score improvement (non-responder, mild, moderate, marked responders), the improvement directly correlated with a decrease in ATP levels (Figure 1). CONCLUSIONS: Decreases in urine ATP levels after BTX-A injections were associated with clinical improvement in OAB symptoms. This may be the result of a direct or indirect effect of BTX-A on mechanisms of urothelial ATP release and signaling. Quantification of urine ATP may provide a valuable tool to assess treatment response and guide further management. Source of Funding: N/A © 2021 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 206Issue Supplement 3September 2021Page: e103-e104 Advertisement Copyright & Permissions© 2021 by American Urological Association Education and Research, Inc.MetricsAuthor Information Samantha Freeman More articles by this author Laura Tellechea More articles by this author Ilir Agalliu More articles by this author Melissa Laudano More articles by this author Nitya Abraham More articles by this author Sylvia Suadicani More articles by this author Expand All Advertisement Loading ...

RETRACTED ARTICLE: ATP level in red blood cells improves by altering the low-level DPSS laser irradiation condition

The effects of DPSS laser irradiating stored blood at 405, 589, and 670 nm on the ATP levels in human red blood cells have been studied in comparison. Human blood samples were taken from healthy donors’ whole blood and stored. A DPSS laser with different wavelengths was used as a radiation source, with an output power of 10 mW and a fluence of 70 J/cm2 per irradiated sample. The CellTiter-GloLuminescent Assay kit (Promega) was used to determine ATP levels in RBCs. The blood was kept for a maximum of 20 days, and samples were evaluated on days 0, 5, 10, 15, and 20 before and after irradiation. The red blood cell ATP level was calculated by optical density using an ELISA reader to test the emission intensity. No significant differences in RBCs counts were found between pre- and post-irradiated cells at various storage periods with different laser wavelengths. In contrast, at a laser wavelength of 589 nm with storage times of 5, 10, 15, and 20 days, the RBCs had more significant differences (P < 0.05, P < 0.05, P < 0.03, P < 0.02, respectively) between pre- and post-irradiated cells. Moreover, various DPSS lasers were applied and were found to induce significant changes in ATP levels (P < 0.0003, P < 0.04, and P < 0.01, respectively). The highest increase in the ATP level (4.9%) was observed when the laser dose of 70 J/cm2 was delivered at a 589 nm wavelength. Our results clearly demonstrate that red blood cell radiation with low-power lasers of 405, 589, and 670 nm increases the ATP level in RBCs compared with their non-irradiated counterparts at various storage times. This, it is suggested that laser DPSS can cause structural transformations of the red blood cell membrane or components of the membrane.

Mitochondrial destiny in type 2 diabetes: the effects of oxidative stress on the dynamics and biogenesis of mitochondria.

BackgroundOne reason for the development of insulin resistance is the chronic inflammation in obesity.Materials & MethodsScientific articles in the field of knowledge on the involvement of mitochondria and mitochondrial DNA (mtDNA) in obesity and type 2 diabetes were analyzed.ResultsOxidative stress developed during obesity contributes to the formation of peroxynitrite, which causes cytochrome C-related damage in the mitochondrial electron transfer chain and increases the production of reactive oxygen species (ROS), which is associated with the development of type 2 diabetes. Oxidative stress contributes to the nuclease activity of the mitochondrial matrix, which leads to the accumulation of cleaved fragments and an increase in heteroplasmy. Mitochondrial dysfunction and mtDNA variations during insulin resistance may be connected with a change in ATP levels, generation of ROS, mitochondrial division/fusion and mitophagy. This review discusses the main role of mitochondria in the development of insulin resistance, which leads to pathological processes in insulin-dependent tissues, and considers potential therapeutic directions based on the modulation of mitochondrial biogenesis. In this regard, the development of drugs aimed at the regulation of these processes is gaining attention.ConclusionChanges in the mtDNA copy number can help to protect mitochondria from severe damage during conditions of increased oxidative stress. Mitochondrial proteome studies are conducted to search for potential therapeutic targets. The use of mitochondrial peptides encoded by mtDNA also represents a promising new approach to therapy.

Connecting the microbiome to host metabolites: understanding how the microbiome controls immune activity in birds

The gastrointestinal (GI) microbiome plays an important role in the development and function not only of the GI, but also the immune system. Products such as probiotics represent promising treatments for improving animal health through the GI, but their modes of action are largely still unknown. Previous research in our laboratory has demonstrated supplementation with a lactic acid bacteria probiotic product alters host energy partitioning in immune tissues, including increased ATP production and consumption in circulating leukocytes; and was associated with a more rapid antibody response to antigen. To better understand the communication between probiotics and the immune system our laboratory has focused on characterizing how supplementation affects the microbiome and host systems. We hypothesized that the changes previously observed in immune tissue activity and energy metabolism were regulated by a probiotic‐stimulated factor in the serum. We examined the ability of serum isolated from probiotic‐fed animals to augment ATP production in vitro. Chicken lymphocyte cell lines were cultured for 4 days in media supplemented with serum from treated and control birds. Cells cultured in serum from probiotic‐fed birds had higher levels of ATP (P &lt; 0.05) compared to controls. Transcriptomic analysis of these cells suggest an increase in genes associated with cell survival and differentiation, and signaling via TGF‐β, IFN‐γ, IL‐7, and IL‐1β. Analysis of the metabolites in serum and digesta from these animals identified several metabolite changes correlate with the change in ATP levels in lymphocytes and putative changes in pro‐ and anti‐inflammatory cytokine production.

Regulation of TRPC5 currents by intracellular ATP: Single channel studies.

TRPC5 channels are nonselective cation channels activated by G-protein-coupled receptors. It was previously found that recombinant TRPC5 currents are inhibited by intracellular ATP, when studied by whole-cell patch-clamp recording. In the present study, we investigated the mechanism of ATP inhibition at the single-channel levelusing patches from HEK-293 cells transiently transfected with TRPC5 and the M1 muscarinic receptor. In inside-out patches, application of ATP to the intracellular face of the membrane reduced TRPC5 channel activity at both positive and negative potentials without affecting the unitary current amplitude or open dwell time of the channel. The effect of ATP was rapidly reversible. These results suggest that ATP may bind to the channel protein and affect the ability of the channel to open or to remain in an open, nondesensitized state. The activity of TRPC5 channels may be influenced by cellular metabolism via changes in ATP levels.

Novel Evidence that Purinergic Signaling - Nlrp3 Inflammasome Axis Regulates Circadian Rhythm of Hematopoietic Stem/Progenitor Cells Circulation in Peripheral Blood

We found that circadian changes in ATP level in peripheral blood (PB) activate the Nlrp3 inflammasome, which triggers diurnal release of hematopoietic stem/progenitor cells (HSPCs) from murine bone marrow (BM) into PB. Consistent with this finding, we observed circadian changes in expression of mRNA for Nlrp3 inflammasome-related genes, including Nlrp3, caspase 1, IL-1β, IL-18, gasdermin (GSDMD), HMGB1, and S100A9. Circadian release of HSPCs from BM into PB as well as expression of Nlrp3-associated genes was decreased in mice in which pannexin 1-mediated secretion of ATP was inhibited by the blocking peptide 10Panx and in animals exposed to the specific small-molecule inhibitor of the Nlrp3 inflammasome MCC950. In addition to HSPCs, a similar decrease in diurnal cell counts was observed for mesenchymal stromal cells (MSCs), endothelial progenitor cells (EPCs), and very small embryonic-like stem cells (VSELs). These results shed more light on the complexity of circadian regulation of HSPC release into PB, which is coordinated in a purinergic signaling-, innate immunity-dependent manner. Moreover, in addition to circadian changes in expression of the Nlrp3 inflammasome we also observed diurnal changes in expression of other inflammasomes, including Aim2, Nrp1a, and Nlrp1b.

Mode-of-action profiling reveals glutamine synthetase as a collateral metabolic vulnerability of M. tuberculosis to bedaquiline

Combination chemotherapy can increase treatment efficacy and suppress drug resistance. Knowledge of how to engineer rational, mechanism-based drug combinations, however, remains lacking. Although studies of drug activity have historically focused on the primary drug-target interaction, growing evidence has emphasized the importance of the subsequent consequences of this interaction. Bedaquiline (BDQ) is the first new drug for tuberculosis (TB) approved in more than 40 y, and a species-selective inhibitor of the Mycobacterium tuberculosis (Mtb) ATP synthase. Curiously, BDQ-mediated killing of Mtb lags significantly behind its inhibition of ATP synthase, indicating a mode of action more complex than the isolated reduction of ATP pools. Here, we report that BDQ-mediated inhibition of Mtb's ATP synthase triggers a complex metabolic response indicative of a specific hierarchy of ATP-dependent reactions. We identify glutamine synthetase (GS) as an enzyme whose activity is most responsive to changes in ATP levels. Chemical supplementation with exogenous glutamine failed to affect BDQ's antimycobacterial activity. However, further inhibition of Mtb's GS synergized with and accelerated the onset of BDQ-mediated killing, identifying Mtb's glutamine synthetase as a collateral, rather than directly antimycobacterial, metabolic vulnerability of BDQ. These findings reveal a previously unappreciated physiologic specificity of ATP and a facet of mode-of-action biology we term collateral vulnerability, knowledge of which has the potential to inform the development of rational, mechanism-based drug combinations.

Design and Application of A Bioluminescent Biosensor for Detection Of Toxicity Using Huh7-CMV-Luc Cell Line.

Cell-based biosensors (CBBs) are becoming important tools for biosecurity a lications and rapid diagnostics in food microbiology for their unique capability of detecting hazardous materials. Pollutants, such as heavy metals and chemicals, are now considered as a global threat and are associated with detrimental health outcomes. Fast and accurate detection of pollutants is essential to reduce these threats. In this study, the enhancer sequence of human cytomegalovirus (hCMV) IE genes cloned upstream of luc gene in PGL4.26 plasmid, in order to increase basal luciferase activity. This recombinant vector was transfected into Huh7 cell line and after 21 days of treatment with Hygromycin B selectable marker, stable cell line was generated. After several passages, cells containing this vector showed high luciferase activity in normal conditions without any induction following to overexpression of luc gene. Huh7-CMV-luc cell line was able to detect the slightest changes in ATP level, due to the effect of different toxins on the cell which disrupt cellular respiration and ATP production processes. In order to investigate the sensitivity of the cell line, the cells were incubated with 0.1-10 μM of chemical toxins affecting ATP production. These toxins affect luciferase activity in a dose dependent manner, with maximal sensitivity approximately about 0.2 μM to toxin concentrations. Additionally, this biosensor provided a rapid detection as early as 4 h in response to the toxicants. Whole cell biosensors like huh7-CMV-luc cell line can be considered as a powerful tool for the sensitive and efficient monitoring of general toxins, drugs, and environmental pollutants.

Dissecting the regulation and function of ATP at the single-cell level.

Regulation of cellular ATP level is critical for diverse biological processes and may be defective in diseases such as cancer and mitochondrial disorders. While mitochondria play critical roles in ATP level regulation, we still lack a systematic and quantitative picture of how individual mitochondrial-related genes contribute to cellular ATP level and how dysregulated ATP levels may affect downstream cellular processes. Advances in genetically encoded ATP biosensors have provided new opportunities for addressing these issues. ATP biosensors allow researchers to quantify the changes of ATP levels in real time at the single-cell level and characterize corresponding effects at the cellular, tissue, and organismal level. Along this direction, several recent single-cell studies using ATP biosensors, including the work by Mendelsohn and colleagues, have started to uncover the principles for how genetic and nongenetic parameters may modulate ATP levels to affect cellular functions and human health.

The Effects of Hypoxia and Inflammation on Claudin‐1 in CaCo‐2 Cells as a Model Intestinal Epithelium.

BackgroundThe use of hydrocortisone in association with indomethacin is a known risk factor for spontaneous intestinal perforation in extremely low birthweight infants. Hypoxia, inflammation and non‐steroidal anti‐inflammatory drugs independently alter apical junctional complexes of epithelial cells. We are interested in understanding the combined effects of hypoxia, inflammation and non‐steroidal anti‐inflammatory drugs on the apical junctional complex and barrier integrity in a gastrointestinal epithelial cell model.MethodsThrough the use of CaCo‐2 BBe monolayers, an established in vitro model of the intestinal epithelium, we analyzed the effects of the pro‐inflammatory agents (TNF and IFN‐gamma) and anti‐inflammatory agents (indomethacin and hydrocortisone) on junctional protein expression and localization, cell proliferation using Edu labeling, cellular ATP concentration and barrier function.ResultsProtein expression analyzed by Western blotting techniques demonstrated an alteration of claudin‐1, a tight junction sealing protein. The soluble and insoluble fractions of the cells were analyzed in order to determine the location of these proteins. We determined inflammatory conditions produced a higher concentration in the soluble fraction indicating impaired function. Confocal analysis after 24‐hour exposure to hypoxia showed that the combined use of hypoxia and inflammatory agents elicited no significant change in cell proliferation levels. Cellular ATP levels were analyzed to corroborate proliferation studies. Confocal imaging under the same experimental conditions was also conducted in order to probe for claudin‐1 localization within the cells.ConclusionsThere was no observed significant difference in the cell proliferation rate or marked changes in ATP levels in our model. Therefore, we hypothesize that disturbance in barrier function may be related to specific junctional mechanisms. We found claudin‐1 protein expression and localization to be sensitive to both hypoxia and inflammatory cytokines contributing to impaired barrier integrity. These cellular studies suggest claudin‐1 stability may be a potential target in the progression of spontaneous intestinal perforation.Disclosure: The view(s) expressed herein are those of the author(s) and do not reflect the official policy or position of Brooke Army Medical Center, the U.S. Army Medical Department, the U.S. Army Office of the Surgeon General, the Department of the Air Force and Army, Department of Defense or the U.S. Government.Support or Funding InformationSan Antonio Medical Foundation Grant, Trinity University, US Air Force 59th Medical Wing Clinical Research WingThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

HSP90 Facilitates Oncogene-Induced Metabolic Reprogramming in B-Cell Lymphomas

The chaperone HSP90 is used by B-cell lymphomas to support the stability of proteins involved in oncogenic processes such as signaling and anti-apoptosis. While HSP90 inhibitors decrease the levels of these client proteins favoring cell death they also prompt cellular counter-regulatory mechanisms that diminish the efficacy of these drugs. Improving the clinical activity of HSP90 inhibitors will depend on understanding the complexity of HSP90 functions. Here we show that HSP90 facilitates the function of MYC by improving the efficiency of metabolic pathways through the orchestration of enzymatic networks, and that HSP90 inhibition impairs the metabolic fitness of DLBCLs without client protein degradation. Moreover, drugs inducing sub-lethal metabolic stress in DLBCL cells cause apoptosis upon HSP90 inhibition.To identify metabolic enzymes actively chaperoned by HSP90 we integrated the information from proteomics and metabolomics in DLBCL cell lines. Proteomics was performed from the cytoplasmic fraction of OCI-Ly1 and OCI-Ly7 cells chemically precipitated with PU-H71, an HSP90 inhibitor that selectively binds to HSP90 contained in active multi-chaperone complexes.STRING network analysis of the metabolic client proteins identified several hubs highly enriched for enzymes involved in metabolism of nucleotides (e.g. IMPDH2, CTPS1, CAD), carbohydrates (e.g. G6PD, HK2) and proteins (e.g. MTHFR, ASNS). Functionality of the network was assessed by metabolomics from OCI-Ly1 cells treated with PU-H71 500 nM for 6 h (sub-lethal). This dose and timing assured HSP90 inhibition but no client protein degradation. The proteomics and metabolomics mapping into KEGG pathways showed a significant overlap, indicating that HSP90 preferentially interacts with proteins representing regulatory hubs to coordinate their committed activity and thus secure the flow of the pathway.We quantified the effect of HSP90 on the activity of metabolic networks by measuring glycolysis (by lactate production and medium acidification) and mitochondrial respiration (by oxygen consumption) in OCI-LY1 and OCI-LY7 cell lines upon PU-H71. We found that inhibition of HSP90 decreased glycolysis by 20-25% and respiration by 25% (p<0.01 for both). There were no changes in ATP levels. Given that in proliferating cells respiration serves intermediates for crucial anabolic roles, we assessed nucleotides and protein syntheses by using uridine and methionine analogs, respectively. We found that inhibition of HSP90 decreases biosynthesis of nucleotides by 10-20% and proteins by 20-30% (p<0.05 for both). Altogether these results suggest that HSP90 contributes to the channeling of metabolic intermediates into the mitochondria and from there to critical biosynthetic pathways. Further supporting this notion, we found HSP90 inhibition caused an increase in the number of the so-called “rods and rings”, enzyme assemblies composed of CTPS1 and IMPDH2, two key enzymes in the synthesis of GTP and CTP. These enzymatic “polymers” form under metabolic stress conditions to increase the cell's nucleotides biosynthetic efficiency.To understand the mechanistic relevance of these findings to lymphomagenesis, we analyzed the HSP90 metabolic proteome for common features and found it was significantly enriched (chi-square p<0.0001) for MYC target genes. Moreover, it correlated to MYC expression in DLBCL and Burkitt lymphoma (BL) cell lines and patient samples. Chemical precipitation of active HSP90 in BL and DLBCL patient samples showed that HSP90 does not interact directly with MYC but with enzymes that are MYC target genes such as CTPS1 and CAD, in agreement with HSP90 supporting MYC oncogenesis by improving the efficiency of metabolic networks. Remarkably, the expression of MYC (by IHC) in 18 DLBCL tumors associated with only nucleotides (e.g. IMP) and amino acids (e.g. glutamine) (by intracellular metabolomics), which are more reliant on HSP90 as we showed before. We capitalized on this by xenografting a MYC-amplified DLBCL cell line into 20 mice and treating them with the IMPDH2 inhibitor MMF to induce nucleotides stress in presence or absence of PU-H71. We found that under HSP90 inhibition, MMF significantly decreased lymphoma volume better than each drug alone (p<0.001). In sum, we describe a novel function of HSP90 in establishing higher efficiency anabolic networks to support the metabolic stress imposed by MYC in lymphomas. DisclosuresYang:Regeneron Pharmaceuticals: Employment. Cerchietti:Lymphoma Research Foundation: Research Funding; Leukemia and Lymphoma Society: Research Funding; Weill Cornell Medicine - New York Presbyterian Hospital: Employment; Celgene: Research Funding.

The in vitro comparative study of the effect of BPA, BPS, BPF and BPAF on human erythrocyte membrane; perturbations in membrane fluidity, alterations in conformational state and damage to proteins, changes in ATP level and Na+/K+ ATPase and AChE activities

Bisphenols are massively used in the industry, and thus the exposure of biota including humans to these substances has been noted. In this study we have assessed the effect of BPA and its selected analogs, i.e. BPS, BPF and BPAF on membrane of human red blood cells, which is the first barrier that must be overcome by xenobiotics penetrating the cell, and is commonly utilized as a model in the investigation of the effect of different xenobiotics on various cell types. Red blood cells were incubated with BPA and its analogs in the concentrations ranging from 0.1 to 250 μg/ml for 4 h and 24 h. We have noted that the compounds studied altered membrane fluidity at its hydrophobic region, increased internal viscosity and osmotic fragility of the erythrocytes and altered conformational state of membrane proteins. Moreover, bisphenols examined increased thiol groups level, caused oxidative damage to membrane proteins, decreased ATP level, depleted the activity of Na+/K + ATPase and changed the activity of AChE in human red blood cells. It has been shown that the strongest changes were noted in cells treated with BPAF, while BPS caused the weakest (or none) alterations in the parameters studied.

Monitoring ATP dynamics in electrically active white matter tracts.

In several neurodegenerative diseases and myelin disorders, the degeneration profiles of myelinated axons are compatible with underlying energy deficits. However, it is presently impossible to measure selectively axonal ATP levels in the electrically active nervous system. We combined transgenic expression of an ATP-sensor in neurons of mice with confocal FRET imaging and electrophysiological recordings of acutely isolated optic nerves. This allowed us to monitor dynamic changes and activity-dependent axonal ATP homeostasis at the cellular level and in real time. We find that changes in ATP levels correlate well with compound action potentials. However, this correlation is disrupted when metabolism of lactate is inhibited, suggesting that axonal glycolysis products are not sufficient to maintain mitochondrial energy metabolism of electrically active axons. The combined monitoring of cellular ATP and electrical activity is a novel tool to study neuronal and glial energy metabolism in normal physiology and in models of neurodegenerative disorders.

Mild Hypothermia Attenuates the Anesthetic Isoflurane-Induced Cytotoxicity.

The commonly used inhalation anesthetic isoflurane has been reported to induce DNA damage and cytotoxicity. However, the methods to attenuate these effects remain largely to be determined. Mild hypothermia has neuroprotective effects. We therefore set out to assess whether mild hypothermia could protect the isoflurane-induced DNA damage and cytotoxicity. Moreover, we investigated the underlying mechanisms by assessing the effects of mild hypothermia on the isoflurane-induced changes in ATP levels. H4 human neuroglioma cells were treated with 2% isoflurane for 3 or 6 h with and without mild hypothermia (35°C). We assessed the cell viability by using 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) and lactate dehydrogenase (LDH) assay. We determined DNA damage by measuring levels of phosphorylation of the histone protein H2A variant X at Ser139 (γH2A.X), the marker of DNA damage. We also measured ATP levels in the cells. Here we showed that the treatment with 2% isoflurane for 6 h induced cytotoxicity and DNA damage in the cells. Moreover, the treatment with 2% isoflurane for 3 h decreased ATP levels without inducing cytotoxicity. Mild hypothermia attenuated the isoflurane-induced cytotoxicity, DNA damage, and ATP reduction in the cells. Taken together, these data suggest that the isoflurane-induced reduction in ATP levels occurred before the isoflurane-induced cytotoxicity. Isoflurane may induce DNA damage and cause cytotoxicity through reducing ATP levels. Mild hypothermia would ameliorate isoflurane-induced DNA damage and cytotoxicity by attenuating the isoflurane-induced reduction in ATP levels. These pilot studies have established a system and will promote the future investigations of anesthesia neurotoxicity.