Rate Of Phosphorylation Research Articles

Longitudinal mitochondrial bioenergetic signatures of blood monocytes and lymphocytes improve during treatment of drug-susceptible pulmonary tuberculosis patients Monocyte/lymphocyte bioenergetic signatures post-TB treatment

The impact of human pulmonary tuberculosis (TB) on the bioenergetic metabolism of circulating immune cells remains elusive, as does the resolution of these effects with TB treatment. In this study, the rates of oxidative phosphorylation (OXPHOS) and glycolysis in circulating lymphocytes and monocytes of patients with drug-susceptible TB at diagnosis, 2 months, and 6 months during treatment, and 12 months after diagnosis were investigated using extracellular flux analysis. At diagnosis, the bioenergetic parameters of both blood lymphocytes and monocytes of TB patients were severely impaired in comparison to non-TB and non-HIV-infected controls. However, most bioenergetic parameters were not affected by HIV status or glycemic index. Treatment of TB patients restored the % spare respiratory capacity (%SRC) of the circulating lymphocytes to that observed in non-TB and non-HIV infected controls by 12 months. Treatment also improved the maximal respiration of circulating lymphocytes and the %SRC of circulating monocytes of the TB patients. Notably, the differential correlation of the clinical and bioenergetic parameters of the monocytes and lymphocytes from the controls and TB patients at baseline and month 12 was consistent with improved metabolic health and resolution of inflammation following successful TB treatment. Network analysis of the bioenergetic parameters of circulating immune cells with serum cytokine levels indicated a highly coordinated immune response at month 6. These findings underscore the importance of metabolic health in combating TB, supporting the need for further investigation of the bioenergetic immunometabolism associated with TB infection for novel therapeutic approaches aimed at bolstering cellular energetics to enhance immune responses and expedite recovery in TB patients.

Combined transcriptome and proteome profiling reveal cell-type-specific functions of Drosophila garland and pericardial nephrocytes

Drosophila nephrocytes are specialised cells that share critical functional, morphological, and molecular features with mammalian podocytes. Accordingly, nephrocytes represent a preferred invertebrate model for human glomerular disease. Here, we established a method for cell-specific isolation of the two types of Drosophila nephrocytes, garland and pericardial cells, from animals of different developmental stages and ages. Mass spectrometry-based proteomics and RNA-Seq-based transcriptomics were applied to characterise the proteome and transcriptome of the respective cells in an integrated and complementary manner. We observed characteristic changes in the proteome and transcriptome due to cellular ageing. Furthermore, functional enrichment analyses suggested that larval and adult nephrocytes, as well as garland and pericardial nephrocytes, fulfil distinct physiological functions. In addition, the pericardial nephrocytes were characterised by transcriptomic and proteomic profiles suggesting an atypical energy metabolism with very low oxidative phosphorylation rates. Moreover, the nephrocytes displayed typical signatures of extensive immune signalling and showed an active antimicrobial response to an infection. Factor-specific comparisons identified novel candidate proteins either expressed and secreted by the nephrocytes or sequestered by them. The data generated in this study represent a valuable basis for a more specific application of the Drosophila model in analysing renal cell function in health and disease.

Targeting Oxidative Phosphorylation with a Novel Thiophene Carboxamide Increases the Efficacy of Imatinib against Leukemic Stem Cells in Chronic Myeloid Leukemia

Patients with chronic myeloid leukemia (CML) respond to tyrosine kinase inhibitors (TKIs); however, CML leukemic stem cells (LSCs) exhibit BCR::ABL kinase-independent growth and are insensitive to TKIs, leading to disease relapse. To prevent this, new therapies targeting CML-LSCs are needed. Rates of mitochondria-mediated oxidative phosphorylation (OXPHOS) in CD34+CML cells within the primitive CML cell population are higher than those in normal undifferentiated hematopoietic cells; therefore, the inhibition of OXPHOS in CML-LSCs may be a potential cure for CML. NK-128 (C33H61NO5S) is a structurally simplified analog of JCI-20679, the design of which was based on annonaceous acetogenins. NK-128 exhibits antitumor activity against glioblastoma and human colon cancer cells by inhibiting OXPHOS and activating AMP-activated protein kinase (AMPK). Here, we demonstrate that NK-128 effectively suppresses the growth of CML cell lines and that the combination of imatinib and NK-128 is more potent than either alone in a CML xenograft mouse model. We also found that NK-128 inhibits colony formation by CD34+ CML cells isolated from the bone marrow of untreated CML patients. Taken together, these findings suggest that targeting OXPHOS is a beneficial approach to eliminating CML-LSCs, and may improve the treatment of CML.

Quantitative proteomics and phosphoproteomics profiling of meiotic divisions in the fission yeast Schizosaccharomyces pombe

In eukaryotes, chromosomal DNA is equally distributed to daughter cells during mitosis, whereas the number of chromosomes is halved during meiosis. Despite considerable progress in understanding the molecular mechanisms that regulate mitosis, there is currently a lack of complete understanding of the molecular mechanisms regulating meiosis. Here, we took advantage of the fission yeast Schizosaccharomyces pombe, for which highly synchronous meiosis can be induced, and performed quantitative proteomics and phosphoproteomics analyses to track changes in protein expression and phosphorylation during meiotic divisions. We compared the proteomes and phosphoproteomes of exponentially growing mitotic cells with cells harvested around meiosis I, or meiosis II in strains bearing either the temperature-sensitive pat1-114 allele or conditional ATP analog-sensitive pat1-as2 allele of the Pat1 kinase. Comparing pat1-114 with pat1-as2 also allowed us to investigate the impact of elevated temperature (25 °C versus 34 °C) on meiosis, an issue that sexually reproducing organisms face due to climate change. Using TMTpro 18plex labeling and phosphopeptide enrichment strategies, we performed quantification of a total of 4673 proteins and 7172 phosphosites in S. pombe. We found that the protein level of 2680 proteins and the rate of phosphorylation of 4005 phosphosites significantly changed during progression of S. pombe cells through meiosis. The proteins exhibiting changes in expression and phosphorylation during meiotic divisions were represented mainly by those involved in the meiotic cell cycle, meiotic recombination, meiotic nuclear division, meiosis I, centromere clustering, microtubule cytoskeleton organization, ascospore formation, organonitrogen compound biosynthetic process, carboxylic acid metabolic process, gene expression, and ncRNA processing, among others. In summary, our findings provide global overview of changes in the levels and phosphorylation of proteins during progression of S. pombe cells through meiosis at normal and elevated temperatures, laying the groundwork for further elucidation of the functions and importance of specific proteins and their phosphorylation in regulating meiotic divisions in this yeast.

Nucleoside Scaffolds and Carborane Clusters for Boron Neutron Capture Therapy: Developments and Future Perspective.

Nucleosides containing carboranes are one of the most important boron delivery agents for boron neutron capture therapy, BNCT, which are good substrates of hTK1. The development of several nucleosides containing carboranes at early stages led to the discovery of the first generation of 3CTAs by incorporating a hydrocarbon spacer between the thymidine scaffold and carborane cluster and attaching dihydroxylpropyl group on the second carbon (C2) atom of the carborane cluster (e.g., N5 and N5-2OH). Phosphorylation rate, tumor cellular uptake, and retention have been evaluated in parallel to change the length of the tether arm of spacers in these compounds. Many attempts were reported and discussed to overcome the disadvantage of the first generation of 3CTAs by a) incorporating modified spacers between thymidine and carborane clusters, such as ethyleneoxide, polyhydroxyl, triazole, and tetrazole units, b) attaching hydrophilic groups at C2 of the carborane cluster, c) transforming lipophilic closo-carboranes to hydrophilic nidocarborane. The previous modifications represented the second generation of 3CTAs to improve the hydrogen bond formation with the hTK1 active site. Moreover, amino acid prodrugs were developed to enhance biological and physicochemical properties. The structure-activity relationship (SAR) of carboranyl thymidine analogues led to the roadmap for the development of the 3rd generation of the 3CTAs for BNCT.

The LAT Rheostat as a Regulator of Megakaryocyte Activation.

Specifically positioned negatively charged residues within the cytoplasmic domain of the adaptor protein, linker for the activation of T cells (LAT), have been shown to be important for efficient phosphorylation of tyrosine residues that function to recruit cytosolic proteins downstream of immunoreceptor tyrosine-based activation motif (ITAM) receptor signaling. LAT tyrosine 132-the binding site for PLC-γ2-is a notable exception, preceded instead by a glycine, making it a relatively poor substrate for phosphorylation. Mutating Gly131 to an acidic residue has been shown in T cells to enhance ITAM-linked receptor-mediated signaling. Whether this is generally true in other cell types is not known. To examine whether LAT Gly131 restricts ITAM signaling in cells of the megakaryocyte lineage, we introduced an aspartic acid at this position in human induced pluripotent stem cells (iPSCs), differentiated them into megakaryocytes, and examined its functional consequences. iPSCs expressing G131D LAT differentiated and matured into megakaryocytes normally, but exhibited markedly enhanced reactivity to glycoprotein VI (GPVI)-agonist stimulation. The rate and extent of LAT Tyr132 and PLC-γ2 phosphorylation, and proplatelet formation on GPVI-reactive substrates, were also enhanced. These data demonstrate that a glycine residue at the -1 position of LAT Tyr132 functions as a kinetic bottleneck to restrain Tyr132 phosphorylation and signaling downstream of ITAM receptor engagement in the megakaryocyte lineage. These findings may have translational applications in the burgeoning field of in vitro platelet bioengineering.

ATM kinase phosphorylates Ser15 of p53 in a pH-dependent manner

The phosphorylation of Ser15 in the transactivation domain (TAD) of the tumor suppressor protein 53 (p53) by ataxia-telangiectasia mutated (ATM) kinase is a crucial step in the tumor suppressor function of p53. An understanding of the factors that affect the rate of Ser15 phosphorylation may provide new strategies for the manipulation of the ATM-p53 pathway in cancer therapy. In this study, the effect of electrostatic interactions between ATM and p53 was investigated by measuring the phosphorylation of Ser15 at varying pH ranges from 5 to 9. To achieve this, two different kinase assay methods were utilized: the ELISA technique, which directly quantifies the phosphorylated Ser15, and the Universal Kinase Assay, which assesses the formation of ADP. The results revealed that Ser15 phosphorylation was pH-dependent, with higher phosphorylation rates observed in the alkaline range. To ascertain whether the lower phosphorylation rates observed at acidic pH were due to protein denaturation, a pH-dependent solubility profile was generated using the CamSol server. The obtained results demonstrated comparable solubility rates within the pH range of the kinase assays performed. Furthermore, the significance of negatively charged residues in TAD1-39 was evaluated by substituting Asp and Glu residues with hydrophobic and uncharged hydrophilic residues in TAD1-39 using ChimeraX and subsequently comparing their interactions with the ATM using the protein-protein docking server HADDOCK2.4. The results of the docking simulations indicated that the alteration of negatively charged residues with uncharged ones resulted in a reduction in the efficiency of the interaction between the ATM and TAD1-39. In conclusion, it can be stated that electrostatic interactions between the ATM and TAD are important for optimal Ser15 phosphorylation.

Effect of the addition of exogenous progesterone and the progesterone receptor inhibitor (RU 486) on boar cryopreservation semen extenders

Cryopreservation of porcine spermatozoa is detrimental due to their high sensitivity to cold shock, leading to changes akin to capacitation, known as cryocapacitation. These changes, including the acrosomal reaction, hypermotility induction, and protein phosphorylation, might be influenced by the presence of progesterone in seminal plasma and egg yolk, used in most freezing extenders. We tested the effect of various progesterone concentrations added to the freezing extenders (1, 10, and 100 μg/ml). At 100 μg/mL, progesterone decreased the proportion of straightness and tended to reduce viability and the proportion of progressive motility (p < 0.1). At 10 μg/mL, it increased reacted acrosomes in dead sperm (p < 0.05), protein phosphorylation rate (p < 0.05), and tended (p < 0.1) to enhance linear movement compared to the control. To counteract the capacitating effect of progesterone, we examined the effect of antiprogesterone mifepristone (RU 486) at concentrations of 5, 10, 20, 50, 100, and 200 μM, and co-incubated 10 μM of RU 486 with 10 μg/mL of progesterone. RU 486 maintained capacitation levels and motility parameters similar to the control, although high concentrations (100 μM) tended (p = 0.152) to increase protein phosphorylation. Co-incubation reduced the acrosome reaction in dead sperm, and RU 486 appeared to prevent hypermotility stabilizing motility and viability parameters compared to samples with progesterone alone. Protein phosphorylation increased and RU 486 could not restore capacitation to control levels due to its competitive antagonism for progesterone receptors, having less affinity than progesterone, which displaces RU 486 at high concentrations, allowing normal sperm capacitation.

The Role of Sirtuin-1 Isoforms in Regulating Mitochondrial Function.

The sirtuin-1 (SIRT1) gene contains multiple exons that usually undergo alternative splicing. The exclusion of one or more exons causes domain loss in the alternatively spliced isoforms and may change their functions. However, it is not completely established to what extent the loss of a non-catalytic domain could affect its regulatory function. Using muscle cells and SIRT1-knockout cells, we examined the function of the constitutively spliced isoform (SIRT1-v1) versus the alternatively spliced isoforms SIRT1-v2 and SIRT1-v3 that had lost part of the N-terminal region. Our data indicate that partial loss of the N-terminal domains in SIRT1-v2 and SIRT1-v3 attenuated their function. The full-length SIRT1-v1 significantly increased the oxidative phosphorylation and ATP production rate. Furthermore, SIRT1-v1 specifically upregulated the mitochondrial respiratory complex I without affecting the activity of complexes II, III, and IV. Additionally, domain loss affected the regulation of site-specific lysine acetylation in the histone H4 protein, the gene expression of respiratory complex I subunits, and the metabolic balance of oxidative phosphorylation versus glycolysis. Since alternatively spliced isoforms tend to increase with advancing age, the impact of SIRT1 isoforms on mitochondrial respiratory complexes warrants further investigation.

Assessing Onsager’s reciprocity difference in signal transduction cascade model: a new metric for evaluating cellular response dynamics

The cell signaling system translates extracellular changes into biochemical reactions within the cell, ultimately affecting gene expression. This system is crucial for responding to environmental stressors through a series of chemical interactions among intracellular molecules. Although Onsager’s reciprocity theorem is foundational in analyzing non-equilibrium systems, its limitations are evident in the nonlinear responses characteristic of the chemical reactions essential for signal transduction. In this study, we demonstrate that the reciprocity coefficients between signal molecule Xj, Lj:j+1 in the transmission from the jth step to the j + 1th step and molecule Xj+1, Lj+1:j from the j + 1th step to the jth step are not necessarily equal due to nonlinear interactions. This disparity, quantified as Jj:j+1 = −Lj:j+1 + Lj+1:j, reflects the amount of signal transduced between these steps, coupled with the phosphorylation rate of signaling molecules. Through kinetic model simulations, we analyzed the cellular response to external radiation exposure, monitoring how signal transduction progresses via phosphorylation reactions over time. The simulation showed a transition from an unstable to a stable, unimodal signaling pattern at varying radiation doses, correlating well with actual cellular responses. In conclusion, our findings suggest that the discrepancy in Onsager’s reciprocity coefficients correlates with signal transduction velocity, providing novel insights into the thermodynamic underpinnings of cellular signaling mechanisms.

Hypoxic extracellular vesicles from hiPSCs protect cardiomyocytes from oxidative damage by transferring antioxidant proteins and enhancing Akt/Erk/NRF2 signaling

BackgroundStem cell-derived extracellular vesicles (EVs) are an emerging class of therapeutics with excellent biocompatibility, bioactivity and pro-regenerative capacity. One of the potential targets for EV-based medicines are cardiovascular diseases (CVD). In this work we used EVs derived from human induced pluripotent stem cells (hiPSCs; hiPS-EVs) cultured under different oxygen concentrations (21, 5 and 3% O2) to dissect the molecular mechanisms responsible for cardioprotection.MethodsEVs were isolated by ultrafiltration combined with size exclusion chromatography (UF + SEC), followed by characterization by nanoparticle tracking analysis, atomic force microscopy (AFM) and Western blot methods. Liquid chromatography and tandem mass spectrometry coupled with bioinformatic analyses were used to identify differentially enriched proteins in various oxygen conditions. We directly compared the cardioprotective effects of these EVs in an oxygen-glucose deprivation/reoxygenation (OGD/R) model of cardiomyocyte (CM) injury. Using advanced molecular biology, fluorescence microscopy, atomic force spectroscopy and bioinformatics techniques, we investigated intracellular signaling pathways involved in the regulation of cell survival, apoptosis and antioxidant response. The direct effect of EVs on NRF2-regulated signaling was evaluated in CMs following NRF2 inhibition with ML385.ResultsWe demonstrate that hiPS-EVs derived from physiological hypoxia at 5% O2 (EV-H5) exert enhanced cytoprotective function towards damaged CMs compared to EVs derived from other tested oxygen conditions (normoxia; EV-N and hypoxia 3% O2; EV-H3). This resulted from higher phosphorylation rates of Akt kinase in the recipient cells after transfer, modulation of AMPK activity and reduced apoptosis. Furthermore, we provide direct evidence for improved calcium signaling and sustained contractility in CMs treated with EV-H5 using AFM measurements. Mechanistically, our mass spectrometry and bioinformatics analyses revealed differentially enriched proteins in EV-H5 associated with the antioxidant pathway regulated by NRF2. In this regard, EV-H5 increased the nuclear translocation of NRF2 protein and enhanced its transcription in CMs upon OGD/R. In contrast, inhibition of NRF2 with ML385 abolished the protective effect of EVs on CMs.ConclusionsIn this work, we demonstrate a superior cardioprotective function of EV-H5 compared to EV-N and EV-H3. Such EVs were most effective in restoring redox balance in stressed CMs, preserving their contractile function and preventing cell death. Our data support the potential use of hiPS-EVs derived from physiological hypoxia, as cell-free therapeutics with regenerative properties for the treatment of cardiac diseases.

Integration of transcriptomics and spatial biology analyses reveals Galactomyces ferment filtrate promotes epidermal interconnectivity via induction of keratinocyte differentiation, proliferation and cellular bioenergetics.

Human skin is the first line of defence from environmental factors such as solar radiation and is susceptible to premature ageing, including a disruption in epidermal differentiation and homeostasis. We evaluated the impact of a Galactomyces Ferment Filtrate (GFF) on epidermal differentiation and response to oxidative stress. We used transcriptomics, both spatial and traditional, to assess the impact of GFF on epidermal biology and homeostasis in keratinocytes (primary or immortalized) and in exvivo skin explant tissue. The effect of GFF on cell adhesion rates, cellular ATP levels and proliferation rates were quantitated. Oxidative phosphorylation and glycolytic rates were measured under normal and stress-induced conditions. Transcriptomics from keratinocytes and exvivo skin explants from multiple donors show GFF induces keratinocyte differentiation, skin barrier development and cell adhesion while simultaneously repressing cellular stress and inflammatory related processes. Spatial transcriptomics profiling of exvivo skin indicated basal keratinocytes at the epidermal-dermal junction and cornifying keratinocytes in the top layer of the epidermis as the primary cell types influenced by GFF treatment. Additionally, GFF significantly increases crosstalk between suprabasal and basal keratinocytes. To support these findings, we show that GFF can significantly increase cell adhesion and proliferation in keratinocytes. GFF also protected overall cellular bioenergetics under metabolic or oxidative stress conditions. Our findings provide novel insights into cellular differences and epidermal spatial localization in response to GFF, supporting previous findings that this filtrate has a significant impact on epidermal biology and homeostasis, particularly on spatially defined crosstalk. We propose that GFF can help maintain epidermal health by enhancing keratinocyte crosstalk and differentiation/proliferation balance as well as promoting an enhanced response to stress.

Pan-Cancer, Genome-Scale Metabolic Network Analysis of over 10,000 Patients Elucidates Relationship between Metabolism and Survival.

Despite the high variability in cancer biology, cancers nevertheless exhibit cohesive hallmarks across multiple cancer types, notably dysregulated metabolism. Metabolism plays a central role in cancer biology, and shifts in metabolic pathways have been linked to tumor aggressiveness and likelihood of response to therapy. We therefore sought to interrogate metabolism across cancer types and understand how intrinsic modes of metabolism vary within and across indications and how they relate to patient prognosis. We used context specific genome-scale metabolic modeling to simulate metabolism across 10,915 patients from 34 cancer types from The Cancer Genome Atlas and the MMRF-COMMPASS study. We found that cancer metabolism clustered into modes characterized by differential glycolysis, oxidative phosphorylation, and growth rate. We also found that the simulated activities of metabolic pathways are intrinsically prognostic across cancer types, especially tumor growth rate, fatty acid biosynthesis, folate metabolism, oxidative phosphorylation, steroid metabolism, and glutathione metabolism. This work shows the prognostic power of individual patient metabolic modeling across multiple cancer types. Additionally, it shows that analyzing large-scale models of cancer metabolism with survival information provides unique insights into underlying relationships across cancer types and suggests how therapies designed for one cancer type may be repurposed for use in others.

LL37/self-DNA complexes mediate monocyte reprogramming

LL37 alone and in complex with self-DNA triggers inflammatory responses in myeloid cells and plays a crucial role in the development of systemic autoimmune diseases, like psoriasis and systemic lupus erythematosus. We demonstrated that LL37/self-DNA complexes induce long-term metabolic and epigenetic changes in monocytes, enhancing their responsiveness to subsequent stimuli. Monocytes trained with LL37/self-DNA complexes and those derived from psoriatic patients exhibited heightened glycolytic and oxidative phosphorylation rates, elevated release of proinflammatory cytokines, and affected naïve CD4+ T cells. Additionally, KDM6A/B, a demethylase of lysine 27 on histone 3, was upregulated in psoriatic monocytes and monocytes treated with LL37/self-DNA complexes. Inhibition of KDM6A/B reversed the trained immune phenotype by reducing proinflammatory cytokine production, metabolic activity, and the induction of IL-17-producing T cells by LL37/self-DNA-treated monocytes. Our findings highlight the role of LL37/self-DNA-induced innate immune memory in psoriasis pathogenesis, uncovering its impact on monocyte and T cell dynamics.

Abstract PO3-06-08: Epithelial to Mesenchymal Transition Confers Sensitivity to Cytotoxic Agent Ophiobolin A via Alterations in Mitochondrial Function and Metabolic Pathways

Abstract Epithelial to Mesenchymal Transition Confers Sensitivity to Cytotoxic Agent Ophiobolin A via Alterations in Mitochondrial Function and Metabolic Pathways Haleigh Parker1, Kayla Haberman1, Alexander Kornienko2, Antonio Evidente3, Daniel Romo4, Benny Abraham Kaipparettu5,6, Joseph Taube1,6 1 Department of Biology, Baylor University, Waco, TX, USA 2 Department of Chemistry and Biochemistry, Texas State University, San Marcos, TX, USA 3 Department of Chemical Sciences, University of Naples Federico II, Complesso Universitario Monte Sant’Angelo, Naples, Italy 4 Department of Chemistry and Biochemistry, Baylor University, Waco, TX, USA 5 Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA 6 Dan L. Duncan Cancer Center, Baylor College of Medicine, Houston, TX, USA Background Metastatic progression in patients with triple negative breast cancer (TNBC) occurs in approximately half of all patients, reducing median overall survival. Metastasis may be facilitated through the epithelial to mesenchymal transition (EMT), which generates cancer cells with enhanced metastatic capacity and resistance to chemotherapeutics, features which are partially mediated by alterations in metabolic pathways and mitochondrial function. Here, we show a drug-like small molecule possesses EMT-specific cytotoxic activity via EMT-imparted variations in metabolic and mitochondrial functions. The fungus-derived sesterterpenoid, Ophiobolin A (OpA), possesses nanomolar cytotoxic activity and a high therapeutic index, though its target and mechanism of action remain unknown. Objectives The objectives of this study are to characterize alterations in metabolic pathways and mitochondrial function in the context of EMT, determine OpA induced alterations in specific metabolic pathways involved in metastasis and identify metabolic targets for the treatment of chemotherapeutic-resistant, metastatic triple negative breast cancer. Methods This study identifies distinct, novel metabolic alterations associated with EMT status and specific changes in mitochondrial and metabolic function associated with OpA treatment via analysis of intermediate metabolites, rates of oxidative phosphorylation and glycolysis, metabolic pathway capacity and dependency, protein expression, iron localization, reactive oxygen species accumulation and transmission electron microscopy. Results Our analysis indicates that OpA acts in a mitochondria-specific manner to cause a loss of membrane potential in EMT-positive, but not EMT-negative, cells with specific effects on complex III of the electron transport chain and oxidative phosphorylation as a whole. Furthermore, distinct shifts in glycolysis not only support previous data, but also provides insight as to mitochondrial functionality. Cells were also found to have distinct metabolic pathway capacities/dependencies and ROS and iron accumulation, depending on EMT status and OpA treatment. Finally, specific morphological alterations were uncovered via TEM image analysis. Therefore, we conclude that EMT imparts alterations in mitochondrial function and metabolic pathways, conferring sensitivity to the cytotoxic effects of OpA. Citation Format: Haleigh Parker, Kayla Haberman, Joseph Taube, Alexander Kornienko, Antonio Evidente, Daniel Romo, Benny Kaipparettu. Epithelial to Mesenchymal Transition Confers Sensitivity to Cytotoxic Agent Ophiobolin A via Alterations in Mitochondrial Function and Metabolic Pathways [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-06-08.

Quantifying in vivo Mitochondrial Effciency at Rest and during Exercise in Human Skeletal Muscle

Introduction: Mitochondrial effciency provides an additional mechanism to fine-tune oxidative phosphorylation rate to match ATP demand in skeletal muscle with far-reaching consequences on exercise tolerance. However, the direct measurement of both oxygen consumption (VO2) and ATP production from the same region of the contracting muscle in vivo remains a technical challenge. Purpose: Using an innovative approach combining noninvasive phosphorus and proton magnetic resonance spectroscopy (31P/1H-MRS), the present study aimed to characterize skeletal muscle ATP synthesis rate and muscle VO2 at rest and during a graded dynamic plantar flexion exercise to determine mitochondrial effciency in the gastrocnemius muscle. Method: We assessed mitochondrial effciency in the gastrocnemius muscles of 12 healthy adults (21± 1 yrs.). 31P and 1H magnetic resonance spectroscopy (MRS) was performed using a whole-body 3T MR system (Skyra, Siemens) at rest and during a series of constant workloads coupled to brief transient ischemia to quantify both oxidative ATP synthesis (ATPox) rate and myoglobin-derived oxygen consumption (Mb-derived VO2). The MRS data were acquired with a dual-tuned 31P/1H surface coil with linear polarization positioned over the gastrocnemius muscle. Results: As expected, the end-exercise PCr concentration decreased from 29 ± 2 mM of WRmax to 18 ± 1 mM with increasing workload. Likewise, the end-exercise Mb oxygenation level declined linearly with increasing workload. As the exercise workload increased, the ATPox synthesis rate increased linearly (r2 = 0.45), whereas there was no significant change in Mb-derived VO2. At rest, the estimated P/O ratio in the gastrocnemius muscle was 1.95 ± 0.68, whereas non-physiological values were obtained during exercise. Conclusion: During exercise, Mb-derived VO2 was likely limited by O2 availability with the methodology employed (30-sec occlusion, spectrum time resolution of 10 sec), which precluded the accurate quantification of mitochondrial effciency. However, mitochondrial effciency calculated at rest is in agreement with previously documented values ex vivo and thus provides an additional parameter to evaluate mitochondrial function in vivo. This work was funded in part by National Heart, Lung, and Blood Institute Grant R00HL125756. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Abstract SY35-02: Spatial mapping of mitochondrial networks in lung cancer

Abstract Mitochondria are critical to the governance of metabolism and bioenergetics in cancer cells. The mitochondria form highly organized networks, in which their outer and inner membrane structures define their bioenergetic capacity. However, in vivo studies delineating the relationship between the structural organization of mitochondrial networks and their bioenergetic activity have been limited. Here we present an in vivo structural and functional profiling of mitochondrial networks and bioenergetic phenotypes in non-small cell lung cancer (NSCLC) using an integrated platform comprised of positron emission tomography (PET) imaging, respirometry and 3-dimensional scanning block-face electron microscopy (3D SBEM). Interestingly, the diverse bioenergetic phenotypes and metabolic dependencies we identified in NSCLC tumors align with distinct structural organization of mitochondrial networks present. Further, we discovered that mitochondrial networks are organized into distinct compartments within tumor cells. In tumors with high rates of oxidative phosphorylation (OXPHOSHI) and fatty acid oxidation we identified peri-droplet mitochondrial networks wherein mitochondria contact and surround lipid droplets. In contrast, we discovered that in tumors with low rates of OXPHOS (OXPHOSLO), high glucose flux regulated i) peri-nuclear localization of mitochondria, ii) structural remodeling of cristae and iii) mitochondrial respiratory capacity. Our findings suggest that in NSCLC, mitochondrial networks are compartmentalized into distinct subpopulations that govern the bioenergetic capacity of tumors. Structure-and-function studies defining the relationship between mitochondrial architecture and metabolic dependencies may hold promise as strategies to profile metabolic liabilities unique to lung cancer subtypes. We anticipate that coupling PET imaging with 3D SBEM will have dynamic applications beyond that of lung cancer and enrich our understanding of how mitochondrial bioenergetics impact human disease. Citation Format: David B. Shackelford. Spatial mapping of mitochondrial networks in lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr SY35-02.

Mevalonate kinase-deficient THP-1 cells show a disease-characteristic pro-inflammatory phenotype.

Bi-allelic pathogenic variants in the MVK gene, which encodes mevalonate kinase (MK), an essential enzyme in isoprenoid biosynthesis, cause the autoinflammatory metabolic disorder mevalonate kinase deficiency (MKD). We generated and characterized MK-deficient monocytic THP-1 cells to identify molecular and cellular mechanisms that contribute to the pro-inflammatory phenotype of MKD. Using CRISPR/Cas9 genome editing, we generated THP-1 cells with different MK deficiencies mimicking the severe (MKD-MA) and mild end (MKD-HIDS) of the MKD disease spectrum. Following confirmation of previously established disease-specific biochemical hallmarks, we studied the consequences of the different MK deficiencies on LPS-stimulated cytokine release, glycolysis versus oxidative phosphorylation rates, cellular chemotaxis and protein kinase activity. Similar to MKD patients' cells, MK deficiency in the THP-1 cells caused a pro-inflammatory phenotype with a severity correlating with the residual MK protein levels. In the MKD-MA THP-1 cells, MK protein levels were barely detectable, which affected protein prenylation and was accompanied by a profound pro-inflammatory phenotype. This included a markedly increased LPS-stimulated release of pro-inflammatory cytokines and a metabolic switch from oxidative phosphorylation towards glycolysis. We also observed increased activity of protein kinases that are involved in cell migration and proliferation, and in innate and adaptive immune responses. The MKD-HIDS THP-1 cells had approximately 20% residual MK activity and showed a milder phenotype, which manifested mainly upon LPS stimulation or exposure to elevated temperatures. MK-deficient THP-1 cells show the biochemical and pro-inflammatory phenotype of MKD and are a good model to study underlying disease mechanisms and therapeutic options of this autoinflammatory disorder.

Effects of Aspirin Eugenol Ester on Liver Oxidative Damage and Energy Metabolism in Immune-Stressed Broilers.

The aim of this study was to investigate the effects of aspirin eugenol ester (AEE) on liver oxidative damage and energy metabolism in immune-stressed broilers. In total, 312 broilers were divided into 4 groups (saline, LPS, SAEE, and LAEE). Broilers in the saline and LPS groups were fed a basal diet; the SAEE and LAEE groups had an added 0.01% AEE in their diet. Broilers in the LPS and LAEE groups were injected with lipopolysaccharides, while the saline and SAEE groups were injected with saline. Results showed that AEE increased the body weight, average daily gain, and average daily feed intake, as well as decreasing the feed conversion ratio of immune-stressed broilers. AEE protects against oxidative damage in immune-stressed broiler livers by elevating the total antioxidant capacity, superoxide dismutase activity, and glutathione S-transferase alpha 3 (GSTA3) and glutaredoxin 2 (GLRX2) expression, while decreasing malondialdehyde content. AEE lessened inflammation by reducing prostaglandin-F2α production and prostaglandin-endoperoxide synthase 2 (PTGS2) and interleukin-1beta (IL-1β) expression. AEE decreased oxidative phosphorylation rates by increasing succinic acid levels and lowering both adenosine diphosphate (ADP) levels and ceroid lipofuscinosis neuronal 5 (CLN5) expression. AEE modulated the metabolism of phenylalanine, tyrosine, lipids, and cholesterol by reducing the phenyllactate and L-arogenate levels, lowering dopachrome tautomerase (DCT) and apolipoprotein A4 (APOA4) expression, and increasing phenylpyruvic acid and dopa decarboxylase (DDC) expression. In summary, AEE can effectively alleviate liver oxidative damage and energy metabolism disorders in immune-stressed broilers.

Kinetic 18F-FDG PET/CT imaging of hepatocellular carcinoma: a dual input four-compartment model.

The endoplasmic reticulum plays an important role in glucose metabolism and has not been explored in the kinetic estimation of hepatocellular carcinoma (HCC) via 18F-fluoro-2-deoxy-D-glucose PET/CT. A dual-input four-compartment (4C) model, regarding endoplasmic reticulum was preliminarily used for kinetic estimation to differentiate 28 tumours from background liver tissue from 24 patients with HCC. Moreover, parameter images of the 4C model were generated from one patient with negative findings on conventional metabolic PET/CT. Compared to the dual-input three-compartment (3C) model, the 4C model has better fitting quality, a close transport rate constant (K1) and a dephosphorylation rate constant (k6/k4), and a different removal rate constant (k2) and phosphorylation rate constant (k3) in HCC and background liver tissue. The K1, k2, k3, and hepatic arterial perfusion index (HPI) from the 4C model and k3, HPI, and volume fraction of blood (Vb) from the 3C model were significantly different between HCC and background liver tissues (all P < 0.05). Meanwhile, the 4C model yielded additional kinetic parameters for differentiating HCC. The diagnostic performance of the top ten genes from the most to least common was HPI(4C), Vb(3C), HPI(3C), SUVmax, k5(4C), k3(3C), k2(4C), v(4C), K1(4C) and Vb(4C). Moreover, a patient who showed negative findings on conventional metabolic PET/CT had positive parameter images in the 4C model. The 4C model with the endoplasmic reticulum performed better than the 3C model and produced additional useful parameters in kinetic estimation for differentiating HCC from background liver tissue.