Petras Dzeja

Mayo Clinic - Rochester · Department of Cardiovascular Medicine

Aims: Recent studies have revealed a close connection between cellular metabolism and the chronic inflammatory process of atherosclerosis. While the link between systemic metabolism and atherosclerosis is well established, the implications of altered metabolism in the artery wall are less understood. Pyruvate dehydrogenase kinase (PDK)-dependent i...

Changes in dynamics of ATP γ- and β-phosphoryl turnover and metabolic flux through phosphotransfer pathways in cancer cells are still unknown. Using 18O phosphometabolite tagging technology, we have discovered phosphotransfer dynamics in three breast cancer cell lines: MCF7 (non-aggressive), MDA-MB-231 (aggressive), and MCF10A (control). Contrary t...

Changes in dynamics of ATP γ- and β-phosphoryl turnover and metabolic flux through phosphotransfer pathways in cancer cells are still unknown. Using 18O phosphometabolite tagging technology, we have discovered phosphotransfer dynamics in three breast cancer cell lines: MCF7 (non-aggressive), MDA-MB-231 (aggressive), and MCF10A (control). Contrary t...

Adenylate kinase2 (AK2) catalyzes trans-compartmental nucleotide exchange, but the functional implications of this mitochondrial intermembrane isoform is only partially understood. Here, transgenic AK2−/− null homozygosity was lethal early in embryo, indicating a mandatory role for intact AK2 in utero development. In the adult, conditional organ-sp...

New technologies permit determining metabolomic profiles of human diseases by fingerprinting metabolites levels. However, to fully understand metabolomic phenotypes, metabolite levels and turnover rates are necessary to know. Krebs cycle is the major hub of energy metabolism and cell signaling. Traditionally, ¹³C stable isotope labeled substrates w...

Alzheimer’s Disease (AD) is a devastating neurodegenerative disorder without a cure. Here we show that mitochondrial respiratory chain complex I is an important small molecule druggable target in AD. Partial inhibition of complex I triggers the AMP-activated protein kinase-dependent signaling network leading to neuroprotection in symptomatic APP/PS...

Alzheimer’s Disease (AD) is a devastating neurodegenerative disorder without a cure. Here we show that mitochondrial respiratory chain complex I is an important small molecule druggable target in AD. Partial inhibition of complex I triggers the AMP-activated protein kinase-dependent signaling network leading to neuroprotection in symptomatic APP/PS...

Axonemal dynein ATPases direct ciliary and flagellar beating via adenosine triphosphate (ATP) hydrolysis. The modulatory effect of adenosine monophosphate (AMP) and adenosine diphosphate (ADP) on flagellar beating is not fully understood. Here, we describe a deficiency of cilia and flagella associated protein 45 (CFAP45) in humans and mice that pre...

We demonstrate that mitochondrial respiratory chain complex I is an important small molecule druggable target in Alzheimers Disease (AD). Partial inhibition of complex I triggers the AMP-activated protein kinase-dependent signaling network leading to neuroprotection in symptomatic APP/PS1 mice, a translational model of AD. Treatment of APP/PS1 mice...

A hallmark of cancer cells is the ability to rewire their bioenergetics and metabolic signaling circuits to fuel their uncontrolled proliferation and metastasis. Adenylate kinase (AK) is the critical enzyme in the metabolic monitoring of cellular adenine nucleotide homeostasis. It also directs AK→ AMP→ AMPK signaling controlling cell cycle and prol...

Depleting creatine levels in thermogenic adipocytes by inhibiting creatine biosynthesis reduces thermogenesis and causes obesity. However, whether creatine import from the circulation affects adipocyte thermogenesis is unknown. Here we show that deletion of the cell-surface creatine transporter (CrT) selectively in fat (AdCrTKO) substantially reduc...

Figure S3. PFKFB3 synergizes with chemoresistant ovarian cancer cells. Combination of PFK158 with cisplatin and with carboplatin in equipotent combinations (IC50 over IC50 ratio) was assessed for synergy using the Chou‐Talalay methodology. The cells were exposed to each drug alone and in combination per protocol for 48 h. The combination indices (...

Figure S4. PFK158 treatment inhibits LD biogenesis. A. OV90 cells were treated with PFK158 (0‐10 μM) followed by Bodipy staining to detect LDs. B. Immunoblot analysis shows the protein expression of p‐PFKFB3, t‐PFKFB3, p‐cPLA2 and t‐cPLA2 after PFK158 (0‐10 μM) treatment in OV90 cells. (C‐D) Transient downregulation of PFKFB3 in OV90 cells shows a...

Figure S5. Autophagy inhibition confers resistance to PFK158 plus carboplatin‐mediated synergy. Cell viability assays were performed with a combination of increasing concentrations of carboplatin with 1x IC50 of PFK158 with and without bafilomycin A (BafA) pretreatment in replicates of 4. Cells were pretreated with 50 nM BafA for 2 h followed by d...

Table S1: Antibodies and Reagents

Figure S1: Higher p‐PFKFB3 in OV tumors Representative images of immunohistochemical staining of phospho (p)‐PFKFB3 (ser461) and total (t)‐PFKFB3 performed in three ovarian tumor samples.

Figure S2A‐C. Mitochondrial respiration is compromised in resistant cells. A‐B. Oxygen consumption rate (OCR) was measured with an XF24 extracellular flux analyzer (Seahorse Bioscience) after protocols recommended by the manufacturer. Resistant cells (C13 and HeyMDR) showed lower OCR compared to OV2008 and HeyA8. Data represent mean ± SD, two‐tail...

Figure S8. p62 co‐localizes with LDs in ovarian cancer cells. Co‐localization of p62 and LD were evaluated by immuno‐fluorescence analysis in OVCAR5 cells. Co‐localization of p62‐HA with Bodipy (Fig.S8, second row) is attenuated with EBSS treatment (Fig.S8, third row), while treatment with bafilomycin rescued the phenotype (Fig.S8, last row).

Figure S6. PFK158 mediated inhibition of cPLA2 activity and degradation of LDs is autophagy‐dependent. The arachidonic acid release was evaluated in C13 and HeyA8MDR cells in the presence of 5 μM PFK158 along with 50 nM Bafilomycin with untreated cells as controls. Cells were incubated with 3H‐AA under serum‐starved condition for 24 h. Fresh mediu...

Figure S7. Reduction in neutral lipids in PFK158 treated and PFKFB3 knockdown OVCAR5 cells. OVCAR5 cells stably downregulated with shRNA‐PFKFB3 (A) or treated with 5 μM of PFK158 for 12 h and 24 h (B) were subjected to analysis for neutral lipids including cholesteryl ester and triacylglycerols The samples were extracted using Metabolon's standard...

Figure S9. p62 associates with cPLA2 in ovarian cancer cells. A. HeyA8MDR cell lysates were co‐immunoprecipitated with either p62/SQSTM1 or t‐cPLA2 antibody and subsequently immunoblotted to determine the protein expression of t‐cPLA2, and p62/SQSTM1. B. Immunofluorescence analysis shows co‐localization of t‐cPLA2 with p62/SQSTM1.

Metabolic alterations are increasingly recognized as important novel anti‐cancer targets. Amongst several regulators of metabolic alterations, fructose 2,6 bisphosphate (F2,6BP) is a critical glycolytic regulator. Inhibition of the active form of PFKFB3ser461 using a novel inhibitor, PFK158 resulted in reduced glucose uptake, ATP production, lactat...

Enhanced glucose uptake and a switch to glycolysis are key traits of M1 macrophages, whereas enhanced fatty acid oxidation and oxidative phosphorylation are the main metabolic characteristics of M2 macrophages. Recent studies challenge this traditional view, indicating that glycolysis may also be critically important for M2 macrophage differentiati...

Background: Routine prenatal care fails to identify a large proportion of women at risk of fetal growth restriction (FGR). Metabolomics, the comprehensive analysis of low molecular weight molecules (metabolites) in biological samples, can provide new and earlier biomarkers of prenatal health. Recent research has suggested possible predictive first...

Classical activation of M1 macrophages with lipopolysaccharide (LPS) is associated with a metabolic switch from oxidative phosphorylation to glycolysis. However, the generalizability of such metabolic remodeling to other modes of M1 macrophage stimulation, e.g. type II interferons (IFNs) such as IFNγ, has remained unknown as has the functional sign...

(Cell Metabolism 26, 1–12; October 3, 2017) In the originally published version of this article, in the last sentence of the Introduction subsection, there was a typo in the following sentence: “Our findings indicate that adipose tissue creatine energetics suppresses diet-induced thermogenesis and combats obesity.” Instead of “suppresses,” the sent...

Metabolic processes and signalling are crucially important for the healthy functioning and energy homeostasis of the human body. Dr Petras Dzeja, currently working at Mayo Clinic College of Medicine in Rochester, Minnesota, focuses his studies on cardiovascular metabolomics, phosphotransfer circuits and system bioenergetics, aiming to elucidate the...

Diet-induced thermogenesis is an important homeostatic mechanism that limits weight gain in response to caloric excess and contributes to the relative stability of body weight in most individuals. We previously demonstrated that creatine enhances energy expenditure through stimulation of mitochondrial ATP turnover, but the physiological role and im...

Metabolic signaling mechanisms of tissue regeneration is still an enigma. Energy state and metabolite signals regulate cell commitment to self-renewal, lineage specification, differentiation and regeneration. In a favorable metabolic environment, cells can grow and proliferate, however when energy is low - metabolic monitoring system sends signals...

Background: Heart failure (HF) is associated with changes in cardiac substrate utilization and energy metabolism, including a decline in high-energy phosphate content, mitochondrial dysfunction, and phosphotransfer enzyme deficiency. Methods and results: A shift toward glucose metabolism was noted in the end stage of HF in animals, although HF i...

Integration of mitochondria with cytosolic ATP-consuming/ATP-sensing and substrate supply processes is critical for muscle bioenergetics and electrical activity. Whether age-dependent muscle weakness and increased electrical instability depends on perturbations in cellular energetic circuits is unknown. To define energetic remodeling of aged atrial...

Energy metabolism and metabolic signaling circuits orchestrate cell commitment to self-renewal, lineage specification, differentiation and regeneration. When energy resources are plenty, cell can grow, proliferate and regenerate, however when energy is low - augmented adenylate kinase (AK)-mediated AMP signaling turns on AMPK which silences p53/p21...

Heart failure (HF) is associated with ventricular dyssynchrony and energetic inefficiency, which can be alleviated by cardiac resynchronization therapy (CRT). The aim of this study was to determine the metabolomic signature in HF and its prognostic value for the response to CRT. This prospective study consisted of 24 patients undergoing CRT for adv...

Development of therapeutic strategies to prevent Alzheimer's disease (AD) is of great importance. We show that mild inhibition of mitochondrial complex I with small molecule CP2 reduces levels of amyloid beta and phospho-Tau and averts cognitive decline in three animal models of familial AD. Low-mass molecular dynamics simulations and biochemical s...

Comprehensive characterization of disease-related metabolomic phenotypes and drug effects requires monitoring metabolite levels and their turnover rates. Tandem application of stable isotope 18O-assisted 31P NMR and mass spectrometric techniques uniquely allow simultaneous measurements of phosphorus-containing metabolite levels and their dynamics i...

Guzun, R., Kaambre, T., Bagur, R., Grichine, A., Usson, Y., Varikmaa, M. Anmann, T., Tepp, K., Timohhina, N., Shevchuk, I., Chekulayev, V., Boucher, F., Dos Santos, P., Schlattner, U., Wallimann, T., Kuznetsov, A. V., Dzeja, P., Aliev, M., Saks, V. "Modular organization of cardiac energy metabolism: energy conversion, transfer and feedback regulati...

The adenylate kinase isoform network is integral to the cellular energetic system and a major player in AMP metabolic signaling circuits. Critical in energy state monitoring and stress response, the dynamic behavior of the adenylate kinase network in governing intracellular, nuclear, and extracellular nucleotide signaling processes has been increas...

To meet high cellular demands, the energy metabolism of cardiac muscles is organized by precise and coordinated functioning of Intra-Cellular Energetic Units (ICEUs). ICEUs represent structural and functional modules integrating multiple fluxes at sites of ATP generation in mitochondria and ATP utilization by myofibrillar, sarcoplasmic reticulum an...

Multiple sclerosis (MS) is a chronic inflammatory and demyelinating disease of the CNS. Although, MS is well characterized in terms of the role played by immune cells, cytokines and CNS pathology, nothing is known about the metabolic alterations that occur during the disease process in circulation. Recently, metabolic aberrations have been defined...

Technological innovations and translation of basic discoveries to clinical practice drive advances in medicine. Today's innovative technologies enable comprehensive screening of the genome, transcriptome, proteome, and metabolome. The detailed knowledge, converged in the integrated "omics" (genomics, transcriptomics, proteomics, and metabolomics),...

Plasticity in energy metabolism allows stem cells to match the divergent demands of self-renewal and lineage specification. Beyond a role in energetic support, new evidence implicates nutrient-responsive metabolites as mediators of crosstalk between metabolic flux, cellular signaling, and epigenetic regulation of cell fate. Stem cell metabolism als...

The mammalian heart loses its regenerative potential within a week after birth. Although under certain circumstances adult cardiomyocytes can re-enter the cell cycle, initiate DNA synthesis and progress towards mitosis, however, they do not complete a conventional mitosis cycle and proceed towards endomitosis resulting in polyploidy. This suggests...

Metabolic drugs frequently target mitochondrial functions and AMP signaling. Metformin, one of the most prescribed anti-diabetic medications, exerts its glucose lowering effect through mild interference with mitochondrial energy metabolism triggering homeostatic adenylate kinase (AK)-catalyzed AMP and downstream AMPK signaling. However, metabolomic...

The perpetual and vigorous nature of heart muscle work requires efficient myocardial energetics. This depends not only on adequate ATP production, but also on efficient delivery of ATP to muscle ATPases and rapid removal of ADP and other by-products of ATP hydrolysis. Indeed, recent evidence indicates that defects in communication between ATP-produ...

Engineering pluripotency through nuclear reprogramming and directing stem cells into defined lineages underscores cell fate plasticity. Acquisition of and departure from stemness are governed by genetic and epigenetic controllers, with modulation of energy metabolism and associated signaling increasingly implicated in cell identity determination. T...

A new method was here developed for the determination of 18O-labeling ratios in metabolic oligophosphates, such as ATP, at different phosphoryl moieties (α-, β-, and γ-ATP) using sensitive and rapid electrospray ionization mass spectrometry (ESI-MS). The ESI-MS-based method for monitoring of 18O/16O exchange was validated with gas chromatography–ma...

The identification of early mechanisms underlying Alzheimer's Disease (AD) and associated biomarkers could advance development of new therapies and improve monitoring and predicting of AD progression. Mitochondrial dysfunction has been suggested to underlie AD pathophysiology, however, no comprehensive study exists that evaluates the effect of diff...

Axonal trafficking of mitochondria in primary neuron from NTG mouse. Visualization of mitochondria in E17 Hip neuron was done using TMRM. 600 frames were acquired by imaging the axon every second using LSM 510 confocal microscope. Imaging was done focusing on the axon with the cell body located at the top of the image. Resulting movie was analyzed...

Animated 3D reconstruction of mitochondrial structure in Hip tissue of APP/PS1 mouse 24 weeks of age. For 3D reconstruction of the mitochondrial structure, the grayscale of the individual EM section images was first inverted so that the organelle became bright objects. The inverted images were then sequentially co registered using the Normalized Mu...

The behavior of the mitochondrial permeability transition pore has been linked to mitochondrial maturation underlying cardiomyocyte differentiation in the embryo. Mitochondrial signaling in heart development has direct implications for cardiogenesis and stem cell lineage specification.

Next-generation screening of disease-related metabolomic phenotypes requires monitoring of both metabolite levels and turnover rates. Stable isotope (18)O-assisted (31)P nuclear magnetic resonance (NMR) and mass spectrometry uniquely allows simultaneous measurement of phosphometabolite levels and turnover rates in tissue and blood samples. The (18)...