Publications by authors named "Wolfgang F Graier"

134 Publications

Assessment of Mitochondrial Ca Uptake.

Methods Mol Biol 2021 ;2276:173-191

Gottfried Schatz Research Center for Cell Signaling, Metabolism and Aging, Molecular Biology and Biochemistry, Medical University of Graz, Graz, Austria.

Mitochondrial Ca uptake regulates mitochondrial function and contributes to cell signaling. Accordingly, quantifying mitochondrial Ca signals and elaborating the mechanisms that accomplish mitochondrial Ca uptake are essential to gain our understanding of cell biology. Here, we describe the benefits and drawbacks of various established old and new techniques to assess dynamic changes of mitochondrial Ca concentration ([Ca]) in a wide range of applications.
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http://dx.doi.org/10.1007/978-1-0716-1266-8_13DOI Listing
January 2021

Effect of hypoxia factors gene silencing on ROS production and metabolic status of A375 malignant melanoma cells.

Sci Rep 2021 May 14;11(1):10325. Epub 2021 May 14.

Department of Medical and Clinical Biochemistry, Faculty of Medicine, Pavol Jozef Šafárik University in Košice, Trieda SNP 1, 04011, Košice, Slovakia.

The innate response of melanocytes to exogenous or endogenous stress stimuli like extreme pH and temperature, metabolite and oxygen deficiency or a high UV dose initiates a cellular stress response. This process activates adaptive processes to minimize the negative impact of the stressor on the pigment cell. Under physiological conditions, a non-cancer cell is directed to apoptosis if the stressor persists. However, malignant melanoma cells will survive persistent stress thanks to distinct "cancerous" signaling pathways (e.g. MEK) and transcription factors that regulate the expression of so-called "survival genes" (e.g. HIF, MITF). In this survival response of cancer cells, MEK pathway directs melanoma cells to deregulate mitochondrial metabolism, to accumulate reduced species (NADH), and to centralize metabolism in the cytosol. The aim of this work was to study the effect of gene silencing in malignant melanoma A375 cells on metabolic processes in cytosol and mitochondria. Gene silencing of HIF-1α, and miR-210 in normoxia and pseudohypoxia, and analysis of its effect on MITF-M, and PDHA1 expression. Detection of cytosolic NADH by Peredox-mCherry Assay. Detection of OCR, and ECAR using Seahorse XF96. Measurement of produced O with MitoTracker Red CMXRos. H NMR analysis of metabolites present in cell suspension, and medium. By gene silencing of HIF-1α and miR-210 the expression of PDHA1 was upregulated while that of MITF-M was downregulated, yielding acceleration of mitochondrial respiratory activity and thus elimination of ROS. Hence, we detected a significantly reduced A375 cell viability, an increase in alanine, inositol, nucleotides, and other metabolites that together define apoptosis. Based on the results of measurements of mitochondrial resipiratory activity, ROS production, and changes in the metabolites obtained in cells under the observed conditions, we concluded that silencing of HIF-1α and miR-210 yields apoptosis and, ultimately, apoptotic cell death in A375 melanoma cells.
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http://dx.doi.org/10.1038/s41598-021-89792-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8121821PMC
May 2021

Potassium ions promote hexokinase-II dependent glycolysis.

iScience 2021 Apr 22;24(4):102346. Epub 2021 Mar 22.

Gottfried Schatz Research Center, Molecular Biology and Biochemistry, Medical University of Graz, Neue Stiftingtalstraße 6/6, 8010 Graz, Austria.

High expression levels of mitochondria-associated hexokinase-II (HKII) represent a hallmark of metabolically highly active cells such as fast proliferating cancer cells. Typically, the enzyme provides a crucial metabolic switch towards aerobic glycolysis. By imaging metabolic activities on the single-cell level with genetically encoded fluorescent biosensors, we here demonstrate that HKII activity requires intracellular K. The K dependency of glycolysis in cells expressing HKII was confirmed in cell populations using extracellular flux analysis and nuclear magnetic resonance-based metabolomics. Reductions of intracellular K by gramicidin acutely disrupted HKII-dependent glycolysis and triggered energy stress pathways, while K re-addition promptly restored glycolysis-dependent adenosine-5'-triphosphate generation. Moreover, expression and activation of K1.3, a voltage-gated K channel, lowered cellular K content and the glycolytic activity of HEK293 cells. Our findings unveil K as an essential cofactor of HKII and provide a mechanistic link between activities of distinct K channels and cell metabolism.
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http://dx.doi.org/10.1016/j.isci.2021.102346DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8047173PMC
April 2021

Lysophosphatidic Acid Induces Aerobic Glycolysis, Lipogenesis, and Increased Amino Acid Uptake in BV-2 Microglia.

Int J Mol Sci 2021 Feb 17;22(4). Epub 2021 Feb 17.

Division of Molecular Biology and Biochemistry, Gottfried Schatz Research Center, Medical University of Graz, 8010 Graz, Austria.

Lysophosphatidic acid (LPA) species are a family of bioactive lipids that transmit signals via six cognate G protein-coupled receptors, which are required for brain development and function of the nervous system. LPA affects the function of all cell types in the brain and can display beneficial or detrimental effects on microglia function. During earlier studies we reported that LPA treatment of microglia induces polarization towards a neurotoxic phenotype. In the present study we investigated whether these alterations are accompanied by the induction of a specific immunometabolic phenotype in LPA-treated BV-2 microglia. In response to LPA (1 µM) we observed slightly decreased mitochondrial respiration, increased lactate secretion and reduced ATP/ADP ratios indicating a switch towards aerobic glycolysis. Pathway analyses demonstrated induction of the Akt-mTOR-Hif1α axis under normoxic conditions. LPA treatment resulted in dephosphorylation of AMP-activated kinase, de-repression of acetyl-CoA-carboxylase and increased fatty acid content in the phospholipid and triacylglycerol fraction of BV-2 microglia lipid extracts, indicating de novo lipogenesis. LPA led to increased intracellular amino acid content at one or more time points. Finally, we observed LPA-dependent generation of reactive oxygen species (ROS), phosphorylation of nuclear factor erythroid 2-related factor 2 (Nrf2), upregulated protein expression of the Nrf2 target regulatory subunit of glutamate-cysteine ligase and increased glutathione synthesis. Our observations suggest that LPA, as a bioactive lipid, induces subtle alterations of the immunometabolic program in BV-2 microglia.
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http://dx.doi.org/10.3390/ijms22041968DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7923140PMC
February 2021

Dynamic Control of Mitochondrial Ca Levels as a Survival Strategy of Cancer Cells.

Front Cell Dev Biol 2021 4;9:614668. Epub 2021 Feb 4.

Molecular Biology and Biochemistry, Gottfried Schatz Research Center, Medical University of Graz, Graz, Austria.

Cancer cells have increased energy requirements due to their enhanced proliferation activity. This energy demand is, among others, met by mitochondrial ATP production. Since the second messenger Ca maintains the activity of Krebs cycle dehydrogenases that fuel mitochondrial respiration, proper mitochondrial Ca uptake is crucial for a cancer cell survival. However, a mitochondrial Ca overload induces mitochondrial dysfunction and, ultimately, apoptotic cell death. Because of the vital importance of balancing mitochondrial Ca levels, a highly sophisticated machinery of multiple proteins manages mitochondrial Ca homeostasis. Notably, mitochondria sequester Ca preferentially at the interaction sites between mitochondria and the endoplasmic reticulum (ER), the largest internal Ca store, thus, pointing to mitochondrial-associated membranes (MAMs) as crucial hubs between cancer prosperity and cell death. To investigate potential regulatory mechanisms of the mitochondrial Ca uptake routes in cancer cells, we modulated mitochondria-ER tethering and the expression of UCP2 and analyzed mitochondrial Ca homeostasis under the various conditions. Hence, the expression of contributors to mitochondrial Ca regulation machinery was quantified by qRT-PCR. We further used data from The Cancer Genome Atlas (TCGA) to correlate these findings with expression patterns in human breast invasive cancer and human prostate adenocarcinoma. ER-mitochondrial linkage was found to support a mitochondrial Ca uptake route dependent on uncoupling protein 2 (UCP2) in cancer cells. Notably, combined overexpression of Rab32, a protein kinase A-anchoring protein fostering the ER-mitochondrial tethering, and UCP2 caused a significant drop in cancer cells' viability. Artificially enhanced ER-mitochondrial tethering further initiated a sudden decline in the expression of UCP2, probably as an adaptive response to avoid mitochondrial Ca overload. Besides, TCGA analysis revealed an inverse expression correlation between proteins stabilizing mitochondrial-ER linkage and UCP2 in tissues of human breast invasive cancer and prostate adenocarcinoma. Based on these results, we assume that cancer cells successfully manage mitochondrial Ca uptake to stimulate Ca-dependent mitochondrial metabolism while avoiding Ca-triggered cell death by fine-tuning ER-mitochondrial tethering and the expression of UCP2 in an inversed manner. Disruption of this equilibrium yields cancer cell death and may serve as a treatment strategy to specifically kill cancer cells.
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http://dx.doi.org/10.3389/fcell.2021.614668DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7889948PMC
February 2021

Targeting cellular senescence based on interorganelle communication, multilevel proteostasis, and metabolic control.

FEBS J 2020 Nov 17. Epub 2020 Nov 17.

Institute for Biomedical Aging Research, Leopold-Franzens Universität Innsbruck, Austria.

Cellular senescence, a stable cell division arrest caused by severe damage and stress, is a hallmark of aging in vertebrates including humans. With progressing age, senescent cells accumulate in a variety of mammalian tissues, where they contribute to tissue aging, identifying cellular senescence as a major target to delay or prevent aging. There is an increasing demand for the discovery of new classes of small molecules that would either avoid or postpone cellular senescence by selectively eliminating senescent cells from the body (i.e., 'senolytics') or inactivating/switching damage-inducing properties of senescent cells (i.e., 'senostatics/senomorphics'), such as the senescence-associated secretory phenotype. Whereas compounds with senolytic or senostatic activity have already been described, their efficacy and specificity has not been fully established for clinical use yet. Here, we review mechanisms of senescence that are related to mitochondria and their interorganelle communication, and the involvement of proteostasis networks and metabolic control in the senescent phenotype. These cellular functions are associated with cellular senescence in in vitro and in vivo models but have not been fully exploited for the search of new compounds to counteract senescence yet. Therefore, we explore possibilities to target these mechanisms as new opportunities to selectively eliminate and/or disable senescent cells with the aim of tissue rejuvenation. We assume that this research will provide new compounds from the chemical space which act as mimetics of caloric restriction, modulators of calcium signaling and mitochondrial physiology, or as proteostasis optimizers, bearing the potential to counteract cellular senescence, thereby allowing healthy aging.
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http://dx.doi.org/10.1111/febs.15631DOI Listing
November 2020

Metabolic Profiling of Single Cancer Cells Using Mitochondrial ATP Probes.

STAR Protoc 2020 Sep 8;1(2):100048. Epub 2020 Jun 8.

Molecular Biology and Biochemistry, Gottfried Schatz Research Center, Medical University of Graz, Neue Stiftingtalstraße 6/6, Graz 8010, Austria.

The metabolic activity of cells is interrelated with cell signaling, functions, and fate. Uncontrolled cancer cell proliferation requires metabolic adaptations. Research focusing on understanding the characteristics of cell metabolism is crucial for the development of novel diagnostic and therapeutic strategies. Here, we describe protocols for the ATP profiling of single cancer cells by fluorescence live-cell imaging. In response to distinct metabolic inhibitions, we record individual mitochondrial ATP dynamics using established Förster resonance energy transfer-based genetically encoded fluorescent ATP probes. For complete details on the use and execution of this protocol, please refer to Depaoli et al. (2018).
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http://dx.doi.org/10.1016/j.xpro.2020.100048DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7580088PMC
September 2020

ER-to-Golgi Transport in HeLa Cells Displays High Resilience to Ca and Energy Stresses.

Cells 2020 10 17;9(10). Epub 2020 Oct 17.

Molecular Biology and Biochemistry, Gottfried Schatz Research Center, Medical University of Graz, Neue Stiftingtalstraße 6/6, 8010 Graz, Austria.

One third of all human proteins are either transmembrane or soluble secretory proteins that first target the endoplasmic reticulum (ER). These proteins subsequently leave the ER and enter the Golgi apparatus via ER-Golgi intermediate vesicular structures. Live-cell imaging of cargos fused to fluorescent proteins (FPs) enables the high-resolution visualization and characterization of secretory transport processes. Here, we performed fluorescence time-lapse imaging to assess the Ca and energy dependency of ER-to-Golgi transport in living HeLa cells, a cancer cell model which has been well investigated. Our data revealed that ER-to-Golgi transport remained highly efficient in the absence of ATP-generating substrates, despite clear reductions in cytosolic and mitochondrial ATP levels under these energy stress conditions. However, cell treatment with 2-deoxy-D-glucose (2-DG), which severely diminished subcellular ATP levels, abolished ER-to-Golgi transport. Interestingly, while 2-DG elevated cytosolic Ca levels and reduced long-distance movements of glycosylphosphatidylinositol (GPI)-positive vesicles, robust short-term ER Ca mobilizations, which strongly affected the motility of these vesicles, did not considerably impair ER-to-Golgi transport. In summary, we highlight that ER-to-Golgi transport in HeLa cells remains functional despite high energy and Ca stress levels.
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http://dx.doi.org/10.3390/cells9102311DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7603030PMC
October 2020

The contribution of uncoupling protein 2 to mitochondrial Ca homeostasis in health and disease - A short revisit.

Mitochondrion 2020 11 15;55:164-173. Epub 2020 Oct 15.

Molecular Biology and Biochemistry, Gottfried Schatz Research Center, Medical University of Graz, Neue Stiftingtalstraße 6/6, 8010 Graz, Austria; BioTechMed, Graz, Austria. Electronic address:

Considering the versatile functions attributed to uncoupling protein 2 (UCP2) in health and disease, a profound understanding of the protein's molecular actions under physiological and pathophysiological conditions is indispensable. This review aims to revisit and shed light on the fundamental molecular functions of UCP2 in mitochondria, with particular emphasis on its intricate role in regulating mitochondrial calcium (Ca) uptake. UCP2's modulating effect on various vital processes in mitochondria makes it a crucial regulator of mitochondrial homeostasis in health and disease.
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http://dx.doi.org/10.1016/j.mito.2020.10.003DOI Listing
November 2020

Yes (again) to local NO.

Nat Chem Biol 2020 06;16(6):606-607

Molecular Biology and Biochemistry, Gottfried Schatz Research Center, Medical University of Graz, Graz, Austria.

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http://dx.doi.org/10.1038/s41589-020-0552-7DOI Listing
June 2020

Nonclassical nuclear localization signals mediate nuclear import of CIRBP.

Proc Natl Acad Sci U S A 2020 04 31;117(15):8503-8514. Epub 2020 Mar 31.

Gottfried Schatz Research Center for Cell Signaling, Metabolism and Aging, Molecular Biology & Biochemistry, Medical University of Graz, 8010 Graz, Austria;

The specific interaction of importins with nuclear localization signals (NLSs) of cargo proteins not only mediates nuclear import but also, prevents their aberrant phase separation and stress granule recruitment in the cytoplasm. The importin Transportin-1 (TNPO1) plays a key role in the (patho-)physiology of both processes. Here, we report that both TNPO1 and Transportin-3 (TNPO3) recognize two nonclassical NLSs within the cold-inducible RNA-binding protein (CIRBP). Our biophysical investigations show that TNPO1 recognizes an arginine-glycine(-glycine) (RG/RGG)-rich region, whereas TNPO3 recognizes a region rich in arginine-serine-tyrosine (RSY) residues. These interactions regulate nuclear localization, phase separation, and stress granule recruitment of CIRBP in cells. The presence of both RG/RGG and RSY regions in numerous other RNA-binding proteins suggests that the interaction of TNPO1 and TNPO3 with these nonclassical NLSs may regulate the formation of membraneless organelles and subcellular localization of numerous proteins.
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http://dx.doi.org/10.1073/pnas.1918944117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7165476PMC
April 2020

Myeloperoxidase and Septic Conditions Disrupt Sphingolipid Homeostasis in Murine Brain Capillaries In Vivo and Immortalized Human Brain Endothelial Cells In Vitro.

Int J Mol Sci 2020 Feb 9;21(3). Epub 2020 Feb 9.

Division of Molecular Biology and Biochemistry, Gottfried Schatz Research Center, Medical University of Graz, Graz 8010, Austria.

During inflammation, activated leukocytes release cytotoxic mediators that compromise blood-brain barrier (BBB) function. Under inflammatory conditions, myeloperoxidase (MPO) is critically involved in inflicting BBB damage. We used genetic and pharmacological approaches to investigate whether MPO induces aberrant lipid homeostasis at the BBB in a murine endotoxemia model. To corroborate findings in a human system we studied the impact of sera from sepsis and non-sepsis patients on brain endothelial cells (hCMEC/D3). In response to endotoxin, the fatty acid, ceramide, and sphingomyelin content of isolated mouse brain capillaries dropped and barrier dysfunction occurred. In mice, genetic deficiency or pharmacological inhibition of MPO abolished these alterations. Studies in metabolic cages revealed increased physical activity and less pronounced sickness behavior of MPO compared to wild-type mice in response to sepsis. In hCMEC/D3 cells, exogenous tumor necrosis factor α (TNFα) potently regulated gene expression of pro-inflammatory cytokines and a set of genes involved in sphingolipid (SL) homeostasis. Notably, treatment of hCMEC/D3 cells with sera from septic patients reduced cellular ceramide concentrations and induced barrier and mitochondrial dysfunction. In summary, our in vivo and in vitro data revealed that inflammatory mediators including MPO, TNFα induce dysfunctional SL homeostasis in brain endothelial cells. Genetic and pharmacological inhibition of MPO attenuated endotoxin-induced alterations in SL homeostasis in vivo, highlighting the potential role of MPO as drug target to treat inflammation-induced brain dysfunction.
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http://dx.doi.org/10.3390/ijms21031143DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7037060PMC
February 2020

Endothelial lipase increases eNOS activating capacity of high-density lipoprotein.

Biochim Biophys Acta Mol Cell Biol Lipids 2020 04 7;1865(4):158612. Epub 2020 Jan 7.

Gottfried Schatz Research Center, Molecular Biology and Biochemistry, Medical University of Graz, Neue Stiftingtalstraße 6/6, 8010 Graz, Austria; BioTechMed-Graz, Mozartgasse 12/II, 8010 Graz, Austria. Electronic address:

Endothelial lipase (EL) changes structural and functional properties of high-density lipoprotein (HDL). HDL is a relevant modulator of endothelial nitric oxide synthase (eNOS) activity, but the effect of EL on HDL induced eNOS-activation has not yet been investigated. Here, we examined the impact of EL-modified HDL (EL-HDL) on eNOS activity, subcellular trafficking, and eNOS- dependent vasorelaxation. EL-HDL and empty virus (EV)-HDL as control were isolated from human serum incubated with EL-overexpressing or EV infected HepG2 cells. EL-HDL exhibited higher capacity to induce eNOS phosphorylation at Ser1177 and eNOS activity in EA.hy 926 cells, as well as eNOS-dependent vasorelaxation of mouse aortic rings compared to control HDL. As revealed by confocal and structured illumination-microscopy EL-HDL-driven induction of eNOS was accompanied by an increased eNOS-GFP targeting to the plasma membrane and a lower eNOS-GFP colocalization with Golgi and mitochondria. Widefield microscopy of filipin stained cells revealed that EL-HDL lowered cellular free cholesterol (FC) and as found by thin-layer chromatography increased cellular cholesterol ester (CE) content. Additionally, cholesterol efflux capacity, acyl-coenzyme A: cholesterol acyltransferase activity, and HDL particle uptake were comparable between EL-HDL and control HDL. In conclusion, EL increases eNOS activating capacity of HDL, a phenomenon accompanied by an enrichment of the plasma membrane eNOS pool, a decreased cell membrane FC and increased cellular CE content.
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http://dx.doi.org/10.1016/j.bbalip.2020.158612DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7116681PMC
April 2020

HDAC inhibition improves cardiopulmonary function in a feline model of diastolic dysfunction.

Sci Transl Med 2020 01;12(525)

Cardiovascular Research Center, Lewis Katz School of Medicine, Temple University, Philadelphia, PA 19140, USA.

Heart failure with preserved ejection fraction (HFpEF) is a major health problem without effective therapies. This study assessed the effects of histone deacetylase (HDAC) inhibition on cardiopulmonary structure, function, and metabolism in a large mammalian model of pressure overload recapitulating features of diastolic dysfunction common to human HFpEF. Male domestic short-hair felines ( = 31, aged 2 months) underwent a sham procedure ( = 10) or loose aortic banding ( = 21), resulting in slow-progressive pressure overload. Two months after banding, animals were treated daily with suberoylanilide hydroxamic acid (b + SAHA, 10 mg/kg, = 8), a Food and Drug Administration-approved pan-HDAC inhibitor, or vehicle (b + veh, = 8) for 2 months. Echocardiography at 4 months after banding revealed that b + SAHA animals had significantly reduced left ventricular hypertrophy (LVH) ( < 0.0001) and left atrium size ( < 0.0001) versus b + veh animals. Left ventricular (LV) end-diastolic pressure and mean pulmonary arterial pressure were significantly reduced in b + SAHA ( < 0.01) versus b + veh. SAHA increased myofibril relaxation ex vivo, which correlated with in vivo improvements of LV relaxation. Furthermore, SAHA treatment preserved lung structure, compliance, blood oxygenation, and reduced perivascular fluid cuffs around extra-alveolar vessels, suggesting attenuated alveolar capillary stress failure. Acetylation proteomics revealed that SAHA altered lysine acetylation of mitochondrial metabolic enzymes. These results suggest that acetylation defects in hypertrophic stress can be reversed by HDAC inhibitors, with implications for improving cardiac structure and function in patients.
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http://dx.doi.org/10.1126/scitranslmed.aay7205DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7065257PMC
January 2020

Visualization of Sirtuin 4 Distribution between Mitochondria and the Nucleus, Based on Bimolecular Fluorescence Self-Complementation.

Cells 2019 12 6;8(12). Epub 2019 Dec 6.

Gottfried Schatz Research Center, Chair of Molecular Biology and Biochemistry, Medical University of Graz, Neue Stiftingtalstraße 6/6, 8010 Graz, Austria.

Mitochondrial sirtuins (Sirts) control important cellular processes related to stress. Despite their regulatory importance, however, the dynamics and subcellular distributions of Sirts remain debatable. Here, we investigate the subcellular localization of sirtuin 4 (Sirt4), a sirtuin variant with a mitochondrial targeting sequence (MTS), by expressing Sirt4 fused to the superfolder green fluorescent protein (Sirt4-sfGFP) in HeLa and pancreatic β-cells. Super resolution fluorescence microscopy revealed the trapping of Sirt4-sfGFP to the outer mitochondrial membrane (OMM), possibly due to slow mitochondrial import kinetics. In many cells, Sirt4-sfGFP was also present within the cytosol and nucleus. Moreover, the expression of Sirt4-sfGFP induced mitochondrial swelling in HeLa cells. In order to bypass these effects, we applied the self-complementing split fluorescent protein (FP) technology and developed mito-STAR (mitochondrial sirtuin 4 tripartite abundance reporter), a tripartite probe for the visualization of Sirt4 distribution between mitochondria and the nucleus in single cells. The application of mito-STAR proved the importation of Sirt4 into the mitochondrial matrix and demonstrated its localization in the nucleus under mitochondrial stress conditions. Moreover, our findings highlight that the self-complementation of split FP is a powerful technique to study protein import efficiency in distinct cellular organelles.
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http://dx.doi.org/10.3390/cells8121583DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6953047PMC
December 2019

Calcium Signaling in ß-cell Physiology and Pathology: A Revisit.

Int J Mol Sci 2019 Dec 4;20(24). Epub 2019 Dec 4.

Molecular Biology and Biochemistry, Gottfried Schatz Research Center for Cell Signaling, Metabolism and Aging, Medical University of Graz, Neue Stiftingtalstraße 6/6, 8010 Graz, Austria.

Pancreatic beta (β) cell dysfunction results in compromised insulin release and, thus, failed regulation of blood glucose levels. This forms the backbone of the development of diabetes mellitus (DM), a disease that affects a significant portion of the global adult population. Physiological calcium (Ca) signaling has been found to be vital for the proper insulin-releasing function of β-cells. Calcium dysregulation events can have a dramatic effect on the proper functioning of the pancreatic β-cells. The current review discusses the role of calcium signaling in health and disease in pancreatic β-cells and provides an in-depth look into the potential role of alterations in β-cell Ca homeostasis and signaling in the development of diabetes and highlights recent work that introduced the current theories on the connection between calcium and the onset of diabetes.
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http://dx.doi.org/10.3390/ijms20246110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6940736PMC
December 2019

Tracking intra- and inter-organelle signaling of mitochondria.

FEBS J 2019 11 11;286(22):4378-4401. Epub 2019 Nov 11.

Gottfried Schatz Research Center, Molecular Biology and Biochemistry, Medical University of Graz, Austria.

Mitochondria are as highly specialized organelles and masters of the cellular energy metabolism in a constant and dynamic interplay with their cellular environment, providing adenosine triphosphate, buffering Ca and fundamentally contributing to various signaling pathways. Hence, such broad field of action within eukaryotic cells requires a high level of structural and functional adaptation. Therefore, mitochondria are constantly moving and undergoing fusion and fission processes, changing their shape and their interaction with other organelles. Moreover, mitochondrial activity gets fine-tuned by intra- and interorganelle H , K , Na , and Ca signaling. In this review, we provide an up-to-date overview on mitochondrial strategies to adapt and respond to, as well as affect, their cellular environment. We also present cutting-edge technologies used to track and investigate subcellular signaling, essential to the understanding of various physiological and pathophysiological processes.
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http://dx.doi.org/10.1111/febs.15103DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6899612PMC
November 2019

-acetylaspartate availability is essential for juvenile survival on fat-free diet and determines metabolic health.

FASEB J 2019 12 1;33(12):13808-13824. Epub 2019 Nov 1.

Institute of Biochemistry, Graz University of Technology, Graz, Austria.

-acetylaspartate (NAA) is synthesized by aspartate -acetyltransferase (gene: ) from acetyl-coenzyme A and aspartate. In the brain, NAA is considered an important energy metabolite for lipid synthesis. However, the role of NAA in peripheral tissues remained elusive. Therefore, we characterized the metabolic phenotype of knockout (ko) and adipose tissue-specific (ako) -ko mice as well as NAA-supplemented mice on various diets. We identified an important role of NAA availability in the brain during adolescence, as 75% of -ko mice died on fat-free diet (FFD) after weaning but could be rescued by NAA supplementation. In adult life, NAA deficiency promotes a beneficial metabolic phenotype, as -ko and -ako mice showed reduced body weight, increased energy expenditure, and improved glucose tolerance on chow, high-fat, and FFDs. Furthermore, -deficient adipocytes exhibited increased mitochondrial respiration, ATP synthesis, and an induction of browning. Conversely, NAA-treated wild-type mice showed reduced adipocyte respiration and lipolysis and increased lipogenesis, culminating in reduced energy expenditure, glucose tolerance, and insulin sensitivity. Mechanistically, our data point to a possible role of NAA as modulator of pancreatic insulin secretion and suggest NAA as a critical energy metabolite for adipocyte and whole-body energy homeostasis.-Hofer, D. C., Zirkovits, G., Pelzmann, H. J., Huber, K., Pessentheiner, A. R., Xia, W., Uno, K., Miyazaki, T., Kon, K., Tsuneki, H., Pendl, T., Al Zoughbi, W., Madreiter-Sokolowski, C. T., Trausinger, G., Abdellatif, M., Schoiswohl, G., Schreiber, R., Eisenberg, T., Magnes, C., Sedej, S., Eckhardt, M., Sasahara, M., Sasaoka, T., Nitta, A., Hoefler, G., Graier, W. F., Kratky, D., Auwerx, J., Bogner-Strauss, J. G. -acetylaspartate availability is essential for juvenile survival on fat-free diet and determines metabolic health.
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http://dx.doi.org/10.1096/fj.201801323RDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6894082PMC
December 2019

Development and Application of Sub-Mitochondrial Targeted Ca Biosensors.

Front Cell Neurosci 2019 4;13:449. Epub 2019 Oct 4.

Molecular Biology and Biochemistry, Gottfried Schatz Research Center, Medical University of Graz, Graz, Austria.

Mitochondrial Ca uptake into the mitochondrial matrix is a well-established mechanism. However, the sub-organellar Ca kinetics remain elusive. In the present work we identified novel site-specific targeting sequences for the intermembrane space (IMS) and the cristae lumen (CL). We used these novel targeting peptides to develop green- and red- Ca biosensors targeted to the IMS and to the CL. Based on their distinctive spectral properties, and comparable sensitivities these novel constructs were suitable to visualize Ca-levels in various (sub) compartments in a multi-chromatic manner. Functional studies that applied these new biosensors revealed that knockdown of MCU and EMRE yielded elevated Ca levels inside the CL but not the IMS in response to IP-generating agonists. Knockdown of VDAC1, however, strongly impeded the transfer of Ca through the OMM while the cytosolic Ca signal remained unchanged. The novel sub-mitochondrially targeted Ca biosensors proved to be suitable for Ca imaging with high spatial and temporal resolution in a multi-chromatic manner allowing simultaneous measurements. These informative biosensors will facilitate efforts to dissect the complex sub-mitochondrial Ca signaling under (patho)physiological conditions.
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http://dx.doi.org/10.3389/fncel.2019.00449DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6788349PMC
October 2019

Presenilin-1 Established ER-Ca Leak: a Follow Up on Its Importance for the Initial Insulin Secretion in Pancreatic Islets and β-Cells upon Elevated Glucose.

Cell Physiol Biochem 2019 ;53(3):573-586

Molecular Biology and Biochemistry, Gottfried Schatz Research Center for Cellular Signaling, Metabolism & Aging, Medical University of Graz, Graz, Austria.

Background/aims: In our recent work, the importance of GSK3β-mediated phosphorylation of presenilin-1 as crucial process to establish a Ca leak in the endoplasmic reticulum and, subsequently, the pre-activation of resting mitochondrial activity in β-cells was demonstrated. The present work is a follow-up and reveals the importance of GSK3β-phosphorylated presenilin-1 for responsiveness of pancreatic islets and β-cells to elevated glucose in terms of cytosolic Ca spiking and insulin secretion.

Methods: Freshly isolated pancreatic islets and the two pancreatic β-cell lines INS-1 and MIN-6 were used. Cytosolic Ca was fluorometrically monitored using Fura-2/AM and cellular insulin content and secretion were measured by ELISA.

Results: Our data strengthened our previous findings of the existence of a presenilin-1-mediated ER-Ca leak in β-cells, since a reduction of presenilin-1 expression strongly counteracted the ER Ca leak. Furthermore, our data revealed that cytosolic Ca spiking upon administration of high D-glucose was delayed in onset time and strongly reduced in amplitude and frequency upon siRNA-mediated knock-down of presenilin-1 or the inhibition of GSK3β in the pancreatic β-cells. Moreover, glucose-triggered initial insulin secretion disappeared by depletion from presenilin-1 and inhibition of GSK3β in the pancreatic β-cells and isolated pancreatic islets, respectively.

Conclusion: These data complement our previous work and demonstrate that the sensitivity of pancreatic islets and β-cells to glucose illustrated as glucose-triggered cytosolic Ca spiking and initial but not long-lasting insulin secretion crucially depends on a strong ER Ca leak that is due to the phosphorylation of presenilin-1 by GSK3β, a phenomenon that might be involved in the development of type 2 diabetes.
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http://dx.doi.org/10.33594/000000158DOI Listing
December 2019

Publisher Correction: IRE1α modulates ER and mitochondria crosstalk.

Nat Cell Biol 2019 Oct;21(10):1300

Molecular Biology and Biochemistry, Gottfried Schatz Research Center, Medical University of Graz, Graz, Austria.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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http://dx.doi.org/10.1038/s41556-019-0400-8DOI Listing
October 2019

MICU1 controls cristae junction and spatially anchors mitochondrial Ca uniporter complex.

Nat Commun 2019 08 19;10(1):3732. Epub 2019 Aug 19.

Gottfried Schatz Research Center, Molecular Biology and Biochemistry, Medical University of Graz, Neue Stiftingtalstraße 6/6, 8010, Graz, Austria.

Recently identified core proteins (MICU1, MCU, EMRE) forming the mitochondrial Ca uniporter complex propelled investigations into its physiological workings. Here, we apply structured illumination microscopy to visualize and localize these proteins in living cells. Our data show that MICU1 localizes at the inner boundary membrane (IBM) due to electrostatic interaction of its polybasic domain. Moreover, this exclusive localization of MICU1 is important for the stability of cristae junctions (CJ), cytochrome c release and mitochondrial membrane potential. In contrast to MICU1, MCU and EMRE are homogeneously distributed at the inner mitochondrial membrane under resting conditions. However, upon Ca elevation MCU and EMRE dynamically accumulate at the IBM in a MICU1-dependent manner. Eventually, our findings unveil an essential function of MICU1 in CJ stabilization and provide mechanistic insights of how sophistically MICU1 controls the MCU-Complex while maintaining the structural mitochondrial membrane framework.
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http://dx.doi.org/10.1038/s41467-019-11692-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6700202PMC
August 2019

ATGL/CGI-58-Dependent Hydrolysis of a Lipid Storage Pool in Murine Enterocytes.

Cell Rep 2019 08;28(7):1923-1934.e4

Gottfried Schatz Research Center, Molecular Biology and Biochemistry, Medical University of Graz, 8010 Graz, Styria, Austria; BioTechMed-Graz, 8010 Graz, Styria, Austria. Electronic address:

As circulating lipid levels are balanced by the rate of lipoprotein release and clearance from the plasma, lipid absorption in the small intestine critically contributes to the maintenance of whole-body lipid homeostasis. Within enterocytes, excessive triglycerides are transiently stored as cytosolic lipid droplets (cLDs), and their mobilization sustains lipid supply during interprandial periods. Using mice lacking adipose triglyceride lipase (ATGL) and its coactivator comparative gene identification-58 (CGI-58) exclusively in the intestine (intestine-specific double KO [iDKO]), we show that ATGL/CGI-58 are not involved in providing substrates for chylomicron synthesis. Massive intestinal cLD accumulation in iDKO mice independent of dietary lipids together with inefficient lipid incorporation into cLDs in the early absorption phase demonstrate the existence of a secretion/re-uptake cycle, corroborating the availability of two diverse cLD pools. This study identified ATGL/CGI-58 as critical players in the catabolism of basolaterally (blood) derived lipids and highlights the necessity to modify the current model of intestinal lipid metabolism.
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http://dx.doi.org/10.1016/j.celrep.2019.07.030DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6713565PMC
August 2019

Mitochondrial-Endoplasmic Reticulum Interplay: A Lifelong On-Off Relationship?

Contact (Thousand Oaks) 2019 19;2:2515256419861227. Epub 2019 Jul 19.

Gottfried Schatz Research Center, Molecular Biology and Biochemistry, Medical University of Graz, Graz, Austria.

This article comments recent publications that highlight an intriguing importance of specific settings in the interaction between the mitochondria and the endoplasmic reticulum to ensure cell-specific functions like the responsiveness to elevated glucose in pancreatic β-cells. Hence, alterations of the mitochondria-endoplasmic reticulum communications under various pathological conditions like aging or cancer often come with enhanced Ca transfer that, in turn, yields stimulation of basal mitochondrial activity to meet the increasing adenosine triphosphate demand of the very cell. Such observations identify mitochondria-associated membranes as potential target for new therapeutic strategies against aging or cancer.
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http://dx.doi.org/10.1177/2515256419861227DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6687505PMC
July 2019

Live cell imaging of signaling and metabolic activities.

Pharmacol Ther 2019 10 7;202:98-119. Epub 2019 Jun 7.

Molecular Biology and Biochemistry, Gottfried Schatz Research Center, Medical University of Graz, Neue Stiftingtalstraße 6/6, 8010 Graz, Austria; BioTechMed Graz, Austria. Electronic address:

The interplay of metabolic and signaling processes is prerequisite for the functionality of cells. Any disturbances may have severe consequences, resulting in the development of diseases. However, the complex coordination of metabolism and signaling events makes it difficult to decipher the link between molecular irregularities and pathogenesis. An excellent way to provide more clarity is to see into the living cell and watch cellular processes in real-time, with the add-on of being able to manipulate certain processes. Live cell imaging enables us to do exactly that, with steadily improving spatial and temporal resolution. Modern genetically encoded fluorescent probes in combination with state-of-the-art high-resolution imaging devices have proven themselves as a valuable approach for monitoring, manipulating and ultimately understanding the interaction of cell metabolism and signaling. These probes also represent powerful tools for detecting biomarkers of disease, identifying new drug targets and elucidating drug actions at the cellular to the molecular level.
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http://dx.doi.org/10.1016/j.pharmthera.2019.06.003DOI Listing
October 2019

Live-Cell Imaging of Physiologically Relevant Metal Ions Using Genetically Encoded FRET-Based Probes.

Cells 2019 05 22;8(5). Epub 2019 May 22.

Gottfried Schatz Research Center, Chair of Molecular Biology and Biochemistry, Medical University of Graz, Neue Stiftingtalstraße 6/6, 8010 Graz, Austria.

Essential biochemical reactions and processes within living organisms are coupled to subcellular fluctuations of metal ions. Disturbances in cellular metal ion homeostasis are frequently associated with pathological alterations, including neurotoxicity causing neurodegeneration, as well as metabolic disorders or cancer. Considering these important aspects of the cellular metal ion homeostasis in health and disease, measurements of subcellular ion signals are of broad scientific interest. The investigation of the cellular ion homeostasis using classical biochemical methods is quite difficult, often even not feasible or requires large cell numbers. Here, we report of genetically encoded fluorescent probes that enable the visualization of metal ion dynamics within individual living cells and their organelles with high temporal and spatial resolution. Generally, these probes consist of specific ion binding domains fused to fluorescent protein(s), altering their fluorescent properties upon ion binding. This review focuses on the functionality and potential of these genetically encoded fluorescent tools which enable monitoring (sub)cellular concentrations of alkali metals such as K, alkaline earth metals including Mg and Ca, and transition metals including Cu/Cu and Zn. Moreover, we discuss possible approaches for the development and application of novel metal ion biosensors for Fe/Fe, Mn and Na.
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http://dx.doi.org/10.3390/cells8050492DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6562680PMC
May 2019

IRE1α modulates ER and mitochondria crosstalk.

Nat Cell Biol 2019 06;21(6):667-668

Molecular Biology and Biochemistry, Gottfried Schatz Research Center, Medical University of Graz, Graz, Austria.

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http://dx.doi.org/10.1038/s41556-019-0338-xDOI Listing
June 2019

Lipid-independent control of endothelial and neuronal TRPC3 channels by light.

Chem Sci 2019 Mar 15;10(9):2837-2842. Epub 2019 Jan 15.

Gottfried Schatz Research Center - Biophysics , Medical University of Graz , Neue Stiftingtalstraße 6/D/04 , 8010 Graz , Austria . Email:

Lipid-gated TRPC channels are highly expressed in cardiovascular and neuronal tissues. Exerting precise pharmacological control over their activity in native cells is expected to serve as a basis for the development of novel therapies. Here we report on a new photopharmacological tool that enables manipulation of TRPC3 channels by light, in a manner independent of lipid metabolism and with higher temporal precision than lipid photopharmacology. Using the azobenzene photoswitch moiety, we modified GSK1702934A to generate light-controlled TRPC agonists. We obtained one light-sensitive molecule (OptoBI-1) that allows us to exert efficient, light-mediated control over TRPC3 activity and the associated cellular Ca signaling. OptoBI-1 enabled high-precision, temporal control of TRPC3-linked cell functions such as neuronal firing and endothelial Ca transients. With these findings, we introduce a novel photopharmacological strategy to control native TRPC conductances.
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http://dx.doi.org/10.1039/c8sc05536jDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6427946PMC
March 2019

pH-Lemon, a Fluorescent Protein-Based pH Reporter for Acidic Compartments.

ACS Sens 2019 04 30;4(4):883-891. Epub 2019 Mar 30.

Molecular Biology and Biochemistry, Gottfried Schatz Research Center , Medical University of Graz , Neue Stiftingtalstraße 6/6 , 8010 Graz , Austria.

Distinct subcellular pH levels, especially in lysosomes and endosomes, are essential for the degradation, modification, sorting, accumulation, and secretion of macromolecules. Here, we engineered a novel genetically encoded pH probe by fusing the pH-stable cyan fluorescent protein (FP) variant, mTurquoise2, to the highly pH-sensitive enhanced yellow fluorescent protein, EYFP. This approach yielded a ratiometric biosensor-referred to as pH-Lemon-optimized for live imaging of distinct pH conditions within acidic cellular compartments. Protonation of pH-Lemon under acidic conditions significantly decreases the yellow fluorescence while the cyan fluorescence increases due to reduced Förster resonance energy transfer (FRET) efficiency. Because of its freely reversible and ratiometric responses, pH-Lemon represents a fluorescent biosensor for pH dynamics. pH-Lemon also shows a sizable pH-dependent fluorescence lifetime change that can be used in fluorescence lifetime imaging microscopy as an alternative observation method for the study of pH in acidic cellular compartments. Fusion of pH-Lemon to the protein microtubule-associated protein 1A/1B-light chain 3B (LC3B), a specific marker of autophagic membranes, resulted in its targeting within autolysosomes of HeLa cells. Moreover, fusion of pH-Lemon to a glycophosphatidylinositol (GPI) anchor allowed us to monitor the entire luminal space of the secretory pathway and the exoplasmic leaflet of the plasma membrane. Utilizing this new pH probe, we revealed neutral and acidic vesicles and substructures inside cells, highlighting compartments of distinct pH throughout the endomembrane system. These data demonstrate, that this novel pH sensor, pH-Lemon, is very suitable for the study of local pH dynamics of subcellular microstructures in living cells.
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http://dx.doi.org/10.1021/acssensors.8b01599DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6488996PMC
April 2019