New cellular/physiologic roles of G6PD · hemolytic anemia in G6PD-deficient patients upon...

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New cellular/physiologic roles of G6PD 趙 崇 義 特聘教授兼研發長 Dr. Daniel Tsun-Yee Chiu, Distinguished Professor Graduate Institute of Medical Biotechnology, Dean of Research & Development, Chang Gung University(長庚大學), Taiwan Research Prof., Dept. of Pediatric Hematology, Chang Gung Memorial Hospital (Linkou), Taiwan Past President, Society Free Radical Research-Asia(2014, 2015) Email: [email protected] July 14, 2016
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  • New cellular/physiologic roles of G6PD

    Dr. Daniel Tsun-Yee Chiu, Distinguished Professor

    Graduate Institute of Medical Biotechnology,

    Dean of Research & Development,

    Chang Gung University(), TaiwanResearch Prof., Dept. of Pediatric Hematology,

    Chang Gung Memorial Hospital (Linkou), Taiwan

    Past President, Society Free Radical Research-Asia(2014, 2015)

    Email: [email protected]

    July 14, 2016

    mailto:[email protected]

  • 2

    A Brief History of Chang Gung University

    ( , CGU) of Taiwan- Established in April 1987 as Chang Gung Medical College

    associated with the largest medical facilities (Chang Gung Memorial Hospitals) in Taiwan(perhaps the largest in the world, with 7 hospitals in Taiwan and 2 hospitals in China).

    - Changed our name to Chang Gung College of Medicine and

    Technology in 1993.

    - Upgraded the College to Chang Gung University in July, 1997

    consisting the Colleges of Medicine, College of Engineering and

    College of Management .

    - Currently has approximately 614 full-time faculty members and

    7,145 students (including undergraduates 5,063 and graduates 2,082).

    - Ranked 5th in Taiwan and approximately 400th world-wide.

  • Redox Homeostasis (Balanced Redox Status)

    (A balance between Yin & Yan, ?)

    Antioxidants = oxidants,

    Oxidative stress

    Antioxidant

    Oxidant

    Antioxidants < Oxidants

    In contrast, then Reductive Stress

  • The Classical roles of G6PD:

    to regenerate NADPH as antioxidant to produce Ribose

    Glucose

    G6PD

    O2-

    (superoxide anion) HK

    Glucose-6-phosphateNADP

    NADPH

    2GSH

    GSSG

    SOD

    H2O2

    H2O

    CAT

    H2O + O21

    2

    Glutathione reductaseGPx

    6-phosphoglucono--lactone

    6PGL

    6-phosphogluconate

    6PGD

    Ribulose-5-phosphate

    Ru5PI

    Ribose-5-phosphate

    NADP

    NADPH

    Glutathione reductase

    2GSH

    GSSG

    GPxH2O2

    H2O

    NADPHIsocitrate dehydrogenase (ICDH)

    Malic enzyme (ME)NADP

    4

    Pentose phosphate pathway, PPP

  • 5

    G6PD deficiency (Also known as Favism)

    Glucose-6-phosphate dehydrogenase (G6PD) deficiency, a most

    common enzyme deficiency affecting over 400 million people

    worldwide, causes a spectrum of diseases including, acute and

    chronic hemolytic anemia, neonatal jaundice and etc.

    J Med Screen 19:103-104, 2012

    Lancet 371: 64-74, 2008

    G6PD deficiency

    in Taiwan:Male 3%

    Female 0.9%

    Redox Rep 12: 109-18, 2007

    Free Rad Res 48: 1028-48, 2014

    Free Rad Res (in press), 2016

  • G6PDNADPH

    NOS

    Nitric oxide

    SuperoxideNOX

    NADP

    NOSNitric oxide synthase

    NOXNADPH oxidase

    Pro-oxidant role of G6PD :

    Provides substrate to generate free radicals

    (% of resting cells)

    Decreased NO & Superoxide production FEBS Lett . 436:411-4, 1998

    But Effective Neutrophil Extra-cellular Trap Formation Free Rad Res 47:699-709, 2013

    Oxidants

    6

  • Th

    resh

    old

    Dual roles of G6PD in redox homeostasis affecting cellular function

    cyto-regulatory cyto-toxic

    Approximate concentration of oxidants

    Level of

    Oxidative Stress

    oxidant

  • G6PD Deficiency

    (Redox Report 12: 109, 2007; Free Rad Res 48: 1028, 2014; Free Rad Res in press, 2016)

    Decreased NADPH ProductionDecreased NO &

    Superoxide production

    FEBS Letters 436:411-414, 1998,

    But Effective Neutrophil Extra-cellular Trap Formation Free Rad Res 47:699, 2013 Redox Imbalance

    Accelerated SenescenceFree Rad Biol Med 29: 156-169, 2000;

    FEBS Letters 475: 257-262, 2000

    Retarded Cell GrowthFree Rad Biol Med 29: 156-169, 2000

    Increased susceptibilityto certain diseases

    Jpn J Canc Res 92: 576-581, 2001

    Endocrine 19: 191-196, 2002

    [Ophthalmic Epid. 13: 109-114, 2006]

    Increased susceptibility to:

    1.Corona Virus infection.

    J Infect Dis. 197:812-6, 2008

    2. Enterovirus infection .

    J Gen. Virol.89:2080-9, 2008

    3. Protection by EGCGJ Agr Food Chem 57:6140-7, 2009

    Increased Susceptibility to Oxidative Insult

    Free Rad Biol Med 36: 580, 2004; Cytometry Part A 69: 1054, 2006

    J Agr Food Chem 54: 1638, 2006; Free Radic Res. 41:571, 2007

    Free Rad Biol Med 47: 529, 2009

    G6PD deficiency-induced cellular abnormalities &

    related phenotypes (beyond RBCs abnormalities)

    Alterations in 1. Signal transduction(MAPK)Free Rad Biol Med 49: 361, 2010

    2. Proteomic J Proteom Res 12: 3434, 2013

    3.Metabonomic Free Rad Biol Med 54: 71, 2013

    4. C. elegans model. Cell Death Disease 4, e616, 2013

  • Inability to maintain GSH pool in G6PD-deficient

    red cells causes futile AMPK activation and

    irreversible metabolic disturbance

    Tang Hsiang Yu et al()

    (Antioxid Redox Sign 22: 744-759, 2015;

    IF=7.407)

    9

    This work is, in part, based on the publication of

    Ho HY, Cheng ML, Shiao MS and Chiu DTY.

    Characterization of global metabolic responses

    of G6PD-deficient hepatoma cells to diamide-

    induced oxidative stress.

    Free Radic Biol Med 54: 71-84, 2013; IF=5.736

    (Applying New Technology to tackle traditional problems)

  • Typical workflow for MS-based metabolic

    profiling TOF

    Sample

    collection and

    pretreatment

    Data analysis

    (Big data)Data collection

    Statistical analysis / Bioinformatics

    Volcano plot

    Heat map (ANOVA)

    Principal

    component analysis

    (PCA)

    Condition tree

    (Clustering)

    Metabolome: Metabolic

    pathways and interactionFunction and

    dysfunction

    Targeted Metabolite

    identification

    10

  • Principal component analysis (PCA) in

    G6PD deficient and control RBCs

    with or without diamide-treatment

    N

    N_1 diamide

    P_1 mM

    diamide

    P

    Principal component analysis (PCA) of metabolomes in control and G6PDdeficient RBCs with or without diamide treatment. Both groups were un- or treatedwith 1mM of diamide for various time period. Features were acquired in ESI positiveion mode. Antiox. Redox Signaling 22: 744-759, 2015

    27.31 %

    37.78 %

    11

  • Cysteine

    Ophthalmic acid

    Altered glutathione metabolism in G6PD

    deficient RBCs leading to the formation of

    ophthalmic acid upon diamide treatment

    -glutamylcysteine

    synthetase

    Normal GSH Synthetic Pathway

    Altered GSH Synthetic

    Pathway

    Methionine

    Cycle

    Ophthalmic acid has never been reported in human RBCs before

    Antiox. Redox Signaling 22: 744-759, 2015

    2-aminobutyrate

    12

  • Antiox. Redox Signaling 22: 744-759, 2015

    Cysteine

    Ophthalmic acid

    Alterations are mainly due to the reprogramming

    from GSH regeneration via the glutathione

    reductase system to GSH synthesis via

    -glutamylcysteine synthetase

    -glutamylcysteine

    synthetase

    Normal GSH Synthetic Pathway

    Altered GSH Synthetic

    Pathway

    Methionine

    Cycle

    GR

    NADPH NADP

    G6PD

    Reprogramming from GSH

    regeneration to synthesisX

    X

    2-aminobutyrate

    13

    Blockade

    of GSH

    regeneration

  • 1. Diamide treatment induces major alterations in GSH related metabolites in G6PD

    deficient RBCs including the appearance of unusual metabolites such as

    opthalmic acid which has never been reported in human RBCs before.

    2. Such impairment in GSH related metabolism is mainly due to the reprogramming

    from GSH regeneration to GSH synthesis and is accompanied by exhaustive

    ATP consumption and enhanced glycolytic activities in G6PD deficient RBCs.

    Unfortunately, the last step in glycolysis catalyzed by pyruvate kinase(PK) to

    produce ATP is blocked in G6PD-deficient RBCs due to the inactivation of PK

    by oxidative stress.

    3. Changes in metabolic activities cause functional defects such as membrane

    protein aggregation and decreased in RBC deformability of G6PD-deficient

    RBCs and these new findings provide additional explanation concerning acute

    hemolytic anemia in G6PD-deficient patients upon encountering oxidative stress

    such as favism and infection.

    In conclusion, this metabolomic study shows that G6PD-deficient RBCs

    desperately struggle to maintain redox homeostasis upon oxidant challenge to

    avoid cell death but without success.

    Tang et al. Antiox. Redox Signaling 22: 744-759, 2015

    Summary & Conclusion from our metabonomic study

    14

  • Th

    resh

    old

    Dual roles of G6PD in redox homeostasis affecting cellular function

    cyto-regulatory cyto-toxic

    Approximate concentration of oxidants

    Level of

    Oxidative Stress

    oxidant

    (One major

    aspect of

    research

    is to prove

    New Concepts)

  • A novel protective role for G6PD in

    aflatoxin B1-mediated cytotoxicity

    revealed by proteomic study

    Hsin-Ru Lin(), Chih-Ching Wu()et al.

    J Proteome Res. 12: 3434-3448, 2013

    (IF=5.001)

    16

  • Lin et al.

    (A)

    (B)

    0.0

    0.5

    1.0

    1.5

    2.0

    2.5

    3.0

    3.5

    G6P

    D a

    ctiv

    ity

    (IU

    /mg)

    Ctrl G6PD-kd Ctrl G6PD-kd

    Single clone A549 Mixed clone A549

    Ctrl G6PD-kd Ctrl G6PD-kd

    Single clone A549 Mixed clone A549

    Actin

    G6PD

    1.00 0.22 0.81 0.18

    Fig.1. Reduced G6PD status in G6PD-knockdown cells. (A) G6PD activities in A549 single and mixed clone cell lysates were

    assayed, and expressed in IU/mg of protein. (B) Expression of G6PD protein in A549 single and mixed clone cell lysates was

    analyzed by western bloting. Data are meanSD, N=2, ** P0.001. (J Proteome Res. 12: 3434-3448, 2013)

    * ** *

    Generation of G6PD-knockdown A549 cells by shRNA

    For subsequent protein analysis by proteomic technique

    17

  • Verification of Proteins Differentially Expressed in

    G6PD-Knockdown A549 Cells

    18

    The mRNA levels of EPHX1 and AKR1C3 were decreased in G6PD-knockdown A549 cells

    compared with control cells, in which mRNA fold changes were positively correlated to the

    iTRAQ ratios. (J Proteome Res. 12: 3434-3448, 2013)

    Table -2

    18

  • Pathway analysis of differentially expressed

    proteins in G6PD-knockdown A549 cells Table.3.

    J Proteome Res. 12: 3434-3448, 2013 19

  • Protective Role of G6PD in Aflatoxin B1 (AFB1)

    Induced Cytotoxicity

    Figure 4. G6PD deficiency augments AFB1-

    induced cell death. (A) G6PD-kd and Ctrl

    A549 cells were treated with 25, 50M AFB1

    for the indicated time intervals. Cell

    viabilities were evaluated with MTT assay

    and calculated as a ratio relative to cells

    without AFB1 treatment. Results were

    expressed as mean values SD from four independent experiments (*P < 0.01; paired t-

    test). (B) Cell death was monitored with

    annexin V/PI staining. Either annexin V- or

    PI-positive cells are classified as dead cells.

    Results were expressed as mean values SD from three independent experiments (*P <

    0.01; paired t-test).(B)

    G6PD may perform a

    protective role in AFB1-induced

    cytotoxicity via regulation of

    EPHX1 expression.

    J Proteome Res. 12: 3434-3448, 2013

    20

  • G6PD Activity Is Required for ROS-Mediated

    EPHX1 Production

    Figure 6. Impairment of nuclear Nrf2 translocation by G6PD

    knockdown. (A) Nuclear localization of Nrf2 was assessed immunochemically by using Nrf2-specific Ab in A549

    cells treated with AFB1.(B) Superoxide level was monitored with DHE and flow cytometry in A549 cells treated

    with 50 M AFB1 for the indicated time intervals. Fold superoxide induction in the AFB1-treated cells was

    calculated as a ratio relative to their own controls.(*P < 0.01; paired t-test).

    J Proteome Res. 12: 3434-3448, 2013

    21

  • Figure. AFB1 treatment induces

    cytotoxicity via production of

    AFB1DNA adducts. Physiologically,

    stimulus by low level of AFB1 causes

    cellular ROS production via oxidation of

    NADPH, the product in the pentose

    phosphate pathway catalyzed by G6PD.

    The induced ROS accordingly leads to

    Nrf2 nuclear translocation and expression

    of EPHX1, an enzyme involved in

    detoxification of AFB1. G6PD knockdown

    results in reduced generation of cellular

    NADPH, thereby causing impairment of

    AFB1-induced ROS production.

    Consequently, EPHX1 expression is less

    active in G6PD-depletion cells, suggesting

    a protective role of G6PD upon AFB1-

    inducible cytotoxicity.

    Model depicting the protective role of G6PD in

    AFB1- mediated cytotoxicity

    Lin HR el. al , J. Proteome Res. 2013, 12, 34343448 22

  • Glucose-6-Phosphate Dehydrogenase Enhances Antiviral

    Response through Downregulation of NADPH Sensor

    HSCARG and Upregulation of NF- B Signaling

    Dr. Yi-Hsuan Wu,

    23

    Viruses 7: 6689-6706, 2015

  • 24

    Summary: Glucose-6-Phosphate Dehydrogenase

    Enhances Antiviral Response through Downregulation

    of NADPH Sensor HSCARG and Upregulation of NF-B

    Signaling

    Viruses 7: 6689-6706, 2015

  • Diminished COX-2/PGE2-mediated antiviral

    response due to impaired NOX/MAPK signaling in

    G6PD-knockdown lung epithelial cells

    Lin HR, Wu YH, Yen WC,

    Yang CM, Chiu DTY

    Plos ONE 11: e0153462, 2016

    Dr. Yi-Hsuan Wu

    ()Dr. Hsin-Ru Lin

    ()

  • Diminished COX-2/PGE2-Mediated Antiviral Response

    Due to Impaired NOX/MAPK Signaling

    in G6PD-Knockdown Lung Epithelial Cells

    (G)

    PLoS One. 2016 Apr 20;11(4):e0153462

  • Glucose 6-phosphate dehydrogenase knockdown

    enhances IL-8 expression in HepG2 cells via

    oxidative stress and NF-kB signaling pathway

    Hung-Chi Yang, Mei-Ling Cheng, Yi-Syuan Hua,

    Yi-Hsuan Wu, Hsin-Ru Lin, Hui-Ya Liu, Hung-Yao Ho,

    Daniel Tsun-Yee Chiu

    Yang, et al., Journal of Inflammation 12:34, 2015

    Dr. Hung Chi Yang

    ()

    27

  • G6PD deficiency up-regulates IL-8 through

    oxidative stress and NF-B pathway

    28

    NF-kB

    knockdown

    suppresses IL-8

    antioxidants

    suppress IL-8

    Yang, et al., Journal of Inflammation 12:34, 2015

  • 29

    Glucose 6-phosphate dehydrogenase

    deficiency enhances germ cell apoptosis

    and causes defective embryogenesis in

    Caenorhabditis elegans

    Dr. Hung Chi Yang

    ()

    Cell Death & Disease 4: e616, 2013

    (IF=5.014)

  • 30

    G6PD knockdown enhances germ cell apoptosis, impairs egg production and

    induces severe hatching defect (A)

    (C)

    (B)

    Germ cell apoptosis

    Egg production

    Hatching

  • 1. G6PD-knockdown C. elegans displays enhanced germ cell

    apoptosis and reduced egg production as well as a severe

    defect in hatching.

    2. In contrast to its toxic effect in egg production, ROS may play

    an important role as signal molecules to mediate certain

    pathway(s) that in turn can affect hatching in C. elegans.

    3. This G6PD-knockdown animal model allows us to delineate

    the chronic effects of G6PD-deficiency at the multicellular

    organism level.

    31

    Summary from G6PD-deficient

    C. elegans study

    Cell Death & Disease 4: e616, 2013

  • g6pd knockdown in zebrafish embryos

    produces pericardial edema

    Wu. et al. Unpublished data

  • G6PD knockdown induces morphological

    changes and cadherin switch in A549 cells

    Wu. et al. Unpublished data

    LS LG

    A549

    G6PD

    E-cadherin

    -actin

    N-cadherin

    LS

    LG

    Scale bar, 50 m

  • AcknowledgementLong time colleagues and collaborators

    Dr. Mei-Ling Cheng, Associate Prof., Chang Gung Univ.

    Dr. Hung-Yao Ho, Associate Prof., Chang Gung Univ.

    Other Collaborators of Chang Gung

    Dr. Ming-Shi Shiao, Prof. , CGU

    Dr. Chih-Ching Wu, Associate Prof., CGU

    Dr. Sze-Cheng John Lo, Prof., CGU

    Collaborators of other Institutes

    Dr. Li-Ping Gao (Lanzhou Univ., China )

    Dr. Chang-Jun Lin (Lanzhou Univ., China )

    Prof. Arnold Stern (NYU, USA)

    Prof. Frans Kuypers(CHORI, Oakland/

    UC Berkeley, USA)

    Dr. Hung-Chi Yang

    Dr. Hsin-Ru Lin Dr. Hsiang-Yu Tang

    Dr. Yi-Hsuan Wu

    34

    Thank you very much for your attention!

    Email: dtychiu @mail.cgu.edu.tw A professor is just as good as his/her

    graduate students and/or postdoctoral fellows

  • ULK1/2 Constitute a Bifurcate Node Controlling

    Glucose Metabolic Fluxes in Addition to Autophagy

    Authors

    Terytty Yang Li, Yu Sun, Yu Liang, ...,

    Shu-Yong Lin, Daniel Tsun-yee Chiu,

    Sheng-Cai Lin

    Correspondence

    [email protected]

    In Brief

    Metabolic reprogramming underlies

    adaptive responses to various stresses.

    Li et al. discover that the autophagy-initiating

    kinases ULK1/2 also phosphorylate multiple

    glycolytic enzymes to sustain glycolysis and

    increase the proportion of glucose flux to

    the pentose phosphate pathway during

    nutritional stresses.

    Molecular Cell. 62:359-70, 2016 (SCI IF=13.958)

    mailto:[email protected]