Brenna O-GlcNAc Final Poster
1
S/T Understanding the Regulation of O-GlcNAc in Mitochondrial Function Brenna Seawalt, Ee Phie Tan, Stefan Graw, Pramod Dhakal, Devin Koestler, Christy Hagan, Russell Swerdlow and Chad Slawson Department of Biochemistry and Molecular Biology, University of Kansas Medical Center, Kansas City, KS 66160 OGT/OGA G Abstract O-linked N-acetylglucosamine (O-GlcNAc) is a post-translational modification (PTM) that involves the addition of a single sugar, N- acetylglucosamine, to serine or threonine residues of protein in the cytoplasm, nucleus, or mitochondria. Two enzymes facilitate this modification: O-GlcNAc transferase (OGT), which adds the modification and O-GlcNAcase (OGA), which removes the modification. The addition and removal of O-GlcNAc by these two enzymes is termed O-GlcNAc cycling and works as a molecular switch. Of note, PTMs are used in regulating protein function and therefore can affect mitochondrial activity. We postulate that O- GlcNAc regulates mitochondrial function. The proper regulation of mitochondrial function is essential for cellular metabolism and respiration. Improper regulation of mitochondria leads to cellular damage and disease development. Here, we examined how elevation of O-GlcNAc affects mitochondrial function. In order to elevate O-GlcNAc levels, we supplemented SH-SY5Y and NT2 neuroblastoma cells with Thiamet-G (TMG), an OGA inhibitor, or Glucosamine (GlcN), which increases OGT substrate availability. We found that both TMG and GlcN treatment increased O- GlcNAc levels and OGA protein and transcript levels. Next, we found that mitochondrial respiration was altered and ATP production was decreased. We found that reactive oxygen species (ROS) production was significantly reduced in treated cells. We also found that pathway activity and protein expression of the master regulator of antioxidant response, the transcription factor NRF2 was down regulated. Finally, the protein expression of NRF2 antioxidant genes such as manganese superoxide dismustase (MnSOD) and thioredoxin reductase 1 (TXNRD1) was lower. Altogether, these data demonstrate that prolonged alterations to the cellular homeostasis of O-GlcNAc affects mitochondrial function by reprogramming metabolic activity. Further understanding of how O-GlcNAc cycling regulates metabolism will provide new insights into metabolic diseases such as Alzheimer’s. Research is supported by: KUMC Alzheimer’s Disease Center Mabel A. Woodyard Fellowship National Institute of Health R01DK100595-01 National Institute of Health COBRE P20GM104936 #845.1 O-GlcNAc Regulation of Mitochondrial Function D-Glucosamine Supplementation Extends Life Span of Nematodes and of Aging Mice GlcN extends C. elegans life span GlcN reduces ATP level GlcN transiently induces ROS formation A B C (Weimer et al., Nature Comm, 2014) O-GlcNAc: Sensor of Cellular Environment Hexosamine Biosynthetic Pathway (Slawson et al., Trends Biochem Sci, 2007) Glucose Glc-6-P Fruc-6-P GlcN-6-P UDP-GlcNAc GlcNAc-6-P GlcNAc-1-P Glutamine GFAT1 Fatty Acid Amino Acid Nucleotide Carbohydrate UTP Acetyl CoA Glucosamine (GlcN) GNPNAT1 AGM1 UAP O-GlcNAc Transferase (OGT) Protein OH O-GlcNAcase (OGA) O-GlcNAc Protein 0 day 21 days Harvest/ Passage Daily 10uM TMG or 0.35mM GlcN treatment in 5mM Galactose medium Method 1: Thiamet-G (TMG) Method 2: ROS H 2 O GPX2, PRDX1, SOD, CAT, TXNRD1 O-GlcNAc ? ? ? NRF2 TMG/GlcN Treatment Alters Mitochondrial Function 0.00 500.00 1000.00 1500.00 2000.00 2500.00 3000.00 3500.00 4000.00 4500.00 Ctrl TMG GlcN AUC OCR (pMoles) P<0.05 P<0.05 P<0.05 A Mitochondrial respiration is altered 0 0.2 0.4 0.6 0.8 1 1.2 Ctrl TMG GlcN Relative ATP Level Treated/Control n = 3 * * B ATP level is significantly reduced Mitochondrial O-GlcNAcylation Increases After TMG/GlcN Treatment C 225 kDa 52 kDa 110 kDa WB: O-GlcNAc WB: OGA WB: OGT WB: Actin M M C M C Ctrl TMG GlcN WB: VDAC C: Cytoplasm M: Mitochondria Copyright © 2009 Pearson Education, Inc., publishing as Pearson Benjamin Cummings A TMG/GlcN Treatment Lowers Cellular Reactive Oxygen Species (ROS) B Total cellular ROS level is lower H 2 O 2 Nucleus H 2 O 2 Fe 2+ HO RNS O 2 - Cyt o plasm Con TMG 0 5000 10000 15000 20000 25000 30000 35000 40000 Non-Induced Fluorescence Intensity (Superoxide Production) P<0.0 5 P<0.05 P<0.05 P<0.05 Con TMG 0 5000 10000 15000 20000 25000 30000 35000 40000 45000 Non-Induced Ros Induced Fluorescence Intensity (Total ROS Production) P<0.05 P<0.05 P<0.05 P<0.05 P<0.05 Superoxide level is lower Altered ATP production Extended mitochondrial shape TMG/GlcN Treatment Changes Nuclear Factor-Like 2 (NRF2) Regulation ROS NRF2 NRF2 KEAP1 Cytoplasm Nucleus NRF2 Ub NRF2 KEAP1 ERK 1/2 Jun ATF4 c-MAF FRA1 c-Fos CBP/p300 SOD PRDX1 CAT GPX2 Antioxidant Genes A The master regulator of antioxidant responses C TXNRD1 Expressions are reduced Ctrl TMG GlcN 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 Relative TXNRD1 mRNA Level TXNRD1/GAPDH * n = 4 WB: TXNRD1 WB: Actin Ctrl TMG GlcN Ctrl TMG GlcN 0 0.2 0.4 0.6 0.8 1 1.2 1.4 TXNRD1/Actin * n = 4 D NRF2 is O-GlcNAcylated Nrf2 IP IgG 1° Ctrl TMG GlcN WB: O-GlcNAc WB: Nrf2 Hypothesis We hypothesized that elevations in cellular O- GlcNAc levels alter metabolic profiles through reprogramming of gene programs. Mitochondrial Morphology is Disrupted in OGT/OGA Gain of Function Cells Canonical Pathway Activity Altered After TMG/GlcN Treatment Canonical Pathway Activity Altered After TMG/GlcN Treatment 0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 3.25 3.50 3.75 4.00 0.00 0.01 0.02 0.03 0.04 0.05 0.06 0.07 0.08 0.09 0.10 0.11 0.12 0.13 -log(p value) Ratio Huntington’s Disease Signaling ERK/MAPK Signaling Protein Kinase A Signaling Synaptic Long Term Potentiation NRF2-mediated Oxidative Stress Response Threshold -log(p value) ERK/MAPK Signaling Huntington’s Disease Signaling NRF2-mediated Oxidative Stress Response Protein Kinase A Signaling Synaptic Long Term Potentiation 0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 Threshold 0.00 0.01 0.02 0.03 0.04 0.05 0.06 0.07 0.08 0.09 0.10 0.11 0.12 0.13 0.14 Ratio NRF2-mediated oxidative stress response is predicted to be down-regulated GlcN TMG A modification of Ser and Thr residues by -N- acetylglucosamine Is NOT elongated to more complex oligosaccharides Localized to the cytoplasm, mitochondria, and nucleus Often reciprocal with phosphorylation Added to proteins by O-GlcNAc Transferase and removed by O- GlcNAcase Highly abundant PTM (>1500 identified proteins) Does NOT have canonical consensus site O-GlcNAc Transferase (OGT) O-GlcNAcase (OGA) What is O-GlcNAc? (Hart et al., An. Rev. Biochem, 2011) 62 14 24 38 TMG 76 GlcN 21 65 157 86 TMG 222 GlcN Up-regulated genes Down-regulated genes Transcription is Altered After TMG/GlcN Treatment TMG/GlcN Treatment Alters O-GlcNAc Cycling Ctrl 225 kDa 52 kDa 110 kDa WB: O-GlcNAc WB: OGA WB: OGT WB: GFAT1 WB: Actin TMG GlcN A O-GlcNAcylation/OGA protein expression is altered B O-GlcNAc/OGA protein expression is altered in NT2 cells Ctrl TMG GlcN WB: Nrf2 WB: Actin B NRF2 expressions are altered WB: Nrf2 WB: Actin WB: O-GlcNAc 225 kDa 52 kDa 110 kDa WB: OGA WB: OGT WB: Actin Ctrl TMG GlcN
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Transcript of Brenna O-GlcNAc Final Poster
S/T
Understanding the Regulation of O-GlcNAc in Mitochondrial FunctionBrenna Seawalt, Ee Phie Tan, Stefan Graw, Pramod Dhakal, Devin Koestler, Christy Hagan, Russell Swerdlow and Chad Slawson
Department of Biochemistry and Molecular Biology, University of Kansas Medical Center, Kansas City, KS 66160
OGT/OGA GAbstractO-linked N-acetylglucosamine (O-GlcNAc) is a post-translational modification (PTM) that involves the addition of a single sugar, N-acetylglucosamine, to serine or threonine residues of protein in the cytoplasm, nucleus, or mitochondria. Two enzymes facilitate this modification: O-GlcNAc transferase (OGT), which adds the modification and O-GlcNAcase (OGA), which removes the modification. The addition and removal of O-GlcNAc by these two enzymes is termed O-GlcNAc cycling and works as a molecular switch. Of note, PTMs are used in regulating protein function and therefore can affect mitochondrial activity. We postulate that O-GlcNAc regulates mitochondrial function. The proper regulation of mitochondrial function is essential for cellular metabolism and respiration. Improper regulation of mitochondria leads to cellular damage and disease development. Here, we examined how elevation of O-GlcNAc affects mitochondrial function. In order to elevate O-GlcNAc levels, we supplemented SH-SY5Y and NT2 neuroblastoma cells with Thiamet-G (TMG), an OGA inhibitor, or Glucosamine (GlcN), which increases OGT substrate availability. We found that both TMG and GlcN treatment increased O-GlcNAc levels and OGA protein and transcript levels. Next, we found that mitochondrial respiration was altered and ATP production was decreased. We found that reactive oxygen species (ROS) production was significantly reduced in treated cells. We also found that pathway activity and protein expression of the master regulator of antioxidant response, the transcription factor NRF2 was down regulated. Finally, the protein expression of NRF2 antioxidant genes such as manganese superoxide dismustase (MnSOD) and thioredoxin reductase 1 (TXNRD1) was lower. Altogether, these data demonstrate that prolonged alterations to the cellular homeostasis of O-GlcNAc affects mitochondrial function by reprogramming metabolic activity. Further understanding of how O-GlcNAc cycling regulates metabolism will provide new insights into metabolic diseases such as Alzheimer’s.
Research is supported by: KUMC Alzheimer’s Disease CenterMabel A. Woodyard FellowshipNational Institute of Health R01DK100595-01National Institute of Health COBRE P20GM104936
#845.1
O-GlcNAc Regulation of Mitochondrial Function
D-Glucosamine Supplementation Extends Life Span of Nematodes and of Aging Mice
GlcN extends C. elegans life span
GlcN reduces ATP level
GlcN transiently induces ROS formation
A B C
(Weimer et al., Nature Comm, 2014)
O-GlcNAc: Sensor of Cellular Environment
Hexosamine Biosynthetic Pathway
(Slawson et al., Trends Biochem Sci, 2007)
Glucose
Glc-6-P
Fruc-6-P
GlcN-6-P
UDP-GlcNAc
GlcNAc-6-P
GlcNAc-1-P
Glutamine
GFAT1
Fatty Acid
Amino Acid
Nucleotide
Carbohydrate
UTP
Acetyl CoA
Glucosamine (GlcN)
GNPNAT1
AGM1
UAP
O-GlcNAc Transferase
(OGT)
Protein
OH
O-GlcNAcase (OGA)
O-GlcNAc
Protein
0 day 21 days
Harvest/Passage
Daily 10uM TMG or 0.35mM GlcN treatment in 5mM
Galactose medium
Method 1:
Thiamet-G (TMG)Method 2:
ROSH2O
GPX2, PRDX1, SOD, CAT, TXNRD1
O-GlcNAc? ?
?
NRF2
TMG/GlcN Treatment Alters Mitochondrial Function
0.00
500.00
1000.00
1500.00
2000.00
2500.00
3000.00
3500.00
4000.00
4500.00
Ctrl TMG GlcN
AU
C O
CR
(pM
oles
)
P<0.05
P<0.05P<0.05
AMitochondrial respiration is altered
0
0.2
0.4
0.6
0.8
1
1.2
Ctrl TMG GlcN
Rel
ativ
e A
TP L
evel
Trea
ted/
Con
trol
n = 3
* *
BATP level is significantly reduced
Mitochondrial O-GlcNAcylation Increases After TMG/GlcN Treatment
C
225 kDa
52 kDa
110 kDa
WB: O-GlcNAc
WB: OGA
WB: OGTWB: Actin
M MC MC
Ctrl TMG GlcN
WB: VDAC
C: CytoplasmM: Mitochondria
Copyright © 2009 Pearson Education, Inc., publishing as Pearson Benjamin Cummings
A
TMG/GlcN Treatment Lowers Cellular Reactive Oxygen Species (ROS)
B
Total cellular ROS level is lower
H2O2
NucleusH2O2
Fe2+
HO
RNS
O2-
Cyto
plas
m
Control TMG GlcN0
5000
10000
15000
20000
25000
30000
35000
40000
Non-InducedRos Induced
Fluo
resc
ence
Inte
nsity
(Sup
erox
ide
Prod
uctio
n)
P<0.05
P<0.05P<0.05
P<0.05
Control TMG GlcN0
5000
10000
15000
20000
25000
30000
35000
40000
45000
Non-InducedRos Induced
Fluo
resc
ence
Inte
nsity
(Tot
al R
OS
Prod
uctio
n)
P<0.05P<0.05
P<0.05
P<0.05
P<0.05
Superoxide level is lower
Altered ATP productionExtended mitochondrial shape
TMG/GlcN Treatment Changes Nuclear Factor-Like 2 (NRF2) Regulation
ROS
NRF2
NRF2
KEAP1
Cytoplasm
Nucleus
NRF2
Ub
NRF2
KEAP1
ERK 1/2
Jun
ATF4
c-MAF
FRA1
c-Fos
CBP/p300
SODPRDX1 CAT GPX2
Antioxidant Genes
AThe master regulator of antioxidant responses
CTXNRD1 Expressions are reduced
Ctrl TMG GlcN0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
Rel
ativ
e TX
NR
D1
mR
NA
Lev
elTX
NR
D1/
GA
PDH
*
n = 4
WB: TXNRD1
WB: Actin
Ctrl TMG GlcN
Ctrl TMG GlcN0
0.2
0.4
0.6
0.8
1
1.2
1.4
TXN
RD
1/A
ctin *
n = 4
DNRF2 is O-GlcNAcylated
Nrf2 IPIgG 1° Ctrl TMG GlcN
WB: O-GlcNAc
WB: Nrf2
Hypothesis We hypothesized that elevations in cellular O-GlcNAc levels alter metabolic profiles through reprogramming of gene programs.
Mitochondrial Morphology is Disrupted in OGT/OGA Gain of Function Cells
Canonical Pathway Activity Altered After TMG/GlcN Treatment
Canonical Pathway Activity Altered After TMG/GlcN Treatment
0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 3.25 3.50 3.75 4.00
0.00 0.01 0.02 0.03 0.04 0.05 0.06 0.07 0.08 0.09 0.10 0.11 0.12 0.13
-log(p value)
Ratio
Huntington’s Disease Signaling
ERK/MAPK Signaling
Protein Kinase A Signaling
Synaptic Long Term Potentiation
NRF2-mediated Oxidative Stress Response
Threshold
-log(p value)
ERK/MAPK Signaling
Huntington’s Disease Signaling
NRF2-mediated Oxidative Stress Response
Protein Kinase A Signaling
Synaptic Long Term Potentiation
0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00
Threshold
0.00 0.01 0.02 0.03 0.04 0.05 0.06 0.07 0.08 0.09 0.10 0.11 0.12 0.13 0.14 Ratio
NRF2-mediated oxidative stress response is predicted to be down-regulated
GlcN
TMG
A modification of Ser and Thr residues by -N-acetylglucosamine Is NOT elongated to more complex oligosaccharides Localized to the cytoplasm, mitochondria, and nucleus Often reciprocal with phosphorylation Added to proteins by O-GlcNAc Transferase and removed by O-GlcNAcase Highly abundant PTM (>1500 identified proteins) Does NOT have canonical consensus site
O-GlcNAc Transferase (OGT)
O-GlcNAcase (OGA)
What is O-GlcNAc?
(Hart et al., An. Rev. Biochem, 2011) 621424
38TMG
76GlcN
21 65 157
86TMG
222GlcN
Up-regulated genes Down-regulated genes
Transcription is Altered After TMG/GlcN Treatment
TMG/GlcN Treatment Alters O-GlcNAc Cycling
Ctrl
225 kDa
52 kDa
110 kDa
WB: O-GlcNAc
WB: OGA
WB: OGT
WB: GFAT1
WB: Actin
TMG GlcN
AO-GlcNAcylation/OGA protein expression is altered
BO-GlcNAc/OGA protein expression is altered in NT2 cells
Ctrl TMG GlcN
WB: Nrf2
WB: Actin
BNRF2 expressions are altered
WB: Nrf2
WB: Actin
WB: O-GlcNAc
225 kDa
52 kDa
110 kDa
WB: OGA
WB: OGT
WB: Actin
Ctrl TMG GlcN
