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 GAbstract
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/Pass
age
Daily 10uM TMG or 0.35mM
GlcN treatment in 5mM
Galactose medium
Method 1:
Thiamet-G (TMG)Method 2:
H2O
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
AUCOCR(pMoles)
Ctrl TMG GlcN
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
RelativeATPLevel
Treated/Control
Ctrl TMG GlcN
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 MC MC
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
0
5000
10000
15000
20000
25000
30000
35000
40000
45000
Control TMG GlcN
FluorescenceIntensity(Superoxide
Production)
Non-Induced
Ros Induced
P<0.05
P<0.05
P<0.05
P<0.05
0
5000
10000
15000
20000
25000
30000
35000
40000
45000
50000
Control TMG GlcN
FluorescenceIntensity(TotalROS
Production)
Non-Induced
Ros Induced
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
0
0.5
1
1.5
2
2.5
Ctrl TMG GlcN
RelativeTXNRD1mRNALevel
TXNRD1/GAPDH
*
n = 4
WB: TXNRD1
WB: Actin
Ctrl TMG GlcN
0
0.2
0.4
0.6
0.8
1
1.2
1.4
Ctrl TMG GlcN
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) 621424
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