Oxidative Stress and JNK Activation Final
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Transcript of Oxidative Stress and JNK Activation Final
Oxidative Stress and JNK Activation Cause Mitochondrial
Dysfunction after VDAC Opening
Diana FangClemson University
Mentors: Dr. John J. Lemasters and Dr. Eduardo N. Maldonado
Department of Drug Discovery & Biomedical Sciences
ΔΨ
Mitochondrial Bioenergetics: Non-proliferating Cells
Maldonado & Lemasters, Mitochondrion 2014
ΔΨ
Warburg Metabolism
Mitochondrial Bioenergetics: Proliferating Cells
Maldonado & Lemasters, Mitochondrion 2014
Erastin & X1 Cause Hyperpolarization and Subsequent Depolarization of Mitochondrial Membrane in HepG2 Cells
Baseline
120 min 240 min
Erastin
X1Baseline
60 min 120 minMaldonado & DeHart et al., unpublished
VDAC Opening ?
VDAC Opening ?
JNK P
c-Jun N-terminal Kinase (JNK)
Mitochondria
ROS
MKK4MKK7
MEKK 1,4MLK3ASK1
JNK1,2,3
Stress(ie. ROS)
ROS
ROS
Translocation
Mitochondrial Dysfunction & Cell Death
ΔΨ
HypothesisWe hypothesize that VDAC-Tubulin antagonist, X1, promotes hyperpolarization and reactive oxygen species (ROS) formation which leads to activation of JNK and cause mitochondrial depolarization and mitochondrial dysfunction.
AimTo evaluate the effects of decreasing ROS and JNK inhibition on late depolarization induced by X1.
JNK Inhibition with SP600125 Delays Mitochondrial Dysfunction after X1
15 min 30 min 60 min
+ SP600125 + X1
0 min 15 min 30 min 60 min
+ X1 0
255
0 min 15 min 30 min 60 min
+ JNKi VIII + X1
+ X1 0
255
JNK Inhibition with JNK Inhibitor VIII Delays Mitochondrial Dysfunction after X1
N-Acetylcysteine does not prevent hyperpolarization but does prevent depolarization of ∆Ψ
+ NAC + X1
0 min 15 min 30 min 60 min0 min 15 min 30 min 60 min
+ X1 0
255
ROS Promotes Mitochondrial Dysfunction after X1
Time (min)
0 30 60
Nuc
lear
TM
RM
Flu
ores
cenc
e (%
of B
asel
ine)
0
500
1000
1500
X1JNKi VIII + X1SP600125 + X1NAC + X1Vehicle
****
****
***
Summary • JNK Inhibitors do not prevent X1 induced hyperpolarization but does
attenuate subsequent depolarization • NAC does not prevent the hyperpolarization of the mitochondrial
membrane but does prevent the late depolarization after X1, blocking mitochondrial dysfunction
ConclusionThe late depolarization following the hyperpolarization induced by X1 is mediated by oxidative stress and JNK activation.
Acknowledgements Lab Members • John J. Lemasters, MD,
PhD• Eduardo N. Maldonado,
DVM, PhD• Monika Gooz, MD, PhD• David N. DeHart, soon PhD • Kareem Heslop• David Johnson
MUSC Summer Undergraduate Program
• Stephanie Brown-Guion, MSM
Questions?
Downstream of JNKKey Players:
- Sab: sequesters SHP-1 (phosphatase) and Binds with JNK
- SHP-1: Tyrosine Phosphatase
- c-Src: Tyrosine Kinase that activates SHP-1, complexes I and II
- DOK-4: Docking protein that is necessary for SHP-1 to dephosphorylate c-Src
Info from Win et al., Hepatology 2016
Compounds used
SP600125 JNK Inhibitor VIII N-Acetylcysteine (NAC)
NAC Prevents ∆
Time (min)
0 30 60
Nuc
lear
TM
RM
Flu
ores
cenc
e (A
U)
0
25
50X1JNKi VIII + X1SP600125 + X1NAC + X1Vehicle