Pathways of Skeletal Muscle Atrophy: HIV as a Model System? Chelsea Bueter, Michelle McKinzey, Chloe...
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Transcript of Pathways of Skeletal Muscle Atrophy: HIV as a Model System? Chelsea Bueter, Michelle McKinzey, Chloe...
Pathways of Skeletal Muscle Atrophy: HIV as
a Model System?
Chelsea Bueter, Michelle McKinzey, Chloe Salzmann, Michael Zorniak
Department of Biology, Lake Forest College, Lake Forest, Illinois 60045 USA
Most Studied Pathway
Stress
PI3K
AKT
FOXO
mTOR
Atrogin-1
Protein degradation
Protein synthesis
S6K
(Nucleus)
pp
Introduction
The Past of HIV
• Vpr protein stops the cell cycle
• Prevents programmed cell death
• Increased replication of HIV
• AIDS muscle wasting symptom
Introduction
HIV to SMA?
• Vpr secreted like a hormone
• Infects other cells and organs
• AIDS wasting syndrome in skeletal muscle
Introduction
Cancer Cachexia
http://www2.msstate.edu/~shbryd/Disorders.html
• Cachexia is a syndrome in cancer patients
• Progressive muscle wasting, weight loss, weakness and fatigue
Introduction
The Past of Cachexia
• Pathway unknown
• Cytokines induced muscle wasting
• One hypothesis: Muscle wasting in cancer due to increased energy consumption
Introduction
What is Oxidative Stress?
• Cancer, Parkinson’s, Diabetes, and SMA• Free Radicals or Reactive Oxygen Species• Steal electrons to restore valence stability
Introduction
The Past of Oxidative Stress
Goldberg et al, 1986
• Calcium activates protein degradation
Appel et al, 1997
• Vitamin E decreases calcium levels
• Vitamin E is an anti-oxidant
• Thus, oxidative stress calcium levels and activates protein degradation.
Introduction
Diabetes
• Characterized by insulin deficiency or insulin intolerance
• Juvenile diabetes (type 1)- genetically linked but also diet linked
• Type 2 - middle-aged people-low insulin levels
• Leads to many other disorders
Introduction
The Past of Diabetes
• 1993- studies showed that in non-diabetics, insulin levels and activity remained high
• Diabetics showed very low insulin levels or activity
• Common symptom in diabetics was SMA
• Hypothesis= Insulin tolerance may be linked to SMA
Introduction
HIV’s Vpr protein
• Two Direct Pathways to SMA
1. Insulin Resistance
2. Glucocorticoid Hypersensitivity
HIV
Effects of Vpr binding
Vpr
Cdc25
Serine/Threonine residues
Cdc25
14-3-3
14-3-3
Vpr
14-3-3
Vpr
Cdc25
HIV
14-3-3
Vpr
Vpr inhibits insulin effects on FOXO
Vpr
Cdc25
14-3-3
Cdc25
Serine/Threonine residues on FOXO
HIV
What is insulin supposed to do?
FOXO
(Nucleus)
p
Insulin
FOXO p14-3-3
FOXO
Insulin
14-3-3
FOXO p14-3-3
FOXO p
14-3-3
HIV
How does Vpr affect glucocorticoid receptors?
Vpr
LQQLL
HIV
•Specific LXXLL motif binds GRE
•Completely different from ability to arrest cell cyle
Kino et al 2000
Vpr as a Co-regulator of GR
Vpr
TATA
Transcription: enhancing glucocorticoid signal
G R
TFIIB
TFIID
RNA polymerase II
GRE
HIV
Kino et al 2000
Summary of Vpr & SMAVPR Protein
Skeletal Muscle Atrophy
VprGRE
Glucocorticoid
14-3-3
Vpr
FOXOnucleus
Atrogin-1
Therapies for HIV muscle wasting
• Steroid hormone receptor antagonists (RU 486)
• Vpr antagonists
• Current antiretroviral therapies
Cai et al. Study
MISR Mouse MIKK Mouse
Constitutively active IκBα
Constitutively active IκB
Cachexia
Inactive NF-κB Always active NF-κB
Tumor Activity
• Presence of a tumor increases the level of NF-κB activity in wild type mice
Cachexia
Tumor Necrosis Factor - TNFα
• In the presence of IκBα, activity decreases
• Without IκBα, inhibitor does not stop production
Cachexia
What else does NF-κB affect?
• TNF activates NF-κB which causes a decrease in MyoD production
Cachexia
Oxidative StressReactive Oxygen Species
Mitochondria
Caspase-3Calpain
Sarcomere
Unit of Myofibril
20S/26S Proteasome
Calcium
Cytochrome c
CalpastatinCalpastatin
NO
Oxidative Stress
Effect of Oxidative Stress
• Increase of oxidative stress increases calsequesterin
• Calsequestrin sequesters intracellular calciumHunter et al, 2001
Laser Densitometry
Oxidative Stress
Effect of Increased Calcium
Type II Diaphragm Muscle Fibers by Immunohistochemistry
• Disuse increases calpain and 20S proteasome activity
Tidball et al, 2002
Oxidative Stress
Calpain Proteolysis
Koh et al, 2000
• Calcium treatment increases protein cleavage
• Protein cleavage can be inhibited by nitric oxide and calpastatin
Oxidative Stress
Caspase Activity
• Caspases inhibit calpastatin
• Calpastatin is a calpain inhibitor
Wang et al, 1998
Oxidative Stress
SummaryOxidative Stress
Increase Ca2+
Calpain & Caspase 3 activity increases
Releases Actomyosin to be degraded in proteasome
Skeletal Muscle Atrophy
Therapies for Oxidative Stress
• NO and Calpastatin Transgene• Vitamin E protects against ROS• Vitamin C restores Vitamin E activity
Cell
Vitamin E
ROS
Oxidative Stress
What activates the Ubiquitin-proteasome pathway?
• No difference in diabetics without vs. diabetics with acidosis
• Acidosis is not a stimulus of ubiquitin dependent atrophy
Diabetes
Price et al. 1999
Effects of Insulin on Ub-proteasome pathway
• Less protein degradation in insulin treated muscles
• Lower insulin levels leads to activation of Ub-proteasome pathway
Diabetes
Price et al., 1999
Results of Pathway Activation
• Levels of Ub-ligase and its coenzyme increase
• Amount of Ub-conjugation by these increases
Diabetes
Goldberg et al., 1999
Proteasome Formation
• mRNA for 19 S and 20 S subunits increases• Formation of 26 S proteasome increases
Diabetes
Attaix et al., 2004
Proteasome Activity
• Flourescence in atrophied muscles higher than control muscles
• Flourescence is analogous to amount of 26 S proteasome activity
Diabetes
Attaix et al., 2004
Summary of Diabetes and SMADiabetes
Insulin decrease/ glucocorticoid increase
E3-alpha ubiquitin ligase increases
26 S Proteasome activity increases
Skeletal Muscle Atrophy
Therapy for Diabetes
• Treatments for diabetes generally focus on maintaining available insulin levels NOT SMA
• Side effect of the insulin treatment, however, is associated with the reverse pathway of atrophy, i.e. hypertrophy
Diabetes
Vpr
VPR Protein
Inhibit insulin effects on FOXO
Glucocorticoid hypersensitivity
Co-activates glucocorticoid receptor
Atrogin-1 induction
Skeletal Muscle Atrophy
Oxidative StressOxidative Stress
Increase Ca2+
Calpain & Caspase 3 activity increases
Releases Actomyosin to be degraded in proteasome
Skeletal Muscle Atrophy
DiabetesDiabetes
Insulin decrease/ glucocorticoid increase
E3-alpha ubiquitin ligase increases
26 S Proteasome activity increases
Skeletal Muscle Atrophy
HIV
Skeletal Muscle Atrophy
VPR Protein
Inhibit insulin effects on FOXO
Glucocorticoid hypersensitivity
Co-activates glucocorticoid receptor
Atrogin-1 induction
Oxidative Stress
Increase Ca2+
Calpain & Caspase 3
activity increases
Releases Actomyosin
to be degraded in proteasome
Cachexia
Murf-1 increase
IKK/NF-kappa B pathway
MyoD mRNA decrease
Diabetes
Insulin decrease/ glucocorticoid
increase
E3-alpha ubiquitin increases
26 S Proteasome activity increases