Transcript HIV’s Vpr protein - Lake Forest College

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
Presentation Pathway
Introduction
Diseases
HIV
Cachexia
Oxidative Stress
Diabetes
Discussion
Introduction
Paradigm of SMA
Hypertrophy vs. Atrophy
http://www.nndb.com
Introduction
Most Studied Pathway
Stress
PI3K
mTOR
AKT
S6K
FOXO
p
p
Atrogin-1
Protein degradation
(Nucleus)
Protein
synthesis
?
?
HIV
Diabetes
?
Cachexia
?
Oxidative
Stress
VPR
Protein
?
?
?
?
Skeletal Muscle Atrophy
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
• Cachexia is a syndrome
in cancer patients
• Progressive muscle
wasting, weight loss,
weakness and fatigue
http://www2.msstate.edu/~shbryd/Disorders.html
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
Presentation Pathway
Introduction
Diseases
HIV
Cachexia
Oxidative Stress
Diabetes
Therapies
Discussion
HIV
?
Skeletal Muscle Atrophy
HIV
HIV’s Vpr protein
• Two Direct Pathways to SMA
1. Insulin Resistance
2. Glucocorticoid Hypersensitivity
HIV
Effects of Vpr binding
Serine/Threonine residues
Vpr
Vpr
14-3-3
14-3-3
Cdc25
Cdc25
Vpr
14-3-3
Cdc25
HIV
Vpr Inhibits Cell Cycle
Triggers mitosis machinery
Alberts et al 2004
HIV
Stopped Cell Cycle
Dividing
G2/M
Dividing
G2/M
•Vpr stops the cell cycle at G2/M
He et al 1995
HIV
Vpr inhibits insulin effects on
FOXO
Serine/Threonine residues on FOXO
Vpr
Vpr
14-3-3
14-3-3
Cdc25
Cdc25
HIV
Effect of Vpr on FOXO
•Vpr doesn’t bind FOXO
Kino et al 2005
HIV
What is insulin supposed to do?
Insulin
Insulin
p
FOXO
FOXO
14-3-3
FOXO
p
14-3-3
14-3-3
FOXO
p
(Nucleus)
14-3-3
FOXO
p
HIV
Vpr won’t let insulin work!
FOXO
No FOXO
Kino et al 2005
HIV
How does Vpr affect glucocorticoid
receptors?
Vpr
LQQLL
Kino et al 2000
•Specific LXXLL motif binds GRE
•Completely different from ability to arrest cell cyle
Vpr as a Co-regulator
Kino et al 2000
HIV
Vpr as a Co-regulator of GR
Vpr
GR
GRE
TFIIB
RNA
polymerase II
TFIID
Transcription: enhancing
glucocorticoid signal
TATA
Kino et al 2000
Summary of Vpr & SMA
VPR
Protein
Vpr
GRE
Vpr
14-3-3
Glucocorticoid
FOXO
nucleus
Atrogin-1
Skeletal Muscle Atrophy
Therapies for HIV muscle wasting
• Steroid hormone receptor antagonists (RU
486)
• Vpr antagonists
• Current antiretroviral therapies
Cancer Cachexia
?
Skeletal Muscle Atrophy
Cachexia
NF-κB
IκB
P
IκBα
NF- κB
Nucleus
Cachexia
Cai et al. Study
MISR Mouse
MIKK Mouse
Constitutively active
IκBα
Constitutively active
IκB
Inactive NF-κB
Always active NF-κB
Cachexia
NF-κB Activity
• NF-κB activity is high in MIKK mice
Cachexia
MIKK Mice vs. MISR Mice
• MIKK mice have a much lower body mass
Cachexia
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 does NF-κB affect?
• MURF1 mRNA is much higher in MIKK mice
than in MISR mice
Cachexia
What else does NF-κB affect?
• TNF activates NF-κB which causes a
decrease in MyoD production
Cachexia
Troponin in Cardiac Muscle
• Troponin-1 is degraded in the presence of
MURF1
Cachexia Pathway
TNF-α
activates
NF-κB
increases
MURF1
decreases
MyoD
Cachexia
Therapy for Cachexia
• Salicylate inhibits the NF-κB pathway, preventing
muscle loss
Oxidative Stress
?
Skeletal Muscle Atrophy
Oxidative Stress
Oxidative Stress
Reactive Oxygen Species
Calcium
Mitochondria
Cytochrome c
Calpastatin
Calpain
Caspase-3
Calpastatin
NO
Sarcomere
Unit of Myofibril
20S/26S Proteasome
Oxidative Stress
Effect of Disuse
• Disuse
increases oxidative stress
Tidball et al, 2002
Oxidative Stress
Effect of Oxidative Stress
Laser Densitometry
• Increase of oxidative stress increases calsequesterin
• Calsequestrin sequesters intracellular calcium
Hunter et al, 2001
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
• Calcium treatment increases protein cleavage
• Protein cleavage can be inhibited by nitric oxide and
calpastatin
Koh et al, 2000
Oxidative Stress
Caspase Activity
• Caspases inhibit calpastatin
• Calpastatin is a calpain inhibitor
Wang et al, 1998
Summary
Oxidative
Stress
Increase Ca2+
Calpain & Caspase 3 activity
increases
Releases Actomyosin to be
degraded in proteasome
Skeletal Muscle Atrophy
Oxidative Stress
Therapies for Oxidative Stress
ROS
Cell
Vitamin E
• NO and Calpastatin Transgene
• Vitamin E protects against ROS
• Vitamin C restores Vitamin E activity
Diabetes
?
Skeletal Muscle Atrophy
Diabetes
Insulin levels
Ub-ligase
E3-α
26 S proteasome
Diabetes
What activates the Ubiquitinproteasome pathway?
• No difference in diabetics without vs. diabetics with
acidosis
• Acidosis is not a stimulus of ubiquitin dependent
atrophy
Price et al. 1999
Diabetes
Effects of Insulin on Ubproteasome pathway
• Less protein degradation in insulin treated
muscles
• Lower insulin levels leads to activation of Ubproteasome pathway
Price et al., 1999
Diabetes
Results of Pathway Activation
• Levels of Ub-ligase and
its coenzyme increase
• Amount of Ub-conjugation
by these increases
Goldberg et al., 1999
Diabetes
Proteasome Formation
• mRNA for 19 S and 20 S subunits increases
• Formation of 26 S proteasome increases
Attaix et al., 2004
Diabetes
Proteasome Activity
• Flourescence in atrophied muscles higher than
control muscles
• Flourescence is analogous to amount of 26 S
proteasome activity
Attaix et al., 2004
Summary of Diabetes and SMA
Diabetes
Insulin decrease/ glucocorticoid
increase
E3-alpha ubiquitin ligase increases
26 S Proteasome activity increases
Skeletal Muscle Atrophy
Diabetes
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
Presentation Pathway
Introduction
Diseases
HIV
Cachexia
Oxidative Stress
Diabetes
Discussion
?
HIV
?
Diabetes
?
Cachexia
VPR
Protein
Skeletal Muscle Atrophy
Oxidative
Stress
Vpr
VPR
Protein
Inhibit insulin
effects on FOXO
Co-activates glucocorticoid
receptor
Glucocorticoid
hypersensitivity
Atrogin-1
induction
Skeletal Muscle Atrophy
Cachexia
Cachexia
IKK/NFkappa B
pathway
Murf-1
increase
MyoD mRNA
decrease
Skeletal Muscle Atrophy
Oxidative Stress
Oxidative
Stress
Increase
Ca2+
Calpain & Caspase 3
activity increases
Releases Actomyosin to be
degraded in proteasome
Skeletal Muscle Atrophy
Diabetes
Diabetes
Insulin decrease/ glucocorticoid increase
E3-alpha ubiquitin ligase increases
26 S Proteasome activity increases
Skeletal Muscle Atrophy
Diabetes
Insulin decrease/
glucocorticoid
increase
E3-alpha
ubiquitin
increases
26 S Proteasome
activity increases
HIV
Cachexia
Oxidative
Stress
VPR
Protein
Co-activates
glucocorticoid
receptor
IKK/NFkappa B
pathway
Murf-1
increase
MyoD mRNA
decrease
Inhibit insulin
effects on FOXO
Glucocorticoid
hypersensitivity
Atrogin-1
induction
Skeletal Muscle Atrophy
Increase
Ca2+
Calpain &
Caspase 3
activity
increases
Releases
Actomyosin
to be
degraded in
proteasome
Acknowledgements
Thanks to Dr. D, Sara Herrera, Tammy
Hibler, Arun Paul, and Chris Prater