linda hendershot brazil pre-meeting course
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LINDA HENDERSHOT MAY 27, 2012
“UPR mechanisms: role in development and disease”
CSSI Workshop on Cell Stress
Pre-meeting Course
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NucleusMitochondria
ER
THE ENDOPLASMIC RETICULUM: A DEDICATED PROTEIN FOLDING COMPARTMENT
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THE ENDOPLASMIC RETICULUM: A DEDICATED PROTEIN FOLDING COMPARTMENT
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THE ENDOPLASMIC RETICULUM: A DEDICATED PROTEIN FOLDING COMPARTMENT
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THE ENDOPLASMIC RETICULUM: A DEDICATED PROTEIN FOLDING COMPARTMENT
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KDELKDEL
BiP
THE ENDOPLASMIC RETICULUM: A DEDICATED PROTEIN FOLDING COMPARTMENT
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THE ENDOPLASMIC RETICULUM: A DEDICATED PROTEIN FOLDING COMPARTMENT
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THE ENDOPLASMIC RETICULUM: A DEDICATED PROTEIN FOLDING COMPARTMENT
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THE ENDOPLASMIC RETICULUM: A DEDICATED PROTEIN FOLDING COMPARTMENT
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Major aspects of the mammalian UPR
• Up-regulation of ER chaperones (i.e., BiP, GRP94, GRP170).- prevents protein aggregation and contributes to
maintaining ER function during stress conditions- promotes refolding when stress subsides
• Transient inhibition of protein translation.- limits the accumulation of unfolded proteins- not restricted to secretory pathway proteins
• Increased degradative capacity of the cell.- reduces load of unfolded proteins
• Arrest of cells in the G1 phase of cell cycle.- ensures cells experiencing stress are not replicated
• Activation of apoptotic pathways if stress persists.- protects organism at the expense of the cell
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The Cellular Response to ER Stress or the Unfolded Protein Response
1. The signal – how damage is assessed
2. The transducers – how the signal is relayed
3. The response – characterized by damage control
4. Defeat – protecting the organism
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1. Signal?2. Transducers?
3. Downstream Elements?
4. Regulatory Elements?
GRPs
Nucleus
ER
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Unfolded proteins that bind to BiP activate the UPR, and over-expression of BiP inhibits activation
CHOP
COS -un
trans
fected
pSG5
- GFP
sorte
d
pSG5-
s G
FP so
rted
BiP
s
Kozutsumi et al, Nature 1988
Morris et al, JBC, 1997
ERp72
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The Signal
1. The signal is the accumulation of unfolded
proteins; specifically those that bind to BiP
(the Hsp70 chaperone of the ER)
2. Levels of BiP and not other chaperones are
monitored
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Yeast BiP promoter linked to E. coli lacZ gene
No stress
+ stress EMS mutagenesis22 bp
UPRElacZ
Introduce into yeast
Ern1p
Ire1p
Mori et al. Cell, 1993
Cox et al. Cell, 1993
Identification of the UPR signaling machinery
Kohno et al. MCB, 1993
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Components of yeast unfolded protein response (UPR)
Ern1p/Ire1p
S/T S/T
P P Rnase L homology domain endonuclease activity
Hac1 pre-mRNA
ER stress ??
tRNA ligase
Hac1p
GRPsUPRE
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Mammalian ER stress transducers
Ire1 /
S/T S/T
P P
↑ degradative capacity
PERK
S/TP
S/TP
Inhibition of protein synthesis and growth arrest
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Identification of mammalian UPR transducers using one-hybrid screen methodology
Human BiP promoter
XBP1
ATF6
Yoshida et al, JBC, 1998
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Tm +-
ER lumen
cytosol
ATF6 is a transmembrane protein with a cytosolicallyoriented transcription factor domain that is liberated upon
UPR activation
Yoshida et al, JBC, 1998
Stress-sensing
Proteolyticcleavage
DTT: 0’ 15’ 30’
Shen et al Dev Cell 2002
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BiP levels regulate activation of ER stress signaling molecules
ER stress signal
- changes in ER environment
- accumulation of unfolded proteins
- binding of unfolded proteins to BiP
Ire1/Ire1
PERK
ATF6
P
S/T
P
S/T
P
S/T
P
S/T
Bertolotti et al, Nat. Cell Biology,
Shen et al Dev Cell 2002
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The Ire1 pathway
ER stress
Rnase L homology domain endonuclease activity
XBP-1 mRNA
S/T S/T
P
RNA ligase
XBP-1(S)EDEM/ERdj3/ERdj4/p58
UPRE
P
- 26 bases
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PERK pathway
S/T S/T
P P
eIF-2P Translation inhibition
ER stress
CHOP/HERPATF composite
Increased degradation
Cell cycle arrest
Cyclin D1
ATF4ORF ORFORF
Protection from oxidative stress
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ER stress induces a transient block in general protein synthesis
0 Tg (h):
Commassie
35S label
0.5 1 2 3 4 6 8
Brewer et al, PNAS 1995
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ATF6 pathway
Golgi
GRPs / XBP-1 / CHOP
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1. ER Stress SignalBiP-associated unfolded proteins
ER
Nucleus
2. Signal TransducersIre1, PERK, ATF6
3. Downstream ElementseIF2- phosphorylationp38 activationATF6 cleavageCHOP inductionNFB activationXBP1 cleavageATF4 induction
eIF-2 P
P
P
PP
Translation inhibitionCell cycle arrestATF4 synthesis
5. DefeatCaspase 12 activationApoptosis
4. Transcriptional Responses
GRPs / XBP-1CHOP
??NFB targets
XBP1 targets
--
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UPR and development
1. Some aspects are required for plasma cell differentiation
2. Essential for regulating normal homeostatsis of the pancreas
3. Required for hepatocyte differentiation
4. Plays a critical role in the normal physiology of osteoblasts
5. Contributes to adipocyte differentiation
6. Two ER stress sensing transgenic mice also show evidence of UPR activation in heart, somites, neural tubes, and skeletal muscle
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Producing a mouse that expresses GFP in response to ER stress
Iwawaki,T. et al. Nat. Med. 2004.
XBP-1 mRNA
XBP-1S
XBP-1 mRNA 26 bases
P P
Ire1 kinase
ATG
actin promoter Venus
Venus
ATG
No stress
UPR activation actin promoter
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Up-regulation of proteins involved in ER expansion occur in waves during plasma cell differentiation
Van Anken, et. al.,Immunity 18:243-53, 2003
B lymphocyte
Plasma cell
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XBP-1 is required for plasma cell differentiation and Ig secretion
Reimold, et. al., Nature 2001
B220
IgM
IgD IgD
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BRdUDividing cells
TUNELApoptotic cells
Reimold, et. al., G&D, 2000
And for normal liver development
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Whereas PERK is required for exocrine pancreatic function
Harding, et. al., Mol. Cell, 2001
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UPR and disease
Cytoprotective Protect, then destroy Cytodestructive
HBV Ischemia Diabetes
HCV glomerular nephritis Neuropathgen. virusMMLV
CMV FrCasE
Simian Virus 5HSV hauntaviruses
Cancers Neurodegen. DiseasesSurvival Alzheimer’sAngiogenesis Parkinson’sChemosensitivity Huntington’s
Lysosomal storage dis.Sandoff-gal deficiencyGaucher’s
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Solid tumors experience an altered physiological environment
Oxygen
Nutrients
Wastes, pH
Hypoxic Resp.
Inhib. Glycosyl.
& ATP production
Alter side chain charges
protein folding UPR
High metabolic state
demands on molecular
chaperones
Hypoxic condition: Distance of tumor cell from functional blood
vessel >0.2mm
Inhib. S-S
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The UPR and cancer
• Some UPR elements have been shown to be induced in breast, esophageal, prostate, pancreatic, hepatocellular, gastric, and multiple myeloma cancers and in a number of xenograft studies.
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Proangiogenicfactors
XBP1(S)ATF4
UPR
Hypoxia
HIF
Tumor growth
Increased Metabolic rate
Proangiogenicfactors
Pereira ER, et al., Endoplasmic Reticulum Stress in Health and Disease, (In press)
Tumors depend on angiogenesis to alleviate stresses caused by an insufficient environment
Rapid cell division Increased Metabolic Rate
UPR ??
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1069 probesDifferential expression
185 Angiogenesisassociated
33 secreted proteinsor transcription factors
↑14/19 positive regulators
↓1 negative regulator
The UPR induces expression of a significant number of proangiogenic factors
Pereira ER, et al., PLoS ONE, 2010
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Gene Symbol Angiogenic Effect Fold change
3 hour ThapsigarginFold change
8 hour Thapsigargin
ANG Positive 2.4 8.1
ANGPT2
Positive 2.2
CTGF Positive 2.2 1.4
EPAS1 Positive 1.8 2.6
EREG Positive 2.3 6.3
FGF2 Positive 1.5 3.1
F3 Positive 2.9 1.6
FGF1 Positive 1.1
IL1A Positive 4.4 10.8
IL6 Positive 4.8 7.0
IL8 Positive 54.25 27.9
KLF5 Positive 2.6 3.5
TGFB2 Positive 2.9
VEGFA Positive 2.7
VASH1 Negative -3.0
The UPR induces expression of a significant number of proangiogenic factors
Pereira ER, et al., PLoS ONE, 2010
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VEGF human
-5k -4k -3k -2k -1k-6k-7k-8k
TSS
VEGF mouse
-5k -4k -3k -2k -1k-6k-7k-8k
VEGF rat
-5k -4k -3k -2k -1k-6k-7k-8k
XBP1 ATF4 NFB HIF1
+1k
+1k
+1k
Potential binding sites for UPR inducible transcription factors in VEGF promoters
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Does the UPR contribute to angiogenesis under normal physiological states? Why should this be a component of
the UPR in normal cells?
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Does the UPR contribute to angiogenesis under normal physiological states? Why should this be a component of
the UPR in normal cells?
-Ire1 KO is lethal at 12.5 days
Zhang, K. et al., JCI, 2005
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Iwawaki, T., et al., PNAS, 2009
XBP-1 is highly expressed in the placenta
And Ire1 KO mice have decreased expression of VEGF in placenta
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Targeted disruption of Ire1 everywhere but in trophoblastsrescued embryonic lethality, so Ire1 expression in trophoblasts
is critical for angiogenesis in mouse placenta!
Iwawaki, T., et al., PNAS, 2009
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Summary
1. Disruption of ER homeostasis adversely affects normal protein folding and results in the activation of a cytoprotective signal transduction response known as the unfolded protein response.
2. This response is characterized by increased expression of molecular chaperones, transient inhibition of protein synthesis, up-regulation of ERAD components, and exit from cell cycle. However prolonged UPR activation can result in induction of apoptotic programs.
3. Three ER localized proteins (Ire1, PERK, and ATF6) signal the response and are coordinately activated by the accumulation of unfolded proteins and decreases in levels of free BiP.
4. A number of normal development and differentiation programs are dependent on UPR activation.
5. A growing list of disease states are associated with UPR activation. In some instances, the prosurvival aspects of the UPR lead to disease (i.e., cancer and some viruses), whereas in other cases the proapoptotic elements of the response are responsible for the disease pathology (i.e., neurodegenerative diseases).