Protein folding: sculpting evolutionary - faseb.org Lindquist Lecture 2009.pdf · Kyowa Hakko...
Transcript of Protein folding: sculpting evolutionary - faseb.org Lindquist Lecture 2009.pdf · Kyowa Hakko...
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Protein folding:
sculpting evolutionary
change, human health,
and disease.
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Hemoglobin GFP
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25 39 °C
hsp104hsp90hsp70
hsp26
hsp60
Heat shock response
Induced heatand
many other stresses
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25 39 °C
hsp104hsp90hsp70
hsp26
hsp60 Master regulatorHSF1
Yeast to man
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Hsp90 facilitates evolution
CancerDrug resistance
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Hsp90 in yeast
• Essential for life
• Very abundant
• Normally don’t need much
• Need much more under stress
• Acts as buffer
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Hsp90 in Mammals (several labs)
• Complexed with inactive hormone
receptors
• Complexed with inactive oncogenic
kinases
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• Complexed with inactive hormone
receptors
• Complexed with inactive oncogenic
kinases
Hypothesis: Hsp90 is a repressor
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Hypothesis: Hsp90 keeps proteins repressed
Inactive
90 9090
v-src GR
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Hypothesis: Hsp90 keeps proteins repressedv-src GR
Active
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v-SrcGAL promoter
accumulation
Wt hsp90
activity
Wt hsp90 Wt
20 x exposure
hsp90
c-Src
activity
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Normal vs. Oncogenic src Kinase
Self-inhibited c-src
YP mutation
P
P
P
P
P
Active mutant v-src
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Normal vs. Oncogenic src Kinase
Self-inhibited wild-type c-src
YP mutation
Misfolded, unstable mutant protein
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Hsp90 Saves it
90
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Hsp90 Promotes Activation
90
But while bound it is inactive
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Hsp90 Promotes Activation
P
P
P
P
P
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Hsp90 drug reverses transformation
V-Src
Luke Whitesell
V-Src + GA
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PharmaDD Online 2006
Vernalis Novartis*
Conforma Biogen-Idec*
Serenex Pfizer*
Kosan BMS*
Kyowa Hakko Kogyo*
Synta*
Infinity/Medimmune*
Sanofi-Aventis
Merck
Others??
June 2008
Hsp90 Inhibitors:
Rapid Progress in
Clinical Development
* Phase I/II clinical trials in progress
Is any of this relevant to humans?
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1. Hsp90 chaperones mutant proteins
Mutations new activities.
But proteins donʼt fold well.
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90
P
Activation & repression of gene expression
Development & morphogenesis
Cell cycle progression
Responses to environment
Cell growth
Apoptosis
Activation & repression of gene expression
Development & morphogenesis
Cell cycle progression
Responses to environment
Cell growth
Apoptosis
Many co-chaperones andVariations on the theme
90
90
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Signal Transduction Networks and Hsp90
Hanahan and Weinberg, Cell 2000
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Drug Resistance in Fungi
ubiquitous
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Emergence of drug resistance in Candida albicans
- + Fluconazole
-
+ Radicicol
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Evolution in a human host
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Resistance of Clinical Isolates
GdA
FL (µg/ml)0 256
Isolates collected by Ted White, Spencer Redding and colleagues.
CAI4
0 256
In v
ivo
sele
ctio
n
CaCi
FL (µg/ml)
39°C
FL (µg/ml)0 256
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Mechanism of Hsp90-Dependent Resistance
Ergosterol
Erg3Ergosterol
Erg11
. .WT
Azoles
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Mechanism of Hsp90-Dependent Resistance
Toxic sterol
WT
Erg3 Erg11Ergosterol
AzolesMutation
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Mechanism of Hsp90-Dependent Resistance
Alternate sterol
WT
Erg3 Erg11Ergosterol
AzolesMutation
. .
HSP90
Calcineurin
Response!
. .
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2. Hsp90 chaperones responders.
mutations simultaneously solve & create problems
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Aspergillus fumigatus
• Diverged ~ 1 BY
• Major human pathogen
• Mortality rates ~ 90 %
• Already resistant to new chemical entity
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Hsp90 Modulates Caspofungin Resistance in A. fumigatus Clinical Isolates
Isolate 1 Isolate 2 Isolate 3
10µM GdA
Cowen LE et al., PNAS 2009
test strip
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Informer Screen 25,000 Compounds
Two hundred hits
most not toxic to human cells
RO3 - Screen up to 500,000 new compounds
NIH Comprehensive Screening Center &
Molecular Libraries Probe Production Center Network
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That is just one wayone hsp interfaces with
disease and how we might manipulate it.
What about others?
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25 39 °C
hsp104hsp90hsp70
hsp26
hsp60
Master regulator
HSF1
Yeast to man
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Heat Shock Transription Factor
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Heat shock/stress response ---a systematic cellular defense to stresses
Protein homeostasis
Not needed without stress!
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Hsf1-/-
Hsf1 enables chemical carcinogenesis
Hsf1+/+
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Ras oncogeneHsf1 Reduces Survival
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Hsf1 Reduces Survival p53+/R172H
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What about humans?
(Mouse ≠ human)
What about maintenance?
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HSF1 requirement correlates with malignant state
normal tumorigenic(primary) (hTERT) (hTERT, LTA and HRAS)
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HSF1 maintains the growth and survival of human breast cancer cells
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HSF1 maintains the growth and survival of human cancer cells withdiverse histopathological origins
(Fibroblasts) (Cervical) (Prostate) (Kidney) (MPNST)
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Hsf1
What aboutneurodegeneration?
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Neurodegenerative Diseases
Adapted from Forman, Trojanowski, and Lee 2004
Alzheimer FTDP-17 Parkinson
Huntington ALS CJD
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Yeast cells as living test tubes
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Parkinsonʼs Disease
• Second most common neurodegenerative disorder.
• Loss of dopamine-producing neurons from substantia nigra. Severe motor defects…...
• Bewildering environmental influences.
• Mostly sporadic but some genetic forms.
• Hallmark of the disease inclusions of aSynuclein
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α-Synuclein Localization
α-synuclein GFPGal
one copy two copies
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glucose
Identify genes that make cells
better or worse
αSyn-GFP
Library of ~5,000 yeast genes
. . . . . .gene1, gene2, gene3, gene4…….gene5000
galactose
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better
worse
No effect
Screened 5,000 ORFs~ 60 modifiers
empty well
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• Galactose induction• Protein trafficking• Manganese transport• Nitrosative & Oxidative stress• Kinases and Phosphatases• Protein quality control• Unknown Function
Genetic hit list
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• Galactose induction• Protein trafficking• Manganese transport• Nitrosative & Oxidative stress• Kinases and Phosphatases• Protein quality control• Unknown Function
Genetic hit list
ER l Golgi Enhancers
ER Golgi Suppressors
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Primary Neurons
Rat embryonic midbrain cultures(Chris Rochet)
Drosophila PD model(Nancy Bonini)
C. Elegans PD model(Guy Caldwell)
Hits from yeast screen
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Rab1 rescues α-Syn-induced loss in primary rat midbrain cultures
With Chris Rochet (Purdue)
P < 0.01
Several others, diverse
functionsas well!
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Enhancers and Suppressors tested in rat primary neurons
Suppressors• Rab3A* & Rab8A*• Pde2/PDE9A*• Cdc5/PLK2*• Hrd1/SYVN1• Ubp3/USP10
Not confirmed• Yck3/CSNK1G3
*also effective in nematode model
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Enhancers and Suppressors tested in rat primary neurons
Suppressors• Rab3A* & Rab8A*• Pde2/PDE9A*• Cdc5/PLK2*• Hrd1/SYVN1• Ubp3/USP10
Not confirmed• Yck3/CSNK1G3
*also effective in nematode modelYeast homolog rescues cell from aSyn
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tip
iceberg
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Huntingtin “OFF” Huntingtin “ON”
25Q
47Q
72Q
103Q
0Q
PolyQ length-dependent toxicity serial dilution
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Virtually everythingfrom high throughputscreens is different.
Except: The Heat Shock Response
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Hsf1 knockouts die faster from prion infection (intracranial)
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Hard Place
Rock
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There is still great hope!
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Short term therapy for cancer?Blood brain barrier?
HSF potentiators for ND rather than inducers?
Specific arms of the response?
Breaking the feedback loop?
Finding the right balance?
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Human Glioma Progenitor Cells – Orthotopic Transplantation Model
Keith Ligon and Santosh Kesari/Stiles Lab - DFCI
!
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Katherine BorkovichDebbie Nathan
Didier PicardKeith Yamamoto
Xang XuMike Singer
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AcknowledgementsLeah Cowen
--------
Theodore White (Seattle Biomedical Ins.)James Anderson (University of Toronto)
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Thanks!
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Aaron GitlerTiago Outiero Anil Cashikar
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Julie Su, Esti Yeger Lotem, Ernest Fraenkel
Esti Yeger-Lotem
Ernest Fraenkel
Linhui Julie Su
Smitha Jagadesh
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Sylvia KrobitschMartin DuennwaldSmitha Jagadish
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Antony Cooper Paul Muchowski Chris Rochet
Nancy Bonini Guy Caldwell Charles Barlowe
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Bhupinder Bhullar Josh LaBaer
Harvard Institute of Proteomics
Harvard Institute Chemical &Cell Biology at Longwood Caroline Shamu
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Chengkai DaiLuke Whitesell
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Sandro Santagata
Leslie Gunatilaka-Natural Products Chemistry
University of Arizona
Dana Farber Cancer InstituteSantosh Kesari/Stiles LabTan Ince
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Hsf-/- micegenerous gift ofIvor Benjamin
Chief of CardiologyUniversity of Utah
and Xianzhong Xiao
Tan InceDepartment of
Pathology
Brigham & WomensHospital