MISFOLDING DISORDERS –A trip to the unknown cause of diseases?
Marc BaumannMeilahti Clinical Proteomics Core Unit and the NeuroMed
Research Program,Biomedicum Helsinki
E-Mail: [email protected]
(http://research.helsinki.fi/corefacilities/proteinchem)
- CONFORMATIONAL ASPECTS OF PROTEINS
- AMYLOIDOSES: WHAT IS AN AMYLOID?
- HOW IS AMYLOID MADE?
- PROTEIN TOXICITY: WHO IS TOXIC, WHO NOT?
Covered in this talk…
- WHAT ABOUT OTHER MISFOLDING DISEASES?
Insight
Protein misfoldingADAM SMITH
Protein folding and misfoldingCHRISTOPHER M. DOBSON
Quality control in the endoplasmic reticulum protein factory 891ROBERTO SITIA AND INEKE BRAAKMAN
Protein degradation and protection against misfolded or damaged proteins 895ALFRED L. GOLDBERG
Folding proteins in fatal waysDENNIS J. SELKOE
Therapeutic approaches to protein-misfolding diseases 905FRED E. COHEN AND JEFFERY W. KELLY
Vol 426 No 6968 pp739-91118/25 December 2003
Protein misfolding
CONFORMATION -> FOLDING
Proteins 2006 Mar 15;62(3):698-707.
The determinants of the stability in the human prion protein: insights into folding and misfoldingCOLACINO S. TIANA G. BROGLIA RA. AND COLOMBO G
Prion disease: The shape of things to comeROXANNE KHAMSI
Nature 439, 134-135 (12 January 2006)
Misfolding is killing! The Prion Case
Non-foldedNon-functional protein
Non-foldedNon-functional protein
Conformationalchange
Folded functionalnative protein
Folded functionalnative protein
Protein non-folding, what’s going on?
(Ubiquitin-Proteasome) degradation Pathway(Ubiquitin-Proteasome) degradation Pathway
A)
Non-foldedNon-functional protein
Non-foldedNon-functional protein
Conformationalchange
B)
Protein folding occur in the ER
Schematic of typical animal cell, showing subcellular components.Organelles: (1) nucleolus (2) nucleus (3) ribosome (4)
vesicle, (5) rough endoplasmic reticulum (ER), (6) Golgi apparatus, (7) Cytoskeleton, (8) smooth ER, (9) mitochondria, (10) vacuole, (11) cytoplasm, (12) lysosome, (13) centrioles
Ubiquitin-Proteasome degradation Pathway
The Nobel Prize in Chemistry 2004
Ciechanover HershkoRose
"for the discovery of ubiquitin-mediatedprotein degradation"
In THE CELL AT THE ER...
We’reProteins,I feel it!
I think that I have to get a better shape!
Ups, I do not know my native structure
RULES AND REGULATIONS CONTROL THE PROTEIN NUMBERS AND FOLDING
Well all I wantTo know is,am I CORRECT?
E3 makesThe deci-sions and fixes the labels
OhNooo
With a label round your neck, there is no going back...
Marked proteins are hacked to bits in the mincer
Are you for the mincer too?
No,Not me!
Order can berestored – forthe time being!
Foldedfunctional protein
Foldedfunctional protein
Conformationalchange Abnormal proteinAbnormal protein
Protein misfolding, what can it do?
Can cause a loss of thephysiological function
Can cause aggregationand deposition
Non-foldedNon-functional protein
Non-foldedNon-functional protein
Can become toxic
Disease Protein involved
Alzheimer's disease Amyloid-ß protein
CJD, FFI , Kuru, BSE Prion protein
Parkinson disease -synuclein, parkin (?)
Huntington disease Huntingtin
Diabetes type II Amylin
Amyotrophic lateral sclerosis Superoxide dismutase
Serpin deficiency, emphysema, cirrhosis Serpins
Haemodialysis amyloidosis, prostatic amyloid ß2-microglobulin
Cystic fibrosis CFTR protein
CADASIL disease Notch3 receptor protein
Etc, etc, etc...
Examples of protein misfolding disorders
Protein involved
E.Coli Curlin protein
P. Falciparum Malarial coat protein
Spider Spider silk
Mammalian melanosomes pMEL
Mammalian brain Apolipoprotein E
Etc, etc, etc...
Some protein misfolding without a disorder
What is the mechanism of proteinmisfolding and aggregation?
Lets take an example:
THE AMYLOID
Why does Amyloid form?
Alzheimer’s amyloid plaques Prion plaques Parkinson’s
Lewy bodies
Protein Aggregates in Conformational Disorders
XAggregates
Amyloid fibers
Ultrastructure of Amyloid
Foldedfunctional protein
Foldedfunctional protein
AmyloidAmyloid
Ultrastructure of aggregates
Very often these aggregates resembleAmyloid fibers although they do not
fulfil the criteria to be Amyloid
The Alzheimer’s Amyloid Precursor Protein
The critical amino acids
40 – 46 (47)amino acids
Physico-chemical properties of the critical amino acids:Alzheimer’s Amyloid
High
Low
Average
….MSSYAFFVQT….
….HQKLVFFAED….
Random database search...
Amphoterin (HMG-1)
Alzheimer’s Amyloid
HMG-1, Aggrecan, Misshapen kinase isoform MINK, MMP-2, TRP3 cation channel…
Physico-chemical properties of the critical amino acids:Amphoterin
High
Average
Low
Amphoterin (HMG-1)
GKGDPKKPRGKMSSYAFFVQTCREEHKKKHPDASVNFSEFSKKCSERWKTMSAKEKGKFE
DMAKADKARYEREMKTYIPPKGETKKKFKDPNAPKRPPSAFFLFCSEYRPKIKGEHPGLS
IGDVAKKLGEMWNNTAADDKQPYEKKAAKLKEKYEKDIAAYRAKGKPDAAKKGVVKAEKSKKKKEEEDDEEDEEDEEEEEEEEDEDEEED
DDDE
EP EP
The fragment of Amphoterin (HMG-1) makes amyloid –like fibrils
Physico-chemical properties of the critical amino acids:Of the known Amyloid Core Sequences
0.5
0.7
0.9
1.1
1.3
1.5
M S S Y A F F V Q T C
0.6
0.8
1
1.2
1.4
S N N F G A I L S S
0.6
0.8
1
1.2
1.4
N G N C F I L D
0.4
0.6
0.8
1
1.2
1.4
1.6
H Q K L V F F A E
Alpha-helix
Beta-turn
Beta-turn
Beta-sheet
Alpha-helix
Beta-sheet Beta-sheet
Alpha-helix
Beta-turn Amphoterin fragment
Islet amyloid fragment Alzheimer’s amyloid fragment
Beta-sheet
Alpha-helix
Beta-turn
Gelsolin fragment
Kallijärvi et al (2001) Biochemistry 40: 10032-10037
GammaD crystallin, SH domain phosphoglycerate kinase (PGK), SH3 domain of the alpha-subunit of bovine phosphatidylinositol-3'-kinase (PI3-SH3)...
Also other non-disease related proteins make amyloid –like fibrils
All those which we have so far tested sharea common ”amyloid core sequence”
which is responsible for their misfolding.
...........AND share the neurotoxic properties of amyloids
Why do misfolded proteins kill??
The “stick to all” theory
Apolipoprotein E4 isA risk factor for AD
A-betaNAC
A-betaAC
ApoE
STD
0,9
1,4
1,9
30000 35000 40000 m/z
inte
nzity
apoE4(34kDa)
ApoE4 + Ab
(38kDa)
0,9
1,4
1,9
30000 35000 40000 m/z
inte
nsity
apoE4(34kDa)
0
5
10
15
20
5500 10500 15500 20500 25500 m/z
intensity
0
5
10
15
20
5500 10500 15500 20500 25500 m/z
intensity
Ab dimer
Ab trimerAb tetramer
0
1
2
3
4
5
6
7
3800 5800 7800 9800
0
1
2
3
4
5
6
7
3800 5800 7800 9800
With ApoE
Without ApoE
MS analysisof the effectof ApoE on
Alzheimer’s Amyloidpolymerization
Physico-chemical properties of some of the ApoE4Sequence epitopes
High
Average
Low
Apolipoprotein E4
num. sequence from-to
1. WKYRRPVTT 21-33
2. KKVFFSTQ 36-48
3. ALTPGVVL 61-78
4. VQLGRDTSV 89-111
5. AASVFTRKLPYT 121-148
6. EAWWTRVFLRE 183-221
Sequences in ApoE predicted to be able to form amyloid-like structures
2
3
4
5
6
7
8
9
1000 6000 11000 16000 21000 26000 31000m/z
intensity
2
3
4
5
6
7
8
9
1000 6000 11000 16000 21000 26000 31000m/z
intensity
0
10
20
30
4000 6000 8000
0
10
20
30
4000 6000 8000
Analysis of the parts on ApoE which are protected
by Alzheimer’s amyloid peptide (limited enzymatic
cleavage assay)
2
36
1 KVEQAVETEPEPELRQQTEWQSGQRWELALGRFWDYLRWVQTLSEQVQEELLSSQVTQELRALMDET68 MKELKAYKSELEEQLTPVAEETRARLSKELQAAQARLGADMEDVCGRLVQYRGEVQAMLGQSTEELR135 VRLASHLRKLRKRLLRDADDLQKRLAVYQAGAREGAERGLSAIRERLGPLVEQGRVRAATVGSLAGQ202 PLQERAQAWGERLRARMEEMGSRTRDRLDEVKEQVAEVRAKLEEQAQQIRLQAEAFQARLKSWFEPL269 VEDMQRQWAGLVEKVQAAVGTSAAPVPSDNH
ApoE sequences protected by Alzheimer’s amyloid fragment
0
2
4
6
8
10
12
14
4000 5000 6000 7000 8000 9000 10000
m/z
intensity
Ab
N-term
C-term
N-term
0
2
4
6
8
10
12
14
4000 5000 6000 7000 8000 9000 10000
m/z
intensity
Protected areas highlighted in red
Predicted sequencesable to form amyloid-like
structures are inside of the measured areas
N-term N-term
ApoE sequences protected by Alzheimer’s amyloid fragment
0
2
4
6
8
10
12
14
4000 5000 6000 7000 8000 9000 10000
m/z
intensity
0
2
4
6
8
10
12
14
4000 5000 6000 7000 8000 9000 10000
m/z
intensity
Inhibition of the binding of Alzheimer’s amyloid
fragment to ApoE by a synthetic beta-sheet
breaker peptide made towards ApoE
(GRFEQWARAVQ)
1 KVEQAVETEPEPELRQQTEWQSGQRWELALGRFWDYLRWVQTLSEQVQEELLSSQVTQELRALMDET68 MKELKAYKSELEEQLTPVAEETRARLSKELQAAQARLGADMEDVCGRLVQYRGEVQAMLGQSTEELR135 VRLASHLRKLRKRLLRDADDLQKRLAVYQAGAREGAERGLSAIRERLGPLVEQGRVRAATVGSLAGQ202 PLQERAQAWGERLRARMEEMGSRTRDRLDEVKEQVAEVRAKLEEQAQQIRLQAEAFQARLKSWFEPL269 VEDMQRQWAGLVEKVQAAVGTSAAPVPSDNH
MS
MS
1 KVEQAVETEPEPELRQQTEWQSGQRWELALGRFWDYLRWVQTLSEQVQEELLSSQVTQELRALMDET68 MKELKAYKSELEEQLTPVAEETRARLSKELQAAQARLGADMEDVCGRLVQYRGEVQAMLGQSTEELR135 VRLASHLRKLRKRLLRDADDLQKRLAVYQAGAREGAERGLSAIRERLGPLVEQGRVRAATVGSLAGQ202 PLQERAQAWGERLRARMEEMGSRTRDRLDEVKEQVAEVRAKLEEQAQQIRLQAEAFQARLKSWFEPL269 VEDMQRQWAGLVEKVQAAVGTSAAPVPSDNH
ApoE interacts with Alzheimer’s amyloid fragment by its own amyloidogenic sequence motifs! What about all the others. If they also posess their own amyloidogenic motifs, why would they not
use the same way to act??
The answer to the question...
Is Alzheimer's disease an apolipoprotein E amyloidosis?Lancet. 1995 Apr 15;345(8955):956-8.
Wisniewski, T. et al
A carboxyl-terminal fragment (residues 216-299) of apolipoprotein E is present in Alzheimer's disease lesions
In vitro this fragment from recombinant apolipoprotein E could form amyloid-like fibrils, which were Congo-red positive
Thus senile plaques contain at least both amyloid beta and apolipoprotein E amyloid fibrils.
Is Alzheimer's disease an apolipoprotein E amyloidosis?Lancet. 1995 Apr 15;345(8955):956-8.
Wisniewski, T. et al
A carboxyl-terminal fragment (residues 216-299) of apolipoprotein E is present in Alzheimer's disease lesions
In vitro this fragment from recombinant apolipoprotein E could form amyloid-like fibrils, which were Congo-red positive
Thus senile plaques may contain both amyloid beta and apolipoprotein E amyloid fibrils.
Is Alzheimer's disease an apolipoprotein E amyloidosis?Lancet. 1995 Apr 15;345(8955):956-8.
Wisniewski, T. et al
A carboxyl-terminal fragment (residues 216-299) of apolipoprotein E is present in Alzheimer's disease lesions
In vitro this fragment from recombinant apolipoprotein E could form amyloid-like fibrils, which were Congo-red positive
Thus senile plaques contain at least both amyloidbeta and apolipoprotein E amyloid fibrils
MISFOLDING DISORDERS – MIGHT THEY BE PROTEIN CONFORMATIONAL DISORDERS
WHERE MORE THAN ONE PROTEIN PARTICIPATE IN THE CASCADE OF MISFOLDING EVENTS?
MYSTERIOUS FACTOR-X IN PRION DISORDERS?
IS IT JUST ANOTHER MISFOLDING PROTEIN?
AE
NMR structures of three single-residue variants of the human prion protein Calzolai et al. PNAS | July 18, 2000 | vol. 97 | no. 15 | 8340-8345
CADASILa Notch3 mutation causing
misfolding
CADASIL
• Name given and linkage established: CADASIL from cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy. Linked to chromosome 19 (q12) by Tournier-Lasserve et al. Nature Genetics 1993; 3:256-59
• Gene defect and defective protein identified: Notch3 at 19p13, by Joutel et al. Nature 383:707-710, 1996
MRI FLAIR: lacunar infarcts
*
* Frontal horn of left lateral ventricle
Electron micrograph of a small dermal artery: widened sub-endothelial space (*), irregularity of vascular smooth muscle cells (VSMCs) and granular osmiophilic material (GOM >). E = endothelium
>
>
Lumen
VSMC
VSMC
VSMC
VSMC
VSMC
*
**
* E
E
E
CADASIL: A vascular dementia the diagnosis of which can bemade on a skin biopsy
Electron microscopy of a dermal artery: deposition of granular osmio-philic material (GOM *) in indentations (notches) of degenerating smooth muscle cells and between these cells. (N = nucleus)
1 m0.4 m
Pathognomonic finding is deposition of Notch3 extra-cellular domain (N3ECD) in the walls of arteries (*)
Skin biopsy: N3ECD immunostaining
nerve*
*
*
*
confocal
Notch3 extracellular domain (N3ECD) is a main component of GOM
Ishiko et al: Acta Neuropathol 2006; 112: 333-9(immunoelectronmicroscopy)
CADASIL WM: arterioles (N3ECD)
CADASIL WM: capillary (N3ECD)
CADASIL cortex: small arterioles and a capillary (N3ECD)
CADASIL cortex: capillary (N3ECD)
CADASIL subarachnoidal space: arteries (N3ECD)
In CADASIL Notch3 extracellular domain(N3ECD/GOM) accumulates not only on WM arterioles but also on WM capillaries(pericytes) as well as on these vessels in cerebral cortex, although corticalarterioles are not equally thickened.
Pathogenesis• Haploinsufficiency/hypomorphic effect unlikely• Gain or loss of a cysteine molecule affects formation of
sulphur bridges and causes conformational change in the Notch3 molecule (protein misfolding and aggregation). Thus, gain of function of the mutated protein most likely.
• Most likely mechanism gain of function:– Dominant negative effect (reduced function of the
wild-type allele): does not appear to occur– Hypermorphic effect (increased function of the
mutated allele): does not appear to occur– Neomorphic effect (mutant protein has new
additional (toxic?) functions (Opherk et al. CADASIL mutations enhance spontaneous multi-merization of NOTCH3. Hum Mol Genet. 2009;18:2761-7)
D. Even after S2 and S3 cleavages mis-folding preventsinternalization of the complex (with orwithout the ligand)
A. Mutation ( ) in the ligand binding area ( )
no ligand bindingor signaling
Vascular smooth muscle cell
Signal sending cell
Notch3
S3 S2
NICD to nucleussignalling
Accumulation
Accumulation
A B C
D
B. Mutation outside the ligand binding area
ligand binds, S3 cleavage and signalingoccur, but…
Delta/Jaggedligand
No internalization signal
No internalization signal S2
S3 S3
Accumulation
C. Mutated misfolded Notch3 does notundergo S2 cleavage and sop up Delta /Jagged ligands, block other ligandsor dimerize with other receptors
Mol Med 2007; 13: 305-314
Mol Med 2007; 13: 305-314
11 differentially expressed proteins discovered• Proteins related to protein degradation and folding and
free radical scavenging – Proteasome components, HSP27 and free radical scavenging
enzymes: Expected consequence due to the unpaired cysteine related misfolding -> unfolderd protein response -> ER stress ; depletion of glutathione and production of reactive oxygen species (ROS)
• Proteins related to vascular smooth muscle cell (VSMC) contraction– accentuated angiotensin II response
We have found in 2D-gel electrophoresis of genuine human vascular smooth muscle cellsfrom a patient with CADASIL and controls, that the expression of the following proteinsinvolved in actin metabolism were different: Rho protein dissociation inhibitor (RhoGDI): upregulated; Profilin: upregulated; HSP27 upregulated; Cysteine and glysine rich protein(CRP): upregulated. (Ihalainen et al. Molec Med 2007; 13:305-14)Hence the actin organization was analysed in VSMCs from different vascular beds in CADASIL patients (pre- and post-mortem). The actin network was altered suggesting that Notch3 is involved in the regulation of actin, the major the contractile protein in VSMCs. (Tikka et al. submitted)
SMA
In Conclusion
Misfolded proteins are quite naturally occuringrisk factors for the life…
which the nature can just sometimesnot deal with.
They form spontaneously by mutationswhich are only controlled by the evolution.
BAD LUCK…or ???
The Yeast story...
Yeast uses a prion-like protein for to control its lifein various environmental conditions.
It keeps this protein in an amyloid-like form if it is notneeded…
When needed, it can produce the same protein in asoluble form.
Protein misfoldingHelsinki-Biomedicum
Marc Baumann
Gibril Fadika (Gelsolin AA)Candace Laverette (SH3)Christina Pompey (Cystatin)Vonda Meeks (Cystatin, SH3)
Can Hekim (Cystatin)Jukka Kallijärvi (Alzheimers AA, Amphoterin)Riikka Nissinen (Alzheimers AA, Melatonin)Petra Gromova (Alzheimers AA and ApoE)
Bronislaw Clod (Polish Acad. of Sci.)(Alzheimers AA, Melatonin)Maciej Lalowski (Polish Acad. of Sci.)(Alzheimers AA)
Katri Niemi (Secondary AA)Kari Eklund (Secondary AA)
Saara Tikka (CADASIL)
Eeva Kauppi (Technical Assistance)Rabah Soliymani (Technical Assistance)
Helsinki-Biomedicum
Marc Baumann
Gibril Fadika (Gelsolin AA)Candace Laverette (SH3)Christina Pompey (Cystatin)Vonda Meeks (Cystatin, SH3)
Can Hekim (Cystatin)Jukka Kallijärvi (Alzheimers AA, Amphoterin)Riikka Nissinen (Alzheimers AA, Melatonin)Petra Gromova (Alzheimers AA and ApoE)
Bronislaw Clod (Polish Acad. of Sci.)(Alzheimers AA, Melatonin)Maciej Lalowski (Polish Acad. of Sci.)(Alzheimers AA)
Katri Niemi (Secondary AA)Kari Eklund (Secondary AA)
Saara Tikka (CADASIL)
Eeva Kauppi (Technical Assistance)Rabah Soliymani (Technical Assistance)
Protein misfolding
NYU Medical CenterNew York, U.S.A.
Blas FrangioneThomas WisniewskiJorge GhisoAsok KumarFrances PrelliMaciej Lalowski
Laura Morelli (Univ. Buenos Aires)
NYU Medical CenterNew York, U.S.A.
Blas FrangioneThomas WisniewskiJorge GhisoAsok KumarFrances PrelliMaciej Lalowski
Laura Morelli (Univ. Buenos Aires)
Texas Univ. MedicalBranch, Galveston, USA
Claudio Soto
Statens Serum InstitutCopenhagen, Denmark
Niels H.H. Heegaard
The Scripps Research InstituteLa Jolla, USA
Jeffery Kelly
Helsinki-HaartmanInstitute
Matti HaltiaHannu Kalimo
Univ of HelsinkiCentral Hospital
Sari Kiuru-Enari
Nordic CADASIL research groupClinical studies– Matti Viitanen U of T, Karol Inst– Susanna Roine U of T– Auli Verkkoniemi U of H
Pathology– Hannu Kalimo U of H– Qing Miao* U of T
Genetics– Minna Pöyhönen U of H – Kati Mykkänen* U of T– Maija Junna * U of T
Proteomics and molecular biology– Marc Baumann U of H– Saara Tikka (nee Ihalainen)* U of H– Yan Peng Ng U of H– Urban Lendahl and team Karol Inst
* PhD or DMedSci student
Imaging and PET studies– Juha Rinne U of T – Susanna Roine U of T
Ophthalmology– Tero Kivelä U of H– Paula Summanen U of H– Mika Harju U of H
Neuropsychology– Kaarina Amberla* Karol InstVasoregulation– Anna Stenborg U of Uppsala– Andreas Terent U of Uppsala– Robert Bergholm U of H
U of H = Univ of Helsinki, FinlandU of T = Univ of Turku, FinlandKarol Inst = Karolinska Institutet, Sweden
Amyloids are always found i two distinct conformations
Amyloidogenicproteins
Pathogenicform
Non-pathogenicform
Amyloidogenicconformation
Non-amyloidogenicconformation
DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGVVIA1 42
A 1-42
N C APPA1 695
Sequences of Alzheimer’s -sheet breaker peptides
iA 11 RDLPFFPVPIDiA 9 RDLPFFPVDiA 7 LPFFPVDiA 6 LPFFVD
iA 4 LPFFiA 3 PFF
iA 5 LPFFD
-sheet breaker peptides
Soto and Baumann (1996) Biochem. Biophys. Res. Commun. 226: 672-680
Activity of a -sheet breaker peptideIn vitro activity Activity in cells
Activity in rat model
TreatmentControl
TreatmentControl
TreatmentControl
