MISFOLDING DISORDERS – A trip to the unknown cause of diseases? Marc Baumann Meilahti Clinical Proteomics Core Unit and the NeuroMed Research Program,Biomedicum Helsinki E-Mail: [email protected](http://research.helsinki.fi/corefacilities/proteinchem)
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Marc Baumannresearch.med.helsinki.fi/corefacilities/proteinchem... · 2012. 10. 8. · Protein degradation and protection against misfolded or damaged proteins 895 ALFRED L. GOLDBERG
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MISFOLDING DISORDERS –A trip to the unknown cause of diseases?
Marc BaumannMeilahti Clinical Proteomics Core Unit and the NeuroMed
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
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.