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Who’s Afraid of the Big, Bad, Misfolded Protein?
Meghan Murphy1, Moses Misplon1, Joel Pollen1, Christine Pollnow1, Maia Stack1, David Stack PhD2 and Anita Manogaran PhD3.
1 Community SMART Team of Southeastern WI. 2 University of Wisconsin–Milwaukee
3 Department of Biological Sciences, University of Illinois at Chicago; Chicago, IL
Abstract
Fatal prion diseases, such as Creutzfeldt-Jakob disease in humans, are
associated with the conversion of the normally folded mammalian PrP
protein to a misfolded prion form that aggregates. Understanding how
prions behave has been greatly facilitated by the study of prions in
Saccharomyces cerevisiae, or baker’s yeast. In yeast, the translation
release factor, Sup35, misfolds to form the [PSI+] prion. Although the
Sup35 protein has a significantly different primary sequence from
PrP, its prion form behaves similarly to human prions. The N-terminus of Sup35 is
Q/N-rich and responsible for prion formation. Determining the structure of this
region has proven difficult since the N-terminus forms aggregates instead of the
ordered crystals required for structural studies. However, a small seven amino
acid sequence (GNNQQNY) from Sup35's N-terminus was crystallized by Nelson
et al. (2005). We have constructed a 3D physical model of GNNQQNY as part of
Prion diseases are fatal, transmissible neurodegenerative
Healthy brain
CJD infected brain
the MSOE SMART Teams program using 3D printing technology.
GNNQQNY's structure suggests that multiple prion molecules assemble
into strong fibrous aggregates through tightly structured interlocking
parallel beta sheets called a cross-β spine. The structure of GNNQQNY
suggests a potential model of how human prions assemble, which could
potentially help in developing therapies that prevent aggregation..
Summary
diseases such as Mad Cow Disease
and Creutzfeldt-Jakob Disease (CJD)
in humans. CJD often has decadelong incubation periods and cannot be
effectively diagnosed while in
incubation. Brain sections of postmortem CJD patients (lower) exhibit
sponge-like holes compared to
healthy brain sections (upper). There
are currently no cures for prion
diseases and prions are resistant to
common methods of sterilization.
The [PSI+] yeast prion can be used as a model of
mammalian prion behavior. Understanding
aggregation of the GNNQQNY sequence may lead
to possible therapies for human prion diseases.
GNNQQNY Aggregate Structure
The Q/N rich N-terminus of Sup35, which is
responsible for [PSI+] formation, cannot form crystals.
Nelson et al. were able to crystallize 7 amino acids
(GNNQQNY)
from the prion
domain. GNNQQNY
binds in interlocking
antiparallel units,
which form a stack
held together by
hydrogen bonds
(left).
Model of Prion Formation
Prions convert
correctly folded
proteins to prion
form
Misfolded proteins
form prion
aggregates
GNNQQNY Sequence from the
N-terminus of the [PSI+] Prion
1YJP.pdb
Introduction of
Foreign Prion
Prion
Aggregation
Prion oligo
formation
Prions are misfolded proteins that escape the cell’s mechanisms
for destroying them. When a prion (red shape, left) is introduced
into a cell, it converts correctly folded proteins (blue ovals, left) into
the prion form (center). These misfolded proteins assemble into
prion aggregates (right). In mammalian prion diseases, the
presence of these aggregates are a hallmark of cell death.
Using Yeast as a Model
Researchers study prions in yeast rather
than mammalian prions because of:
● Similar behavior
● Nominal risk
● Yeast have short life cycle
● Yeast are easy to cultivate
Budding Yeast Cells
(Saccharomyces cerevisiae)
Community SMART
team
Sup35, [PSI+], and Related Research
The translational release factor, Sup35, can misfold to
form the [PSI+] prion. Fusing the Green Fluorescent
Protein (GFP) to Sup35 allows tracking within the cell.
[PSI+] appearance is enhanced by increasing the levels
of Sup35-GFP in the cell. Before prion induction, Sup35GFP is spread evenly (1. left panel). During initial stages
of prion formation, Sup35 forms intermediate ring and
line structures (1. center) that eventually turn into
fluorescent dots (1. right) that indicate [PSI+] aggregates.
1.
2.
When researchers induce [PSI+] formation in cells lacking
a cell polarity gene, BEM1 (2.), the intermediate ring
stages (2. center) appear normally, but cells do not
become [PSI+]. Therefore, bem1 deletion mutants inhibit
[PSI+] prion formation.
Structure of GNNQQNY molecule: Nelson, R., Sawaya, MR., Balbirnie, M., Madsen, AO., Riekel, C., Grothe, R. & Eisenberg, D. (2005). Structure of the crossβ-spine of amyloid-like fibrils. Nature. 435, 773-778. Brain images: Aguzzi, A., Glatzel, M., Montrasio, F., Prinz, M & Heppner FL. (2001).
Interventional strategies against prion disease. Nature Reviews Neuroscience. 2, 745-749.
A SMART Team project supported by the National Institutes of Health (NIH) – National Center for Research Resources Science Education Partnership Award (NCRR-SEPA).