Transcript Acidic pH and Detergents Enhance in Vitro Conversion of

Acidic pH and Detergents Enhance in
Vitro Conversion of Human Brain PrPc
to a PrPSc-like Form
by Wen-Quan Zou and Neil R. Cashman
The Journal of Biological Chemistry
Vol. 277, No. 46, Issue of November 15, pp. 43953-43947,
2002
Useful Terms & Abbreviations
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BSE – Bovine Spongiform Encephalopathy (Mad Cow
Disease)
CJD – Creutzfeldt-Jakob Disease
OSE – Ovine Spongiform Encephalopathy (Scrapie)
PrPC – cellular prion protein (normal)
rPrP – recombinant prion protein
PrPSc – pathogenic prion protein
PK – proteinase K
GdnHCl – guanidine hydrochloride
PMCA – protein misfolding cyclic amplification
PBS – phosphate buffered saline
SDS – sodium dodecyl sulfate
Introduction
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Most diseases can be traced to bacteria, viruses, fungi or
parasites.
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Prions are pieces of protein, containing no DNA or RNA to
direct its activity.
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Prions have been linked to BSE, OSE, CJD and Kuru and
appear to cross between species.
Introduction (continued)
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Interspecies transmission is due to feeding diseased
carcasses of one animal to another, causing progressive
destruction of brain tissue and death.
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Pathogenic prions have the same primary structure
(amino acid sequence) as normal PrP but differ in the
secondary and tertiary structures.
Introduction (continued)
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Pathogenic prions move neuron to neuron destroying
each cell and have the ability to transform normal
prions into pathogenic isoforms.
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Researchers have not discovered how or why some
prions transform into disease-causing vectors or have
the power to convert other prions.
Review of Literature
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Normal soluble, PK sensitive PrPc (rich in α-helices) is
converted to infectious, insoluble, proteinase K-resistant
PrPSc (rich in β-sheets), by a template-directed process
catalyzed by PrPSc. This has been modeled in vitro.
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Kocisko, D.A., et. al., (1994) Nature 370, 471-474
Saborio, G.P., et. al., (1999) Biochem. Biophys. Res. Commun. 258,
470-475
The insoluble, β-sheet form of the prion protein (PrPSc) is
the only known component associated with the group of
transmissible, fatal neurodegenerative diseases found in
humans and other animals. A posttranslational process,
changes the conformation of the protein from normal PrPC
to PrPSc.
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Prusiner, S.B. et. al., (1998) Cell 93, 337-348
Review of Literature (continued)
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The primary structure of PrPC and PrPSc are identical but
secondary and tertiary structures differ, creating different
physicochemical properties. PrPC is soluble in detergent and
can be degraded by proteinase K (PK). PrPSc is insoluble in
detergents and is resistant to PK digestion, forming
aggregates.
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S.B. Prusiner (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 13363-13383
Recombinant PrP (rPrP) have been used to demonstrate
changes of α-helices into β-sheets and concomitant selfassociation with the use of acidic pH when combined with
detergents in vitro.
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W. Swietnicki, et. al., (1997) J. Biol. Chem. 272, 27517-27520
S. Hornemann & R. Glockshuber (1998) Proc. Natl. Acad. Sci. U.S.A. 95,
6010-6014
G.S. Jackson, et. al., (1999) Biochim. Biophys. Acta 1431, 1-13
Review of Literature (continued)
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Conversion of PrPC to PrPSc can be promoted by protein
misfolding cyclic amplification (PMCA).
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G. P. Saborio, et. al., (2001) Nature 411, 810-813
Acid-induced conformational transition and aggregation may
be associated with the protonation of acidic amino acids
aspartate (Asp) and glutamate (Glu) using a peptide (195213) corresponding to the C-terminal region of PrP.
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W.Q. Zou, et. al., (2001) Eur. J. Biochem. 268, 4885-4891
Hypothesis
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Acidic pH and guanidine hydrochloride
(GdnHCl)-treated brain tissue containing
normal PrPC can be converted into
abnormal PrPSc in an in vitro environment
and is a superior substrate to untreated
PrPC.
PrPC
PrPSc
Methodology
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Brain tissue samples were obtained from normal
human brain, prion-infected brain (CJD patient) and
mice (wild-type and PrP-/-).
Samples of brain tissue were treated with GdnHCl at
Ph 3.5 and 7.4 to create Acid/GdnHCl-treated PrP
and mock-treated samples.
Assay of Detergent Insolubility and Proteinase K
resistance was performed using immunoblotting.
Methodology (continued)
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Immunoprecipitation of PrP samples was performed
using anti-PrP monoclonal antibodies 6H4 & 3F4 and
magnetic Dynabeads.
In vitro conversion of Acid/GdnHCl-treated PrP to a
form similar to PrPSc was performed using the CJD
brain tissue as a template.
Data/Results
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The mock-treated samples of
PrP were predominantly in
the detergent-soluble (S)
fraction, and acid/GdnHCltreated PrP in the insoluble
pellet fraction (P).
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Acidic pH and GdnHCl
induces conformational
transition of recombinant PrP
into a detergent-insoluble
PrPSc-like species, even when
the sample is returned to
physiological pH for 7 days.
Data/Results
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At pH < 3.5 PrP is insoluble,
while at pH > 4.5 it is soluble.
This corresponds to the pKa
of the side chains of
Aspartate & Glutamate,
suggesting the solubility of
PrP may be associated with
protonation of acidic residues.
Data/Results
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Most brain proteins are
soluble in acidic
environments.
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GdnHCl makes PrP insoluble
at low pH (3.5); only becomes
soluble as the concentration
increases.
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Acidic pH-treated PrP may
possess a unique structure at
1.5M GdnHCl.
Data/Results
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Partial proteinase K (PK)
resistance is a hallmark of
PrPSc.
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Both mock-treated and
Acid/GdnHCl- treated brain
PrP do not demonstrate this
same resistance at
concentrations > 1μg/ml PK.
Data/Results
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Immunoprecipitation of
treated PrP and PrPSc with
anti-PrP antibodies, 6H4 &
3F4. Lanes 1 & 3 are mocktreated, 2 & 4 acid/GdnHCl
treated, 5 & 7 normal brain, 6
& 8 CJD brain. There was no
significant difference in
binding of 6H4 & 3F4 in mock
or acid-treated samples.
There was a significant
decrease of precipitate in
CJD samples. (Molecular
masses are shown in kDa).
Data/Results
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Lanes 1 & 2: trace PrPSc with mock or
acid pH/GdnHCl treated samples in
SDS (a denaturing anionic detergent)
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Lanes 3 & 4: no PrPSc is added to
samples, SDS is added
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Figure a suggests SDS may induce
conformational change in treated brain
PrP.
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Figure b shows converted PrP display
a protease-induced gel mobility shift
similar to that displayed in CJD brain.
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Figure c shows no amplification of
PrPSc was observed when human
template was incubated with brain
homogenates from treated wild-type or
PrP-/- mice.
Discussion
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This study supports the hypothesis that acidic pH &
GdnHCl induces a physical transition of cellular PrP in
normal brain homogenates.
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Treated PrP becomes detergent-insoluble (similar to
PrPSc) but still remains PK-sensitive and epitope
accessible. Only a small portion of the acidic pH treated
PrP acquires PK resistance if treated with low
concentrations of SDS, which is enhanced if in the
presence of trace amounts of native PrPSc.
Discussion (continued)
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The pH range of 3.5-4.5 in which these transitions occur is
consistent with protonation of acidic amino acids.
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Obscuration in PrPSc of the 3F4 epitope (residues 109-112)
is consistent with the theory that PrPSc formation is
dependent upon structural changes in codons ~90-120.
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PrP is synthesized in the rough endoplasmic reticulum and
transits through the Golgi apparatus to the cell surface. It is
recycled in the endosomal-lysosomal pathway. PrPSc forms
on the cell surface and accumulates in the endosomallysosomal system.
Conclusion/Further Research
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Conversion of PrPC to PrPSc may pass through two discrete
stages:
1. Low pH & denaturants induce the first stage (making it
more “recruitable” than native PrPC.)
2. SDS assists in the second stage of rearrangement in the
presence of a PrPSc template.
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Further research should focus on the possibility that acidic
pH PrP may be useful to determine the conformational
events of underlying prion protein conversion in disease, the
molecular co-factors and posttranslational modifications
critical in conversion and pharmaceutical agents that might
prevent PrPSc formation in vitro and in vivo.