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

1
Acidic pH and Detergents Enhance in Vitro
Conversion of Human Brain PrPc to a PrPSc-like
Formby Wen-Quan Zou and Neil R. CashmanThe
Journal of Biological ChemistryVol. 277, No. 46,
Issue of November 15, pp. 43953-43947, 2002
2
Useful Terms Abbreviations

• 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

3
Introduction

• Most diseases can be traced to bacteria, viruses,
  fungi or parasites.
• Prions are pieces of protein, containing no DNA
  or RNA to direct its activity.
• Prions have been linked to BSE, OSE, CJD and Kuru
  and appear to cross between species.

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Introduction (continued)

• Interspecies transmission is due to feeding
  diseased carcasses of one animal to another,
  causing progressive destruction of brain tissue
  and death.
• Pathogenic prions have the same primary structure
  (amino acid sequence) as normal PrP but differ in
  the secondary and tertiary structures.

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Introduction (continued)

• Pathogenic prions move neuron to neuron
  destroying each cell and have the ability to
  transform normal prions into pathogenic isoforms.
  
• Researchers have not discovered how or why some
  prions transform into disease-causing vectors or
  have the power to convert other prions.

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Review of Literature

• Normal soluble, PK sensitive PrPc (rich in
  a-helices) is converted to infectious, insoluble,
  proteinase K-resistant PrPSc (rich in ß-sheets),
  by a template-directed process catalyzed by
  PrPSc. This has been modeled in vitro.
• 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, ß-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.
• Prusiner, S.B. et. al., (1998) Cell 93, 337-348

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Review of Literature (continued)

• 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.
• S.B. Prusiner (1998) Proc. Natl. Acad. Sci.
  U.S.A. 95, 13363-13383
• Recombinant PrP (rPrP) have been used to
  demonstrate changes of a-helices into ß-sheets
  and concomitant self-association with the use of
  acidic pH when combined with detergents in vitro.
• 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

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Review of Literature (continued)

• Conversion of PrPC to PrPSc can be promoted by
  protein misfolding cyclic amplification (PMCA).
• 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 (195-213)
  corresponding to the C-terminal region of PrP.
• W.Q. Zou, et. al., (2001) Eur. J. Biochem. 268,
  4885-4891

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Hypothesis

• 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
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Methodology

• 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.

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Methodology (continued)

• 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.

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Data/Results

• The mock-treated samples of PrP were
  predominantly in the detergent-soluble (S)
  fraction, and acid/GdnHCl-treated PrP in the
  insoluble pellet fraction (P).
• 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.

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Data/Results

• At pH lt 3.5 PrP is insoluble, while at pH gt 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.

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Data/Results

• Most brain proteins are soluble in acidic
  environments.
• GdnHCl makes PrP insoluble at low pH (3.5) only
  becomes soluble as the concentration increases.
• Acidic pH-treated PrP may possess a unique
  structure at 1.5M GdnHCl.

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Data/Results

• Partial proteinase K (PK) resistance is a
  hallmark of PrPSc.
• Both mock-treated and Acid/GdnHCl- treated brain
  PrP do not demonstrate this same resistance at
  concentrations gt 1µg/ml PK.

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Data/Results

• Immunoprecipitation of treated PrP and PrPSc with
  anti-PrP antibodies, 6H4 3F4. Lanes 1 3 are
  mock-treated, 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).

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Data/Results

• Lanes 1 2 trace PrPSc with mock or acid
  pH/GdnHCl treated samples in SDS (a denaturing
  anionic detergent)
• Lanes 3 4 no PrPSc is added to samples, SDS is
  added
• Figure a suggests SDS may induce conformational
  change in treated brain PrP.
• Figure b shows converted PrP display a
  protease-induced gel mobility shift similar to
  that displayed in CJD brain.
• Figure c shows no amplification of PrPSc was
  observed when human template was incubated with
  brain homogenates from treated wild-type or
  PrP-/- mice.

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Discussion

• This study supports the hypothesis that acidic pH
  GdnHCl induces a physical transition of
  cellular PrP in normal brain homogenates.
• 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.

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Discussion (continued)

• The pH range of 3.5-4.5 in which these
  transitions occur is consistent with protonation
  of acidic amino acids.
• 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.
• 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
  endosomal-lysosomal system.

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Conclusion/Further Research

• 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.
• 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.