From the
Prion diseases are fatal neurodegenerative disorders
afflicting humans and other mammals. Manifest as sporadic, inherited,
and infectious illnesses, they seem to be the result of a
conformational transition in the normal, cellular prion protein
(PrP) (
)whereby the aberrant, diseased protein
isoform (PrP
) is formed(1) . The transmissible
particle or prion is composed largely, if not exclusively, of
PrP
and is resistant to procedures that modify nucleic
acids(2, 3) . No candidate scrapie-specific
polynucleotide has been identified(4) . The name prion was
coined for the proteinaceous infectious
agent (3) , and the designation PrP
was derived
from scrapie, an infectious disease of sheep known for at least 200
years that has been transmitted to hamsters and
mice(5, 6) . PrP
and PrP
have
been purified from the brains of healthy and scrapie-infected Syrian
hamsters, respectively. Among a plethora of early proposals as to the
possible causes of scrapie were suggestions of a protein devoid of
nucleic acid(2, 7, 8, 9) . Over the
last two decades, substantial evidence has been gathered suggesting
that PrP
is likely to be the only component of the
infectious prion particle.
More than 150,000 cattle have died of a
new prion disease called bovine spongiform encephalopathy (BSE) or
``mad cow disease''(10) , believed to have originated
from cattle feed infected with sheep scrapie prions. A 1988 ban on the
feed produced from the offal of domestic animals has begun to control
the epidemic; in 1992, 37,000 cases of BSE were recorded while
only
22,000 were diagnosed in 1994.
Prion diseases of humans
include Creutzfeldt-Jakob disease (CJD),
Gerstmann-Sträussler-Schienker syndrome (GSS), and
fatal familial insomnia (FFI)(11, 12, 13) .
Generally patients with GSS present with ataxia and cerebellar
dysfunction while those with CJD exhibit memory loss and a progressive
decline of intellect. GSS and FFI are familial diseases whereas CJD can
be sporadic, familial, or infectious. The infectious human prion
diseases are rare, but kuru spread through ritualistic cannibalism was
once the most common fatal illness among the Fore people of Papua New
Guinea(11) . Iatrogenic CJD is another infectious prion disease
resulting from inadequately sterilized surgical instruments, dura mater
grafts, and human growth hormone isolated from the pituitaries of
cadavers. Prion diseases vary in their course and rapidity of
development, but they all cause neurodegeneration and ultimately death.
Autopsy generally reveals spongiform degeneration of the brain,
sometimes with amyloid plaques that stain with Congo red and react with
-PrP antibodies.
Identification and Properties of PrP
The protease-resistant fragment of PrP called
PrP 27-30 was discovered in fractions partially purified from
Syrian hamster (SHa) brain by monitoring the enrichment of scrapie
infectivity with respect to
protein(14, 15, 16) . Differential
solubilization with detergents, limited proteolysis to remove
extraneous proteins and centrifugation were used to purify scrapie
prions. The purification was extended using discontinuous sucrose
gradient centrifugation. PrP 27-30 aggregates to form amyloid
rods that stain with Congo red dye to display green-gold birefringence
under polarized light(17) . N-terminal sequencing of PrP
27-30 revealed a ragged N terminus(18) . The N-terminal
amino acid sequence allowed the selection of a clone from a hamster
brain cDNA library and the identification of the PrP gene (19, 20) . Subsequently, both PrP
and
PrP
were found to be encoded within a single exon of the
chromosomal gene as proteins of 254 amino acids(21) , although
they display quite different physical properties(22) .
N-terminal sequencing of full-length PrP(23) and PrP
(23, 24, 25) revealed the
posttranslational loss of an N-terminal 22-residue signal peptide. A
23-residue C-terminal peptide is also lost upon addition of a
glycosylphosphatidylinositol (GPI) anchor, identified by the release of
2.8 mol of ethanolamine/mol of PrP together with myoinositol,
phosphate, and stearic acid in acid hydrolysates of PrP
27-30(26) . Although PrP has a highly conserved
hydrophobic domain, it is not an integral membrane protein but is
attached to cell surface membranes by its GPI, which is unusual in that
it is sialylated(27) . The majority of PrP molecules carry bi-,
tri-, and tetraantennary neutral and N-linked sialylated
oligosaccharides at two sites(28, 29) . A disulfide
bond links the only two cysteines in the mature protein(23) .
Peptide mapping of PrP
confirmed that the amino acid
sequence corresponds to that predicted from the gene
sequence(30) . The major posttranslational modifications
summarized in Fig. 1A are essentially the same for both
PrP
and PrP
, although detailed comparisons of
the heterogeneous N-linked sugars and GPI anchors are not
completed.
Figure 1:
PrP structure. A,
posttranslational modifications; B, normal polymorphisms (underlined) and pathogenic mutations to human PrP (above the line), and the boundaries of the four putative
helices of PrP (below the line).
PrP can be solubilized with low
concentrations of detergents such as Zwittergent 3-12; it has
been purified from hamster and mouse brains by a number of methods,
most recently employing detergent solubilization, centrifugation, metal
ion affinity chromatography, ion exchange, and lectin
affinity(31) . Purified PrP
does not display the
resistance to proteolysis that characterizes PrP
.
Non-denatured PrP
is insoluble but does not form ordered
arrays, whereas PrP 27-30 polymerizes into highly ordered
macroscopic amyloid structures(17) . Solubilization or
disruption of these rod-shaped amyloids by denaturants such as boiling
SDS, alkali, or guanidinium thiocyanate inactivate prion
infectivity(32) . On negatively stained electron micrographs,
PrP
and PrP
appear as amorphous deposits but
were immunolabeled with
-PrP antibodies; PrP 27-30 rods were
readily visualized without immunolabeling (Fig. 2)(31) .
Figure 2:
Electron micrographs of PrP (A), PrP
(B), and PrP 27-30 (C). Immunogold labeling was employed for A and B. (Reproduced from (31) ).
The polymerization of PrP 27-30 into amyloids and the presence
of PrP amyloid plaques in some prion diseases have led to assumptions
that amyloid formation is essential for the formation of
PrP(33, 34, 35, 36, 37, 38) .
However, PrP
can be formed in the absence of amyloid, and
the presence of amyloid plaques is not obligatory for prion diseases.
The existence of different prion isolates or strains with
characteristic incubation times and pathology poses an
enigma(39, 40, 41, 42) . As prion
diseases are apparently caused by an abnormal conformation of
PrP, different strains might correspond to distinct stable
conformations of PrP
. Alternatively, a second component
might mediate the properties of PrP
. Still another
possibility is a labile or infrequent covalent change that induces
different properties in PrP
.
Spectroscopy of PrP and Peptides
The physical properties of PrP have made it
difficult to obtain crystals suitable for x-ray diffraction. Only
recently has a purification of PrP
been developed for
undenatured protein(31) . Fourier transform infrared (FTIR) and
circular dichroism (CD) have provided information on the secondary
structures of PrP 27-30, PrP
, and PrP
(31) (Fig. 3). PrP
is essentially
-helical and devoid of
-sheet, whereas the
-sheet
content of PrP
is
40% (31) and the truncated
form, PrP 27-30, contains more than 50%
-sheet(31, 43, 44, 45) . CD
also demonstrated the remarkable thermal stability of the structure of
PrP
(46) . Procedures that inactivate the
infectivity of PrP 27-30 and PrP
such as
purification by SDS-polyacrylamide gel electrophoresis or treatment
with alkali also cause a substantial reduction in the
-sheet
content(44, 45, 46) . Having identified no
covalent differences between the two PrP isoforms, we suggested that
the conversion of PrP
to PrP
is a
conformational change(30) , involving a transition from
-helical to
-sheet structure(31) .
Figure 3:
A, FTIR spectra of the amide region for
PrP (solid line), PrP
(dashed
line), and PrP 27-30 (dotted line) (reproduced from (31) ). B, CD spectrum of PrP
immunopurified from hamster brain.
Nineteen mutations of the PrP gene are associated with
inherited human prion disease (see Fig. 1B), five being
linked statistically. A Pro Leu mutation at codon 102, the first
point mutation identified(47) , causes spontaneous
transmissible neurodegenerative disease (GSS) in transgenic (Tg)
mice(48) . Inherited prion diseases display different
phenotypes and are often referred to as familial CJD, GSS, and FFI. Of
particular interest is the D178N mutation, which is linked to inherited
prion disease. Patients with a Val-129 (a polymorphic site) on the
mutant allele present with familial CJD but with FFI if they have a
Met-129(49) . The Met/Val polymorphism at 129 does not cause
disease but Met or Val homozygosity appears to predispose to sporadic
prion disease in Caucasians (50) but not Asians (51) .
Alignment of 11 mammalian and 1 avian PrP sequences revealed
several regions of extreme conservation, punctuated by more variable
segments(52, 53) . Structure prediction algorithms
identified four potential regions of secondary structure, which in
PrP might form
-helices suggesting a 4-helix bundle
protein(54) . The four putative
-helical regions
H1-H4 illustrated in Fig. 1B are within the
protease-resistant core of PrP 27-30 (residues 90-231).
Potential hydrophobic helix-helix interaction sites were identified,
and combinatorial packing revealed
300,000 possible
three-dimensional arrangements of the helices. Approximately 200
structures were connected, sterically reasonable, capable of forming
the disulfide bridge between Cys-179 and Cys-214, and compatible with
solvent constraints posed by the glycosylation. Four families of
plausible structures that exhibited a sensible ratio of
solvent-accessible surface area to molecular volume were identified and
analyzed for the spatial clustering of pathogenic point mutations. The X-bundle model illustrated in Fig. 4is most
susceptible to disruption by these mutations (55) and shows
close proximity between the codon 178 mutation that induces familial
CJD or FFI and the codon 129 polymorphism that determines the
phenotype.
Figure 4:
Schematic representations of the
four-helix bundle of PrP 27-30 from modeling studies. Left, predicted helix-helix interaction sites are shown in green. Right, positions of mutations within the
-helices that segregate with inherited prion diseases and the
codon 129 polymorphism are shown in red. (Reproduced from (55) .)
The four putative helices were synthesized as peptides of
13-17 amino acids. Their secondary structures were probed by
attenuated total reflection FTIR spectroscopy. When dried from a
helix-promoting solvent such as 1,1,1,3,3,3-hexafluoroisopropanol, the
peptides displayed spectra indicative of -helices with amide I
bands maximizing at 1650-1660 cm
. However, on
addition of aqueous buffers, only the spectrum of H2 remained
unchanged. The other three immediately adopted
-sheet
conformations, with dramatic shifts of the amide I peaks to
1620-1626 cm
(54) . Further, the three
peptides that favored
-sheet conformation slowly precipitated from
aqueous solution as amyloid fibrils. This conformational instability
was consistent with ambiguities in the secondary structure predictions.
Alternative prediction algorithms consistently identified the same four
regions of secondary structure in PrP, but differed as to whether they
would form
-helices or
-sheets(55) .
Other
synthetic PrP peptides have a high -sheet content and form
amyloid. The sequence 106-126, which embraces H1(109-122),
forms fibrils and is cytotoxic to hippocampal neurons in culture,
particularly at pH 5 compared with pH 7(56, 57) . A
search of protein data bases for the repeating motif (XGXX)
, where X is
small and hydrophobic identified the PrP sequence 118-133 as
amyloidogenic(58) ; fibril formation was favored in the pure
Met-129 peptide compared with mixed Met-129 and Val-129(36) .
The fibrillogenic properties of PrP peptides were reported to be
enhanced by the presence of the mutations D178N and E200K(59) .
An -helix to
-sheet model of PrP
formation is
supported by a consideration of the known PrP point mutations, only one
of which (F198S) involves a change to an amino acid having a lower
-sheet propensity(60, 61) . These predict that
the Pro-102/Leu-105 mutations should give the most dramatic change,
consistent with the codon 102 mutation causing spontaneous
transmissible disease in transgenic mice(62) . It is likely
that key residues can be identified that may have an even greater
influence on the conformational transition which generates prions de novo. It is interesting that all but one of the known point
mutations fall within the region corresponding to PrP 27-30 and
are clustered within or adjacent to the four putative helices (Fig. 1). However, inserts of multiple additional copies of an
8-amino acid repeat outside of the PrP 27-30 region also cause
inherited prion
disease(63, 64, 65, 66) .
Determination of the tertiary structure experimentally may yield
important clues to the quaternary structure. Scrapie infectivity is
enhanced by dispersion of the rods in detergent-protein-lipid complexes (67) ; thus, aggregation is not a necessary requirement for
infectivity. Inactivation of prions by ionizing radiation suggested a
target size of 55 kDa, implying a dimer of PrP as the
fundamental unit (68) . The tertiary structure model for
PrP
reveals hydrophobic faces that could be involved in
dimer formation(55) .
One striking aspect of prion diseases is that they can be
manifest as either genetic or infectious disorders. Infectious prions
consist predominantly or perhaps entirely of PrP. Species
barriers that inhibit interspecies transmission of prions are due, at
least in part, to species-specific variations in PrP amino acid
sequences(7, 69, 70, 71) . Ablation
of the PrP gene in Prn-p
mice does not affect development
of these animals(72) , but renders them resistant to prions and
they do not propagate scrapie infectivity (73, 74) .
The formation of nascent PrP appears to involve
interaction between PrP
and PrP
, which is most
efficient when both have the same primary structure. This interaction,
which induces a transition from an
-helix-rich to a
-sheet-rich conformer, can be modeled with PrP
peptides(75) . For example, the highly conserved hydrophobic
tetradecapeptide H1 (residues 109-122) has the ability to convert
other PrP peptides from
-helix or coil conformations to
-sheet structure, including peptide H2 which does not
spontaneously adopt
-sheet as an isolated peptide in aqueous
buffers. Longer more hydrophilic peptides containing the H1 sequence, e.g. 104H1 (residues 104-122) which is random coil in
aqueous buffer, can also be converted by interaction with H1. Only a
small fraction of 104H1 spontaneously converts to
-sheet in
buffers containing up to 30% acetonitrile, whereas the addition of a
small amount of H1 induces an
shift in a large
fraction of molecules, e.g. in 10% acetonitrile, 1% H1 is
sufficient to convert 25% of the 104H1 to
-sheet and 10% H1
converts all the 104H1(75) .
Barriers to Prion Infection between Species
Difficulties in transmitting scrapie from one animal species
to another (7) were also observed in transmission of human
diseases such as kuru and CJD to various animal species, including
non-human primates (76) . This is partly due to the reduced
efficiency of interactions between endogenous PrP and
exogenous PrP
that differ in their amino acid sequence.
Thus, transgenic mice that express both mouse and hamster PrP can be
infected with either mouse or hamster prions; the prions that result
from these infections retain their specificities. While Prn-p
mice are resistant to scrapie prions, they are rendered
susceptible to hamster prions after being crossed with transgenic mice
expressing hamster PrP
(73, 74) . Species
barriers can be simulated with synthetic peptides, e.g. the
conversion of hamster 104H1 to
-sheet is faster when the
interacting species is hamster H1 rather than mouse H1 which differs at
two positions(75) .
Due to PrP sequence differences between
mouse and human (28 residues), only 10% of mice develop disease
with long incubation times >1 year following intracerebral
inoculation with human CJD prions. Surprisingly, the efficiency of
infection was not improved when the human PrP gene was expressed in Tg
mice. A chimeric PrP gene (MHu2M) having the human sequence in the
central region from codons 96 to 167 was introduced into the Tg mice,
all of which developed a fatal CJD-like illness within
230 days of
inoculation with CJD prions (71) . This suggests that a host
factor(s) such as a chaperone, designated ``protein X'',
plays a role in the conversion of PrP
into
PrP
. The Syrian hamster (SHa) and mouse PrPs are
sufficiently similar that SHaPrP can interact with mouse protein X
while the affinity of human PrP for protein X is probably much lower.
The Tg mice expressing human/mouse chimeric PrP
should
provide a valuable tool for evaluating public health risks posed by
prion-tainted foodstuffs, pharmaceuticals, or other products derived
from potentially contaminated human or animal sources.
Significant
differences observed in the ease of transmission of CJD to various apes
and monkeys do not follow classical phylogenic patterns (77) .
Twenty-five non-human primate PrP genes were sequenced to learn which
amino acid positions were most critical in determining species
barriers(70) . Compared with human PrP, amino acid identities
ranged from 92.9 to 99.6%. The most critical region proved to
correspond to the N terminus of PrP 27-30 and included the
putative helix H1. Three highly susceptible species including
chimpanzees are identical in the region 90-130, whereas two
species that are poor hosts for human prions have a Met Val
substitution at codon 112. It is noteworthy that the majority of the
sequence variations fall outside the predicted regions of secondary
structure, in contrast to the pathogenic mutations, which cluster
within or adjacent to these regions(55) . An understanding of
the molecular basis of species barriers is important in appraising the
risk posed by the epidemic of mad cow disease in Great
Britain(78) .
Our present understanding of prion diseases is attributable to a breadth of studies using a variety of disciplines including genetics, molecular biology, cell biology, and protein chemistry. Molecular modeling and spectroscopic studies of peptides have opened new avenues of investigation. Improvements in synthesis of longer peptides should allow greater portions of the PrP molecule to be studied. Attempts to create infectivity by in vitro conversion of entirely synthetic peptides or proteins may show whether any undetected chemical modification of PrP or a second molecule is required for prion infectivity.
Ultimately an appreciation of the
molecular processes underlying the conversion of PrP to
PrP
could facilitate the design or discovery of
therapeutics to inhibit the development of prion diseases. Intriguing
similarities between prion diseases, Alzheimer's disease, and
Parkinson's disease (79) suggest common mechanisms of
pathogenesis may feature in these illnesses. A process similar to prion
formation may explain cytoplasmic inheritance of some traits in
yeast(80) . Thus, it may transpire that the exciting new field
of prion biology has much wider significance than previously
appreciated.