However, PrP113 – 231 lacked any toxicity suggesting the normal N-terminus of the C-terminal metabolic cleavage product of the prion protein suppressed toxicity.. PrP peptides and protei
Trang 1Toxicity of novel C-terminal prion protein fragments and peptides harbouring disease-related C-terminal mutations
Maki Daniels1, Grazia Maria Cereghetti2and David R Brown1
1 Department of Biochemistry, Cambridge University, UK;2Institute of Molecular Biology and Biophysics, ETH-Hoeneggerberg, Zu¨rich, Switzerland
Mice expressing a C-terminal fragment of the prion protein
instead of wild-type prion protein die from massive neuronal
degeneration within weeks of birth The C-terminal region
of PrPc (PrP121 – 231) expressed in these mice has an
intrinsic neurotoxicity to cultured neurones Unlike PrPSc,
which is not neurotoxic to neurones lacking PrPcexpression,
PrP121 – 231 was more neurotoxic to PrPc-deficient cells
Human mutations E200K and F198S were found to enhance
toxicity of PrP121 – 231 to PrP-knockout neurones and
E200K enhanced toxicity to wild-type neurones The normal
metabolic cleavage point of PrPcis approximately
amino-acid residue 113 A fragment of PrPccorresponding to the
whole C-terminus of PrPc (PrP113 – 231), which is eight
amino acids longer than PrP121 – 231, lacked any toxicity This suggests the first eight amino residues of PrP113 – 121 suppress toxicity of the toxic domain in PrP121 – 231 Addition to cultures of a peptide (PrP112 – 125) correspond-ing to this region, in parallel with PrP121 – 231, suppressed the toxicity of PrP121 – 231 These results suggest that the prion protein contains two domains that are toxic on their own but which neutralize each other’s toxicity in the intact protein Point mutations in the inherited forms of disease might have their effects by diminishing this inhibition Keywords: prion; neurotoxicity; circular dichroism; neuro-degeneration
PrPcis a normal cell surface glycoprotein expressed by many
cells including neurones and astrocytes [1– 3], microglia [4],
oligodendroglia [2], leukocytes [5] and muscle cells [6]
PrPc is attached to the cell membrane via a GPI
(glyco-phosphoinositol) anchor [7] Predominantly expressed at
synapses [8], it has been suggested that PrPcis important for
neuronal activity [9] More recently it has been shown that
PrPcbinds copper via an octameric repeat region [10] PrPc
has been shown to bind significant amounts of copper in
vivo and this copper binding may be necessary for its
normal form [11] Recent work has suggested two functions
for the protein PrPc influences uptake of copper into
neurones [12] where it can be utilized for synaptic release
[12] or incorporation into enzymes such as Cu/Zn
super-oxide dismutase [13] Other data suggest that once PrPchas
bound copper, the protein can act as a superoxide dismutase
or superoxide scavenger [14]
During normal metabolism of PrPc, cleavage occurs in a
region around amino-acid residues 112 – 114 The metabolic
C-terminal fragment of this protein can be detected
nor-mally in brain [15] and the N-terminal fragment, retaining
the copper binding region can be purified by metal affinity
chromatography from brain [16] The rate of cleavage is
regulated by protein kinase C [17] and cleavage has been
suggested to be brought about by the metalloprotease
disintegrins ADAM10 and ADAM17 [18] However, the
cellular fate or function of these fragments after cleavage remains unknown Conformational change in PrPcstructure results in a higher percentage of b sheet structure and increased protease resistance, suggesting that the protein can
no longer be cleaved at this point
Prion diseases are fatal neurodegenerative diseases In these diseases, PrPcis converted to a protease resistant form (PrPSc)that cannot be cleaved at the normal cleavage site PrPScrepresents an altered isoform that differs markedly in conformation and accumulates to high levels in nervous tissue [19] PrPScis either a major part or the sole constituent
of the infectious agent of prion disease [20] and is also neurotoxic when applied to cultured cells [21] PrPSc is evidently the cause of neurodegeneration in vivo However, induction of neuronal loss both in vivo and in vitro requires the expression of PrPc[21,22] Mice lacking expression of PrPc are resistant to both the toxicity of PrPSc and its neurodegenerative effects [22,23]
Attempts to understand the mechanism of PrPSc neuro-toxicity have focussed on a single peptide known as PrP106 – 126 [24,25] This peptide corresponds to the region
of the human protein that is normally cleaved during cellular processing of PrPc but which becomes protease resistant when PrPc is converted to PrPSc This peptide has many features of PrPSc including protease resistance, ability to form fibrils and high b sheet content The mechanism of the action of this peptide has been studied in detail in culture by many groups [24,26,27,28,29,30,31] including our own [25,32,33,34,35,36,37] and has also been shown to be toxic
in vivo [38] The basic mechanism by which PrP106 – 126 kills neurones in cerebellar cell cultures has been shown
to be the same as that by which PrPScacts Both require neuronal expression of PrPc[21,25,32] and the involvement
of a stress event such as superoxide production by activated microglia [21,25] PrP106 – 126 kills the neurones, probably
Correspondence to D R Brown, Department of Biology and
Biochemistry, University of Bath, Bath BA2 7AY, UK.
Fax: 1 44 1225 826 779, Tel.: 1 44 1225 323 133,
E-mail: bssdrb@bath.ac.uk
(Received 14 September 2001, accepted 1 October 2001)
Abbreviations: MTT,
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide.
Trang 2as a result of reduction in neuronal resistance to oxidative
stress [33] Apoptosis induced by the peptide follows as a
result of increased calcium influx throughL-type channels
and NMDA receptors [34] Attempts to define the toxic
region of the protein further have identified the region
AGAAAAGA as being necessary for both fibril formation
and PrPSc-like toxicity [36] The maximum toxicity was
found for a peptide corresponding to amino-acid residues
113 – 126 of the human sequence containing this palindrome
[36]
Mice have been generated that express truncated versions
of PrPc In particular, a mouse expressing a prion protein of
106 amino acids [39] has been used to identify those parts of
the protein necessary for both infection and
neurodegenera-tion in prion disease The truncated version of PrPccan be
converted into a truncated PrPSc capable of infecting the
same transgenic mice and inducing neurodegeneration The
106 amino acids comprise the amino-acid residues 89 – 140
and from 177 to the C-terminus
Although some mice expressing truncated forms of PrPc
do not develop spontaneous disease [39,40] others do [41,42]
Of particular interest are mice with N-terminal deletions
lacking amino-acid residues up to 121 or 134 of the mouse
sequence These mice express truncated protein at the cell
surface and develop degeneration in the cerebellum shortly
after birth This disease is not prion disease but represents a
novel form of cerebellum-specific degeneration Some mice
that have genetic ablations to prevent expression of all PrPc
overexpress the prion protein homologue, Doppel [43] Such
mice show Purkinje cell degeneration Doppel is
homo-logous to the N-terminally truncated PrP that induces
cerebellar cell loss Co-expression of full length PrPcwith
either the N-terminally truncated PrPcin mice [42] or
re-introduced into the Doppel expressing PrPc-knockout mice
[44] prevents neurodegeneration Therefore expression of
Doppel or a C-terminal region of PrPc(PrP121 – 231) in the
absence of the full length prion protein leads to
neurodegeneration The mechanism of this is unknown
The present investigation was carried out to determine if
the C-terminal region of PrPc(PrP121 – 231) has intrinsic
neurotoxicity and whether PrPchas an intrinsic mechanism
to inhibit this Unlike PrPScPrP121 – 231 is more neurotoxic
to PrPc-deficient cells Mutants of PrP121 – 231 carrying
known human mutations in the C-terminal region (E200K,
D178N and F198S) were also investigated In particular,
E200K and F198S were also found to enhance toxicity
However, PrP113 – 231 lacked any toxicity suggesting the
normal N-terminus of the C-terminal metabolic cleavage
product of the prion protein suppressed toxicity
M A T E R I A L S A N D M E T H O D S
Unless stated, pharmacological agents were purchased from
Sigma
Animals
Prion protein knockout mice (Npu-Prnp8/8) used in this study
were those described by Manson et al [45] or for some
experiments a different strain was used (Zrk-Prnp8/8) and
these were as described by Bu¨eler et al [46] The wild-type
mice used were either 129Ola mice (as control for
Npu-Prnp8/8) or descendants of an F1 generation mouse produced
by interbreeding the original parental strains (C57BL/6 J and 129/Sv(ev) mice) used to generate Zrk-Prnp8/8 mice originally
Neuronal cell culture Preparation of cerebellar cells from 6-day-old mice (P6) or cortical cells from newborn mice (P0) was as previously described [25,36] Briefly, the cerebella were dissociated
in Hank’s Solution (Gibco) containing 0.5% trypsin (Sigma) and plated at 1– 2 106cells:cm22in 24-well trays (Falcon) coated with poly D-lysine (50 mg:mL21, Sigma) Cultures were maintained in Dulbecco’s minimal essential medium (Gibco) supplemented with 10% fetal bovine serum, 2 mM glutamine and 1% antibiotics (penicillin, streptomycin, fungizone; Gibco) Cultures were maintained at 37 8C with 5% CO2for 10 days The neuronal nature of these cultures was confirmed by immunostaining of parallell wells with neurofilament as previously described [3]
Peptides or proteins were added to cultures initially and on the third day Cell survival was determined on day
5 or 7 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl-tetrazo-lium bromide (MTT; Sigma) was diluted to 200 mM in Hanks’ solution (Gibco) and added to cultures for 1 h at
37 8C The MTT formazan product was released from cells
by addition of dimethylsulfoxide (Sigma) and measured at
570 nm in a Unicam Helios spectrophotometer (ATI Unicam) Relative survival in comparison to untreated control cultures could then be determined
For coculture experiments, cerebellar cells were plated as normal in 24-well trays Microglia were plated at 10 000 cells per well in Falcon insets with 0.4-mm pore diameter Peptides could then be applied directly to the lower wells and to the insert After peptide treatments, the inserts were removed and MTT assays carried out on the cerebellar cells
as before
Glia cell culture Microglia were isolated as described previously [25] Briefly, cortices from newborn mice were dissociated with trypsin and seeded into tissue culture flasks (Falcon) When cultures were confluent, microglia were isolated by rapid shaking for 2 hours Microglia were isolated by shaking mixed glial cultures for 2 h The floating cells were collected and plated for 30 min The nonadhering cells were discarded The adhering cells were identified as pure cultures of micro-glia by immunostaining for ferritin as previously described [25]
PrP peptides and protein Mouse prion protein peptides (PrP121 – 231), both wild-type and those carrying human mutations, were generated as previously described [47] The wild-type peptide (PrP121 – 231) was mutated to carry amino-acid substitutions equivalent to the human mutations E200K, D178N and F198S as previously described [47] The numbering refers to the human sequence but the equivalent amino-acid residue
of the mouse sequence was altered (one codon proximal in each case)
Purification of these mutants from inclusion bodies was as previously described [47] except that the bacteria were
Trang 3grown at 26 8C to avoid major degradation of the protein and
no gradient was applied to the DE52/CM52 column during
purification Full length protein (PrP23 – 231) and other
recombinant proteins equivalent to the complete metabolic
C-terminal cleavage product of PrP (PrP113 – 231) or
dele-tion mutants, PrP23 – 112, PrP79 – 231 and PrP105 – 231,
were also purified and refolded as previously described [14]
PrP23 – 112, PrP113 – 231, PrP105 – 231 and PrP79 – 231
were generated by PCR-based mutagenesis using splint
oligonucleotides to introduce a restriction site before codon
113, 105 or 79 or after codon 112 Following digestion of the
pET-PrP construct with Nde I the N-terminus encoding
region was removed and religation of the vector lead to a
pET-PrP construct expressing the complete metabolic
C-terminal fragment of PrP as determined by sequencing
The N-terminal fragment was prepared by digestion of the
mutated plasmid with Xho I, removing the C-terminal
encoding fragment and religating the plasmid Fidelity of
the N-terminus of these deletion mutants was determined by
mass spectroscopy analysis
Shorter peptides were synthesized in house by the PNAC
facility These peptides based on the mouse prion protein
sequence with corresponding amino-acid residues were
PrP112 – 125(AGAAAAGAVVGGLG), 121 – 146(AVVGG
LGYMLGSAMSRPIIHFGSDYED), PrP147 – 171(RYYRE
NMYRYPNQVYYRPVDQYSNQ), PrP163 – 184(RPVDQ
YSNQNNFVHDCVNITIK), PrP180 – 198(NITIKQHTVTT
KGENFT) and PrP196 – 220(NFTETDVKMMERVVRQM
CVTQYQKE)
CD spectroscopy and analysis
CD spectra were recorded for prion proteins and peptides
using a CD6 spectropolarimeter (Jobin Yvon, Division
d’Instrumente S.A.) or a Jasco J-810 spectropolarimeter,
calibrated with ammonium (1)-camphor-10-sulfonate by a
method similar to that described previously [36] Peptide
samples were diluted from high concentration
dimethylsulf-oxide stocks to a concentration of 2 mg:mL21 in 10 mM
sodium phosphate (pH 7.4) These samples were measured
in cuvettes of 1 mm or 0.5 mm pathlength (Hellma) The
spectrum from 190 nm to 250 nm was analysed with step
resolution of 0.5 nm at a temperature of 23 8C Five scans
were averaged and the background from buffer was
subtracted Spectra are presented as molar ellipticity (u)
R E S U L T S
PrP121 – 231 is toxic to neurones
Prion proteins of different lengths were tested for toxicity by
application to cultures of cerebellar neurones Cerebellar
cultures were prepared from 6-day-old mice The proteins
were applied to the cultures and the cultures were
maintained in serum-free medium for 7-days The medium
was changed every 3 days and replenished with prion
proteins At the end of that time, survival was determined
using an MTT assay Full length recombinant PrPc, PrP79 –
231, PrP105 – 231 and PrP113 – 231 showed no toxicity to
wild-type cultures of cerebellar cells (Fig 1A) However,
PrP121 – 231 was toxic to these cells in a dose-dependent
manner The proteins were also applied to cultures of
Npu-Prnp8/8 cerebellar cells and the same results were obtained
(Fig 1B) However, PrP121 – 231 was more toxic to Prnp8/8 cerebellar cells than to wild-type cells (Student’s t-test,
P , 0.05) Additionally, PrP121 – 231 was more toxic to Zrk-Prnp8/8 cerebellar cells than wild-type cerebellar cells
Fig 1 Toxicity of prion proteins Cerebellar cell cultures from wild-type (A) and Npu-Prnp8/8 (B) cerebellar cells were grown in serum-free medium and treated with prion proteins at different concentrations for
7 days After that time the survival of the cultures was measured with an MTT assay The values were compared to those for untreated cultures as
a percentage Shown are PrP23 – 231 (W) PrP79– 231 (O), PrP105–231 (A), PrP113–231 (K) and PrP121–231 (X) Bovine serum albumin (B) was used as a negative control (C) Wild-type and Zrk Prnp8/8 cerebellar cells were either treated with L -leucine methyl ester (grey bars) or cocultured with additional microglia (black bars) Cerebellar cells were treated with 1 mg:mL21PrP23– 231 or PrP121 – 231 for 7 days After this time, the survival was measured using an MTT assay and comparing values to those of untreated cultures (open bars) Shown are the mean ^ SEM of four experiments (different cultures) with three determinations (separate wells) each.
Trang 4(Fig 1C) This confirms that the increased toxicity to
Prnp8/8 cerebellar cells is a result of the lack of PrPc
expression and not the genetic background of the mice
These results suggest that the toxicity of PrP121 – 231 does
not require the expression of native PrPc
Previously, it has been shown that the synthetic peptide
fragment PrP106 – 126 is toxic and that this toxicity requires
microglia L-Leucine methyl ester selectively destroys
microglia [25] Cultures of wild-type and Zrk- Prnp8/8
cerebellar cells were treated with 50 mM L-leucine methyl
ester for 2 h before application of PrP121 – 231 or were
cocultured continuously with wild-type microglia during
application of PrP121 – 231 for 7 days At the end of this
time an MTT assay was carried out Treatment with
L-leucine methyl ester had only a minor effect on the
toxicity of the protein Addition of microglia had no
signifi-cant effect on the toxicity of PrP121 – 231 These results
suggest that the toxicity of PrP121 – 231 does not require
involvement of microglia
In order to verify that PrP121 – 231 is toxic to neurones,
staining with Hoechst 33342 reagent was carried out during
treatment of cerebellar cell cultures Dying neurones could
be determined by the presence of condensed and/or
frag-mented nuclei (Fig 2) Treatment with PrP121 – 231 caused
a great increase in the number of fragmented nuclei present
after 4 days of treatment as compared to treatment with full
length protein (PrP23 – 231) The quantitation of this is
shown in Fig 2C
Peptide dissection of PrP121 – 231 toxicity
Five synthetic peptides corresponding to the majority of
PrP121 – 231 were synthesized These peptides were
PrP121 – 146, PrP147 – 171, PrP163 – 184, PrP180 – 198 and
PrP196 – 220 Initially we attempted to synthesize a set of
four nonoverlapping peptides However, one of these
peptides self terminated during synthesis Therefore two
overlapping peptides were produced to cover the region
172 – 180 As for the recombinant proteins, the toxicity of
these peptides was tested on cultures of wild-type and
Npu-Prnp8/8 cerebellar cells (Fig 3) Peptides PrP121 – 146,
PrP147 – 171 and PrP180 – 198 showed little or no toxicity to
wild-type cells whereas PrP196 – 220 and PrP163 – 184 were
both toxic to wild-type cerebellar cells (Fig 3A) However,
when applied to Npu-Prnp8/8 cerebellar cells PrP147 – 171
showed toxicity This suggests that this peptide is toxic in
the absence of PrPcexpression PrP163– 184 showed reduced
toxicity However, PrP196 – 220 also remained similarly
toxic (Fig 3B) These results suggest that the toxicity of the
C-terminal domain of PrPcin particular is associated with
amino-acid residues 163 – 184 but that amino-acid residues
further N-terminal to this may also participate in the toxicity
to PrPcdeficient cells Additionally residues of PrP196 – 220
might also participate in this toxicity
The peptides used in this analysis were studied using
the CD spectroscopic technique The analysis of the
struc-tural content was determined by the curve fitting program
CNNR The CD spectra of the five peptides appears in Fig 4
The analysis of the structural content of the peptides appears
in Table 1 As can be seen in Fig 4, PrP180 – 198 has a
noticeably different spectrum with an unusual minimum at
230 nm It is currently unclear what this minimum
represents PrP196 – 200 has a high percentage of b sheet
structure This peptide readily forms fibrils (data not shown)
Mutants of PrP121 – 231 Further recombinant mouse proteins based on PrP121 – 231 were analysed for toxicity These new proteins contained the equivalent of one of three human point mutations associated with human prion disease The mutations analysed were E200K, D178N and F198S For simplicity the mutants of PrP121 – 231 shall be referred to by these mutation assign-ments even though in the mouse sequence the location of the residue is numerically one place closer to the N-terminus in the sequence (e.g E200K in human but in mouse the E is at 199) PrP121 – 231 will be referred to as wild-type protein Wild-type cerebellar cells were treated with the four
Fig 2 PrP121–231 causes apoptotic cell death Cerebellar cells from wild-type mice were exposed to PrP23 – 231 (A) or PrP121 – 231 (B) for 2 days The cells were then stained with the Hoechst reagent to detect fragment or condensed nuclei An increase in aberrant nuclei (bright condensed, fragmented) was only detected in cultures treated with PrP121 – 231 Scale bar, 50 m M (C) Quantitation of aberrant nuclei
in culture treated with either PrP23 – 231, PrP121 – 231 or the control Ten fields were counted on three coverslips for four separate experiments Shown and mean and SEM.
Trang 5different proteins for 7 days, after which time an MTT assay
was used to quantitate survival The E200K mutation was
the only mutation to increase the toxicity of PrP121 – 231
significantly above that of the wild-type protein (P , 0.05)
(Fig 5A) Zrk-Prnp8/8 cerebellar cells were also treated with
the proteins and assayed for survival in the same manner
(Fig 5B) Both E200k and F198S showed significantly
(P , 0.05) more toxicity to Zrk-Prnp8/8 cerebellar cells than
the wild-type PrP121 – 231 The implication of these results
is that the mutations in the region of amino-acid residues
198 – 200 enhance the toxicity of the peptide PrP121 – 231
Peptide inhibition of PrP121 – 231 toxicity
In the paper by Shmerling et al [42] it was suggested that
the in vivo toxicity of PrP121 – 231 was inhibited by the
coexpression of full length PrPc(PrP23 – 231) Therefore we tested whether PrP23 – 231, PrP23 – 112, PrP105 – 231 or PrP113 – 231 would inhibit the toxicity of PrP121 – 231 to Npu-Prnp8/8 cerebellar cells Of these proteins only PrP23 –
231 inhibited toxicity completely as determined by MTT assay (Fig 5C) However, PrP105 – 231 showed a low level
of inhibition This result suggests that PrP23 – 231 contains a domain inhibitory to the toxicity of PrP121 – 231, but this domain does not lie in the unstructured N-terminus; possibly
it lies in the hydrophobic domain of PrPc
A recent study on optimization of the neurotoxicity of the well characterized prion protein peptide PrP106 – 126 [36] indicated that optimal toxicity of this region is located in amino-acid residues 113 – 126 of the human sequence This region is identical to amino-acid residues 112 – 125 of the
Fig 4 Circular dichroism study of peptides Peptides were freshly prepared in 10 m M phosphate buffer pH 7.4 at 2 mg:mL21 The peptides shown are PrP112 – 125, PrP121 – 146, PrP147 – 171, PrP163 –
184, PrP180 – 198 and PrP196– 220 Five sweeps were collected at 0.5-nm intervals Spectra are presented as molar ellipticity (u).
Fig 3 Toxicity of peptides to cerebellar neurones Cerebellar cell
cultures from wild-type (A) and Npu-Prnp8 / 8 (B) cerebellar cells were
grown in serum free medium and treated with prion protein peptides at
different concentrations for 5 days After that time the survival of the
cultures was measured with an MTT assay The values were compared
to those for untreated cultures as a percentage Shown are PrP121 – 146
(W) PrP147–171 (X), PrP163–184 (A), PrP180–198 (K) and PrP196–
220 (O) Shown are the mean ^ SEM of four experiments with three
determinations each.
Table 1 Analysis of CD of peptides Values determined using the
CNNR program Analysis based on the spectra between 190 and 250 nm.
Helix b Sheet b Turn Random coil
Trang 6mouse sequence This peptide is highly toxic to wild-type
cerebellar cells but is not at all toxic to Zrk-Prnp8/8
cerebellar cells [37] The ability of this peptide to inhibit
the toxicity of PrP121 – 231 was analysed Increasing
amounts of PrP112 – 125 were mixed with fixed amounts
of E200K, D178N, F198S and wild-type PrP121 – 231 and
applied to Npu-Prnp8/8 cerebellar cells for 5 days After
this time, an MTT assay was used to measure survival
(Fig 5D) PrP112 – 125 suppressed the toxicity of
wild-type PrP in a dose-dependent manner reaching saturation
above a 1 : 1 molar ratio PrP112 – 125 was not able to
completely suppress the toxicity of the mutant proteins
There was no further effect of toxicity above a 1: 1 molar
ratio The relative order of effective suppression was
wild-type F198S E200K D178N Indeed, for
D178N there was no significant inhibition of toxicity
(P 0.05)
CD analysis of peptide – protein interaction
The CD spectra of PrP121 – 231 and its mutants (in the
presence or absence of an equimolar amount of PrP112 –
125) were measured (Fig 6) In general, and as previously
described [47], the three point mutations did not alter the CD
spectra markedly (Fig 6A) PrP112 – 125 has previously been shown to have a predominantly random coiled structure with some amount of b sheet (Fig 4) The mixture of wild-type PrP121 – 231 and PrP112 – 125 resulted
in a unique CD spectrum that did not resemble that of PrP121 – 231, PrP112 – 125 or an arithmetic sum of the two
Fig 5 Toxicity of PrP121 – 231 mutants Cerebellar cell cultures
from wild-type (A) and Zrk-Prnp8/8 (B) cerebellar cells were grown in
serum free medium and treated with mutants of PrP121 – 231 at
different concentrations for 7 days After that time the survival of the
cultures was measured with an MTT assay The values were compared
to those for untreated cultures as a percentage Shown are wild-type (W)
D178N (K), F198S (O) and E200K (X) (C) Npu-Prnp8/8 cerebellar
cells were either treated with 50 m M of PrP121 – 231 and with
increasing concentrations of PrP23 – 231 (X), PrP23–112 (W), PrP105–
231 (K) or PrP112–231 (O) After 5 days treatment the survival was
measured using an MTT assay and comparing values to those of
untreated cultures Shown are the mean ^ SEM of four experiments
with three determinations each (D) Npu-Prnp8 / 8 cerebellar cells were
either treated with PrP112 – 125 at varying concentrations alone (B) or
with the addition of 50 m M of PrP121 – 231 (W) or PrP121–231
mutants, D178N (K), F198S (O) or E200K (X) After 5 days treatment
the survival was measured using an MTT assay and comparing values to
those of untreated cultures Shown are the mean ^ SEM of four
experiments with three determinations each.
Fig 6 Circular dichroism study of prion proteins Proteins were freshly prepared in 10 m M phosphate buffer pH 7.4 at 0.1 mg:mL21 Wild-type PrP121– 231 (WT) and three mutants (E200K, F198S, D178N) were measured either on their own (compared in A) or mixed with an equimolar amount of PrP112 – 125 (B – E) The spectra of the wild-type form of PrP121 – 231 and the mutants on their own (thin line) are compared to spectra of the same proteins mixed with PrP112 – 125 (thick line) In addition the spectra for PrP105 – 231 (F) and PrP113 –
231 (G) were also determine on their own (thin line) or mixed with PrP112 – 125 (thick line) The increased levels of structural elements were determined by a CD spectra analysis program Five sweeps were collected at 0.5-nm intervals Spectra are presented as molar ellipticity [u] The increased helical content in all the proteins induced by PrP112 – 125 was determined and plotted in (H).
Trang 7PrP112 – 125 had similar but lesser effect on the mutant
proteins E200K and F198S There was little effect on the
spectrum of D178N In particular, the mixtures showed
relative increases in helical content (Fig 6H) This change
in spectrum possibly represents a change in the structure of
the protein as a result of an interaction between the protein
and the peptide
In order to determine if the structure produced by the
interaction of PrP112 – 125 and PrP121 – 231 represents a
‘true’ PrP structure, the CD spectra of PrP105 – 231 and
PrP113 – 231 was determined (Fig 6) The CD spectra of
both proteins appear similar but dissimilar to the mixtures
between PrP121 – 231 and PrP112 – 125 The spectra of
PrP113 – 231 and PrP105 – 231 were similar to that of
PrP121 – 231 However, both spectra show a loss of a strong
minima at 222 nm and an increase at 230 nm However,
mixing these proteins with PrP112 – 125 did not cause any
change in the spectrum except a slight increase at 230 nm
There was no increase in helical content (Fig 6H)
D I S C U S S I O N
The precise mechanism of neurodegeneration in prion
disease is unknown Much research has concentrated on a
peptide PrP106 – 126 to understand neuronal death in
models of these diseases Until this report there has been
no complete investigation of the prion protein for toxic
domains It has been known for some time that the
N-terminus (amino-acid residues 23 – 90) is not associated
with disease We aimed to investigate whether the
C-terminal domain has inherent toxicity to neurones
because a recent publication by Shmerling et al [42] has
shown that mice expressing a C-terminal fragment of PrPc
(either deletion of amino-acid residues 32 – 121 or 32 – 134)
die within several weeks of birth due to massive
neuro-degeneration in the granule cell layer of the cerebellum A
different C-terminal fragment of PrPc (PrP112 – 231) is
generated by normal cleavage of the protein [15] It has been
suggested that this fragment can be cycled back to the
cell surface where it may have a function independent of the
full length molecule [48] We found that the fragment
PrP121 – 231 was highly neurotoxic This seemed to us to be
paradoxical as one would not expect a normal cleavage
product to be potently neurotoxic However, slightly longer
peptides (PrP105 – 231 and PrP113 – 231) showed no
toxicity The region of the mouse protein containing
amino-acid residues 105 – 125 is almost identical to the
sequence of human PrP, which is known as PrP106 – 126, the
neurotoxic peptide used by many laboratories as a model of
the neurotoxicity of PrPSc However, this region of the
protein, as part of a C-terminal fragment of PrPc, has no
demonstrable toxicity Furthermore, addition of a peptide
with amino-acid residues 112 – 125 of the mouse PrPc
sequence to cerebellar cultures in parallel with PrP121 – 231
neutralized the toxicity of PrP121 – 231 The same result
was obtained with mouse PrP121 – 231 and the human
PrP106 – 126 (data not shown) Under these conditions, the
PrP106 – 126 toxicity was fully inhibited Additionally we
also showed that PrP23 – 231 inhibits toxicity but that
neither the N-terminal fragment (PrP23 – 112) nor the full
C-terminal fragment (PrP113 – 231) inhibit the toxicity
Studies of the N-terminus have shown that it is unstructured
and therefore it is unlikely to undergo a conformational
change when PrP23 – 231 is cleaved However, PrP113 – 231 contains a very hydrophobic N-terminus (amino-acid residues 113 – 135) and it is quite possible that this region would be masked if it interacted with other hydrophobic residues in the rest of the protein
The normal cleavage product of PrPc metabolism is a protein cleaved at around amino-residue 112 – 114 and not
121 – 122 [15,17,18] Therefore separation of the hydro-phobic, palindromic region of 112 – 120 from the rest of the C-terminus is artefactual Such a fragment of the protein has never been shown to exist in vivo Furthermore, the paper by Shmerling et al [42] demonstrates that expression of a fuller C-terminal cleavage product with a deletion only of residues
32 – 106 does not develop pathology The toxicity of the C-terminal part of PrPchas never been apparent before the publication by Shmerling et al [42] and most interest has focussed on the known neurotoxic region PrP106 – 126 whose toxicity came to attention because of study of the toxicity of PrPSc[24] If deletion of PrPcup to residue 121 creates a toxic protein then one would expect that deletion of the C-terminus of the protein should similarly cause prob-lems Muramato et al [41] showed that deletion of amino-acid residues 177 – 200 or 201 – 217 results in neurodegen-erative disease in mice expressing such modified PrPs However, the disease was not as severe as that described by Shmerling et al [42] and the cell death does not appear to be due to the toxicity of the hydrophobic domain (amino-acid residues 112 – 135) Our findings regarding PrP121 – 231 mediated neurotoxicity might explain the cerebellar degeneration described by Shmerling et al [42] However, although our results suggest that the neurodegeneration in this model could be the result of direct neurotoxic effects of PrP121 – 231, the full mechanism of this effect needs to be elucidated
The neurodegeneration seen in the 32 – 121 deleted mice [42] was prevented by the presence of coexpressed full-length PrPc The previous interpretation of this result was that PrPchas a ligand that interacts with it at two domains When the protein only interacts with PrPcat the C-terminal domain, the resulting signal transduction leads to death However, the problem with this model is that the metabolic C-terminal domain of PrPc(PrP112 – 231) exists in neurones all the time Even if it has a much lower affinity for the
‘ligand’ than full length PrPcthen one would nevertheless be causing the death of cerebellar granular cells A better hypothesis according to our data is that the C-terminal domain (PrP121 – 231) as expressed by Shmerling et al [42] can interact with full length PrPc This interaction via binding to the hydrophobic domain of PrPcthen inhibits the toxicity of the C-terminus
The normal N-terminal domain of the C-terminal cleavage product of PrPcis conserved so highly as to be unchanged from reptile to man [49,50] This is very unusual, especially for a region of the protein associated with neurotoxicity Therefore it is clearly essential to the normal metabolism of PrPc We have shown previously [11,37] that this region of the protein is the site of interaction between PrPc with PrPSc Therefore it is possible, under normal metabolic conditions, that the hydrophobic-palindromic region of PrPcinteracts directly with the globular domain of the C-terminus of another molecule of PrPc However, we consider it more likely that each molecule folds in such a way that the very N-terminus of PrP’s C-terminal cleavage
Trang 8product folds into the globular domain Due to the very
hydrophobic nature of this region, this may occurs directly
after cleavage of the protein
Our studies of toxicity used wild-type neurones and
neurones deficient in PrPc expression in parallel This
allowed us to distinguish fragments or peptides that were
toxic in absence of PrPc This also allowed us to demonstrate
that the toxicity of PrP121 – 231 is different to that of PrPSc,
as PrPScis nontoxic to cells lacking PrPcexpression [21] but
the toxicity of PrP121 – 231 is increased to Prnp8/8 cerebellar
cells In particular, the region of amino-acids 147 – 171
shows toxicity only to Prnp8/8 cerebellar cells and might
explain the increased toxicity of PrP121 – 231 to Prnp8/8
cerebellar cells PrP peptides based on the hydrophobic
domain of PrPchave also been suggest to have effects not
mediated directly through PrPc[28,30] but this is the first
time the peptides based other regions of PrP have been
shown to be toxic to Prnp8/8 neurones
Of importance are the studies with peptides that highlight
the region of the C-terminus involved in the toxicity of
PrP121 – 231 to cerebellar neurones Two peptides, PrP163 –
184 and PrP196 – 220, appeared to be the most toxic This is
of considerable interest as the majority of point mutations
associated within inherited human prion disease are found
within these regions (e.g D178N, V180I, T183A, F198S,
E200K, R208H, V201I, Q217R) Indeed, the two point
mutations studied in this paper, E200K and F198S, both map
to PrP196 – 220 and both enhanced the toxicity of PrP121 –
231 It is quite possible that these point mutations alter the
ability of the globular domain to interact with the conserved
hydrophobic region This was demonstrated here by a
decreased ability of PrP112 – 125 to inhibit the toxicity of
the PrP121 – 231 mutants
Many of these point mutations lie along a hydrophobic
cleft in the protein [47,51] It is quite possible that the
N-terminal domain of the C-terminal cleavage product will
fold into this cleft However, this report does not give any
information on this aspect of the structure of the protein
Previously, NMR studies of PrPchave used either PrP121 –
231 or PrP23 – 231 as the protein for analysis [47,52 – 55]
The general finding of these reports was that the C-terminal
of the protein contains three helices that fold together to
make a globular domain while the N-terminus has little
structure For both the full length protein and the C-terminal
fragment the globular domain was very similar However,
the protein PrP121 – 231 does not represent the true
C-terminal fragment of PrP as it does not include the
perfectly conserved amino-acid residues 112 – 120 which
constitute the true N-terminus Therefore it is probably of
vital importance that the C-terminal fragment containing
these additional amino acids be studied further, as the true
C-terminal fragment of PrPcmay form a novel conformation
as yet unknown Support for this comes from the analysis by
CD of two proteins PrP105 – 231 and PrP113 – 231 that have
moderately different spectra to that of PrP121 – 231 It has
previously been suggested that such proteins prepared
from bacteria are sensitive to protease digestion and the
amino-acid residues up to 121 are cleaved off [53] We took
particular care to ensure that our proteins were not degraded
in this way
Analysis of the effect of point mutations on the structure
of PrPchave focussed on their effect on the protein PrP121 –
231 We have observed that two mutations alter the toxicity
of this protein but this is not likely to have direct relevance
to prion disease as the protein PrP121 – 231 does not normally exist However, if the amino-acid residues where these mutations occur are central to refolding of the protein after cleavage then it is possible that C-terminus could be toxic in the inherited prion diseases Alternatively, or in addition, a misfolded C-terminal protein could somehow influence formation of PrPSc Another alternative is that interaction between the globular domain and the hydro-phobic-palindromic region influences cleavage of the protein Then, a large amount of uncleaved mutant PrPc might remain and be sufficient to eventually aggregate and begin conversion to PrPSc
In summary, we have shown that a C-terminal fragment of PrPccommonly used in the investigation of PrPcstructure and known to induce neurodegeneration in mice lacking expression of the full length PrPcis neurotoxic This neuro-toxicity does not require expression of PrPc The neuro-toxicity can be inhibited by the presence of the hydrophobic-palindromic region (amino-acid residues 112 – 125) of the protein either applied as a separate peptide or attached to the protein Known human point mutations enhance the toxicity
of this PrPc fragment However, PrP121 – 231 might not represent the true C-terminal cleavage produce of PrPc metabolism and re-investigation of the biology of the C-terminus of PrPcis necessary
A C K N O W L E D G E M E N T S
The authors than Charles Weissmann for the Zrk1 prion protein knockout mice and Jean Manson for the Npu prion protein knockout mice Thanks also to Rudi Glockshuber for PrP121 – 231 mutant proteins In addition we thank Bill Broadhurst and Tim Daffron for help with the CD analysis.
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