How do PrP mutations lead to prion disease?

How do PrP mutations lead to prion disease?

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My understanding is:

The PrP gene in human cells is expressed as both PrP-c (normal protein) and PrP-sc (prion disease protein). This happens post transcriptionally, that is, the normal and the diseased protein are not distinguished based on genetic mutations, rather they are synthesized based on the same gene, but differ in their tertiary structures, which makes their functions also different.

If the amino acid sequence of both proteins is the same (same gene), then what determines whether the synthesized protein will take the disease-causing tertiary structure or the normal one? Is it due to post-translational modifications?

And finally (I apologize for advance for multiple questions): how do prions, which are void of nucleic acids (being proteins), integrate into the genome of a newly infected host by reverse translation, and become a familial disease?

If the amino acid sequence of both proteins is the same, what determines whether the synthesized protein will take the disease-causing tertiary structure or the normal one?

PrP-C and PrP-Sc do indeed have the same primary structure. However, they differ in secondary and tertiary structure. The protein can take more than one shape (conformation), where the likely conformation spaces taken by the protein are due to chemical and physical interactions. Many proteins can change their shape, often this is not well understood in detail. However, we know that the conformational space of some proteins allows for several, stable conformations of a single protein; the factors which stabilize it in a conformation or cause it to switch are difficult to understand in vivo.

PrP-Sc tends to accumulate in compact, protease-resistant aggregates. Circular dichroism shows that normal PrPC had 43% alpha helical and 3% beta sheet content, whereas PrPSc was only 30% alpha helix and 43% beta sheet. Although the exact 3D structure of PrPSc is not known, it has a higher proportion of β-sheet structure in place of the normal α-helix structure. The end of each fiber acts as a template onto which free protein molecules may attach, allowing the fiber to grow.

Prion proteins therefore propagate accumulation of more and more prion proteins.

Is it due to post-translational modifications?


How do prions, which are void of nucleic acids, integrate into the genome of a newly infected host by reverse translation, and become a familial disease?

They do not enter the genome through reverse translation. The DNA sequence itself is mutated and the mutation drifts across generations for as long as it is being passed on through the germline.

In Vitro Seeding Activity of Glycoform-Deficient Prions from Variably Protease-Sensitive Prionopathy and Familial CJD Associated with PrP V180I Mutation

Both sporadic variably protease-sensitive prionopathy (VPSPr) and familial Creutzfeldt-Jakob disease linked to the prion protein (PrP) V180I mutation (fCJD V180I ) have been found to share a unique pathological prion protein (PrP Sc ) that lacks the protease-resistant PrP Sc glycosylated at residue 181 because two of four PrP glycoforms are apparently not converted into the PrP Sc from their cellular PrP (PrP C ). To investigate the seeding activity of these unique PrP Sc molecules, we conducted in vitro prion conversion experiments using serial protein misfolding cyclic amplification (sPMCA) and real-time quaking-induced conversion (RT-QuIC) assays with different PrP C substrates. We observed that the seeding of PrP Sc from VPSPr or fCJD V180I in the sPMCA reaction containing normal human or humanized transgenic (Tg) mouse brain homogenates generated PrP Sc molecules that unexpectedly exhibited a dominant diglycosylated PrP isoform along with PrP monoglycosylated at residue 181. The efficiency of PrP Sc amplification was significantly higher in non-CJDMM than in non-CJDVV human brain homogenate, whereas it was higher in normal TgVV than in TgMM mouse brain homogenate. PrP C from the mixture of normal TgMM and Tg mouse brain expressing PrP V180I mutation (Tg180) but not TgV180I alone was converted into PrP Sc by seeding with the VPSPr or fCJD V180I . The RT-QuIC seeding activity of PrP Sc from VPSPr and fCJD V180I was significantly lower than that of sCJD. Our results suggest that the formation of glycoform-selective prions may be associated with an unidentified factor in the affected brain and the glycoform-deficiency of PrP Sc does not affect the glycoforms of in vitro newly amplified PrP Sc .

Keywords: Humanized transgenic mice Polymorphism Prion Prion disease Real-time quaking-induced conversion (RT-QuIC) Serial protein misfolding cyclic amplification (sPMCA) Variably protease-sensitive prionopathy (VPSPr).

Prion pathogenesis, diagnostics, and therapy: where do we stand?

Prion diseases, also known as transmissible spongiform encephalopathies (TSEs), are invariably fatal neurodegenerative disorders affecting a broad spectrum of host species and arise via genetic, infectious, or sporadic mechanisms (Table ​ (Table1). 1 ). In humans, prion diseases result from infectious modes of transmission (variant Creutzfeldt-Jakob disease [vCJD], iatrogenic CJD, Kuru) inherited modes of transmission in which there is nonconservative germ line mutation of the PRNP gene open reading frame (familial CJD, Gerstmann-Sträussler-Scheinker Syndrome, Fatal Familial Insomnia) (1, 2) and modes of transmission that have as yet been neither determined nor understood (sporadic CJD [sCJD]). The clinical symptoms associated with each of the human prion disease forms vary dramatically (2).

Table 1

Spectrum of prion diseases of humans and animals

Nomenclature applied to prion biology continues to be complex and confusing to nonspecialists. Here we utilize the term “prion” to denote the causative agent of prion diseases, without implying associated structural properties. We refer to the disease-associated prion protein (PrP Sc ), a disease-specific isoform of the host-encoded cellular prion protein (PrP C ), which accumulates in individuals affected with most forms of TSE (Figure ​ (Figure1) 1 ) (3). While PrP Sc is classically defined as partially protease-resistant, aggregated PrP, it has recently been shown that PrP C may undergo disease-associated structural modifications that do not impart properties of inherent protease resistance (4). In light of this, it is advisable that PrP Sc be defined on the basis of disease-associated structural modifications rather than properties of protease resistance.

Models of PrP C to PrP Sc conversion. (A) The heterodimer model proposes that upon infection of an appropriate host cell, the incoming PrP Sc (orange) starts a catalytic cascade using PrP C (blue) or a partially unfolded intermediate arising from stochastic fluctuations in PrP C conformations as a substrate, converting it by a conformational change into a new β-sheet–rich protein. The newly formed PrP Sc (green-orange) will in turn convert new PrP C molecules. (B) The noncatalytic nucleated polymerization model proposes that the conformational change of PrP C into PrP Sc is thermodynamically controlled: the conversion of PrP C to PrP Sc is a reversible process but at equilibrium strongly favors the conformation of PrP C . Converted PrP Sc is established only when it adds onto a fibril-like seed or aggregate of PrP Sc . Once a seed is present, further monomer addition is accelerated.

Prion diseases are conceptually recent the first cases of Creutzfeldt-Jakob disease were described eight decades ago (5, 6), yet the protein-only theory of prion infection was originally formulated in 1967 (7) and later refined and the term “prion” coined in 1982 (8). The precise physical nature of the prion agent is still the subject of intense scientific controversy. PrP Sc may or may not be congruent with the infectious agent. It remains to be formally proven whether the infectious unit consists primarily or exclusively of: (a) a subspecies of PrP Sc (b) an intermediate form of PrP (PrP*) (9) (c) other host-derived proteins (10) or (d) nonprotein compounds (which may include glycosaminoglycans and maybe even nucleic acids) (11). We still do not know, therefore, whether the prion hypothesis is correct in its entirety.

As with any other disease, a thorough mechanistic understanding of pathogenesis is the best foundation for devising sensitive predictive diagnostics and efficacious therapeutic regimens.

The purpose of the present article is to discuss some aspects of the state of the art in prion science and their impact on prion diagnostics, primarily with respect to peripherally acquired prion disease. As of now, no causal therapies can be offered to prion disease victims. Yet we are witnessing the emergence of an impressive wealth of therapeutic approaches, some of which certainly deserve to be tested for their validity.


Location and nature of mutations

The P101L and H186R mutations are associated with familial prion disease in humans P101 and H186 are both highly conserved between mammalian species. These mutations occur in very different regions of the protein: P101 is located in the unstructured tail of the PrP 27� sequence (modeled in Fig. 1 ), while H186 is packed into the core of the folded C-terminal domain, between 㬒, 㬓 and 㬢. It appears likely that the reasons why these mutations should favor conversion of PrP C to other forms, including pathogenic forms, might differ. The dominant negative mutations Q167R and Q218K both occur in regions of the protein that are solvent-exposed, in the loop between 㬢 and 㬒 for Q167 and towards the end of the 㬓 helix for Q218. Neither of these residues is highly conserved among mammalian species: Q167 is replaced by Glu in the human sequence while Q218 is replaced by Glu in primate sequences.

Structural changes of PrP caused by mutations and pH change

The chemical shift perturbations caused at pH 5.5 by the P101L, Q167R, and Q218K mutations are small and are strictly localized to the mutation site and immediate neighbor residues ( Fig. 2A ), consistent with the positions of these mutation sites in solvent-exposed areas of the protein that are low in secondary and tertiary structure (5,6). By contrast, the H186R mutation causes large chemical shift changes for the linker between 㬑 and 㬢 (Y156, Q159, Y162), 㬒 (Q185, T191, G194) and 㬓 (F197, E206), and minor chemical shift changes around 㬡. The relative magnitude of these changes is consistent with the position of residue 186 in the middle of 㬒, and the packing of the side chain into the core of the molecule. Nevertheless, even for H186R, the majority of the chemical shifts were virtually unaffected by the mutations, indicating that the overall structure of mouse PrP was not substantially altered in any of the mutations. Variations in the chemical shift upon changing conditions such as pH (3.5 − 5.5), KCl (0 − 150mM), urea (0 − 1.5M) and temperature (25 − 40ଌ) showed that structural perturbations were most significant when the pH was changed (Supplementary Fig. S2). Chemical shift differences between mutant and wild type proteins at pH 3.5 ( Fig. 2B ) showed similar trends as those at pH 5.5 Since the chemical shift differences between mutants and wild type at pH 5.5 and pH 3.5 were similar, we inferred that all of the proteins were affected by acidic pH in similar ways.

Perturbations caused by each mutation on the chemical shifts of 15 N and 1 H. Each panel shows the absolute value of an average chemical shift difference (Δδ) calculated by averaging amide 15 N and 1 H chemical shift differences using the empirical equation, Δδavg = [Δδ( 1 H) 2 +Δδ( 15 N) 2 ] ½ , plotted as a function of primary sequence, and includes bars representing the positions of the α-helices (red) and β-strands (blue) in the 3D structure of the wild type protein. A. pH 5.5 B. pH 3.5. Δδ data are not available for residues 168�, for which no backbone resonances are observed (5,26).

Since acidic pH increases the probability of conversion to the pathogenic form (10-15), we anticipated that the inherited pathogenic PrP mutants might display different structural perturbations from the dominant negative PrP mutant or wild type proteins upon acidification. However, the chemical shift differences between pH 5.5 and pH 3.5 were the same for wild type, P101L, Q167R and Q218K, all of which showed similar and large chemical shift changes for residues from K184 to T198 ( Fig. 3 ). This region encompasses the C-terminal half of 㬒 and the connecting loop between helices 㬒 and 㬓. Among the affected residues, H186, T191, and K193 undergo the largest chemical shifts changes, suggesting that the C-terminal part of 㬒 plays a dominant role in the structural changes that occur at acidic pH. This region also has the largest chemical shift differences between pH 7.0 and pH 4.5 in human PrP(121�) (44). In sharp contrast, the H186R mutant shows only small chemical shift differences for helix 㬒 ( Fig. 3 ), implying that this mutant does not undergo pH dependent structural perturbations. These observations identify histidine 186 as responsible for the pH dependent changes in the NMR spectra of the wild type, P101L, Q167R and Q218K mutant proteins. In addition, wild type and all mutants share significant pH-dependent chemical shift changes for residues 141� and 156� at the N- and C-termini of the 㬒 and 㬢 and residues 205� in 㬓 ( Fig. 3 ), implicating a second titratable group.

Perturbations caused by a change of pH from 5.5 to 3.5 for the wild type and each of the mutant proteins. Δδavg = [Δδ( 1 H) 2 +Δδ( 15 N) 2 ] ½ .

A further decrease of pH to ≈ 2.1 resulted in severe line broadening of all resonances except for the N-terminal unfolded region (data not shown) this behavior was similar to that of the β-oligomer form of human PrP under moderately denaturating conditions (1 M urea, 0.2 M NaCl, 20 mM sodium acetate pH 3.6) (45).

Backbone dynamics of wild type and mutant PrPs

15 N T1, 15 N T2, and [ 1 H]- 15 N NOE data sets were acquired at 298 K in 20mM sodium acetate (pH 5.5 and pH 3.5) (Supplementary Fig. S3). Due to the presence of ≈ 35 unstructured residues at the N-terminus in PrP(89�), which affects the tumbling of the folded domain, the rotational diffusion tensor could not be derived directly from the structure, but was obtained from fitting the relaxation data sets to the wild type mouse PrP(121�) structure (PDB id: 1xyx) (26) assuming that the overall structures of wild type and mutant PrPs are not substantially different. Rotational correlation times calculated from the relaxation data were 9.8�.9 ns, much larger than those expected from the empirical Stokes-Einstein estimation (≈ 8.4 ns) (46), and reflect the influence of the N-terminal unfolded region on the rotational tumbling of the C-terminal folded domain (38). The molecular tumbling of wild type and mutant PrPs is slightly anisotropic (DD ≈ 1.4𢄡.7) so that axially symmetric diffusion models fit the experimental data much better than an isotropic diffusion model (Supplementary Table S1). In the fitting, the axis of the longest helix 㬓 coincides with the major principal axis of the rotational diffusion tensor. The dominant effect of 㬓 on the anisotropy of mouse PrP(89�) is consistent with previous observations on the Syrian hamster proteins PrP(23� and 90�) (47). Using fitted rotational diffusion tensors, the backbone dynamics of each residue were determined by model free analysis (35-37). Previous attempts at model free analysis of the Syrian hamster PrP (23� and 90�) resulted in invalid order parameters (S 2 > 1) for many residues (25,47). The current analysis uses a non-isotropic rotational diffusion tensor (48) and a Bayesian information criterion (39,40) for model selection in conjunction with elimination of unrealistic models, which gives physically meaningful S 2 values for all of the fitted residues in wild type and mutants. Notably, a recent model free analysis of a truncated form of the mouse PrP(113�) using the isotropic rotational diffusion tensor has also resulted in valid S 2 values (49).

The motions of the N-terminal unfolded residues were analyzed with a local rotational diffusion model since their rotational tumbling should be independent of the C-terminal folded domain of the protein. The S 2 values in this region are ≈ 0.4, consistent with the highly flexible nature of the N-terminal terminal region (6). However, for all PrPs, there is a cluster of residues around H95 for which S 2 (≈ 0.6𢄠.8) ranges above the rest of the N-terminal unfolded region ( Fig. 4 ).

Order parameters (S 2 ) at pH 5.5 (green) and pH 3.5 (red) for wild type and mutant proteins, calculated from model free analysis of 500 and 600 MHz 15 N T1, 15 N T2, and [ 1 H]- 15 N NOE data sets measured at 298 K (data shown in Supplementary Fig. S3). For the N-terminal unfolded region (89�), a local rotational diffusion model was used. For the folded region (127�), a global axially symmetric rotational diffusion model was optimized using two-field relaxation data sets and the mouse PrP structure (PDB ID: 1xyx) (26). S 2 values are not available for residues 168� for which no backbone resonances are observed (5,26).

At pH 5.5 (green bars in Fig. 4 ), the C-terminal folded region of all the proteins have an S 2 larger than 0.85 indicative of restricted backbone motion. However, two broad regions from 㬡 to 㬑 (residues ≈ 134�) and from the C-terminal half of the 㬒 to the beginning of 㬓 (residues ��) have lower S 2 values, indicative of backbone flexibility. All of the proteins, both wild type and mutants, share a similar S 2 pattern except that a short segment in the H186R mutant following residue 186 (≈ 187�) shows a sharp decrease in S 2 while in the other proteins, the decrease was more gradual ( Fig. 4 ). At pH 3.5 (red bars in Fig. 4 ), all proteins except H186R showed a substantial decrease in S 2 in the same region (residues 187�) while the rest of the protein was essentially unaffected by a decrease of pH. S 2 for the H186R mutant was similar to the values observed for the N-terminal disordered tail. The variations in S 2 between pH 5.5 and 3.5 for the wild type PrP are shown in Fig. 5A, B and compared with those for the H186R mutant in Fig. 5C . These observations suggest that residues 187� are disordered in the H186R mutant protein even at neutral pH.

S 2 values are shown mapped on the mouse PrP structure (PDB ID: 1xyx) (26) as a continuous color scale: red for S 2 < 0.6, red to yellow for 0.6 ≤ S 2 < 0.8, and yellow to blue for 0.8 ≤ S 2 < 1.0. Prolines and residues for which S 2 is not determined due to spectral overlap, absence of data or failure in fitting are shown in gray. A. Wild type PrP at pH 5.5. B. Wild type PrP at pH 3.5. C. H186R mutant PrP at pH 5.5. The side chain of H186 is shown in green.

Internal motions on the ns time scale appear in both of the flexible segments (residues ≈ 134� and ≈ 187�) ( Fig. 6 ). The ns internal motions in the first segment propagate toward the N-terminus as far as A116. Reduced spectral densities J(ωN) and J(0.89ωH) could be used without the necessity for the assumptions made in the model free analysis, and provide further evidence for significant flexibility in these regions (Supplementary Fig. S4). The time scales of the internal motions are virtually unaffected by pH except for wild type and P101L. It is intriguing that only the wild type and P101L proteins have extensive ns internal motions throughout helices 㬑-㬓 at pH 5.5 ( Fig. 6 ), even though high S 2 values indicate restricted backbone motion on the ps-ns time scale. Many of these ns internal motions disappear at pH 3.5 ( Fig. 6 ). The combination of low amplitudes of ps-ns backbone motion with extensive ns internal motion in the same region has been reported in a number of cases (50). Partial aggregation of the wild type and P101L mutant proteins could in principle be responsible for these apparently anomalous internal motions (51). However, on the basis of translational diffusion measurements, we could exclude this possibility: at the same concentration as the NMR relaxation measurements (0.55 mM), wild type, P101L and Q218K have the same translational diffusion coefficients within experimental error (1.06 ± 0.01, 1.08 ± 0.01, and 1.08 ± 0.02 × 10 𢄦 cm 2 s 𢄡 , respectively, at 298 K).

Correlation time of internal motion (τe) at pH 5.5 (green) and pH 3.5 (red). τe was calculated from model free analysis of 500 and 600 MHz 15 N T1, 15 N T2, [ 1 H]- 15 N NOE data sets measured at 298 K. τe data are not available for residues 168� for which no backbone resonances are observed (5,26).

All wild type and mutant proteins show sharp increases in the R2 relaxation rate near 㬡 and 㬢, at G130, V165, and D166 (Supplementary Fig. S3) such an increase is usually interpreted as evidence for conformational exchange on a μs-ms time scale. In order to separate the exchange contribution from R2, exchange-free R2 (R20) was determined from transverse cross correlation (ηxy) rates (52). Our findings show that these sites indeed undergo significant conformational exchange ( Fig. 7A ). The Carr-Purcell-Meiboom-Gill (CPMG) 15 N R2 relaxation dispersion data of G130, V165 and D166 show distinct differences between R2 relaxation rates at 500 and 800 MHz even though each has small R2 dispersion (< 5 s 𢄡 ) ( Fig. 7B ). The exchange rate and the populations of the states were estimated from simultaneous fitting of the 15 N R2 dispersion data of G130, V165 and D166 using the measured R20 and a two-site exchange model. These residues undergo fast exchange (7000 ± 2000 s 𢄡 ), where the population of the less favorable conformation is ≈ 0.4%. The fast conformational exchange of G130, V165 and D166 might be related to an intermediate time scale (μs to ms) conformational fluctuation in the loop connecting 㬢 and 㬒 (residues 168�) which is most likely responsible for the severe line broadening of backbone resonances of these residues in wild type and all four mutant PrPs (5,26). The loop between residues 170� has been implicated in disease (53,54) overexpression of PrP(170N, 174T) causes spongiform encephalopathy disease in the mouse (55). Interestingly, this loop region of human, cow, mouse, dog, and cat PrP is flexible, whereas that of elk, Syrian hamster and bank vole is rigid (6,26,54,56-58), providing insights into species barriers for prion disease.

Conformational exchange. A. Contribution of conformational exchange to R2 relaxation. The transverse cross-correlation rate, ηxy, between the 15 N- 1 H dipole-dipole interaction and the 15 N chemical shift anisotropy (CSA) was measured at 500 MHz for wild type (black) and H186R (red) PrP(89�) in pH 5.5 at 298 K. Exchange-free R2 (R20) was calculated by R20 = -ηxy𢆣[(4c 2 +3d 2 )]/[12cdP2(cos(β))] (52) in which c = (ωN/𢆣)Δσ, d = [μ0hγNγH/(8π 2 )](1/r 3 NH), P2(x) = (3x 2 -1)/2 is the second-rank Legendre polynomial, h is Planck's constant, μ0is the permeability of free space, Δσ is the 15 N CSA, rNH is the amide NH bond length, and β is the angle between the principal axis of the 15 N CSA tensor and the amide NH bond vector. 15 N CSA, rNH and β were assumed to be � ppm, 1.02 Å and 20°, respectively. B. Carr-Purcell-Meiboom-Gill (CPMG) based 15 N R2 relaxation dispersion of wild type PrP(89�) at pH 5.5 and 298 K. 15 N R2 relaxation dispersion data of G130, V165 and D166 at 500 and 800 MHz were simultaneously fitted to the general equation for two-site exchange (kex=kA𡤫+kB𡤪, pA(=1- pB), pB, Δω) (42) using the measured R20. Data (filled circle) and fitted curves (solid line) at 500 MHz (black) and 800 MHz (red) are shown for G130, V165 and D166.

Unlike wild type and other mutants, the H186R mutant has two sets of resonances at residues Y162 and R163 in 㬢 at pH 5.5, with one set having chemical shifts similar to those of the other mutants and wild type proteins (Supplementary Fig. S1). M128, L129 and G130 in 㬡 do not exhibit this slow conformational exchange although they have slightly broader resonances than other residues. We attribute these observations to a slow exchange between two conformations (the rate of exchange is too slow to be detected in the 15 N longitudinal 2-spin-order exchange (zz-exchange) experiments data not shown). In the conformation with chemical shifts different from wild type and other mutants Y162 shows significantly more flexibility:([ 1 H]- 15 N NOEs are 0.19 ± 0.02 and 0.29 ± 0.04 at 500 and 600 MHz, respectively (Fig. S3G) and S 2 is 0.34 ± 0.01) than the conformation with chemical shifts similar to wild type and the other mutants: ([ 1 H]- 15 N NOEs are 0.72 ± 0.09 and 0.61 ± 0.06 at 500 and 600 MHz, respectively and S 2 is 0.92 ± 0.01). The H186R mutation appears to destabilize the β2 region, resulting in an equilibrium between ordered and disordered states. Interestingly, the Y162 peak originating from the more rigid conformation disappears at pH 3.5, while the other Y162 peak originating from the more flexible conformation remains (S 2 = 0.30 ± 0.01), suggesting that the conformational equilibrium in the 㬢 region of H186R shifts toward the disordered state at lower pH.

Protonation of H186

Since our results indicated that H186 is involved in the destabilization of PrP that occurs as the pH is lowered, the protonation states of H186 were examined at pH 5.5 and 3.5 by 15 N㭁 and 15 N㭒 chemical shifts (assignments for the side chain resonances of the 5 histidines are shown in Supplementary Fig. S5). The resonance frequencies of 15 N㭁 and 15 N㭒 are at ≈ 168 and ≈ 250 ppm respectively in a neutral imidazole and they both resonate at ≈ 177 ppm when completely protonated (29). Intermediate chemical shift values are indicative of fast exchange between protonated and deprotonated states. Among the five histidines in wild type mouse PrP(89�), H95, H110, H139 and H176 are exposed on the surface and H186 is partially buried and surrounded by hydrophobic residues (5). The 15 N㭁 and 15 N㭒 chemical shifts indicate that H110 and H176 are protonated at pH 5.5 H95, H139 and H186 are partially deprotonated at pH 5.5 but become protonated at pH 3.5 (Supplementary Table S2 and Fig. 8 ), indicating that the pKas of H95, H139 and H186 imidazole side chain are substantially lower than the typical pKa value (6.6 ± 1) significantly lowered pKa values are frequently observed for histidines located in the interior of proteins (59-61). Theoretical pKa calculations also estimate a consistently low pKa for H186 (62), supporting the notion that the low pKa of H186 is due to its partially buried environment. In addition, the broad lines of the H186 imidazole resonances in the HMQC spectrum at both pH 5.5 and pH 3.5 ( Fig. 8 ) suggest the presence of chemical exchange on a μs-ms time scale.

Long-range 1 H- 15 N HMQC spectra of the histidine residues of wild type PrP(89�) at pH 5.5 (black) and pH 3.5 (red). Protonation states of histidines of wild type mouse PrP(89�) in pH 5.5 and 3.5 at 292 K are inferred from 15 N㭁 and 15 N㭒 chemical shifts. The positions of the cross peaks expected between the histidine ring nuclei when the ring is fully protonated are mapped in the top left corner. The 15 N㭒 resonance of H186 was not observed at either pH. Assignments of the five histidines are outlined in red (H95), orange (H110), green (H139), blue (H176) and black (H186).

It is surprising that H95 and H139 display pKas as low as H186, despite their apparent surface exposure. There do not appear to be substantial chemical shift differences in the vicinity of these residues upon pH change, in contrast to H186 ( Fig. 3 ). The low pKas of the H95 and H139 imidazoles may be due to dynamic effects such as transient hydrophobic or electrostatic interactions.

First all-human mouse model of inherited prion disease

Human prion diseases include Creutzfeldt-Jakob disease (CJD) and Gerstmann-Sträussler-Scheinker disease (GSS). A new study in the open-access journal PLOS Biology reports a significant advance in the development of mouse models of human prion diseases. The study, by Emmanuel Asante and colleagues of the Medical Research Council Prion Unit at University College London, demonstrates spontaneous formation of disease-relevant, transmissible prion protein assemblies in mice bearing only human forms of the prion protein.

Prion diseases are due to the misfolding and cell-to-cell transmission of prion proteins, which go on to induce misfolding in the recipient cell. A significant feature of prion diseases is that different mutations give rise to diseases with strikingly different clinical manifestations. In studying these diseases, the faithful creation and propagation of distinct disease-specific strains has been essential to understanding transmission and pathogenesis.

The prion diseases have largely been modeled in mice by introducing the gene for a prion protein bearing a disease-causing mutation. In previous studies the disease-causing mutations were not studied directly on the human prion protein gene, but instead the equivalent mutations were introduced into the mouse prion protein gene. This complication can cause formation and propagation of a strain of misfolded protein that is not found in human disease, thereby limiting our understanding of the human prion disease.

To overcome this problem, the research team introduced a mutant human prion gene into mice carrying no mouse prion gene. As the mice aged over a year and a half, they spontaneously developed clusters of misfolded prion protein, something never observed before. When those clusters were used to inoculate younger mice carrying the same mutation, those mice developed misfolded prion protein clusters as well, directly demonstrating infectivity of the mutant protein, and mimicking the infectivity of patient-derived clusters of the same mutant protein. This is the first time that a spontaneous infection due entirely to mutant human prion protein has been shown in mice.

"This new model of an inherited prion disease is likely to provide important insights into human disease that we have previously been unable to study in the mouse," Dr Asante said, including events of disease initiation and spread that may inform development of therapies.

How do PrP mutations lead to prion disease? - Biology

Healthy proteins misfold into potentially deadly prions

Prion diseases eat away at the brain. At top, a normal neuron, and below, an infected neuron with an accumulation of the abnormal, scrapie form of the prion protein. Scrapie is a fatal disease affecting sheep.

Humans get prion disorders from inherited mutations, through contami-nation during a medical procedure or, in very rare instances, from consumption of infected animals.

Like any genuinely new area of research, prions seem to have a habit of throwing unexpected surprises at scientists.

Connecting prion disease to other more common disorders, such as Alzheimer’s
or other neuro-degenerative disorders, may expand the number of investigators interested in conducting prion research.

FOR RESEARCHERS LIKE David A. Harris, MD, PhD, the long, slow exit from the twilight zone is all but over. Harris has been studying prions, a new kind of infectious agent thought to be at the heart of several rare neurodegenerative disorders that devastate the brains of humans, cows and sheep.

Prions are weird — unlike any other infectious agent ever identified before. Harris, professor of cell biology and physiology, remembers a time when describing his research sometimes gave him the impression other scientists thought he had “gone to outer space” or was working on “black magic.”

Seven years after the Nobel Prize went to a prion researcher, Harris admits that an ironclad proof of prion theory has yet to be produced. But the skeptics are finding it harder and harder to make their case, and Harris now has a colleague in prion research in his own department, Heather L. True-Krob, PhD, assistant professor of cell biology and physiology.

Harris and True-Krob are gathering new insights into how prions form and cause disease, and as they do, tantalizing hints are starting to emerge that prions may be connected to a much wider range of biological phenomena than the rare brain disorders that first led to their discovery.

Heather L. True-Krob, PhD, and David A. Harris, MD, PhD

Until prions came along, infectious agents always contained some type of genetic material. That material carried the linchpin of the infection cycle: instructions for hijacking host cells to produce new copies of the infectious agent and begin the cycle anew.

Not so the prion — it consists entirely of a misfolded protein. The prion perpetuates itself by influencing nearby normal copies of the same protein, somehow increasing the chances they will misfold and become prions. In cows with mad cow disease, sheep with scrapie, and humans with Creutzfeldt-Jakob disease, this causes a chain reaction that leaves the brain a spongy, hole-filled mess.

Humans get prion disorders from inherited mutations, through contamination during a medical procedure or, in very rare instances, from consumption of infected animals. In addition, some “spontaneous” cases of human prion disease currently can’t be tracked to any genetic or environmental cause. The disorders have no treatment and are fatal in months to several years.

The first part of prion theory, the idea that a change in folding can radically change a protein’s properties, is well-established biological fact. Proteins are long, complex chains, and as those chains fold up, they form specialized structures that can perform various functions. Rearranging the way a protein folds can eliminate those structures, create new structures, or change their accessibility.

The process is roughly comparable to a Swiss Army knife: fold the protein in one configuration, and the can opener sticks out and can be used fold it into another, and the can opener vanishes, a screwdriver sticks out, and the protein has suddenly become a tool used for a very different purposes.

Much of the resistance among scientists to accepting prions springs from the second part of prion theory: the idea that interaction with a misfolded protein can cause another copy of the same protein to become badly folded. The details of how this unprecedented conversion takes place are still a mystery.

“ The problem is that no one knows the exact three-dimensional structure of the prion,” Harris explains. “We know the normal structure of the protein that becomes a prion, but not the structure of the prion itself, and that’s left the process by which prions spread a kind of black box.”

The normal function of the protein that becomes a prion also remains a mystery. Scientists named the protein PrP: The normally folded copy is referred to as PrPC (for cellular PrP), while the prion form is known as PrPSc (for scrapie PrP).

Recent evidence even has scientists questioning one of their most basic assumptions about prions: the idea that PrPSc is the form of the prion protein that kills brain cells. Studies by Harris have shown that transgenic mice with a mutant form of PrP prone to forming prions will get symptoms like those in human prion disorders, but the disease is not infectious to other animals.

“ In terms of the different forms of PrP, we have early evidence that what’s needed to kill a neuron may be different from what’s needed to pass on an infection,” Harris notes.

Like any genuinely new area of research, prions seem to have a habit of throwing unexpected surprises at scientists. One such surprise has actually boosted acceptance of prions among the research community: the identification of prions in yeast cells.

True-Krob specializes in the study of yeast prions, which don’t affect humans and other mammals but have similar structural elements. Yeast prions spread the same way as mammalian prions, with proximity to misfolded copies of the yeast prion protein, Sup35, somehow increasing the chances that normal copies of the same protein will become prions.

During her postdoctoral studies, True-Krob led a project that uncovered another major prion surprise: a positive role for yeast prions. Sup35 normally helps yeast read protein-building instructions from its DNA, a process called translation. True-Krob showed that the prion form of Sup35 disrupted this process. As a result, new material was added to proteins.

The switch to prion-prone Sup35, which occurs spontaneously about once in every million generations of yeast, often has harmful effects. But in about 20 percent of test cases, the disruptions gave the yeast a survival advantage in an environment in which temperature, the availability of food or other factors had changed.

“This system is advantageous for the yeast because they have a way of turning prions on and off,” True-Krob notes. “And that gives us hope that what we learn from yeast may help us find a way to turn prions off in humans.”

Working with prions in yeast lets True-Krob conduct studies that would be prohibitively complex or even impossible in mammalian cells. She can simultaneously expose many different yeast cell lines to a wide range of environmental conditions and genetic variables and see how these factors influence the likelihood that prions will form.

True-Krob is active in the search for additional yeast prions, which has netted a second yeast prion also linked to the translation of information in DNA.

“ People have speculated that there may be up to 100 different prions in yeast,” True-Krob says. “What we learn in yeast will help us search for prions in other systems.”

Harris, who studies mammalian prions, describes his lab’s interests as the molecular and cellular biology of the prion protein: What do both forms of PrP do in the nerve cell, where do they do it, and what do they interact with?

Harris conducts the bulk of his research in approximately 50 lines of mice genetically modified to produce prions and symptoms similar to human prion diseases. In recent years, they’ve produced important clues about what PrPC and PrPSc may be doing in the brain.

Work in mouse models has shown that PrP scrapie builds up in clumps in the brain similar to those seen in more common neurodegenerative disorders like Alzheimer’s disease, Parkinson’s disease, and Huntington’s disease.

Another similarity to these disorders emerged in a recent study, led by Harris, of a cellular suicide switch known as Bax. Harris had read about experiments from other researchers linking Bax to nerve cell suicide in other neurodegenerative disorders, so he decided to see what would happen in one of his mouse models if the Bax gene was knocked out.

As he had hoped, the alteration saved a class of mouse brain cells normally killed off in dramatic fashion in the mouse model of the prion disorder. But the mice still developed movement disorders and other symptoms that were characteristic of their condition when they had a functioning Bax gene.

Further investigation revealed extensive damage to the synapses, areas where branches of two brain cells come together to communicate.

“ This connects prion diseases to other more common disorders because it shows nerve cell death isn’t the only thing we have to worry about in these conditions,” Harris explains. “We have to be concerned about damage to the synapse too, and there’s increasing evidence that is the case in other disorders like Alzheimer’s disease.”

That may make a big difference for therapeutics currently in development, Harris notes.

“ Our results suggest that if we just prevent cell death without doing something to maintain the functionality of the synapse, patients may still get sick,” he says.

Although they work on very different aspects of prion research, True-Krob and Harris collaborate on projects, have a monthly joint lab meeting, and interact frequently.

Harris jokes that he and True-Krob make up “the largest center of prion research within 1,000 miles or so.” True-Krob notes when she was looking for her first faculty position, the possibility of coming to a department with another faculty member studying prions had “definite appeal.”

Their field may soon be getting much less lonely. Connections to more common neurodegenerative disorders are increasing, and other researchers (including True-Krob’s postdoctoral mentor) recently proposed that prions may help store memories in brain cells.

“ That theory’s got a long way to go,” True-Krob says, “but it’s indicative of a new willingness to think about the possibility that prions could have a beneficial role in other systems besides yeast. More and more people are becoming aware of the prion and considering it as a possible explanation for puzzling results.”

PrP Sc

The abnormal, disease-producing protein

  • is called PrP Sc (for scrapie)
  • has the same amino acid sequence as the normal protein that is, their primary structures are identical but
  • its secondary structure is dominated by beta conformation
  • is insoluble in all but the strongest solvents
  • is highly resistant to digestion by proteases
  • When PrP Sc comes in contact with PrP C , it converts the PrP C into more of itself (even in the test tube).
  • These molecules bind to each other forming aggregates.
  • It is not yet clear if these aggregates are themselves the cause of the cell damage or are simply a side effect of the underlying disease process.

Inherited Prion Diseases

Creutzfeldt-Jakob Disease (CJD)

10&ndash15% of the cases of CJD are inherited that is, the patient comes from a family in which the disease has appeared before. The disease is inherited as an autosomal dominant. The patients have inherited at least one copy of a mutated PRNP gene. Some of the most common mutations are:

  • a change in codon 200 converting glutamic acid (E) at that position to lysine (K) (thus designated "E200K")
  • a change from aspartic acid (D) at position 178 in the protein to asparagine (D178N) when it is accompanied by a polymorphism in both PRNP genes that encodes valine at position 129. When the polymorphism at codon 129 is Met on both genes, the D178N mutation produces Fatal Familial Insomnia instead.
  • a change from valine (V) at position at position 210 to isoleucine (V210I)

Extracts of autopsied brain tissue from these patients can transmit the disease to

  • apes (whose PRNP gene is probably almost identical to that of humans).
  • transgenic mice who have been given a Prnp gene that contains part of the human sequence.

These results lead to the important realization that prion diseases can only be transmitted to animals that already carry a PRNP gene with a sequence that is at least similar to the one that encoded the PrP Sc. In fact, knockout mice with no Prnp genes at all cannot be infected by PrP Sc .

Gerstmann-Sträussler-Scheinker disease (GSS)

This prion disease is caused by the inheritance of a PRNP gene with a mutations encoding most commonly

  • leucine instead of proline at position 102 (P102L) or
  • valine instead of alanine at position 117 (A117V)

Again, the disease is also strongly associated with homozygosity for a polymorphism at position 129 (both residues being methionine).

Brain extracts from patients with GSS can transmit the disease to

Transgenic mice expressing the P102L gene develop the disease spontaneously.

Fatal Familial Insomnia (FFI)

People with this rare disorder have inherited

  • a PRNP gene with asparagine instead of aspartic acid encoded at position 178 (D178N)
  • the susceptibility polymorphism of methionine at position 129 of the PRNP genes.

Extracts from autopsied brains of FFI victims can transmit the disease to transgenic mice.

Infectious Prion Diseases

Kuru was once found among the Fore tribe in Papua New Guinea whose rituals included eating the brain tissue of recently deceased members of the tribe. Since this practice was halted, the disease has disappeared. Before then, the disease was studied by transmitting it to chimpanzees using injections of autopsied brain tissue from human victims.


This disease of sheep (and goats) was the first TSE to be studied. It seems to be transmitted from animal to animal in feed contaminated with nerve tissue. It can also be transmitted by injection of brain tissue.

Bovine Spongiform Encephalopathy (BSE) or "Mad Cow Disease"

An epidemic of this disease began in Great Britain in 1985 and before it was controlled, some 800,000 cattle were sickened by it. Its origin appears to have been cattle feed that contained brain tissue from sheep infected with scrapie and had been treated in a new way that no longer destroyed the infectiousness of the scrapie prions.

The use of such food was banned in 1988 and after peaking in 1992, the epidemic declined quickly.

Creutzfeldt-Jakob Disease (CJD)

A number of humans have acquired CJD through accidental exposure to material contaminated with CJD prions.

  • Grafts of dura mater taken from patients with inherited CJD have transmitted the disease to 228 recipients.
  • Corneal transplants have also inadvertently transmitted CJD.
  • Instruments used in brain surgery on patients with CJD have transmitted the disease to other patients. Two years after their supposed sterilization, these instruments remained infectious.
  • 226 people have acquired CJD from injections of human growth hormone (HGH) or human gonadotropins prepared from pooled pituitary glands that inadvertently included glands taken from humans with CJD.

Now that both HGH and human gonadotropins are available through recombinant DNA technology, such disastrous accidents need never recur.

Variant Creutzfeldt-Jakob Disease (vCJD)

This human disorder appeared some years after the epidemic of BSE (Mad Cow Disease) swept through the cattle herds in Great Britain. Even though the cow and human PRNP genes differ at 30 codons, the sequence of their prions suggests that these patients (155 by 2005) acquired the disease from eating contaminated beef.

All the patients are homozygous for the susceptibility polymorphism of methionine at position 129. The BSE epidemic has waned, and slaughter techniques that allow cattle nervous tissue in beef for human consumption have been banned since 1989. Now we must wait to see whether more cases of vCJD are going to emerge or whether the danger is over.

Miscellaneous Infectious Prion Diseases

A number of TSEs have been found in other animals. Cats are susceptible to Feline Spongiform Encephalopathy (FSE). Mink are also susceptible to a TSE. Even though mad cow disease has not been seen in North America, a similar disease is found in elk and mule deer in the Rocky Mountains of the U.S.

Sporadic Prion Diseases

CJD and FFI occasionally occur in people who have no family history of the disease and no known exposure to infectious prions. The cause of their disease is uncertain.

  • Perhaps a spontaneous somatic mutation has occurred in one of the PRNP genes in a cell.
  • Perhaps their normal PrP C protein has spontaneously converted into the PrP Sc form.
  • Or perhaps the victims were simply unknowingly exposed to infectious prions, and sporadic prion diseases do not exist!

Whatever the answer, all the cases are found in people with a susceptibility polymorphism in their PRNP genes.

Prions in Yeast

Two proteins in yeast (Saccharomyces cerevisiae)

are able to form prions that is, they can exist either

  • in a PrP C-like form that is functional or
  • in a PrP Sc-like form that is not.

The greater ease with which yeast can be studied has proved that only protein is involved in prion formation and provided insight into the need for PrP Sc to find PrP C molecules of a similar primary structure in order to be able to convert them into the PrP Sc form.

Evidence that prions are a "protein-only" phenomenon

  • A few molecules of a PrP Sc form of the Sup35 protein, when introduced into yeast cells, convert the yeast cell's own Sup35 protein into prion aggregates. The resulting "disease" phenotype is then passed on to the cell's daughters.

The introduced protein was synthesized in bacteria making it unlikely that it could be contaminated by any gene-containing infectious agent of yeast.

Possible basis of species specificity of prions

  • A particular PrP Sc can only convert PrP C molecules of the same or at least similar primary structure.
  • This requirement of "like-with-like" resides in a short sequence at the N-terminal of the protein (rather like an antibody epitope).
  • Yeasts engineered to form two types of prion form two types of "pure" aggregates within the cell.
  • Even in the test tube, each type of prion finds and aggregates with others of its own type.

So the picture that emerges is that a molecule of PrP Sc acts as a "seed" providing a template for converting PrP C to more PrP Sc. These interact with each other to form small soluble aggregates. These interact with each other to form large insoluble deposits. Although only a small portion of the prion protein is responsible for its specificity, other parts of the molecule are needed for flipping the molecule from the alpha-helical to the beta conformation. All prion proteins contain tracts of repeated Gln-Asn residues which appear to be essential for the conversion process.

Other Pathogenic Prion-like Proteins

The deposits of PrP Scin the brain are called amyloid. Amyloid deposits are also found in other diseases.

  • Alzheimer's disease is characterized by amyloid deposits of
    • the peptide amyloid-beta (A&beta)
    • the protein tau

    With all of these diseases there is evidence that their amyloid-forming proteins, like PrP Sc , can act as a "seed" converting a correctly-folded protein into an incorrectly-folded one and have this effect spread from cell to cell. However, they do not seem to be able to be spread from person to person (unlike the TSEs). Perhaps this is because they are not so incredibly resistant to degradation as PrP Sc is.

    Most cells, including neurons in the brain, contain proteasomes that are responsible for degrading misfolded or aggregated proteins. In the various brain diseases characterized by a build-up of amyloid deposits, it appears that as the small insoluble amyloid precursors accumulate, they bind to proteasomes but cannot be degraded by them. Furthermore, this binding blocks the ability of the proteasomes to process other proteins that are normal candidates for destruction. Because of the critical role of proteasomes in many cell functions, such as mitosis, it is easy to see why this action leads to death of the cell.

    Prion-like proteins not always harmful

    CPEB ("cytoplasmic polyadenylation element binding protein") is a protein that

    • is found in neurons of the central nervous system (as well as elsewhere)
    • stimulates messenger RNA (mRNA) translation
    • is needed for long-term facilitation (LTF)
    • accumulates at activated (by serotonin) synapses
    • has the ability to undergo a change in tertiary structure that
      • persists for long periods
      • induces the same conformational change in other molecules of CPEB forming prion-like aggregates

      Perhaps the accumulation of these aggregates at a stimulated synapse causes a long-term change in its activity (memory).

      Transmembrane PrP

      Three topological forms of PrP

      Most PrP C molecules are attached to the outer leaflet of the plasma membrane through a C-terminal glycosyl-phosphatidylinositol anchor (this topology is designated Sec PrP see Fig. 3). However, when PrP is synthesised in vitro, in transfected cells or in mouse brain, some of the molecules assume a transmembrane orientation 59 –66 . These species, designated Ntm PrP and Ctm PrP, span the lipid bilayer once via a highly conserved hydrophobic region in the centre of the molecule (amino acids 111–134), with either the N-terminus or C-terminus, respectively, on the extracytoplasmic side of the membrane ( Fig. 3).

      Three topological forms of PrP.

      Three topological forms of PrP.

      Ntm PrP and Ctm PrP are generated in small amounts (< 10% of the total) as part of the normal biosynthesis of wild-type PrP in the endoplasmic reticulum. However, mutations within or near the transmembrane domain, including A117V and P105L mutations linked to GSS as well as several ‘artificial’ mutations not seen in human patients, increase the relative proportion of Ctm PrP to as much as 20–30% of the total 59 ,61 ,63 –65 . Current evidence 61 ,64 indicates that the membrane topology of PrP is determined by competition at the translocon between two conflicting topological determinants in the polypeptide chain: (i) the signal sequence (residues 1–22) that directs translocation of the N-terminus of the polypeptide chain across the membrane to produce Sec PrP or Ntm PrP and (ii) the central hydrophobic domain (residues 111–134) that acts as a type II signal-anchor sequence, directing translocation of the C-terminus across the membrane to produce Ctm PrP.

      A proposed pathogenic role of Ctm PrP

      It has been hypothesised that Ctm PrP is a key pathogenic intermediate in both familial and infectiously acquired prion diseases. One piece of evidence for a role in familial forms comes from transgenic mice that synthesise PrP molecules carrying the A117V mutation, or one of the other Ctm PrP-favouring mutations 59 ,63 . Animals expressing these mutant proteins above a threshold level synthesise Ctm PrP in their brains, and spontaneously develop a scrapie-like neurological illness but without PrP Sc detectable by Western blotting or infectivity assays. Evidence for a role of Ctm PrP in infectiously acquired prion diseases comes from mice in which a wild-type hamster PrP transgene serves as a reporter of Ctm PrP formation 63 . When these animals are inoculated with mouse prions, the amounts of Ctm PrP as well as PrP Sc in the brain are found to increase during the course of the infection. This result has been interpreted to indicate that PrP Sc induces formation of Ctm PrP, which is then the proximate cause of neurodegeneration. Thus, the amount of Ctm PrP can be increased either directly by mutations in the PrP molecule, or indirectly via formation of PrP Sc .

      The cell biology of Ctm PrP

      To investigate further the hypothesis that Ctm PrP plays an important role in the pathogenesis of prion diseases, it is necessary to characterise the cell biological properties of this form, since very little is known about its localisation, metabolism, or mode of synthesis and processing in cells. Part of the difficulty in addressing these issues has been that it was not possible to produce Ctm PrP in the absence of the other two topological variants ( Ntm PrP and Sec PrP). We have overcome this limitation by identifying mutations in PrP that cause the protein to be synthesised exclusively with the Ctm PrP topology.

      The starting point for these studies was our discovery of a novel structural feature of Ctm PrP that had not been previously appreciated – Ctm PrP has an uncleaved, N-terminal signal peptide 66 . This feature can be rationalised by the fact that the N-terminus of the polypeptide chain does not enter the ER lumen where signal peptidase is located. We reasoned that mutations in the signal peptide itself might influence the amount of Ctm PrP. Consistent with this idea, we found that the substitution of a charged residue for a hydrophobic residue within the signal sequence (L9R) markedly increased the proportion of Ctm PrP to ∼50% after in vitro translation. Combining this mutation with 3AV, a mutation within the transmembrane domain, to create L9R/3AV resulted in a protein that was synthesised exclusively as Ctm PrP, in both in vitro translation reactions and transfected cells 66 .

      The availability of L9R/3AV PrP provided us for the first time the ability to analyse the properties of Ctm PrP in a cellular context in the absence of the other two topological variants 66 . By labelling cells expressing L9R/3AV PrP with [ 3 H]-palmitate, we demonstrated that Ctm PrP contains a GPI anchor. This result implies that Ctm PrP has an unusual, dual mode of membrane attachment, including both a membrane-spanning domain and a C-terminal GPI anchor. We also found that L9R/3AV PrP (and hence Ctm PrP) is absent from the cell surface, and is completely retained in the ER when expressed in transfected cells. This observation suggests the hypothesis that Ctm PrP is toxic because it stimulates the activation of pro-apoptotic, ER stress-response pathways.

      Most pathogenic mutations do not alter the membrane topology of PrP

      Mutations associated with familial prion diseases are found throughout the length of the PrP sequence 32 . Although mutations in or around the central, hydrophobic region were known to increase the amount of Ctm PrP, the effect of mutations outside of this area had not been examined. Therefore, we carried out in vitro translations of PrP mRNA encoding disease-associated mutations that lie both N- and C-terminal to the central, hydrophobic segment 65 . We found that the proportion of Ctm PrP was not increased over wild-type levels by any of the mutations outside of the central, hydrophobic domain. These results argue against the idea that Ctm PrP is an obligate toxic intermediate in all forms of familial prion diseases.

      What does it mean to have a genetic prion disease?

      In the last two posts we introduced the concept of a prion and introduced the human prion diseases, better known as Creutzfeldt-Jakob disease (CJD), fatal familial insomnia (FFI), and Gertsmann-Straussler-Scheinker syndrome (GSS). Altogether these diseases are pretty rare, with an incidence of about 1 in 1 million people falling ill each year [Holman 2010]. The genetic forms of prion disease are even more rare, accounting for probably just 15% of all cases [Appleby & Lyketsos 2011].

      To understand how these diseases arise, we’ll need to go back to some biology basics. The DNA in the nucleus of your cells is made up of 2 copies each of chromosomes 1-22, plus 2 copies of X (if you’re female) or 1 X and 1 Y (if you’re male). On chromosome 20 there’s a gene called the pr io n p rotein gene, or PRNP. Since you have two copies of chromosome 20 you have two copies of PRNP. You got one of those two copies of PRNP from your mother, and the other one from your father.

      DNA contains instructions that have to be converted into RNA, and then the message in the RNA is translated into protein. So the gene PRNP in your DNA spells out instructions for making, ultimately, the prion protein (PrP). Everyone has PrP on the surface of their cells, and most of the time this is a perfectly good and healthy thing.

      Genetic mutations – think of these as typos in DNA – change the instructions and end up producing a slightly different protein. Most of the time that’s fine, but some mutations are really bad.

      There are two main ways that mutations can be bad. First, they can make a protein that doesn’t do its job correctly. This is called a loss-of-function mutation. The other option is that they can make a protein that does do something new that it shouldn’t be doing. This is called a gain-of-function mutation. Genetic prion diseases are caused by a gain-of-function mutation in PRNP. They produce a slightly altered version of prion protein (PrP). A person can be pretty much healthy and fine with this version of PrP for decades, but as people get older, this protein is likely to eventually misfold and form a prion which can then attack other PrP and spread across the brain.

      Prion protein mutations follow a dominant inheritance pattern. That just means that, unfortunately, it only takes one bad copy of the gene to cause the disease. If you have a parent who had a genetic prion disease, they probably had one bad copy of PRNP, and there’s a 50% chance you inherited that one, and a 50% chance that you inherited the normal, good copy of PRNP.

      You’ll also hear genetic prion diseases described as being autosomal. That just means that they’re not sex-linked – PRNP is on chromosome 20, not on a sex chromosome. Both sexes are affected equally by genetic prion disease.

      Another term you’ll hear tossed around is penetrance. Penetrance is the percentage of people with the bad copy of the gene who will get the disease. Most of the genetic prion diseases that we know of are completely penetrant in the sense that everyone who has the mutation will get the disease eventually, if they live long enough. Some of the genetic prion diseases tend to strike people in their 40s and 50s, and so (as far as we know) virtually everyone who has the mutation dies of it. Other genetic prion diseases tend to have an older age of onset, so that some percentage of people will end up dying of some other cause – heart attacks, cancer, and so on – before they ever have symptoms of prion disease.

      There are over 40 different mutations that can cause genetic prion disease [Beck 2010], and each mutation tends to have its own particular symptoms, disease course, age of onset, and so on [Kong 2003, Mastrianni 2010 (full text)]. In other words, the mutation defines the disease.

      Therefore it’s important to have a system for naming mutations. The system that we use involves a lot of seemingly random numbers and letters such as “E200K.” Let’s break down what this code means.

      Proteins are made of amino acids, and PrP in particular is made of 253 amino acids, which are referred to in order. So the 200 in E200K means there’s a mutation in amino acid #200. E is shorthand for the amino acid glutamate, which is the amino acid that should normally be at position #200. K is the shorthand for the amino acid lysine, which is the amino acid that appears at position #200 instead of glutamate. Putting it all together, “E200K” means lysine instead of glutamate at amino acid #200.

      Sometimes there are even more numbers and letters. For instance, fatal familial insomnia is caused by a mutation called D178N 129M. Here, D178N means asparagine (N) instead of aspartate (D) at amino acid #178, and 129M means … and then methionine (M) at amino acid #129. Fatal familial insomnia is unusual in that the D178N mutation causes the disease, but the exact type of disease is determined by which amino acid is found at position #129 [Goldfarb 1992].

      Still other cases of genetic prion disease are not caused by a change in just one amino acid. One part of PrP has a repeating stretch of the same 8 amino acids over and over again, and this is called the “octapeptide repeat”. Some genetic prion diseases are caused by having too many copies of this repeat, and these are named by the number of extra copies – for instance, 𔄞-OPRI” means 6 extra copies of the octapeptide repeat inserted.

      Genetic counselors give people with genetic mutations advice on what their mutation means. Often this might include some statement about the typical age of disease onset for a person’s mutation. Estimates like these can be useful, but it is worth taking them with a bit of caution. For most genetic prion disease mutations, the age of onset can vary by a few decades. The statistics are usually based on a review of patients’ cases that doctors have reported in medical journals [Kong 2003] and there are many possible biases here. Younger patients might be more likely to be reported, people who die of something else first are almost never reported, and the statistics often fail to take into account the people still alive with the mutation. Moreover, the predicted age of disease onset for any one person really depends on how old they are today – after all, a person who is 60 today can’t have an age of onset of 50, even if that is the average for their mutation. For all of these reasons, statistics like “average age of onset” should never be taken as a hard-and-fast prediction about one’s own life.

      Even more importantly, there are good reasons for optimism that things will change, and that the diseases that we consider fatal and untreatable today will eventually be things of the past. We at Prion Alliance are working hard for a future where prion diseases don’t kill people. And we’re not alone – there are lots of bright scientists working in the prion field, and potential treatments at a variety of stages in the research pipeline. We can’t say exactly when treatments will become available, but lots of excellent science is being done every day, and we think right now is a good time in history to be an optimist.

      Evolution without genes – prions can evolve and adapt too

      If you search for decent definitions of evolution, the chances are that you’ll see genes mentioned somewhere. The American Heritage Dictionary talks about natural selection acting on “genetic variation”, Wikipedia discusses “change in the genetic material of a population… through successive generations”, and TalkOrigins talks about changes that are inherited “via the genetic material”. But, as the Year of Darwin draws to a close, a new study suggests that all of these definitions are too narrow.

      Jiali Li from the Scripps Institute in Florida has found that prions – the infectious proteins behind mad cow disease, CJD and kuru – are capable of Darwinian evolution, all without a single strand of DNA or its sister molecule RNA.

      Prions are rogue version of a protein called PrP. Like all proteins, they are made up of chains of amino acids that fold into a complex three-dimensional structure. Prions are versions of PrP that have folded incorrectly and this misfolded form, called PrPSc, is social, evangelical and murderous. It converts normal prion proteins into a likeness of its abnormal self, and it rapidly gathers together in large clumps that damage and kill surrounding tissues.

      Li has found that variation can creep into populations of initially identical prions. Their amino acid sequence stays the same but their already abnormal structures become increasingly twisted. These “mutant” forms have varying degrees of success in different environments. Some do well in brain tissue others thrive in other types of cell. In each case, natural selection culls the least successful ones. The survivors pass on their structure to the “next generation”, by altering the folds of normal prion proteins.

      This process follows the principles of Darwinian evolution, the same principles that shape the genetic material of viruses, bacteria and other living things. In DNA, mutations manifest as changes in the bases that line the famous double helix. In prions, mutations are essentially different styles of molecular origami. In both cases, they are selectively inherited and they can lead to adaptations such as drug resistance. In prions, it happens in the absence of any genetic material.

      If prions can evolve, and if they can show the same sort of adaptive resistance as bacteria or fungi, does this mean that they are alive? Charles Weissman, who heads up Li’s lab, doesn’t think so on the grounds that prions are completely dependent on their hosts for reproduction. They need normal proteins that are encoded within the genome of their host to make more copies of themselves.. He says, “The remarkable finding that prions can mutate and adapt to their environment imbues them with a further attribute of living things, without however elevating them to the status of being ‘alive’.”

      There are many distinct strains of prion. Each is a version of PrPSc folded in a subtly different way, and new strains can arise out of the blue. Working out their exact structure has been difficult and they’re usually characterised by the symptoms and disease they cause, and how long it takes for these to become apparent.

      Li found that prions taken from brain tissue are different to those grown in cells cultured in a laboratory. The brain-adapted prions are capable of infected nerve tissue and they’re resistant to a drug called swainsonine (swa) that completely blocks the growth of other strains. The cell-adapted prions lack both these abilities but they’re better at growing in cell cultures.

      When Li transferred brain prions into cell cultures, she found that they gradually adapted to their new environment. By the 12th ‘generation’, they were indistinguishable from cell-adapted prions. They had lost their ability to infect nerve tissue in favour of the ability to grow faster in cultured cells. When Li returned these prions back to brain tissue, the brain-adapted forms once again rose to dominance.

      Li also found that prions are capable to evolving resistance to drugs. She treated the cell-prions with swa. At first, the drug blitzed the prion population, slashing the proportion of infected cells by five times from 35% to 7%. But the rogue proteins staged a resurgence, bouncing back to infect around 25% of the cells. After just two rounds of growth, prions from cells that were exposed to swa completely resisted the drug. If the drug was removed, they faded into the background once more as the non-resistant forms took over again.

      Further experiments showed that the resistant strains were already there in the population. But their slower growth rates mean that they’re typically in the minority, accounting for just 1 in 200 prions. When swa blasted the population, these resistant few rose to dominance. Li says that prion populations consist of a multitude of strains and substrains, all of which are different ways of folding the same sequence of amino acids. Evolutionary pressures from the environment determine which of these strains is in power.

      But mutants can arise out of the blue too. Even if a population consists entirely of the same strain (which you can set up through cloning), resistant or sensitive mutants develop spontaneously in a very short span of time. Prions, it seems, are very quick to adapt.

      The fact that prions can evolve drug resistance so quickly is important news for scientists trying to find new treatments for prion diseases, such as Creutzfeld-Jacob Disease (CJD) and bovine spongiform encephalitis (BSE). Rather than trying to target the abnormal proteins themselves, it might be better to reduce the levels of the production of the normal PrP in the first place. The former tactic could be easily thwarted by the rise of resistant strains, while the latter tactic denies natural selection of raw materials to work with.

      Reference: Li et al. 2009. Darwinian Evolution of Prions in Cell Culture. Science DOI: 10.1126/science.1183218