Review
Proteomics applications in prion biology and structure Expert Review of Proteomics Downloaded from informahealthcare.com by Nanyang Technological University on 04/26/15 For personal use only.
Expert Rev. Proteomics 12(2), 171–184 (2015)
Roger A Moore*, Robert Faris and Suzette A Priola Laboratory of Persistent Viral Diseases, Rocky Mountain Laboratories, NIH, NIAID, Hamilton, MT 59840, USA *Author for correspondence: [email protected]
Prion diseases are a heterogeneous class of fatal neurodegenerative disorders associated with misfolding of host cellular prion protein (PrPC) into a pathological isoform, termed PrPSc. Prion diseases affect various mammals, including humans, and effective treatments are not available. Prion diseases are distinguished from other protein misfolding disorders – such as Alzheimer’s or Parkinson’s disease – in that they are infectious. Prion diseases occur sporadically without any known exposure to infected material, and hereditary cases resulting from rare mutations in the prion protein have also been documented. The mechanistic underpinnings of prion and other neurodegenerative disorders remain poorly understood. Various proteomics techniques have been instrumental in early PrPSc detection, biomarker discovery, elucidation of PrPSc structure and mapping of biochemical pathways affected by pathogenesis. Moving forward, proteomics approaches will likely become more integrated into the clinical and research settings for the rapid diagnosis and characterization of prion pathogenesis. KEYWORDS: cerebrospinal fluid . Creutzfeld–Jakob disease . mass spectrometry . prion diagnostics . prion disease .
prion structure . proteomics
.
quaking-induced conversion
Prion diseases, also known as transmissible spongiform encephalopathies, are a group of rare yet universally fatal neurodegenerative diseases that affect humans and other mammals [1,2]. A critical event in prion pathogenesis involves the conversion of normal, proteasesensitive prion protein (PrPC) into an aggregated, pathological isoform, termed PrPSc, which is much higher in b-sheet structure than soluble PrPC. Normal PrPC is expressed most prominently as a glycophosphatidylinositol (GPI)-anchored protein on the cell surface. Although the specific function of PrPC is not yet fully characterized, reports substantiate a role for PrPC in a variety of cellular processes including synaptic transmission and cell signaling [3,4]. The neuropathology of prion diseases in humans and in animals is typically characterized by gliosis and gray matter vacuolation, with deposition of PrPSc in brain tissue as insoluble, partially protease-resistant non-amyloid aggregates high in b-sheet structure [5–7]. It was postulated almost 50 years ago that the infectious agent in these diseases might actually be a self-replicating protein aggregate [8]. Today most researchers believe this to be the case, given the apparent lack of any viral or bacterial component directing disease informahealthcare.com
10.1586/14789450.2015.1019481
transmission. In fact, there are now multiple reports describing the de novo formation of infectious prions from PrPC and a minimal set of molecular components lacking any infectivity [9–11]. The theory that PrPSc is the sole infectious component [12] of the prion poses a challenge to the phenomenon of prion strains [13], which are prion isolates causing distinct pathologies with consistent characteristics such as incubation period, region-specific PrPSc deposition and severity of spongiform degeneration. It is thought that the information responsible for unique strain properties and disease phenotypes is encoded exclusively by PrPSc conformation [7,14], although it has also been proposed that small, soluble b-sheetcontaining oligomers play an important role in prion pathogenesis [15,16]. Characterization of PrPSc structure is thus an important focus of prion research. Most of the documented prion disorders in humans are defined as sporadic Creutzfeld– Jakob disease (sCJD), which typically occurs at a rate of 1–2 individuals per million in a given population [17,18]. Inherited forms of prion disease such as Gerstmann–Stra¨ussler– Scheinker syndrome [19], fatal familial insomnia [20] and hereditary forms of CJD [21] are ISSN 1478-9450
171
2015 This work was authored as part of the Contributor’s official duties as an Employee of the United States Government and is therefore a work of the United States Government. In accordance with 17 USC. 105, no copyright protection is available for such works under US Law.
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Moore, Faris & Priola
also well known, although they occur much less frequently. Acquired forms of human prion disease have historically been caused by various types of exposure to prion-infected biological materials. The earliest recorded prion epidemic, resulting from ritualistic cannibalism involving brain material, occurred in the late 1950s among the Fore tribe in the Kuru region of Papua-New Guinea [22,23]. Later, in the 1990s, a new form of human prion disease resulting from exposure to cattle material tainted with bovine spongiform encephalopathy emerged. This new human prion phenotype, termed variant CJD, received worldwide attention and brought the prion phenomenon to the forefront of public awareness [24]. Although the incidence of acquired prion diseases are generally declining, cases such as those transmitted by blood transfusion [25] continue to occur. Iatrogenic prion infections from ingestion of cadaver-sourced human growth hormone [26] and tainted surgical procedures [27] are also still of concern. Prion diseases are universally fatal with no effective treatment options. There are still significant obstacles to overcome before a cure for prion disease is realized. Reliable antemortem detection of PrPSc and other biomarkers will be of major importance in the clinical setting. Identification of potential interacting partners of pathological PrPSc should also help to determine what role those proteins might play in pathogenesis. Further structural elucidation of PrPSc is critically important, given the increasing consensus among researchers that PrPSc conformation greatly influences prion pathogenesis. Finally, it is important to more clearly map the biochemical pathways affected by pathogenesis in order to gain a better mechanistic understanding of the disease and to facilitate the search for better treatments. Proteomics techniques and the use of mass spectrometry (MS) in particular will continue to play an important role in understanding all of these aspects of prion pathogenesis. Prion disease diagnostics
Diagnosis of prion disease has historically been difficult and time-consuming. The most definitive diagnosis of prions in humans and other non-laboratory mammals relies upon postmortem confirmation of spongiform degeneration by histological methods and/or detection of pathological PrPSc deposition using immunohistochemistry [28–30]. In many instances of human prions, a double evaluation by two highly qualified experts is required for a definitive diagnosis [31]. Formation of PrPSc is thought to be one of the earliest events in prion pathogenesis [12]. Mice infected with the mouse 22L prion strain die after approximately 150 days, yet PrPSc can readily be found by immunoblot within approximately 40 days postinfection [32]. Likewise, PrPSc has been detected by immunoblot in the thalamus of hamsters infected with the scrapie strain 263K at 2 weeks post-inoculation [33] and possibly as early as only 1 day post-infection [34]. If there are other critical biochemical events that precede PrPC misfolding, they have yet to be fully explained. Thus, PrPSc is a key indicator for prion disease. 172
Detection of PrPSc with in vitro conversion assays
Conversion of PrPC into PrPSc using in vitro conversion assays has recently enabled the highly sensitive detection of PrPSc from various tissues. The protein misfolding cyclic amplification (PMCA) assay is based upon the ability of PrPSc to seed the conversion of native PrPC into a proteinase K (PK)-resistant isoform [35]. Potentially infected samples are introduced into uninfected brain homogenates containing PrPC. If infectivity is present, new PrPSc formation is triggered and PrPSc is amplified during serial rounds of intermittent sonication using fresh PrPC-containing brain homogenate. The PMCA has been used to examine multiple aspects of prion disease, including de novo PrPSc formation [9,10]. A key adaption of the PMCA technique utilizes bacterially expressed recombinant (rPrP) as a substrate for conversion into fibrillar aggregates that only takes place in the presence of prion-infected tissue [36]. The PMCA reaction was soon modified to induce rPrP fibril formation by shaking, or quaking-induced conversion (QuIC) in lieu of sonication [37]. Both the original PMCA and the QuIC assays have rapidly evolved with multiple reports demonstrating PrPSc detection capabilities at concentrations of £1 attogram (10–18 g) with high specificity [38–41]. For example, a recent modification of the original QuIC assay, termed real-time or RT-QuIC, was used to detect PrPSc in nasal brushings from sCJD patients with 97% sensitivity and 100% specificity [38]. Detection of PrPSc by targeted MS
Targeted MS approaches aimed at the identification of PrP peptides have also been reported [34,42–44]. This technique detects precursor and fragment ion pairs from a complex proteome digest with extremely high selectivity and sensitivity, thus making it ideal for assay development. In 2007, Onisko et al. developed a selected/multiple reaction monitoring (SRM/ MRM) assay [42] using nanospray liquid chromatography with tandem MS (LC-MS/MS) for the detection of the tryptic peptide VVEQMCTTQYQK, spanning PrP residues 209-220, in attomole amounts (TABLE 1). Importantly, this methodology was used to quantitate PrPSc in the brains of terminally ill Syrian hamsters at 19 ± 2 mg PrP/g of brain; an amount consistent with the 21 mg PrP/g estimated by Diringer et al. [45] 10 years earlier. Detection of PrPSc was even reported after only 1 day post-inoculation in the brains of hamsters that had been injected with the prion strain 263K [34]. Interestingly, human PrP209-220 was also found in urine-derived chorionic gonadotropin preparations, although it is not clear whether these samples were infectious [46]. This approach was later extended to prion-infected mouse and sheep samples, showing that the method could be widely applicable to multiple species, all with low attomole limits of detection [43]. These MRM methods for PrPSc detection relied heavily upon the analysis of the tryptic PrP209-220 peptide VVEQMCTTQYQK, which contains chemically active cysteine (Cys) and methionine (Met) residues. In 2012, Sturm et al. reported another quantitative MRM assay [44] that focused on the peptide YRPVDQY, derived from a chymotrypsin digest of bacterially expressed PrP. The Expert Rev. Proteomics 12(2), (2015)
Proteomics in prion biology
Review
Table 1. Selected SRM/MRM transitions used for PrP identification and quantification. Peptide† 77
109
GQPHGGGWGQPHGGGGWGQGGSHSQWNKPSKPK
81
109
GGGWGQPHGGGGWGQGGSHSQWNKPSKPK
85
GQPHGGGGWGQGGSHSQWNKPSKPK109
89
109
GGGGWGQGGSHSQWNKPSKPK
94
109
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GQGGSHSQWNKPSKPK
96
109
GGSHSQWNKPSKPK
152
156
ENMYR
163
169
YRPVDQY
164
169
RPVDQY
186
194
QHTVTTTTK
195
204
GENFTETDIK
195
GENFTETDVK204
209
220
VVEQMCTTQYQK
209
220
VVEQM(SO)CTTQYQK
208
219
VVEQMCVTQYQK
208
219
VVEQM(SO)CVTQYQK
209
220
VVEQMCITQYQR
209
220
VVEQMCITQYER
209
220
VVEQM(SO)CITQYQR
209
220
VVEQMCITQYER
209
VVEQM(SO)CITQYER220
221
ESQAYYDGR229
221
228
ESQAYYQR
Species
Precursor ion (m/z)
Product ion (m/z)
Ref.
Sh
556.8 (+6)
630.6 (y31)
[47]
Sh
583.8 (+5)
640.2 (y25)
[47]
Sh
512.3 (+5)
593.8 (y23)
[47]
Sh
535.3 (+4)
575.3 y16)
[47]
Sh
431.7 (+4)
513.4 (y14)
[47]
Sh
385.4 (+4)
494.5 (y13)
[47]
Sh
357.0 (+2)
469.2 amu
[49]
Ha, Bo
470.7 (+2)
759.3 (b6)
[44]
Bo
389.2 (+2)
596.3 (b5)
[44]
Sh
339.6 (+3)
450.2 amu
[49]
Ha, Sh
577.3 (+2)
706.4 (y6)
[43]
Hu, Mo
570.3 (+2)
692.3 (y6)
[43]
Ha
757.8 (+2)
438.2 (y3)
[34,42]
Ha
765.8 (+2)
171.1 (a2)
[43]
Mo
756.8 (+2)
171.1 (a2)
[43]
Mo
764.8 (+2)
171.1 (a2)
[43]
Sh
778.1 (+2)
171.1 (a2)
[43]
Hu
778.4 (+2)
696.4 (y5)
[46]
Sh
786.1 (+2)
171.1 (a2)
[43]
Hu
778.6 (+2)
171.1 (a2)
[43]
Hu
786.6 (+2)
171.1 (a2)
[43]
Ha, Mo
544.7 (+2)
510.4 (y4)
[43]
Sh
522.8 (+2)
466.1
[49]
†
Residue numbers shown with the peptide sequence.
potential advantage of targeting this peptide was reportedly based upon its highly conserved nature across multiple species of interest to prion biologists and its lack of chemically active Cys and Met residues. One targeted MS approach toward strain differentiation based upon the analysis of PrPSc is termed N-terminal amino acid profiling (N-TAAP) [47–49]. Given that PrPSc samples from different prion strains can be variably cleaved at N-terminal PK cleavage sites, the identities of the resulting N-terminal tryptic peptides are also variable (TABLE 1). MS has the capacity to differentiate single amino acid differences at the PK cleavage sites. In the N-TAAP method, quantification of selected N-terminal tryptic peptides by an SRM/MRM approach was used to differentiate various prion strains [47,48]. It was found that ratios of certain peptides within and between samples often varied based upon the strain. For example, the PrP peptide spanning residues 96-109 (GGSHSQWNKPSKPK) was reproducibly more abundant in sheep-passaged bovine informahealthcare.com
spongiform encephalopathy than it was in a classical sheep scrapie strain [47]. The N-TAAP approach should eventually prove to be highly useful in the analysis of various sCJD types, which are also known to contain a similar array of N-terminal protease cleavage sites [50,51]. Most of the targeted PrP MS reported to date has been typically utilized with either bacterially expressed rPrP or PrPScderived post-mortem. In order to take a more prominent role in prion diagnostics, MS-based methods need to be utilized with samples that have already been used successfully antemortem with in vitro assays such as cerebrospinal fluid (CSF) [52–54], blood [55], urine [39,56,57] and, most recently, nasal brushings [38] (FIGURE 1). However, a key obstacle for the development of MS-based techniques for PrPSc detection is the need to first remove protease-sensitive PrPC from a sample, given that the proteotypic peptide sequences are the same in the two isoforms. Thus, current SRM/MRM methods are tethered to time-consuming and potentially yield-reducing treatment with 173
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Moore, Faris & Priola
14-3-3 protein for the detection of prion disease in humans is controversial [64], with specificity results as low as 28% B) PrPSc, apoE, reported in a cohort of 420 confirmed Nasal brushings multiple proteins CJD patients [65]. Furthermore, 14-3-3 A) RT-QulC Cerebral spinal fluid protein concentrations are not always B) PrPSc consistent between major sCJD types nor A) RT-QulC, PMCA among the many known subtypes [66]. B) PrPSc, tau, p-tau, 14-3-3, neuron Consequently, clinicians have recomspecific enolase, mended a combination of more than one multiple proteins CSF marker to improve the accuracy of diagnostic testing for sCJD against other Blood serum and plasma neurological conditions [67]. For example, the protein desmoplakin is often elevated A) RT-QulC, PMCA in sCJD patients and has been proposed B) PrPSc, apoE, clusterin, complements, multiple as a supportive CSF marker to help proteins decrease the rate of 14-3-3 false-positive cases in sCJD diagnosis [68]. Elevated total tau protein (t-tau) concentrations with the presence of 14-3-3 protein in Urine A) RT-QulC, PMCA, the CSF has been reported to yield 84% SRM/MRM sensitivity and 96% specificity for sCJD B) PrPSc, α1-antichymotrypsin, patients [67]. The p-tau/t-tau (phosphorymultiple proteins lated/total) ratio, which is much lower in sCJD patients than it is in Alzheimer’s Sc patients, may help to discriminate sCJD Figure 1. Comparison of in vitro and SRM/MRM detection of PrP and the from other neurodegenerative dissource of other major prion disease biomarkers. (A) Major techniques and assays most commonly used for detection and (B) Selected proteins which have diagnostic eases [69,70]. The presence of other neurovalue are identified from various tissues and body fluids. logical disease-associated biomarkers, such as neuron-specific enolase [67] and S100B PK. Circumvention of this bottleneck could facilitate even protein [71], have also provided diagnostic value with prion dismore sensitive, selective and quantitative detection of PrPSc. ease. Thus, the simultaneous detection of multiple prion biomarkers [72] would greatly enhance the accuracy of prion Prion biomarkers used in the clinical setting disease diagnosis. The technological platform of choice to While PrPSc remains a primary target for early prion detection, quantify hundreds of possible prion biomarkers at once with other biomarkers could also play key roles in diagnosis, disease extremely high specificity will likely be the SRM/MRM monitoring and characterization of different prion phenotypes. techniques. A biomarker is generally defined as some measurable indicator of a particular disease state. The number of prion-associated Protein profiling of PrPSc mixtures biomarkers reported in genomic and proteomic studies is One strategy employed to learn more about prion infectivity impressive [53,58,59]. Many of these putative biomarkers, such as has been to inventory the molecular species that co-purify with clusterin or transthyretin, have limited usefulness because they PrPSc mixtures. If PrPSc could be purified while retaining a are so ubiquitous in multiple disease processes including those high infectivity titer, then those components removed during not related to prions. Consequently, the vast majority of the enrichment process are not needed for prion transmission. reported prion biomarkers [58,59] remain in the academic realm The simplest possible mixture that nonetheless contained all and have not always been practical in the clinical setting. elements required for infectivity might be more amenable to The first major protein biomarkers to be used effectively in mechanistic analysis than a highly complex mixture like brain the diagnosis of human prion diseases were the 14-3-3 proteins, homogenate. Unfortunately, infectious PrPSc has never been a ubiquitous family of neuronal proteins often found elevated purified to complete homogeneity, thereby making the minimal in the CSF of patients with neurological disease [53,60,61]. Typi- component analysis strategy more difficult. Over 30 years of cally, the b-isoform of 14-3-3 is detected by immunoblot [62] effort have been devoted toward developing methods of PrPSc and has been found to be more useful for detecting sporadic enrichment [45,73–81]. Despite this effort, enriched PrPSc is typiand familial forms of CJD than it has been for Gerstmann– cally heterogeneous, containing nucleic acids [45,82], lipids [83], Stra¨ussler–Scheinker and fatal familial insomnia [63]. Despite its polysaccharides [84] and probably metal ions [85,86]. These PrPSc widespread use in the clinical setting, the efficacy of preparations are known to be highly infectious [15,45,79,81].
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Brain A) RT-QulC, PMCA, SRM/MRM
174
Expert Rev. Proteomics 12(2), (2015)
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Proteomics in prion biology
Therefore, any non-PrP molecular species potentially required as co-factors for prion transmission must be present in the enriched preparations. Recent MS-based protein profiling studies [87–91] have identified non-PrP proteins that copurify with prion infectivity and therefore might be important in prion pathogenesis. Consistent with early observations [45], more recent studies [87–89] have found that ferritin co-purifies with PrPSc. However, ferritin was also found in control preparations of non-infected mice. This suggests that it is the oligomeric nature of ferritin, rather than any association with PrPSc, which determines its presence during the enrichment process [88]. Moreover, it has been found that at least 30% of the proteins identified with PrPSc could also be found in uninfected preparations, suggesting that these proteins might have been artifacts of the isolation procedure used. The majority of such proteins, like ferritin, collagen, actin and proteasomal subunits, are oligomeric or polymeric, while other proteins such as calcium/calmodulin-dependent protein kinase are highly abundant in brain tissue [92]. The tendency to aggregate and the high abundance of these proteins may explain their presence in mock PrPSc preparations. Proteomicsbased approaches such as these demonstrate that significant levels of non-PrP proteins are present even in some of the best PrPSc preparations. Moving forward, the existing PrPSc enrichment procedures that are largely based upon repeated centrifugation could be modified by the incorporation of a mechanistically distinct step, such as phosphotungstic acid precipitation [93], in order to remove another subset of impurities from the PrPSc mixture. This strategy may result in more homogeneous samples for use in studies such as PrPSc-labeling and structure analysis. A comparison of three recent protein profiling studies in which various proteins that co-purified with PrPSc were characterized reveals a remarkable diversity in the identity of the proteins identified. After proteins found in non-infected preparations [88] were removed from consideration, approximately 40 reported proteins could be compared between studies. Even PrPSc from the same 22L mouse prion strain purified 3 years apart differed significantly in non-PrP protein composition [88]. However, differences in PrPSc protein profiles between the mouse prion strains 22L and RML were remarkably similar when the purifications were done in parallel in a single laboratory [88]. These profiling studies thus suggest that details of the enrichment procedure can play a large role in what proteins are captured during the procedure, supporting the possibility that PrPSc itself carries all of the conformational information needed for prion transmission and determination of disease phenotype. However, it is not yet known to what extent non-protein molecules such as lipids and metal ions might contribute to PrP misfolding and therefore pathogenesis. Interestingly, only PrP and apoE were implicated in all three studies as potentially interacting with PrPSc, revealing that these two proteins were ubiquitous in PrPSc preparations but not in age-matched uninfected control samples (FIGURE 2). ApoE has informahealthcare.com
Giorgi 2009
Review
Moore 2010 0
12
8 2 0
Common identifications Prion protein Apolipoprotein E
0 18
Graham 2011
Figure 2. Common protein identifications in three profiling studies of enriched PrPSc. A comparison of three recent studies in which PrPSc mixtures were profiled reveals a wide diversity of protein content. Proteins that were also found with uninfected preparations were removed from the list. PrPSc and apolipoprotein E consistently copurify with prion infectivity across multiple studies.
been consistently implicated in neurodegenerative disease across many studies. ApoE is a component of the very low-density lipoproteins that transport cholesterol from the blood to the liver. In the nervous system, apoE is thought to support neuronal repair, growth and regeneration [94]. It was found that apoE is upregulated in the brains of prion-infected mice with multiple prion strains [95,96] and is co-localized with various types of amyloid plaque [97,98], including PrPSc deposits [99]. It has also been implicated as a potential prion biomarker since it is found elevated in the CSF of prion-affected patients [100] and in the plasma of prion-infected mice [101]. Using tandem mass spectroscopy, it was found that apoE co-purified in PrPSc mixtures derived from four different mouse prion strains (22L, 22A, 79A, RML) and from hamster prion strain 263K, while none of the uninfected preparations contained apoE [88,89]. Thus, while molecular details of the interaction between apoE and PrPSc remain obscure, it is plausible that apoE plays a role in prion pathogenesis. When selected rodent-passaged prion strains were comparatively profiled for their protein content, no strain-associated differences were detected in PrPSc samples derived from the mouse prion strains 22L, RML, 79A, 22A or the hamster strain 263K [88,89]. Similarly, Graham et al. profiled the protein content of Me7, 22F and 79A mouse-passaged prion strains and did not detect any definitive strain-associated differences in protein composition either [90]. Thus, it appears that if there are non-PrP proteins that facilitate PrPSc-associated strain characteristics, then they must be either present in substoichiometric amounts or recalcitrant to MS-based detection. Further studies are needed to expand the PrPSc profiling approach to search for a wider range of molecules, such as metal ions, lipids, glycans and nucleic acids. Structural analysis of PrPSc
It is now widely believed that PrPSc structure dictates the phenotypic and strain characteristics of prion diseases [14]. For this reason, continued effort is needed to help elucidate PrPSc structure. There are no high-resolution PrPSc models yet available, 175
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Moore, Faris & Priola
perhaps due to the lack of homogeneity in samples utilized. It has to date been impossible to obtain high-resolution x-ray crystallographic or NMR data for any sample of infectious PrPSc derived from mammalian tissue. Thus, lower resolution methods are employed and PrPSc models must be derived from the structural constraints provided by years of experimental data from multiple laboratories. Early ultrastructural studies using negative stain techniques on hamster-passaged PrPSc preparations revealed fibrillary structures that bore a striking resemblance to amyloid fibrils [102]. The 1990s heralded a recognition that PrPSc was no longer monomeric and contained a much higher b-sheet content than PrPC [5,6]. Attenuated total-reflectance Fourier-transform infrared spectroscopy has been an invaluable tool for the study of PrP secondary structure, since this is one of the few methods that provides successful analysis of otherwise intractable aggregates and precipitates [103,104]. Despite the poor resolution offered by protein Fourier-transform infrared spectroscopy, it has been used to differentiate PrPSc samples from various prion strains based upon their reproducibly unique b-sheet banding patterns [89,105–108]. The diverse components that copurify with PrPSc almost certainly affect the structural characteristics exhibited by that mixture. For example, it is possible for a PrPSc preparation based upon standard methodology [88] to be composed of up to approximately 40% ferritin, a protein which is known to contain mostly a-helical secondary structure [89]. In fact, protein impurities such as the a-helical ferritin can contribute to the secondary structural features that have often been attributed to PrPSc itself [89]. Since some studies have reported that PrPSc contains a high degree of a-helical character [5,6,109] while other studies conclude the opposite [7,108,110,111], it is thus important to consider how impurities in a PrPSc sample might contribute to the structural characteristics of PrPSc reported over the years. Experimental validation of structural models
The most widely referenced structural models propose that PrPSc retains a large proportion of its original a-helical content in the C-terminal region [112,113]. Recently, Caughey et al. proposed, based upon molecular dynamics simulations and constraints from experimental data, that PrPSc is composed primarily of a parallel in-register b-sheet core with very little, if any, a-helical content [7]. A mostly b-sheet architecture is consistent with various biophysical studies conducted with fibrils derived from bacterially expressed PrP [114–117], although such fibrils tend to be either non-infectious [118] or many orders of magnitude less infectious than PrPSc derived from infected tissue [11,119–121]. Stronger evidence to support a parallel in-register b-sheet architecture was obtained by performing hydrogendeuterium exchange MS (HDX MS) on real infectious PrPSc amyloid derived from mice lacking the GPI anchor on PrP [111]. The HDX MS experiments suggested that the entire protease-resistant region of PrPSc was composed of b-strands connected by short turns without any residual native a-helices [111]. The same HDX MS approach proved more difficult in 176
the examination of PrPSc derived from GPI-anchored wild-type mice, perhaps due to interference from glycosylation [111]. A parallel in-register b-sheet alignment is also consistent with a report in which PrPSc formed cross-linked dimers and trimers connected via glycine residue 90 through a short crosslinking agent [122]. Once linked, matrix-assisted laser desorption ionization (MALDI)-time-of-flight and nanospray LC-ESI-QqTOF analyses were performed after proteolytic digestion of the PrPSc and it was found that cross-linking between N-terminal Gly-90 residues had to be
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Expert Rev. Proteomics 12(2), 171–184 (2015)
Roger A Moore*, Robert Faris and Suzette A Priola Laboratory of Persistent Viral Diseases, Rocky Mountain Laboratories, NIH, NIAID, Hamilton, MT 59840, USA *Author for correspondence: [email protected]
Prion diseases are a heterogeneous class of fatal neurodegenerative disorders associated with misfolding of host cellular prion protein (PrPC) into a pathological isoform, termed PrPSc. Prion diseases affect various mammals, including humans, and effective treatments are not available. Prion diseases are distinguished from other protein misfolding disorders – such as Alzheimer’s or Parkinson’s disease – in that they are infectious. Prion diseases occur sporadically without any known exposure to infected material, and hereditary cases resulting from rare mutations in the prion protein have also been documented. The mechanistic underpinnings of prion and other neurodegenerative disorders remain poorly understood. Various proteomics techniques have been instrumental in early PrPSc detection, biomarker discovery, elucidation of PrPSc structure and mapping of biochemical pathways affected by pathogenesis. Moving forward, proteomics approaches will likely become more integrated into the clinical and research settings for the rapid diagnosis and characterization of prion pathogenesis. KEYWORDS: cerebrospinal fluid . Creutzfeld–Jakob disease . mass spectrometry . prion diagnostics . prion disease .
prion structure . proteomics
.
quaking-induced conversion
Prion diseases, also known as transmissible spongiform encephalopathies, are a group of rare yet universally fatal neurodegenerative diseases that affect humans and other mammals [1,2]. A critical event in prion pathogenesis involves the conversion of normal, proteasesensitive prion protein (PrPC) into an aggregated, pathological isoform, termed PrPSc, which is much higher in b-sheet structure than soluble PrPC. Normal PrPC is expressed most prominently as a glycophosphatidylinositol (GPI)-anchored protein on the cell surface. Although the specific function of PrPC is not yet fully characterized, reports substantiate a role for PrPC in a variety of cellular processes including synaptic transmission and cell signaling [3,4]. The neuropathology of prion diseases in humans and in animals is typically characterized by gliosis and gray matter vacuolation, with deposition of PrPSc in brain tissue as insoluble, partially protease-resistant non-amyloid aggregates high in b-sheet structure [5–7]. It was postulated almost 50 years ago that the infectious agent in these diseases might actually be a self-replicating protein aggregate [8]. Today most researchers believe this to be the case, given the apparent lack of any viral or bacterial component directing disease informahealthcare.com
10.1586/14789450.2015.1019481
transmission. In fact, there are now multiple reports describing the de novo formation of infectious prions from PrPC and a minimal set of molecular components lacking any infectivity [9–11]. The theory that PrPSc is the sole infectious component [12] of the prion poses a challenge to the phenomenon of prion strains [13], which are prion isolates causing distinct pathologies with consistent characteristics such as incubation period, region-specific PrPSc deposition and severity of spongiform degeneration. It is thought that the information responsible for unique strain properties and disease phenotypes is encoded exclusively by PrPSc conformation [7,14], although it has also been proposed that small, soluble b-sheetcontaining oligomers play an important role in prion pathogenesis [15,16]. Characterization of PrPSc structure is thus an important focus of prion research. Most of the documented prion disorders in humans are defined as sporadic Creutzfeld– Jakob disease (sCJD), which typically occurs at a rate of 1–2 individuals per million in a given population [17,18]. Inherited forms of prion disease such as Gerstmann–Stra¨ussler– Scheinker syndrome [19], fatal familial insomnia [20] and hereditary forms of CJD [21] are ISSN 1478-9450
171
2015 This work was authored as part of the Contributor’s official duties as an Employee of the United States Government and is therefore a work of the United States Government. In accordance with 17 USC. 105, no copyright protection is available for such works under US Law.
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also well known, although they occur much less frequently. Acquired forms of human prion disease have historically been caused by various types of exposure to prion-infected biological materials. The earliest recorded prion epidemic, resulting from ritualistic cannibalism involving brain material, occurred in the late 1950s among the Fore tribe in the Kuru region of Papua-New Guinea [22,23]. Later, in the 1990s, a new form of human prion disease resulting from exposure to cattle material tainted with bovine spongiform encephalopathy emerged. This new human prion phenotype, termed variant CJD, received worldwide attention and brought the prion phenomenon to the forefront of public awareness [24]. Although the incidence of acquired prion diseases are generally declining, cases such as those transmitted by blood transfusion [25] continue to occur. Iatrogenic prion infections from ingestion of cadaver-sourced human growth hormone [26] and tainted surgical procedures [27] are also still of concern. Prion diseases are universally fatal with no effective treatment options. There are still significant obstacles to overcome before a cure for prion disease is realized. Reliable antemortem detection of PrPSc and other biomarkers will be of major importance in the clinical setting. Identification of potential interacting partners of pathological PrPSc should also help to determine what role those proteins might play in pathogenesis. Further structural elucidation of PrPSc is critically important, given the increasing consensus among researchers that PrPSc conformation greatly influences prion pathogenesis. Finally, it is important to more clearly map the biochemical pathways affected by pathogenesis in order to gain a better mechanistic understanding of the disease and to facilitate the search for better treatments. Proteomics techniques and the use of mass spectrometry (MS) in particular will continue to play an important role in understanding all of these aspects of prion pathogenesis. Prion disease diagnostics
Diagnosis of prion disease has historically been difficult and time-consuming. The most definitive diagnosis of prions in humans and other non-laboratory mammals relies upon postmortem confirmation of spongiform degeneration by histological methods and/or detection of pathological PrPSc deposition using immunohistochemistry [28–30]. In many instances of human prions, a double evaluation by two highly qualified experts is required for a definitive diagnosis [31]. Formation of PrPSc is thought to be one of the earliest events in prion pathogenesis [12]. Mice infected with the mouse 22L prion strain die after approximately 150 days, yet PrPSc can readily be found by immunoblot within approximately 40 days postinfection [32]. Likewise, PrPSc has been detected by immunoblot in the thalamus of hamsters infected with the scrapie strain 263K at 2 weeks post-inoculation [33] and possibly as early as only 1 day post-infection [34]. If there are other critical biochemical events that precede PrPC misfolding, they have yet to be fully explained. Thus, PrPSc is a key indicator for prion disease. 172
Detection of PrPSc with in vitro conversion assays
Conversion of PrPC into PrPSc using in vitro conversion assays has recently enabled the highly sensitive detection of PrPSc from various tissues. The protein misfolding cyclic amplification (PMCA) assay is based upon the ability of PrPSc to seed the conversion of native PrPC into a proteinase K (PK)-resistant isoform [35]. Potentially infected samples are introduced into uninfected brain homogenates containing PrPC. If infectivity is present, new PrPSc formation is triggered and PrPSc is amplified during serial rounds of intermittent sonication using fresh PrPC-containing brain homogenate. The PMCA has been used to examine multiple aspects of prion disease, including de novo PrPSc formation [9,10]. A key adaption of the PMCA technique utilizes bacterially expressed recombinant (rPrP) as a substrate for conversion into fibrillar aggregates that only takes place in the presence of prion-infected tissue [36]. The PMCA reaction was soon modified to induce rPrP fibril formation by shaking, or quaking-induced conversion (QuIC) in lieu of sonication [37]. Both the original PMCA and the QuIC assays have rapidly evolved with multiple reports demonstrating PrPSc detection capabilities at concentrations of £1 attogram (10–18 g) with high specificity [38–41]. For example, a recent modification of the original QuIC assay, termed real-time or RT-QuIC, was used to detect PrPSc in nasal brushings from sCJD patients with 97% sensitivity and 100% specificity [38]. Detection of PrPSc by targeted MS
Targeted MS approaches aimed at the identification of PrP peptides have also been reported [34,42–44]. This technique detects precursor and fragment ion pairs from a complex proteome digest with extremely high selectivity and sensitivity, thus making it ideal for assay development. In 2007, Onisko et al. developed a selected/multiple reaction monitoring (SRM/ MRM) assay [42] using nanospray liquid chromatography with tandem MS (LC-MS/MS) for the detection of the tryptic peptide VVEQMCTTQYQK, spanning PrP residues 209-220, in attomole amounts (TABLE 1). Importantly, this methodology was used to quantitate PrPSc in the brains of terminally ill Syrian hamsters at 19 ± 2 mg PrP/g of brain; an amount consistent with the 21 mg PrP/g estimated by Diringer et al. [45] 10 years earlier. Detection of PrPSc was even reported after only 1 day post-inoculation in the brains of hamsters that had been injected with the prion strain 263K [34]. Interestingly, human PrP209-220 was also found in urine-derived chorionic gonadotropin preparations, although it is not clear whether these samples were infectious [46]. This approach was later extended to prion-infected mouse and sheep samples, showing that the method could be widely applicable to multiple species, all with low attomole limits of detection [43]. These MRM methods for PrPSc detection relied heavily upon the analysis of the tryptic PrP209-220 peptide VVEQMCTTQYQK, which contains chemically active cysteine (Cys) and methionine (Met) residues. In 2012, Sturm et al. reported another quantitative MRM assay [44] that focused on the peptide YRPVDQY, derived from a chymotrypsin digest of bacterially expressed PrP. The Expert Rev. Proteomics 12(2), (2015)
Proteomics in prion biology
Review
Table 1. Selected SRM/MRM transitions used for PrP identification and quantification. Peptide† 77
109
GQPHGGGWGQPHGGGGWGQGGSHSQWNKPSKPK
81
109
GGGWGQPHGGGGWGQGGSHSQWNKPSKPK
85
GQPHGGGGWGQGGSHSQWNKPSKPK109
89
109
GGGGWGQGGSHSQWNKPSKPK
94
109
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GQGGSHSQWNKPSKPK
96
109
GGSHSQWNKPSKPK
152
156
ENMYR
163
169
YRPVDQY
164
169
RPVDQY
186
194
QHTVTTTTK
195
204
GENFTETDIK
195
GENFTETDVK204
209
220
VVEQMCTTQYQK
209
220
VVEQM(SO)CTTQYQK
208
219
VVEQMCVTQYQK
208
219
VVEQM(SO)CVTQYQK
209
220
VVEQMCITQYQR
209
220
VVEQMCITQYER
209
220
VVEQM(SO)CITQYQR
209
220
VVEQMCITQYER
209
VVEQM(SO)CITQYER220
221
ESQAYYDGR229
221
228
ESQAYYQR
Species
Precursor ion (m/z)
Product ion (m/z)
Ref.
Sh
556.8 (+6)
630.6 (y31)
[47]
Sh
583.8 (+5)
640.2 (y25)
[47]
Sh
512.3 (+5)
593.8 (y23)
[47]
Sh
535.3 (+4)
575.3 y16)
[47]
Sh
431.7 (+4)
513.4 (y14)
[47]
Sh
385.4 (+4)
494.5 (y13)
[47]
Sh
357.0 (+2)
469.2 amu
[49]
Ha, Bo
470.7 (+2)
759.3 (b6)
[44]
Bo
389.2 (+2)
596.3 (b5)
[44]
Sh
339.6 (+3)
450.2 amu
[49]
Ha, Sh
577.3 (+2)
706.4 (y6)
[43]
Hu, Mo
570.3 (+2)
692.3 (y6)
[43]
Ha
757.8 (+2)
438.2 (y3)
[34,42]
Ha
765.8 (+2)
171.1 (a2)
[43]
Mo
756.8 (+2)
171.1 (a2)
[43]
Mo
764.8 (+2)
171.1 (a2)
[43]
Sh
778.1 (+2)
171.1 (a2)
[43]
Hu
778.4 (+2)
696.4 (y5)
[46]
Sh
786.1 (+2)
171.1 (a2)
[43]
Hu
778.6 (+2)
171.1 (a2)
[43]
Hu
786.6 (+2)
171.1 (a2)
[43]
Ha, Mo
544.7 (+2)
510.4 (y4)
[43]
Sh
522.8 (+2)
466.1
[49]
†
Residue numbers shown with the peptide sequence.
potential advantage of targeting this peptide was reportedly based upon its highly conserved nature across multiple species of interest to prion biologists and its lack of chemically active Cys and Met residues. One targeted MS approach toward strain differentiation based upon the analysis of PrPSc is termed N-terminal amino acid profiling (N-TAAP) [47–49]. Given that PrPSc samples from different prion strains can be variably cleaved at N-terminal PK cleavage sites, the identities of the resulting N-terminal tryptic peptides are also variable (TABLE 1). MS has the capacity to differentiate single amino acid differences at the PK cleavage sites. In the N-TAAP method, quantification of selected N-terminal tryptic peptides by an SRM/MRM approach was used to differentiate various prion strains [47,48]. It was found that ratios of certain peptides within and between samples often varied based upon the strain. For example, the PrP peptide spanning residues 96-109 (GGSHSQWNKPSKPK) was reproducibly more abundant in sheep-passaged bovine informahealthcare.com
spongiform encephalopathy than it was in a classical sheep scrapie strain [47]. The N-TAAP approach should eventually prove to be highly useful in the analysis of various sCJD types, which are also known to contain a similar array of N-terminal protease cleavage sites [50,51]. Most of the targeted PrP MS reported to date has been typically utilized with either bacterially expressed rPrP or PrPScderived post-mortem. In order to take a more prominent role in prion diagnostics, MS-based methods need to be utilized with samples that have already been used successfully antemortem with in vitro assays such as cerebrospinal fluid (CSF) [52–54], blood [55], urine [39,56,57] and, most recently, nasal brushings [38] (FIGURE 1). However, a key obstacle for the development of MS-based techniques for PrPSc detection is the need to first remove protease-sensitive PrPC from a sample, given that the proteotypic peptide sequences are the same in the two isoforms. Thus, current SRM/MRM methods are tethered to time-consuming and potentially yield-reducing treatment with 173
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Moore, Faris & Priola
14-3-3 protein for the detection of prion disease in humans is controversial [64], with specificity results as low as 28% B) PrPSc, apoE, reported in a cohort of 420 confirmed Nasal brushings multiple proteins CJD patients [65]. Furthermore, 14-3-3 A) RT-QulC Cerebral spinal fluid protein concentrations are not always B) PrPSc consistent between major sCJD types nor A) RT-QulC, PMCA among the many known subtypes [66]. B) PrPSc, tau, p-tau, 14-3-3, neuron Consequently, clinicians have recomspecific enolase, mended a combination of more than one multiple proteins CSF marker to improve the accuracy of diagnostic testing for sCJD against other Blood serum and plasma neurological conditions [67]. For example, the protein desmoplakin is often elevated A) RT-QulC, PMCA in sCJD patients and has been proposed B) PrPSc, apoE, clusterin, complements, multiple as a supportive CSF marker to help proteins decrease the rate of 14-3-3 false-positive cases in sCJD diagnosis [68]. Elevated total tau protein (t-tau) concentrations with the presence of 14-3-3 protein in Urine A) RT-QulC, PMCA, the CSF has been reported to yield 84% SRM/MRM sensitivity and 96% specificity for sCJD B) PrPSc, α1-antichymotrypsin, patients [67]. The p-tau/t-tau (phosphorymultiple proteins lated/total) ratio, which is much lower in sCJD patients than it is in Alzheimer’s Sc patients, may help to discriminate sCJD Figure 1. Comparison of in vitro and SRM/MRM detection of PrP and the from other neurodegenerative dissource of other major prion disease biomarkers. (A) Major techniques and assays most commonly used for detection and (B) Selected proteins which have diagnostic eases [69,70]. The presence of other neurovalue are identified from various tissues and body fluids. logical disease-associated biomarkers, such as neuron-specific enolase [67] and S100B PK. Circumvention of this bottleneck could facilitate even protein [71], have also provided diagnostic value with prion dismore sensitive, selective and quantitative detection of PrPSc. ease. Thus, the simultaneous detection of multiple prion biomarkers [72] would greatly enhance the accuracy of prion Prion biomarkers used in the clinical setting disease diagnosis. The technological platform of choice to While PrPSc remains a primary target for early prion detection, quantify hundreds of possible prion biomarkers at once with other biomarkers could also play key roles in diagnosis, disease extremely high specificity will likely be the SRM/MRM monitoring and characterization of different prion phenotypes. techniques. A biomarker is generally defined as some measurable indicator of a particular disease state. The number of prion-associated Protein profiling of PrPSc mixtures biomarkers reported in genomic and proteomic studies is One strategy employed to learn more about prion infectivity impressive [53,58,59]. Many of these putative biomarkers, such as has been to inventory the molecular species that co-purify with clusterin or transthyretin, have limited usefulness because they PrPSc mixtures. If PrPSc could be purified while retaining a are so ubiquitous in multiple disease processes including those high infectivity titer, then those components removed during not related to prions. Consequently, the vast majority of the enrichment process are not needed for prion transmission. reported prion biomarkers [58,59] remain in the academic realm The simplest possible mixture that nonetheless contained all and have not always been practical in the clinical setting. elements required for infectivity might be more amenable to The first major protein biomarkers to be used effectively in mechanistic analysis than a highly complex mixture like brain the diagnosis of human prion diseases were the 14-3-3 proteins, homogenate. Unfortunately, infectious PrPSc has never been a ubiquitous family of neuronal proteins often found elevated purified to complete homogeneity, thereby making the minimal in the CSF of patients with neurological disease [53,60,61]. Typi- component analysis strategy more difficult. Over 30 years of cally, the b-isoform of 14-3-3 is detected by immunoblot [62] effort have been devoted toward developing methods of PrPSc and has been found to be more useful for detecting sporadic enrichment [45,73–81]. Despite this effort, enriched PrPSc is typiand familial forms of CJD than it has been for Gerstmann– cally heterogeneous, containing nucleic acids [45,82], lipids [83], Stra¨ussler–Scheinker and fatal familial insomnia [63]. Despite its polysaccharides [84] and probably metal ions [85,86]. These PrPSc widespread use in the clinical setting, the efficacy of preparations are known to be highly infectious [15,45,79,81].
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Brain A) RT-QulC, PMCA, SRM/MRM
174
Expert Rev. Proteomics 12(2), (2015)
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Proteomics in prion biology
Therefore, any non-PrP molecular species potentially required as co-factors for prion transmission must be present in the enriched preparations. Recent MS-based protein profiling studies [87–91] have identified non-PrP proteins that copurify with prion infectivity and therefore might be important in prion pathogenesis. Consistent with early observations [45], more recent studies [87–89] have found that ferritin co-purifies with PrPSc. However, ferritin was also found in control preparations of non-infected mice. This suggests that it is the oligomeric nature of ferritin, rather than any association with PrPSc, which determines its presence during the enrichment process [88]. Moreover, it has been found that at least 30% of the proteins identified with PrPSc could also be found in uninfected preparations, suggesting that these proteins might have been artifacts of the isolation procedure used. The majority of such proteins, like ferritin, collagen, actin and proteasomal subunits, are oligomeric or polymeric, while other proteins such as calcium/calmodulin-dependent protein kinase are highly abundant in brain tissue [92]. The tendency to aggregate and the high abundance of these proteins may explain their presence in mock PrPSc preparations. Proteomicsbased approaches such as these demonstrate that significant levels of non-PrP proteins are present even in some of the best PrPSc preparations. Moving forward, the existing PrPSc enrichment procedures that are largely based upon repeated centrifugation could be modified by the incorporation of a mechanistically distinct step, such as phosphotungstic acid precipitation [93], in order to remove another subset of impurities from the PrPSc mixture. This strategy may result in more homogeneous samples for use in studies such as PrPSc-labeling and structure analysis. A comparison of three recent protein profiling studies in which various proteins that co-purified with PrPSc were characterized reveals a remarkable diversity in the identity of the proteins identified. After proteins found in non-infected preparations [88] were removed from consideration, approximately 40 reported proteins could be compared between studies. Even PrPSc from the same 22L mouse prion strain purified 3 years apart differed significantly in non-PrP protein composition [88]. However, differences in PrPSc protein profiles between the mouse prion strains 22L and RML were remarkably similar when the purifications were done in parallel in a single laboratory [88]. These profiling studies thus suggest that details of the enrichment procedure can play a large role in what proteins are captured during the procedure, supporting the possibility that PrPSc itself carries all of the conformational information needed for prion transmission and determination of disease phenotype. However, it is not yet known to what extent non-protein molecules such as lipids and metal ions might contribute to PrP misfolding and therefore pathogenesis. Interestingly, only PrP and apoE were implicated in all three studies as potentially interacting with PrPSc, revealing that these two proteins were ubiquitous in PrPSc preparations but not in age-matched uninfected control samples (FIGURE 2). ApoE has informahealthcare.com
Giorgi 2009
Review
Moore 2010 0
12
8 2 0
Common identifications Prion protein Apolipoprotein E
0 18
Graham 2011
Figure 2. Common protein identifications in three profiling studies of enriched PrPSc. A comparison of three recent studies in which PrPSc mixtures were profiled reveals a wide diversity of protein content. Proteins that were also found with uninfected preparations were removed from the list. PrPSc and apolipoprotein E consistently copurify with prion infectivity across multiple studies.
been consistently implicated in neurodegenerative disease across many studies. ApoE is a component of the very low-density lipoproteins that transport cholesterol from the blood to the liver. In the nervous system, apoE is thought to support neuronal repair, growth and regeneration [94]. It was found that apoE is upregulated in the brains of prion-infected mice with multiple prion strains [95,96] and is co-localized with various types of amyloid plaque [97,98], including PrPSc deposits [99]. It has also been implicated as a potential prion biomarker since it is found elevated in the CSF of prion-affected patients [100] and in the plasma of prion-infected mice [101]. Using tandem mass spectroscopy, it was found that apoE co-purified in PrPSc mixtures derived from four different mouse prion strains (22L, 22A, 79A, RML) and from hamster prion strain 263K, while none of the uninfected preparations contained apoE [88,89]. Thus, while molecular details of the interaction between apoE and PrPSc remain obscure, it is plausible that apoE plays a role in prion pathogenesis. When selected rodent-passaged prion strains were comparatively profiled for their protein content, no strain-associated differences were detected in PrPSc samples derived from the mouse prion strains 22L, RML, 79A, 22A or the hamster strain 263K [88,89]. Similarly, Graham et al. profiled the protein content of Me7, 22F and 79A mouse-passaged prion strains and did not detect any definitive strain-associated differences in protein composition either [90]. Thus, it appears that if there are non-PrP proteins that facilitate PrPSc-associated strain characteristics, then they must be either present in substoichiometric amounts or recalcitrant to MS-based detection. Further studies are needed to expand the PrPSc profiling approach to search for a wider range of molecules, such as metal ions, lipids, glycans and nucleic acids. Structural analysis of PrPSc
It is now widely believed that PrPSc structure dictates the phenotypic and strain characteristics of prion diseases [14]. For this reason, continued effort is needed to help elucidate PrPSc structure. There are no high-resolution PrPSc models yet available, 175
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perhaps due to the lack of homogeneity in samples utilized. It has to date been impossible to obtain high-resolution x-ray crystallographic or NMR data for any sample of infectious PrPSc derived from mammalian tissue. Thus, lower resolution methods are employed and PrPSc models must be derived from the structural constraints provided by years of experimental data from multiple laboratories. Early ultrastructural studies using negative stain techniques on hamster-passaged PrPSc preparations revealed fibrillary structures that bore a striking resemblance to amyloid fibrils [102]. The 1990s heralded a recognition that PrPSc was no longer monomeric and contained a much higher b-sheet content than PrPC [5,6]. Attenuated total-reflectance Fourier-transform infrared spectroscopy has been an invaluable tool for the study of PrP secondary structure, since this is one of the few methods that provides successful analysis of otherwise intractable aggregates and precipitates [103,104]. Despite the poor resolution offered by protein Fourier-transform infrared spectroscopy, it has been used to differentiate PrPSc samples from various prion strains based upon their reproducibly unique b-sheet banding patterns [89,105–108]. The diverse components that copurify with PrPSc almost certainly affect the structural characteristics exhibited by that mixture. For example, it is possible for a PrPSc preparation based upon standard methodology [88] to be composed of up to approximately 40% ferritin, a protein which is known to contain mostly a-helical secondary structure [89]. In fact, protein impurities such as the a-helical ferritin can contribute to the secondary structural features that have often been attributed to PrPSc itself [89]. Since some studies have reported that PrPSc contains a high degree of a-helical character [5,6,109] while other studies conclude the opposite [7,108,110,111], it is thus important to consider how impurities in a PrPSc sample might contribute to the structural characteristics of PrPSc reported over the years. Experimental validation of structural models
The most widely referenced structural models propose that PrPSc retains a large proportion of its original a-helical content in the C-terminal region [112,113]. Recently, Caughey et al. proposed, based upon molecular dynamics simulations and constraints from experimental data, that PrPSc is composed primarily of a parallel in-register b-sheet core with very little, if any, a-helical content [7]. A mostly b-sheet architecture is consistent with various biophysical studies conducted with fibrils derived from bacterially expressed PrP [114–117], although such fibrils tend to be either non-infectious [118] or many orders of magnitude less infectious than PrPSc derived from infected tissue [11,119–121]. Stronger evidence to support a parallel in-register b-sheet architecture was obtained by performing hydrogendeuterium exchange MS (HDX MS) on real infectious PrPSc amyloid derived from mice lacking the GPI anchor on PrP [111]. The HDX MS experiments suggested that the entire protease-resistant region of PrPSc was composed of b-strands connected by short turns without any residual native a-helices [111]. The same HDX MS approach proved more difficult in 176
the examination of PrPSc derived from GPI-anchored wild-type mice, perhaps due to interference from glycosylation [111]. A parallel in-register b-sheet alignment is also consistent with a report in which PrPSc formed cross-linked dimers and trimers connected via glycine residue 90 through a short crosslinking agent [122]. Once linked, matrix-assisted laser desorption ionization (MALDI)-time-of-flight and nanospray LC-ESI-QqTOF analyses were performed after proteolytic digestion of the PrPSc and it was found that cross-linking between N-terminal Gly-90 residues had to be
