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The culmination of a lot of work during my postdoctoral fellowship.

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  • First of all I would like to thank everyone here at NGM for inviting me and coming to listen to my talk. Our lab at Case Western is interested in pursuing a really muti-diciplinary approach to understanding both structural and mechanistic aspects of the conformational conversion of the prion protein. As you can see by the title of my talk, my postdoctoral work has focused on the structure of amyloid fibrils formed by the recombinant human prion protein.
  • Prion diseases or transmissible spongiform encephalopathies (TSEs) are a group of fatal neurodegenerative disorders. These disorders are spread over several mammalian species including CJD, GSS and kuru in humans and most notorioulsy in animals, bovine spongiform encephalopathy or ‘mad cow disease’ in cattle and chronic wasting disease in deer and elk It is the consumption of BSE tainted beef by humans which is believed responsible for the outbreak of vCJD in Britain around the turn of this centuryThese disorders may arise spontaneously as in most cases of CJD, may be inherited as with GSS as well as a variety of CJD-related familial point mutations which predispose an individual to disease, or by infection as is the case with animal TSEs and kuru, arising from the ritualistic cannibalism of brains by the Fore tribe of New Guinea
  • This protein is expressed mainly in the CNS and is well conserved over all mammalian speciesThe function of PrPC is still unknown, and has been speculated to be involved in copper homeostasis, cell signaling, cell adhesion, anti-apoptotic, protection against oxidative stress, or modulation of synaptic structure and function
  • In humans, the PrP gene transcribes a 254 amino acid protein containing an N-terminal leader sequence which directs the polypeptide to the cell surface and a C-terminal signal sequence which directs that the attachment of a GPI membrane anchor. Both of these signaling sequences are removed in the mature PrPC resulting which may also be glycosylated at C-terminal asparagines 181 and 197. Here is the solution structure of monomericPrP as determined by NMR using recombinant material. The mature protein consists of an unstructured N-terminal domain which contains the so-called octarepeat region which is able to bind divalent copper, and a globular C-terminal domain which as two short B-sheets, three A-helices and a single disulfide bond between cysteines 179 and 214
  • In the absence of high resolution structural data for the PrPSc agent, we have relied on surrogate data to reveal that conformational conversion of PrPC to PrPSc is closely linked with the TSE diseases.So here we have a normal cow expressing PrPC which exists in its normal cellular form… so what happens in the ‘mad cow’? **click**First brain histology reveals extensive spongiform degeneration, or vacuolarlization, astogliosis as astrocytes are recruited to phagocytoses dying neurons, as well as accumulation of prion protein deposits **click**PrP aggregates also bind the reasonably amyloid specific dyes Congo Red, which results in so-called apple-green birefringence, and Thioflavin T which can be monitored fluormetrically as is the most common way to evaluate the conversion process in vitro. **click**One commonly exploited biochemical difference between PrPC and PrPSc is the resistance of the latter to proteolytic digestion. So in the presence of the non-specific protease Proteinase K, PrPC is completely digested while PrPSc is cleaved with a ragged end at around sites 87-90 and results in an ~140 amino acid protected region **click**Here we see FTIR spectra of hamster PrP. The solid line is normal monomericPrPC and the amide I band here at about 1650 wavenumbers is characteristic of a primarily A-helical protein, while spectra for either PrPSc or Proteinase K digested PrPSc… this PrP27-30… show a red-shifting to 1630, 1640 wavenumbers which is more characteristic of B-sheet…
  • I should also mention the difference between infectivity and toxicity of PrP aggregates in the TSE diseases. It now pretty commonly thought that the infectious and neurotoxic agents are distinct species… as with protein misfolding disorders such as Alzheimer’s the neurotoxic species is proposed to be a some smaller, more structurally fluid oligomer… however, with regards to this talk we will be focusing on the infectious PrPSc and not any putative neurotoxic structural speciesX ray diffraction studies of amyloid fibrils have long since been known to reveal a so called cross-B pattern where individual B-strands lie perpendicular to the long fibril axis.In more recent years we have gotten a higher resolution picture of at least some amyloid folds…in this case solid-state NMR has revealed the fibrillar fold of amyloid-beta fibrils associated with alzheimer’s disease **click**And most recently davideisenberg’s group has been able to get X-ray structures of microcrystals formed by short amyloidogenic peptides… Here is the first such structure formed by a peptide isolated from the yeast prion Sup35. In this case B-strands are stacked in parallel and in-register so that same residues are directly above and below same residues on neighboring molecules. Eisenberg’s group has since discovered a range of packing geometries for these short peptide crystals which can be anti-parallel and out-of-register – however all show this so-called steric zipper which is the anhydrous interface between two B-sheets with tight interdigtation of side chains
  • In my work I am dealing with the in vitro conversion of prion protein to amyloid fibrils. The mechanism by which this occurs is best described by a nucleated polymerization model similar to the formation of crystals. Here we initially observe a lag phase which involves the formation of a stable nucleus, presumably after oligomerization of psome partially unfolded species which is thermodynamically disfavored. After this nucleus is formed, however, it is able to rapidly convert monomeric protein to the amyloid state. If these stable nuclei are added at the beginning of the reaction the lag phase is abolished. Unfortunately, detailed kinetic studies of amyloid fibril growth are far from trivial due to other events which may generate nuclei such as fragmentation of long fibrils or secondary nucleation events, both of which generate additional loci for template-mediated conformational conversion
  • I should state that the experiments I am about to describe were all performed using the PrP fragment 90-231 which reflects the protease-resistant fragment of PrPSc, contains nearly all predisposing point mutations, and is sufficient for disease. Amyloid fibrils formed from this PrP fragment were shown by Stanley Prusiner’s group to cause disease in mice overexpressing this same construct leading them to conclude that these fibrils represent synthetic prions. However, there are some problems with these studies, rPrPamyloid displays a much shorter PrP resistant core region than authentic PrPSc… more importantly, the infectivity titre of the amyloid fibrils is very low, and these transgenic mice also develop the disease later in life…while the infectivity of the fibrils is dubious at best, they do allow us to examine the conformational space of the prion protein with methods that are not amenable to cellular PrP.
  • EPR is really just NMR for unpaired electrons which behave like tiny bar magnets in a strong magnetic field. Where can you find unpaired electrons? Examples include free radicals, particular oxidation states of transition metals, and for the present work, the stable unpaired electron of a nitroxide. Of course an electron is much smaller than a proton which is why an EPR instrument needs a magnet which takes up a corner of the room as opposed to filling a room like a NMR instrumentA magnetic field splits Ms=1/2 electron spin states into two energy levels, because of the differences in masses, a given field will split the electron states ~2000 fold more than proton states.After aligning the electron spin with the applied magnetic field we can excite the electron spin into a resonance condition between the electron spin aligned and opposed spin states with microwave radiation. One should see a nice absorption peak at the resonant frequency but because of technical reasons it is easier to vary the magnetic field than the microwave frequency… also, to improve signal to noise the magnetic field is modulated so that noise is filtered out… this results in the measurement not of absolute absorbance but the slope of the absorbance envelope resulting in a derivative spectrum.
  • In the case of a Nitroxide, the EPR signal is split into a triplet because of the spin 1 Nitrogen nucleus where the unpaired electron residesVery quickly, anytime you see spectra overlaid onto one another, these represent the same molar concentration of nitroxide spins, so it is the overall shape of the line that becomes important – we scale these by normalizing the double intergrals of these spectra to one another, so we have the same area under the curve so to speak, so that broader EPR spectra will display lower amplitude First, these signals give us a site-specific measure of the flexibility of the backbone. This highly isotropic, three line signal here in black would represent a highly mobile backbone, while these broader blue and red signals would indicate increasingly imobilized backbone regions. I should mention that there is also a rotational component to the EPR signal so that a label attached to a small monomeric protein that is tumbling rapidly in solution will show a higher amplitude, more isotropic signal than one that is attached to a protein in a large aggregate such as an amyloid fibrilNext is that you can approximate the distance between spin labels up to about 25 A using conventional EPR… proximity of two labels will result in dipolar broadening of the EPR signal somewhat reminicent of the broadening observed with increasing backbone immobilization… by deconvoluting these signals with a broadening function one can get distance informationThe second proximity regime occurs only when multiple nitroxides are within van derwaal distance of one another and this results in spin-exchange and the emergence of this really striking broad single-line spectrum – experimentally, the only time you ever really can observe these signals are in highly concentrated solutions of nitroxide, crystals of nitroxide… or in certain spin labeled amyloid system
  • Seeing as how I am interviewing for a position as a protein purification scientist, I want to briefly take you through the rPrPpurifcation protocol. We express an N-terminally His-tagged protein in bacteria using the PET vectors. Given the tendency for prion protein to aggregate, we unsuprizinglyfing it in inclusion bodies which we solubilize in our standard buffer containing 6 M GdnHCl and 10 mM glutathione to reduce and disulfide pairings. We load this onto a Ni column and perform the following purification and refolding scheme on a GE AKTA FPLC. First we have a wash in this denaturing solution followed by a gradient whereby we slowly leech out the denaturant and glutathione to properly refold the protein. We wash and apply 50 mMimidazole to further clean up the column and then we elute the protein - for whatever reason I found that somewhat lower concentrations of imidazole in this elute step really increase the final yield for my cysteine variants. Finally we repeat this whole process minus the low imidazole wash – protein from both fractions are identical the splt in yield is generally around 60/40
  • For thecysteine mutants, after elution we dialyze them in acetate pH 4 which is where the protein is really happiest and also prevents any intermolecular disulfide formation between the unlabeled cysteines.
  • Very briefly for any biologist out there I would like to touch upon the potential mechanism of PrPScneuroinvasion, although these processes are still highly controvesialAfter ingestion of the scrapie agent, it may be taken up from the intestinal lumen by microfold cells (i), direct capture by migratory dendritic cells (iii), or by regular intestinal epithelial cells in a complex with ferritin. After this initial uptake, it appears that phagocytosis by macrophages degrades the scrapie agent and are protective, while dendritic cells may transport PrPSc to follicular dendritic cells located in the B-cell rich follicles of peyers patches and gut associated lymphoid tissue. FDCs express PrPC at a relatively high level and may represent the initial location of PrPSc amplification. After incubation in lymphoid tissue in the gut and spleen, PrPSc is presumably able to invade the CNS via enteric nervous system in a process that remains unclear.Then PrPSc invasion of the nervous system occurs in the retrograde direction via sympathetic and parasympathetic nervesIt has been proposed that this retrograde invasion occurs by step-wise interactions of PrPSc with PrPC along the cell surface (i), by fragmentation of large extracellular aggregates, or by some vessicle mediated mechanism (iii). Given that PrPSc spread appears to be directed in the retrograde as opposed to moving to the periphery of the CNS would seem to suggest someform of active transport making this latter mechanism the most likely
  • Prp Presentation

    1. 1. Human Prion Protein Amyloid:<br />A Tale of Structure and Stability<br />Nathan J. Cobb<br />Department of Physiology and Biophysics <br />Case Western Reserve University, Cleveland, OH<br />
    2. 2. The Prion Diseases<br />DiseasePathogenesis<br />Human Diseases<br /><ul><li>Creutzfeldt-Jakobs disease (CJD) Sporadic, iatrogenic
    3. 3. variant CJD Infection from bovine prions?
    4. 4. Familial CJDs Inherited
    5. 5. Kuru Infection
    6. 6. Gerstmann-Straussler-Sheinker (GSS) Inherited
    7. 7. Fatal familial insomnia Sporadic, Inherited</li></ul>Animal Diseases<br /><ul><li>Scrapie (sheep) Sporadic, Infection
    8. 8. Bovine spongiform encephalopathy Sporadic, Infection
    9. 9. Transmissible mink encephalopathy Sporadic, Infection
    10. 10. Chronic wasting disease (cervids) Sporadic, Infection
    11. 11. Feline spongiform encephalopathy Infection</li></li></ul><li>The ‘Protein-Only’ Hypothesis of Prion Diseases<br /><ul><li>The infectious agent in the transmissible spongiform encephalopathies</li></ul> (TSEs) is not a virus, but a misfolded form of the normal (cellular) prion<br /> protein, PrPC<br /><ul><li> This misfolded form, PrPSc(scrapie), is a β-sheet-rich aggregate which arises </li></ul> from conformational conversion of the normally monomeric and primarily <br />α-helical PrPC<br /><ul><li> Like other neurodegenerative diseases such as Alzheimer’s and Parkinson’s, </li></ul> TSEs are often associated with neuronal accumulation of amyloid deposits<br /><ul><li>PrPSc self-perpetuates by first binding PrPC and then inducing conformational </li></ul> conversion of the latter protein to the PrPSc state<br /><ul><li> Although the ‘Protein-only’ nature of the TSEs has yet to be conclusively </li></ul> shown, studies in yeast and other fungi have provided the proof-of-principle <br /> that proteins alone can act as self-perpetuating agents<br /><ul><li> Challenge to the ‘Anfinsen Principle’?</li></li></ul><li>G<br />G<br />H11<br />H2<br />H3<br />G<br />P<br />I<br />S<br />S<br />PHGGGWGQ<br />The Structure of Cellular Prion Protein<br />Leader Sequence: 1-22<br />Octapeptide Repeats: 51-91<br />-Helicies: 144-156,174-194, 200-228<br />-Sheets: 128-131, 161-164<br />Disulfide Bond: 179 and 214<br />N-linked Glycosylation: 181, 197<br />GPI Anchor: 231<br />GPI Anchor Signal: 232-254<br />
    12. 12. PrPC<br />PrPSc<br />Digestion with Proteinase K<br />23<br />231<br />23<br />231<br />S<br />S<br />S<br />S<br />Proteinase K<br />Proteinase K<br />231<br />~87-90<br />S<br />S<br />Surrogates for Structural Information<br />Binding of ‘Amyloid-Specific’ Dyes<br />FTIR spectra of brain-derived PrP<br />— PrPC<br /> - - - PrPSc<br /> ••••• PrP 27-30<br />PrPC<br />PrPSc<br />PK - + - +<br />-39 kDa<br />-28 kDa<br />Staining with <br />Hematoxylin-Eosin<br />-19 kDa<br />-14 kDa<br />Congo Red<br />Thioflavin T<br />Immunostaining for Glial<br />Fibrillary Acidic Protein<br />Immunostaining for PrP<br />Pan et al. (1993) PNAS<br />Aguzzi et al. (2001) Nature<br />
    13. 13. Nelson et al. (2005) Nature<br />Amyloid Fibrils<br />X-ray diffraction structure of microcrystals formed by the peptide GNNQQNY<br />Aβ1-40 structure as determined<br />by solid-state NMR<br />Like other neurodegenerative diseases such as Alzheimer’s and Parkinson’s, <br />TSEs are associated with neuronal accumulation of amyloid deposits<br />Petkova et al. (2004) PNAS<br />
    14. 14. Conversion of rPrP (residues 90-231) into Amyloid Fibrils: ‘Synthetic Prions’<br />Nucleation<br />2% seed<br />No seed<br />Elongation<br />Fragmentation<br />2o Nucleation<br />
    15. 15. What is the Relevance of rPrPAmyloid to PrPSc?<br />Brain-Derived PrPSc‘Synthetic Prions’<br />GlycosylatedUnglycosylated<br /> Membrane-associated NA<br /> 16 kDaPK-resistant core Shorter PK-resistant core<br /><ul><li> It has been shown that in vitro converted amyloidfibrils induce a TSE-like </li></ul> disease in mice overexpressing PrP89-231 (Legname et al. (2004) Science) <br /><ul><li> The infectivity titer is very low, suggesting that only a small fraction of this </li></ul> material is infectious<br /><ul><li>rPrPamyloid allows for structural and biochemical characterization techniques </li></ul> that are not amenable to brain-derived aggregates<br />
    16. 16. Site-Directed Spin Labeling<br />Involves substitution of cysteinefor <br />native residues followed by thiol-specific modification with the nitroxidereagent<br />Nitroxide-labeled proteins yield three important pieces of information<br />1) Distance estimates – dipolar broadening and spin exchange<br />2) Mobility information<br />3) Accessibility<br />
    17. 17. MS = 1/2<br />Hres<br />Hres<br />Hres<br />D(Absorption)/dH<br />MI = 1/2<br />Absorption<br />Energy<br />H<br />H<br />H<br />signal<br />amplitude<br />modulation amplitude<br />EPR spectroscopy<br />Absorption of electromagnetic radiation by an unpaired electron<br />in an applied magnetic field <br />Energy<br />d(Absorption)/dH<br />MI = -1/2<br />Absorption<br />MS = -1/2<br />H<br />H<br />H<br />
    18. 18. +1<br />MI<br />MS<br />+1/2<br />0<br />-1<br />Energy<br />-1<br />-1/2<br />0<br />+1<br />H<br />Nitroxide EPR spectra<br />Distance Estimates<br />Mobility<br />Dipolar Broadening<br />Jayasinghe & Langen (2004) J. Biol. Chem<br />Spin Exchange<br />Margittai & Langen (2004) PNAS<br />
    19. 19. Initial Purification and Refolding of rPrP<br />Denaturing Wash (10 mM reduced glutathione, 6 M GdnHCl, pH 8.0)<br />Gradient from 6 M -> 0 M GdnHCl<br />Wash<br />Low Imidazole (50 mMimidazole, pH 8.0)<br />Collect High Imidazole (350 - 500 mMimidazole, pH 5.8 - 6.4)<br />Standard Buffer: 100 mM phosphate, 10 mMTris, pH 7.0<br />
    20. 20. Final Purification and Labeling of rPrP<br /><ul><li> Dialyze collected fractions into 50 mMactetate, pH 4.0
    21. 21. Pool and concentrate, storage at -80 oC
    22. 22. Incubate with 5x molar equivalents of TCEP at 4 oC, dilute with 50 mM phosphate, </li></ul> pH ~7.0, and add 10x molar equivalents of nitroxide spin-label and thrombin<br /><ul><li> Verify nitroxide-labeling and 6xHis-tag cleavage by MALDI
    23. 23. Remove excess spin label, uncleavedrPrP, rPrP aggregates, and remaining </li></ul> impurities by cation exchange chromatography<br /><ul><li> Measure monomeric EPR signal, concentrate, aliquot and store protein at -80 oC</li></li></ul><li>Characterization of rPrPCysteine Variants<br />Verify native-like α-helical <br />content by CD spectroscopy<br />2) Verify the proper formation <br />of amyloid fibrils by AFM <br />3) Verify native disulfide formation by HPLC/MS of tryptic digests<br />
    24. 24. 1:4 Spin-Diluted<br />Fibril<br />Monomer<br />Undiluted Fibril<br />Fibril<br />Spin Exchange in Labeled rPrPAmyloid Fibrils<br />
    25. 25. EPR Signals for Nitroxide-Labeled rPrP Fibrils<br />Cobb et al. (2007) PNAS<br />
    26. 26. 2 M GdnHCl<br />1 M GdnHCl<br />no denaturant<br />Denaturation of Nitroxide-Labeled rPrP Fibrils<br />Monomer<br />Fibril<br />4 M GdnHCl<br />Fibril (no denaturant)<br />Fibril 4 M GdnHCl<br />Cobb et al. (2007) PNAS<br />
    27. 27. 191 undiluted<br />1:4 dilution<br />190/191, 191/192<br />undiluted<br />191 1:1 dilution<br />1:1 dilution<br />Only a Parallel In-Register -Structure can Describe EPR Data<br />-helix<br />-sheet<br />
    28. 28. N181<br />N197<br />Modeling of Prion Protein Amyloid<br /><ul><li>Residues ~160-220 are in-register and parallel with one another
    29. 29. The native disulfide is maintained in the amyloid structure
    30. 30. Asn 181 and 197 must point toward the outside of the amyloid structure</li></ul>Two-bend model<br />One-bend model<br />N197<br />N181<br />PrP monomer<br />
    31. 31. Molecular Details of the Two-Bend Model<br />N197<br />N181<br />Cobb et al. (2007) PNAS<br />
    32. 32. 231<br />90<br />H/D exchange (170-220)<br />EPR data (160-220)<br />Correlation with H/D Exchange Data<br />Lu et al. (2007) PNAS<br />
    33. 33. Left-handed helical model (89-175)<br />Spiral model (116-164)<br />H/D exchange (170-220)<br />EPR data (160-220)<br />Structural Models of Prion Protein Aggregates<br />Pathogenic Mutations of the Prion Protein<br />E196K<br />T188K<br />T188R<br />F198S<br />E200K<br />D202N<br />H187R<br />V203I<br />Insertion of<br />1,2, or 4-9 repeats<br />R208H<br />T183A<br />V210I<br />253<br />P105L<br />P105T<br />Y145stop<br />M232R<br />V180I<br />E211Q<br />P238S<br />Sim and Caughey <br />(2008) <br />Neurobiol. Aging<br />Wille et al. <br />(2002) PNAS<br />Left-handed<br />β-helical model<br />Govaerts et al. <br />(2004) PNAS<br />Parallel an in-register<br />β-structure of rPrP amyloid<br />Cobb et al. (2007) PNAS<br />Q212P<br />P102L<br />A117V<br />D178N<br />Spiral model<br />DeMarco and <br />Daggett (2004) <br />PNAS<br />Q217R<br />Q160stop<br />1<br />51<br />91<br />23<br />90<br />231<br />
    34. 34. Similar Amyloid Fibrils are Formed under <br />Native Conditions <br />Native conditions <br />50 mM acetate<br />pH 4.0<br />Denaturing Conditions <br />50 mM phosphate <br />2M GdnHCl, pH 7.0<br />Cobb & Surewicz (2008) J. Biol. Chem.<br />
    35. 35. pH-Induced Structural Differences at the <br />N-Terminus are Reversible<br /><ul><li> Fibrils formed under</li></ul> native (pH 4.0) conditions<br /><ul><li> Equilibrated to pH 7.0
    36. 36. Fibrils formed under denaturing </li></ul> (2 M GdnHCl, pH 7.0)conditions<br /><ul><li> Equilibrated to pH 4.0 </li></ul>113<br />193<br />193<br />103<br />103<br />113<br />103<br />192<br />132<br />132<br />Cobb & Surewicz (2008) J. Biol. Chem.<br />
    37. 37. Native and Denaturing Conditions Form rPrP Fibrils of Similar Stability<br />Against pH<br />Against GdnHCl<br />● Denaturing<br />○ Native<br />●EPR<br />● Denaturing<br />○ Native<br />●EPR<br />Monomer<br />Fibril<br />4 M GdnHCl<br />Cobb & Surewicz (2008) J. Biol. Chem.<br />
    38. 38. 179<br />179<br />214<br />214<br />pH 4.0-10.0<br />Highly Acidic Conditions Stabilize rPrP<br />Amyloid Fibrils <br />Fewer Stacked Charges<br />at Very Low pH <br />rPrPAmyloid is more Resistant <br />to GdnHCl at Low pH<br />pH 2.0<br />
    39. 39. Replacement of Hydrophobic with Charged <br />Residues Yields Intriguing Results<br />
    40. 40. Prion Strains and the Species Barrier<br />Cobb & Surewicz (2009) Biochemistry<br />
    41. 41. Structurally Distinct rPrPAmyloid Fibrils <br />2 M GdnHCl<br />500 nm<br />23<br />231<br />4 M GdnHCl<br />S<br />S<br />Proteinase K<br />231<br />~87-90<br />S<br />S<br />
    42. 42. Conclusions<br /><ul><li> As deduced by SDSL/EPR, the amyloid core of rPrPamyloid fibrils is comprised </li></ul> of residues ~160/170-220 - there is no evidence of stable secondary structure <br /> outside this β-core region<br /><ul><li> The molecular architecture of rPrPamyloid fibrils is a parallel and in-register </li></ul> stacking motif, where same residues are in direct contact with their counterparts <br /> on neighboring molecules<br /><ul><li> An identical amyloid core is formed under both denaturing and native conditions,</li></ul> indicating that this region is strongly predisposed to forming amyloid fibrils<br /><ul><li> Stacking of same charges appears to be the main destabilizing force in parallel </li></ul> and in-register amyloid fibrils <br /><ul><li> Substitution at only a single residue can result in structurally distinct amyloid</li></ul> fibrils<br /><ul><li> Varying buffer conditions can result in structurally distinct amyloid fibrils – the N-</li></ul> terminal residues 23-90 are important for amyloid formation at high denaturant <br /> concentrations<br />
    43. 43. Acknowledgements<br />Witold K. Surewicz<br />Frank D. Sönnichsen<br />HassaneMchaourab<br />Adrian C. Apetri<br />Xiaojun Lu<br />This work was funded by NIH grants NS 44158, <br />NS 38604, NS 14359 (to W.K.S.), and <br />NIH Training Grant T32 HL07653 (to N.J.C) <br />
    44. 44. Possible Mechanism of PrPScNeuroinvasion<br />Cobb & Surewicz (2009) Biochemistry<br />