Mammalian Prion Protein Research Articles

Cytosolic aggregates perturb the degradation of nontranslocated secretory and membrane proteins

A wide range of diseases are associated with the accumulation of cytosolic protein aggregates. The effects of these aggregates on various aspects of normal cellular protein homeostasis remain to be determined. Here we find that cytosolic aggregates, without necessarily disrupting proteasome function, can markedly delay the normally rapid degradation of nontranslocated secretory and membrane protein precursors. In the case of mammalian prion protein (PrP), the nontranslocated fraction is recruited into preexisting aggregates before its triage for degradation. This recruitment permits the growth and persistence of cytosolic PrP aggregates, explaining their apparent "self-conversion" seen in earlier studies of transient proteasome inhibition. For other proteins, the aggregate-mediated delay in precursor degradation led to aggregation and/or soluble residence in the cytosol, often causing aberrant cellular morphology. Remarkably, improving signal sequence efficiency mitigated these effects of aggregates. These observations identify a previously unappreciated consequence of cytosolic aggregates for nontranslocated secretory and membrane proteins, a minor but potentially disruptive population the rapid disposal of which is critical to maintaining cellular homeostasis.

Characterizing the Interaction Between Phthalocyanine Tetrasulfonates and Mammalian Prion Protein

Phthalocyanine tetrasulfonates are known to interact with mammalian prion protein, having the ability to act as anti-prion agents and help prevent the conversion of native isoform PrPc to the scrapie isoform PrPsc. However, the interaction between phthalocyanines and PrP remains poorly characterized. We explore the phenomenology of this interaction in detail by examining the binding of phthalocyanines to monomeric native Syrian Hamster prion protein using multiple complementary assays: surface plasmon resonance, FCS, fluorescence quenching, calorimetry, and CD spectroscopy. We determine binding constants, kinetics, stoichiometry, structural and spectroscopic effects of binding, and the influence of buffer ionic strength.

Force Spectroscopy of Mammalian Prion Protein Folding and Misfolding

Prions are unconventional infectious agents that have the capacity to trigger the native prion proteins (PrPC) to adopt a misfolded conformation (PrPSc) which leads to aggregation and neurodegenerative disease. Although the mechanism of PrPSc formation is still unknown, partially-folded intermediate or misfolded states are thought to play important roles in the conversion and amyloid formation processes. Here, we study the folding of the prion protein by single molecule force spectroscopy using optical tweezers and present the first direct observation of partially structured misfolded states. Single molecules of recombinant mammalian prion protein held under tension are repeatedly unfolded and refolded, revealing that the prion protein folds and unfolds cooperatively as a two-state system, with no observable on-pathway intermediates. Intriguingly, we observe multiple off-pathway misfolded states with different lifetimes and extensions. We explore the thermodynamics and kinetics of these states and characterize the different folding pathways of the protein.

Stable Kinesin and Dynein Assemblies Drive the Axonal Transport of Mammalian Prion Protein Vesicles

Kinesin and dynein are opposite-polarity microtubule motors that drive the tightly regulated transport of a variety of cargoes. Both motors can bind to cargo, but their overall composition on axonal vesicles and whether this composition directly modulates transport activity are unknown. Here we characterize the intracellular transport and steady-state motor subunit composition of mammalian prion protein (PrP(C)) vesicles. We identify Kinesin-1 and cytoplasmic dynein as major PrP(C) vesicle motor complexes and show that their activities are tightly coupled. Regulation of normal retrograde transport by Kinesin-1 is independent of dynein-vesicle attachment and requires the vesicle association of a complete Kinesin-1 heavy and light chain holoenzyme. Furthermore, motor subunits remain stably associated with stationary as well as with moving vesicles. Our data suggest a coordination model wherein PrP(C) vesicles maintain a stable population of associated motors whose activity is modulated by regulatory factors instead of by structural changes to motor-cargo associations.

Sequence-dependent Prion Protein Misfolding and Neurotoxicity

Prion diseases are neurodegenerative disorders caused by misfolding of the normal prion protein (PrP) into a pathogenic "scrapie" conformation. To better understand the cellular and molecular mechanisms that govern the conformational changes (conversion) of PrP, we compared the dynamics of PrP from mammals susceptible (hamster and mouse) and resistant (rabbit) to prion diseases in transgenic flies. We recently showed that hamster PrP induces spongiform degeneration and accumulates into highly aggregated, scrapie-like conformers in transgenic flies. We show now that rabbit PrP does not induce spongiform degeneration and does not convert into scrapie-like conformers. Surprisingly, mouse PrP induces weak neurodegeneration and accumulates small amounts of scrapie-like conformers. Thus, the expression of three highly conserved mammalian prion proteins in transgenic flies uncovered prominent differences in their conformational dynamics. How these properties are encoded in the amino acid sequence remains to be elucidated.

Prion disease susceptibility is affected by β-structure folding propensity and local side-chain interactions in PrP

Prion diseases occur when the normally α-helical prion protein (PrP) converts to a pathological β-structured state with prion infectivity (PrP(Sc)). Exposure to PrP(Sc) from other mammals can catalyze this conversion. Evidence from experimental and accidental transmission of prions suggests that mammals vary in their prion disease susceptibility: Hamsters and mice show relatively high susceptibility, whereas rabbits, horses, and dogs show low susceptibility. Using a novel approach to quantify conformational states of PrP by circular dichroism (CD), we find that prion susceptibility tracks with the intrinsic propensity of mammalian PrP to convert from the native, α-helical state to a cytotoxic β-structured state, which exists in a monomer-octamer equilibrium. It has been controversial whether β-structured monomers exist at acidic pH; sedimentation equilibrium and dual-wavelength CD evidence is presented for an equilibrium between a β-structured monomer and octamer in some acidic pH conditions. Our X-ray crystallographic structure of rabbit PrP has identified a key helix-capping motif implicated in the low prion disease susceptibility of rabbits. Removal of this capping motif increases the β-structure folding propensity of rabbit PrP to match that of PrP from mouse, a species more susceptible to prion disease.

Compartment-restricted biotinylation reveals novel features of prion protein metabolism in vivo.

Proteins are often made in more than one form, with alternate versions sometimes residing in different cellular compartments than the primary species. The mammalian prion protein (PrP), a cell surface GPI-anchored protein, is a particularly noteworthy example for which minor cytosolic and transmembrane forms have been implicated in disease pathogenesis. To study these minor species, we used a selective labeling strategy in which spatially restricted expression of a biotinylating enzyme was combined with asymmetric engineering of the cognate acceptor sequence into PrP. Using this method, we could show that even wild-type PrP generates small amounts of the (Ctm)PrP transmembrane form. Selective detection of (Ctm)PrP allowed us to reveal its N-terminal processing, long half-life, residence in both intracellular and cell surface locations, and eventual degradation in the lysosome. Surprisingly, some human disease-causing mutants in PrP selectively stabilized (Ctm)PrP, revealing a previously unanticipated mechanism of (Ctm)PrP up-regulation that may contribute to disease. Thus, spatiotemporal tagging has uncovered novel aspects of normal and mutant PrP metabolism and should be readily applicable to the analysis of minor topologic isoforms of other proteins.

Comparison studies of the structural stability of rabbit prion protein with human and mouse prion proteins

Background: Prion diseases are fatal and infectious neurodegenerative diseases affecting humans and animals. Rabbits are one of the few mammalian species reported to be resistant to infection from prion diseases isolated from other species (I. Vorberg et al., Journal of Virology 77 (3) (2003) 2003–2009). Thus the study of rabbit prion protein structure to obtain insight into the immunity of rabbits to prion diseases is very important.Findings: The paper is a straight forward molecular dynamics simulation study of wild-type rabbit prion protein (monomer cellular form) which apparently resists the formation of the scrapie form. The comparison analyses with human and mouse prion proteins done so far show that the rabbit prion protein has a stable structure. The main point is that the enhanced stability of the C-terminal ordered region especially helix 2 through the D177–R163 salt-bridge formation renders the rabbit prion protein stable. The salt bridge D201–R155 linking helixes 3 and 1 also contributes to the structural stability of rabbit prion protein. The hydrogen bond H186–R155 partially contributes to the structural stability of rabbit prion protein.Conclusions: Rabbit prion protein was found to own the structural stability, the salt bridges D177–R163, D201–R155 greatly contribute and the hydrogen bond H186–R155 partially contributes to this structural stability. The comparison of the structural stability of prion proteins from the three species rabbit, human and mouse showed that the human and mouse prion protein structures were not affected by the removing these two salt bridges. Dima et al. (Biophysical Journal 83 (2002) 1268–1280 and Proceedings of the National Academy of Sciences of the United States of America 101 (2004) 15335–15340) also confirmed this point and pointed out that “correlated mutations that reduce the frustration in the second half of helix 2 in mammalian prion proteins could inhibit the formation of PrPSc”.

Expression and knockdown of cellular prion protein (PrP C) in differentiating mouse embryonic stem cells

The mammalian cellular prion protein (PrP C) is a highly conserved glycoprotein that may undergo conversion into a conformationally altered isoform (scrapie prion protein or PrP Sc), widely believed to be the pathogenic agent of transmissible spongiform encephalopathies (TSEs). Although much is known about pathogenic PrP conversion and its role in TSEs, the normal function of PrP C is poorly understood. Given the abundant expression of PrP C in the developing mammalian CNS and the spatial association with differentiated stages of neurogenesis, recently it has been proposed that PrP C participates in neural cell differentiation. In the present study, we investigated the role of PrP C in neural development during early embryogenesis. In bovine fetuses, PrP C was differentially expressed in the neuroepithelium, showing higher levels at the intermediate and marginal layers where more differentiated states of neurogenesis were located. We utilized differentiating mouse embryonic stem (ES) cells to test whether PrP C contributed to the process of neural differentiation during early embryogenesis. PrP C showed increasing levels of expression starting on Day 9 until Day 18 of ES cell differentiation. PrP C expression was negatively correlated with pluripotency marker Oct-4 confirming that ES cells had indeed differentiated. Induction of ES cells differentiation by retinoic acid (RA) resulted in up-regulation of PrP C at Day 20 and nestin at Day 12. PrP C expression was knocked down in PrP-targeted siRNA ES cells between Days 12 and 20. PrP C knockdown in ES cells resulted in nestin reduction at Days 16 and 20. Analysis of bovine fetuses suggests the participation of PrP C in neural cell differentiation during early embryogenesis. The positive association between PrP C and nestin expression provide evidence for the contribution of PrP C to ES cell differentiation into neural progenitor cells.

Loss of Octarepeats in Two Processed Prion Pseudogenes in the Red Squirrel, Sciurus vulgaris

The N-terminal region of the mammalian prion protein (PrP) contains an ‘octapeptide’ repeat which is involved in copper binding. This eight- or nine-residue peptide is repeated four to seven times, depending on the species, and polymorphisms in repeat number do occur. Alleles with three repeats are very rare in humans and goats, and deduced PrP sequences with two repeats have only been reported in two lemur species and in the red squirrel, Sciurus vulgaris. We here describe that the red squirrel two-repeat PrP sequence actually represents a retroposed pseudogene, and that an additional and older processed pseudogene with three repeats also occurs in this species as well as in ground squirrels. We argue that repeat numbers may tend to contract rather than expand in prion retropseudogenes, and that functional prion genes with two repeats may not be viable.Electronic supplementary materialThe online version of this article (doi:10.1007/s00239-010-9390-7) contains supplementary material, which is available to authorized users.

Horse Prion Protein NMR Structure and Comparisons with Related Variants of the Mouse Prion Protein

The NMR structure of the horse ( Equus caballus) cellular prion protein at 25 °C exhibits the typical PrP C [cellular form of prion protein (PrP)] global architecture, but in contrast to most other mammalian PrP Cs, it contains a well-structured loop connecting the β2 strand with the α2 helix. Comparison with designed variants of the mouse prion protein resulted in the identification of a single amino acid exchange within the loop, D167S, which correlates with the high structural order of this loop in the solution structure at 25 °C and is unique to the PrP sequences of equine species. The β2–α2 loop and the α3 helix form a protein surface epitope that has been proposed to be the recognition area for a hypothetical chaperone, “protein X,” which would promote conversion of PrP C into the disease-related scrapie form and thus mediate intermolecular interactions related to the transmission barrier for transmissible spongiform encephalopathies (TSEs) between different species. The present results are evaluated in light of recent indications from in vivo experiments that the local β2–α2 loop structure affects the susceptibility of transgenic mice to TSEs and the fact that there are no reports on TSE in horses.

Signal sequence insufficiency contributes to neurodegeneration caused by transmembrane prion protein

Protein translocation into the endoplasmic reticulum is mediated by signal sequences that vary widely in primary structure. In vitro studies suggest that such signal sequence variations may correspond to subtly different functional properties. Whether comparable functional differences exist in vivo and are of sufficient magnitude to impact organism physiology is unknown. Here, we investigate this issue by analyzing in transgenic mice the impact of signal sequence efficiency for mammalian prion protein (PrP). We find that replacement of the average efficiency signal sequence of PrP with more efficient signals rescues mice from neurodegeneration caused by otherwise pathogenic PrP mutants in a downstream hydrophobic domain (HD). This effect is explained by the demonstration that efficient signal sequence function precludes generation of a cytosolically exposed, disease-causing transmembrane form of PrP mediated by the HD mutants. Thus, signal sequences are functionally nonequivalent in vivo, with intrinsic inefficiency of the native PrP signal being required for pathogenesis of a subset of disease-causing PrP mutations.

Prions, protein homeostasis, and phenotypic diversity

Prions are fascinating but often misunderstood protein aggregation phenomena. The traditional association of the mammalian prion protein with disease has overshadowed a potentially more interesting attribute of prions: their ability to create protein-based molecular memories. In fungi, prions alter the relationship between genotype and phenotype in a heritable way that diversifies clonal populations. Recent findings in yeast indicate that prions might be much more common than previously realized. Moreover, prion-driven phenotypic diversity increases under stress, and can be amplified by the dynamic maturation of prion-initiating states. In this article, we suggest that these qualities allow prions to act as 'bet-hedging' devices that facilitate the adaptation of yeasts to stressful environments, and might speed the evolution of new traits.

Descent of the Prion Protein Gene Family from the Extracellular Domain of an Ancestral Zip Metal Ion Transporter

Since its discovery over twenty years ago, both the phylogenetic origin and the cellular function of the prion protein (PrP) have remained enigmatic. The subsequent discovery of the PrP paralog Doppel (Dpl) has strengthened the argument that the copper-binding ability of PrP and Dpl may be part of their cellular function.Chemical crosslinking provides insights into a possible function of PrP through the characterization of its molecular neighborhood on the cell surface. Quantitative interactome data demonstrated the spatial proximity of two putative zinc ion transporters of the ZIP family, ZIP6 (Slc39a6) and ZIP10 (Slc39a10), to mammalian prion proteins in vivo. A subsequent bioinformatic analysis revealed the unexpected presence of a PrP-like amino acid sequence within the N-terminal, extracellular domain of a distinct sub-branch of the ZIP protein family, which includes ZIP5, ZIP6 and ZIP10. Structural threading and orthologous sequence alignment analyses argue that the prion protein gene family is phylogenetically derived from a ZIP-like ancestral molecule. The level of sequence homology and the presence of prion protein genes in most chordate species place the split from the ZIP-like ancestor gene at the base of the chordate lineage. This relationship explains structural and functional features found within mammalian prion proteins as elements of an ancient involvement in the transmembrane transport of divalent cations, presumably zinc and/or copper. The phylogenetic and spatial connection to ZIP proteins is expected to open new and thus unexplored avenues of research to elucidate the biology of the prion protein in health and disease.

Possible Pathway between Alpha Helical and Beta Helical Structures of the C-terminal in the Mammalian Prion Protein

The normal form of the prion protein(PrPC) has mostly alpha-helical (AH) secondary structure in the C-terminal region (residues 166-230), while the infectious form (PrPSc) has been proposed to have a left-handed beta helical (LHBH) structure(1). The mechanism of conformational change from PrPC to PrPSc is unknown, but recent electron microscope data(2) and computer modeling(3) of in vitro grown prion fibrils suggest a possible LHBH structure in the C-terminal region. We use high temperature (500K) AMBER molecular dynamics over 10 ns runtimes to study the unfolding transitions commencing from both LHBH and AH C-terminal starting structures. Using stability, contact map, and energetic analyses we find that both structures unfold to very similar AH-like conformations and discuss the potential implications of this result for normal prion cellular function and for prion disease.References1) Govaerts, C., et al. (2004) PNAS101, 8342-8347.2) Tattum, M. H., et al. (2006) J. Mol. Biol.357, 975-985.3) Kunes, K. C., et al. (2008) Prion2, 81-90.The image below compares computational models of the initial LHBH structure(left), the initial AH structure(right), and the unfolded AH-like structure(middle).View Large Image | View Hi-Res Image | Download PowerPoint Slide

Structure-Based Models for Alpha-Helical to Beta-Helical Conformation Change in the C-Terminal of the Mammalian Prion Protein

We employ all atom structure-based models with mixed basis contact maps to explore where there are any significant geometric or energetic constraints limiting conjectured conformational transitions between the alpha-helical (αH) and the left handed beta helical (LHβH) conformations for the C-terminal (residues 166-230) of the mammalian prion protein. The LHβH structure has been proposed to describe infection oligomers(1) and one class of in vitro grown fibrils(2,3), as well as possibly self-templating the conversion of normal cellular prion protein to the infectious form. The structure-based model uses GROMACS based molecular dynamics with a two-dimensional weighted histogram analysis method (WHAM) being applied to study projected energy surfaces. Our preliminary results confirm that the kinetics of the conformation change are not strongly limited by the large scale geometry modification, and evidence exists for a pathway linking the two conformations with a common folding intermediate, also suggested by all atom unfolding simulations(4).(1) Govaerts C., et. al. Evidence for assembly of prions with left-handed beta-helices into trimers. Proc Natl Acad Sci USA 2004; 101; 8342-8347(2) Tattum M. H., et. al. Elongated oligomers assemble into mammalian PrP amyloid fibrils. J. Mol. Biol 2006; 357; 975-985(3) Kunes K., et. al. Left handed β helix models for mammalian prion fibrils. Prion 2008; 2; 81-90(4) See S. Dai and D.L. Cox, abstract elsewhere for this meeting∗Research supported in part by the International Institute for Complex Adaptive Matter, NSF Grant DMR-0844115

Ligand Binding and Hydration in Protein Misfolding: Insights from Studies of Prion and p53 Tumor Suppressor Proteins

Protein misfolding has been implicated in a large number of diseases termed protein- folding disorders (PFDs), which include Alzheimer’s disease, Parkinson’s disease, transmissible spongiform encephalopathies, familial amyloid polyneuropathy, Huntington’s disease, and type II diabetes. In these diseases, large quantities of incorrectly folded proteins undergo aggregation, destroying brain cells and other tissues.The interplay between ligand binding and hydration is an important component of the formation of misfolded protein species. Hydration drives various biological processes, including protein folding, ligand binding, macromolecular assembly, enzyme kinetics, and signal transduction. The changes in hydration and packing, both when proteins fold correctly or when folding goes wrong, leading to PFDs, are examined through several biochemical, biophysical, and structural approaches. Although in many cases the binding of a ligand such as a nucleic acid helps to prevent misfolding and aggregation, there are several examples in which ligands induce misfolding and assembly into amyloids. This occurs simply because the formation of structured aggregates (such as protofibrillar and fibrillar amyloids) involves decreases in hydration, formation of a hydrogen-bond network in the secondary structure, and burying of nonpolar amino acid residues, processes that also occur in the normal folding landscape. In this Account, we describe the present knowledge of the folding and misfolding of different proteins, with a detailed emphasis on mammalian prion protein (PrP) and tumoral suppressor protein p53; we also explore how ligand binding and hydration together influence the fate of the proteins.Anfinsen’s paradigm that the structure of a protein is determined by its amino acid sequence is to some extent contradicted by the observation that there are two isoforms of the prion protein with the same sequence: the cellular and the misfolded isoform. The cellular isoform of PrP has a disordered N-terminal domain and a highly flexible, not-well-packed C-terminal domain, which might account for its significant hydration. When PrP binds to biological molecules, such as glycosaminoglycans and nucleic acids, the disordered segments appear to fold and become less hydrated. Formation of the PrP−nucleic acid complex seems to accelerate the conversion of the cellular form of the protein into the disease-causing isoform. For p53, binding to some ligands, including nucleic acids, would prevent misfolding of the protein. Recently, several groups have begun to analyze the folding−misfolding of the individual domains of p53, but several questions remain unanswered. We discuss the implications of these findings for understanding the productive and incorrect folding pathways of these proteins in normal physiological states and in human disease, such as prion disorders and cancer. These studies are shown to lay the groundwork for the development of new drugs.

Natural and synthetic prion structure from X-ray fiber diffraction

A conformational isoform of the mammalian prion protein (PrP(Sc)) is the sole component of the infectious pathogen that causes the prion diseases. We have obtained X-ray fiber diffraction patterns from infectious prions that show cross-beta diffraction: meridional intensity at 4.8 A resolution, indicating the presence of beta strands running approximately at right angles to the filament axis and characteristic of amyloid structure. Some of the patterns also indicated the presence of a repeating unit along the fiber axis, corresponding to four beta-strands. We found that recombinant (rec) PrP amyloid differs substantially from highly infectious brain-derived prions, both in structure as demonstrated by the diffraction data, and in heterogeneity as shown by electron microscopy. In addition to the strong 4.8 A meridional reflection, the recPrP amyloid diffraction is characterized by strong equatorial intensity at approximately 10.5 A, absent from brain-derived prions, and indicating the presence of stacked beta-sheets. Synthetic prions recovered from transgenic mice inoculated with recPrP amyloid displayed structural characteristics and homogeneity similar to those of naturally occurring prions. The relationship between the structural differences and prion infectivity is uncertain, but might be explained by any of several hypotheses: only a minority of recPrP amyloid possesses a replication-competent conformation, the majority of recPrP amyloid has to undergo a conformational maturation to acquire replication competency, or inhibitory forms of recPrP amyloid interfere with replication during the initial transmission.

Influence of divalent copper, manganese and zinc ions on fibril nucleation and elongation of the amyloid-like yeast prion determinant Sup35p-NM

There is a large body of evidence that divalent metal ions, particularly copper, might play a role in several protein folding pathologies like Alzheimer's disease, Parkinson's disease or the prion diseases. However, contribution of metal ions on pathogenesis and their molecular influence on the formation of amyloid structures is not clear. Therefore, the general influence of metals on the formation of amyloids is still controversially discussed. We have utilized the well established system of yeast Sup35p-NM to investigate the role of three different metal ions, Cu(2+), Mn(2+) and Zn(2+), on amyloidogenesis. Recently, it has been shown that the prion determining region NM of the Saccharomyces cerevisiae prion protein Sup35p, which is responsible for the yeast prion phenotype [PSI(+)], specifically binds Cu(2+) ions. We further characterized the affinity of NM for Cu(2+), which were found to be comparable to that of other amyloidogenic proteins like the mammalian prion protein PrP. The specific binding sites could be located in the aminoterminal N-region which is known to initiate formation of amyloidogenic nuclei. In the presence of Cu(2+), fibril nucleation was significantly delayed, probably due to influences of copper on the oligomeric ensemble of soluble Sup35p-NM, since Cu(2+) altered the tertiary structure of soluble Sup35p-NM, while no influences on fibril elongation could be detected. The secondary structure of soluble or fibrous protein and the morphology of the fibrils were apparently not altered when assembled in presence of Cu(2+). In contrast, Mn(2+) and Zn(2+) did not bind to Sup35p-NM and did not exhibit significant effects on the formation of NM amyloid fibrils.

Evolutionary descent of prion genes from the ZIP family of metal ion transporters.

In the more than twenty years since its discovery, both the phylogenetic origin and cellular function of the prion protein (PrP) have remained enigmatic. Insights into a possible function of PrP may be obtained through the characterization of its molecular neighborhood in cells. Quantitative interactome data demonstrated the spatial proximity of two metal ion transporters of the ZIP family, ZIP6 and ZIP10, to mammalian prion proteins in vivo. A subsequent bioinformatic analysis revealed the unexpected presence of a PrP-like amino acid sequence within the N-terminal, extracellular domain of a distinct sub-branch of the ZIP protein family that includes ZIP5, ZIP6 and ZIP10. Additional structural threading and orthologous sequence alignment analyses argued that the prion gene family is phylogenetically derived from a ZIP-like ancestral molecule. The level of sequence homology and the presence of prion protein genes in most chordate species place the split from the ZIP-like ancestor gene at the base of the chordate lineage. This relationship explains structural and functional features found within mammalian prion proteins as elements of an ancient involvement in the transmembrane transport of divalent cations. The phylogenetic and spatial connection to ZIP proteins is expected to open new avenues of research to elucidate the biology of the prion protein in health and disease.