PrP106-126 peptide disrupts lipid membranes: Influence of C-terminal amidation

PrP106-126 is located within the important domain concerning membrane related conformational conversion of human Prion protein (from cellular isoform PrP^C to scrapie isoform PrP^Sc). Recent advances reveal that the pathological and physicochemical properties of PrP106-126 peptide are very sensitive to its N-terminal amidation, however, the detailed mechanism remains unclear. In this work, we studied the interactions of the PrP106-126 isoforms (PrP106-126_CONH2 and PrP106-126_COOH) with the neutral lipid bilayers by atomic force microscopy, surface plasmon resonance and fluorescence spectroscopy. The membrane structures were disturbed by the two isoforms in a similarly stepwise process. The distinct morphological changes of the membrane were characterized by formation of semi-penetrated defects and sigmoidal growth of flat high-rise domains on the supported lipid bilayers. However, PrP106-126_COOH displayed a higher peptide-lipid binding affinity than PrP106-126_CONH2 (∼2.9 times) and facilitated the peptide-lipid interactions by shortening the lag time. These results indicate that the C-terminal amidation may influence the pathological actions of PrP106-126 by lowering the interaction potentials with lipid membranes.     

PrP106-126 amide causes the semi-penetrated poration in the supported lipid bilayers

A major hallmark of prion diseases is the cerebral amyloid accumulation of the pathogenic PrP(Sc), an abnormally misfolded, protease-resistant, and beta-sheet rich protein. PrP106-126 is the key domain responsible for the conformational conversion and aggregation of PrP. It shares important physicochemical characteristics with PrP(Sc) and presents similar neurotoxicity as PrP(Sc). By combination of fluorescence polarization, dye release assay and in situ time-lapse atomic force microscopy (AFM), we investigated the PrP106-126 amide interacting with the large unilamellar vesicles (LUVs) and the supported lipid bilayers (SLBs). The results suggest that the interactions involve a poration-mediated process: firstly, the peptide binding results in the formation of pores in the membranes, which penetrate only half of the membranes; subsequently, PrP106-126 amide undergoes the poration-mediated diffusion in the SLBs, represented by the formation and expansion of the flat high-rise domains (FHDs). The possible mechanisms of the interactions between PrP106-126 amide and lipid membranes are proposed based on our observations.     

Octapeptide repeat region of prion protein (PrP) is required at an early stage for production of abnormal prion protein in PrP-deficient neuronal cell line

An abnormal isoform of prion protein (PrP^Sc), which is composed of the same amino acids as cellular PrP (PrP^C) and has proteinase K (PK)-resistance, hypothetically converts PrP^C into PrP^Sc. To investigate the region important for PrP^Sc production, we examined the levels of PrP^Sc in PrP gene-deficient cells (HpL3-4) expressing PrP^C deleted of various regions including the octapeptide repeat region (OR) or hydrophobic region (HR). After Chandler or Obihiro prion infection, PrP^Sc was produced in HpL3-4 cells expressing wild-type PrP^C or PrP^C deleted of HR at an early stage and further reduced to below the detectable level, whereas cells expressing PrP^C deleted of OR showed no PrP^Sc production. The results suggest that OR of PrP^C is required for the early step of efficient PrP^Sc production.     

PrP(106-126) does not interact with membranes under physiological conditions

Transmissible spongiform encephalopathies are neurodegenerative diseases characterized by the accumulation of an abnormal isoform of the prion protein PrP^Sc. Its fragment 106-126 has been reported to maintain most of the pathological features of PrP^Sc, and a role in neurodegeneration has been proposed based on the modulation of membrane properties and channel formation. The ability of PrP^Sc to modulate membranes and/or form channels in membranes has not been clearly demonstrated; however, if these processes are important, peptide-membrane interactions would be a key feature in the toxicity of PrP^Sc. In this work, the interaction of PrP(106-126) with model membranes comprising typical lipid identities, as well as more specialized lipids such as phosphatidylserine and GM1 ganglioside, was examined using surface plasmon resonance and fluorescence methodologies. This comprehensive study examines different parameters relevant to characterization of peptide-membrane interactions, including membrane charge, viscosity, lipid composition, pH, and ionic strength. We report that PrP(106-126) has a low affinity for lipid membranes under physiological conditions without evidence of membrane disturbances. Membrane insertion and leakage occur only under conditions in which strong electrostatic interactions operate. These results support the hypothesis that the physiological prion protein PrP^C mediates PrP(106-126) toxic effects in neuronal cells.     

The pathological prion protein forms ionic conductance in lipid bilayer

Transmissible spongiform encephalopathies (TSEs) are neurodegenerative pathologies characterized by the accumulation of amyloid fibrils mainly composed of the pathological isoform of the prion protein (PrP^TSE). PrP^TSE pre-amyloid fibrils are supposed to induce neurodegenerative lesions possibly through the alteration of membrane permeability. The effect of PrP^TSE on cellular membranes has been modeled in vitro by synthetic peptides that are, however, only partially representative of PrP^TSE isoforms found in vivo. In the present work we show that a synthetic membrane exposed to PrP27-30 extracted from TSE-infected hamster brains changes its permeability because of the formation of molecular pores that alter the conductance of the synthetic lipid bilayer. Synthetic membrane challenged with the recombinant prion peptide PrP90-231 shows a much lower conductance. Elevation of calcium ion concentration not only increases the current amplitude due to the action of both PrP27-30 and PrP90-231 on the membrane, but also amplifies the interaction of PrP90-231 with the lipid bilayer.     

Small stress molecules inhibit aggregation and neurotoxicity of prion peptide 106-126

In prion diseases, the posttranslational modification of host-encoded prion protein PrP^c yields a high β-sheet content modified protein PrP^sc, which further polymerizes into amyloid fibrils. PrP106-126 initiates the conformational changes leading to the conversion of PrP^c to PrP^sc. Molecules that can defunctionalize such peptides can serve as a potential tool in combating prion diseases. In microorganisms during stressed conditions, small stress molecules (SSMs) are formed to prevent protein denaturation and maintain protein stability and function. The effect of such SSMs on PrP106-126 amyloid formation is explored in the present study using turbidity, atomic force microscopy (AFM), and cellular toxicity assay. Turbidity and AFM studies clearly depict that the SSMs—ectoine and mannosylglyceramide (MGA) inhibit the PrP106-126 aggregation. Our study also connotes that ectoine and MGA offer strong resistance to prion peptide-induced toxicity in human neuroblastoma cells, concluding that such molecules can be potential inhibitors of prion aggregation and toxicity.     

Molecular model of an alpha-helical prion protein dimer and its monomeric subunits as derived from chemical cross-linking and molecular modeling calculations

Prions are the agents of a series of lethal neurodegenerative diseases. They are composed largely, if not entirely, of the host-encoded prion protein (PrP), which can exist in the cellular isoform PrP^C and the pathological isoform PrP^Sc. The conformational change of the α-helical PrP^C into β-sheet-rich PrP^Sc is the fundamental event of prion disease. The transition of recombinant PrP from a PrP^C-like into a PrP^Sc-like conformation can be induced in vitro by submicellar concentrations of SDS. An α-helical dimer was identified that might represent either the native state of PrP^C or the first step from the monomeric PrP^C to highly aggregated PrP^Sc. In the present study, the molecular structure of these dimers was analyzed by introducing covalent cross-links using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide. Inter- and intramolecular bonds between directly neighboured amino groups and carboxy groups were generated. The bonds formed in PrP dimers of recombinant PrP (90-231) were identified by tryptic digestion and subsequent mass spectrometric analysis. Intra- and intermolecular cross-links between N-terminal glycine and three acidic amino acid side chains in the globular part of PrP were identified, showing the N-terminal amino acids (90-124) are not as flexible as known from NMR analysis. When the cross-linked sites were used as structural constraint, molecular modeling calculations yielded a structural model for PrP dimer and its monomeric subunit, including the folding of amino acids 90-124 in addition to the known structure. Molecular dynamics of the structure after release of the constraint indicated an intrinsic stability of the domain of amino acids 90-124.     

Influence of the pathogenic mutations T188K/R/A on the structural stability and misfolding of human prion protein: Insight from molecular dynamics simulations

Background

Prion diseases are associated with a conformational switch for PrP from PrP^C to PrP^Sc. Many genetic mutations are linked with prion diseases, such as mutations T188K/R/A with fCJD.

Scope of review

MD simulations for the WT PrP and its mutants were performed to explore the underlying dynamic effects of T188 mutations on human PrP. Although the globular domains are fairly conserved, the three mutations have diverse effects on the dynamics properties of PrP, including the shift of H1, the elongation of native β-sheet and the conversion of S2-H2 loop to a 3₁₀ helix.

Major conclusions

Our present study indicates that the three mutants for PrP may undergo different pathogenic mechanisms and the realistic atomistic simulations can provide insights into the effects of disease-associated mutations on PrP dynamics and stability, which can enhance our understanding of how mutations induce the conversion from PrP^C to PrP^Sc.General significanceOur present study helps to understand the effects of T188K/R/A mutations on human PrP: despite the three pathogenic mutations almost do not alter the native structure of PrP, but perturb its stability. This instability may further modulate the oligomerization pathways and determine the features of the PrP^Sc assemblies.     

Prion Protein NMR Structure from Tammar Wallaby (Macropus eugenii) Shows that the β2-α2 Loop Is Modulated by Long-Range Sequence Effects

NMR structures are presented for the recombinant construct of residues 121-230 from the tammar wallaby (Macropus eugenii) prion protein (PrP) twPrP(121-230) and for the variant mouse PrPs mPrP[Y225A,Y226A](121-231) and mPrP[V166A](121-231) at 20 °C and pH 4.5. All three proteins exhibit the same global architecture as seen in other recombinant PrP^Cs (cellular isoforms of PrP) and shown to prevail in natural bovine PrP^C. Special interest was focused on a loop that connects the β2-strand with helix α2 in the PrP^C fold, since there are indications from in vivo experiments that this local structural feature affects the susceptibility of transgenic mice to transmissible spongiform encephalopathies. This β2-α2 loop and helix α3 form a solvent-accessible contiguous epitope, which has been proposed to be the recognition area for a hypothetical chaperone, the “protein X”. This hypothetical chaperone would affect the conversion of PrP^C into the disease-related scrapie form (PrP^Sc) by moderating intermolecular interactions related to the transmission barrier of transmissible spongiform encephalopathies between different species. In contrast to mPrP(121-231) and most other mammalian PrP^Cs, the β2-α2 loop is well defined at 20 °C in tammar wallaby PrP and in the two aforementioned variants of mPrP, showing that long-range interactions with helix α3 can have an overriding influence on the structural definition of the β2-α2 loop. Further NMR studies with two variant mPrPs, mPrP[Y225A](121-231) and mPrP[Y226A](121-231), showed that these interactions are dominantly mediated by close contacts between residues 166 and 225. The results of the present study then lead to the intriguing indication that well-defined long-range intramolecular interactions could act as regulators of the functional specificity of PrP^C.     

Insight into early events in the aggregation of the prion protein on lipid membranes

The key molecular event underlying prion diseases is the conversion of the monomeric and α-helical cellular form of the prion protein (PrP^C) to the disease-associated state, which is aggregated and rich in β-sheet (PrP^Sc). The molecular details associated with the conversion of PrP^C into PrP^Sc are not fully understood. The prion protein is attached to the cell membrane via a GPI lipid anchor and evidence suggests that the lipid environment plays an important role in prion conversion and propagation. We have previously shown that the interaction of the prion protein with anionic lipid membranes induces β-sheet structure and promotes prion aggregation, whereas zwitterionic membranes stabilize the α-helical form of the protein. Here, we report on the interaction of recombinant sheep prion protein with planar lipid membranes in real-time, using dual polarization interferometry (DPI). Using this technique, the simultaneous evaluation of multiple physical properties of PrP layers on membranes was achieved. The deposition of prion on membranes of POPC and POPC/POPS mixtures was studied. The properties of the resulting protein layers were found to depend on the lipid composition of the membranes. Denser and thicker protein deposits formed on lipid membranes containing POPS compared to those formed on POPC. DPI thus provides a further insight on the organization of PrP at the surface of lipid membranes.     

The prion 2018 round tables (I): the structure of PrPSc

Understanding the structure of PrP^Sc is without doubt a sine qua non to understand not only PrP^Sc propagation, but also critical features of that process such as the strain phenomenon and transmission barriers. While elucidation of the PrP^Sc structure has been full of difficulties, we now have a large amount of structural information that allows us to begin to understand it. This commentary article summarizes a round table that took place within the Prion 2018 meeting held in Santiago de Compostela to discuss the state of the art in this matter. Two alternative models of PrP^Sc exist: the PIRIBS and the 4-rung β-solenoid models. Both of them have relevant features. The 4-rung β-solenoid model agrees with experimental constraints of brain derived PrP^Sc obtained from cryo-EM and X-ray fiber diffraction studies. Furthermore, it allows facile accommodation of the bulky glycans that decorate brain-derived PrP^Sc. On the other hand, the infectious PrP23-144 amyloid exhibits a PIRIBS architecture. Perhaps, both types of structure co-exist.     

Cellular uptake of the prion protein fragment PrP106-126 in vitro

The aetiological agent of prion disease is proposed to be an aberrant isoform of the cell surface glycoprotein known as the prion protein (PrP^c). This pathological isoform (PrP^Sc) is abnormally deposited in the extracellular space of diseased CNS. Neurodegeneration in these disease has been shown to be associated with accumulation of PrP^Sc in affected tissue. To investigate the possible uptake mechanisms that may be required for PrP^Sc-induced neurodegeneration we studied the cellular trafficking of the neurotoxic fragment, PrP106-126. We were able to detect, by fluorescence microscopy, PrP106-126 inclusions in murine neurones, astrocytes and microglia in vitro. These inclusions were abundant after 24 hour exposure and still present 48h post-exposure. Shorter exposure times yielded only occasional cells with inclusions. Large extracellular aggregates of PrP106-126 could also be detected, which appeared in a time dependent manner. The appearance of inclusions or aggregates was not dependent on PrP^c expression as determined by exposure of peptides from PrP-null mice. Using transmission electron microscopy and gold particle detection, positively labelled osmiophilic inclusions of peptide could be detected in the cytoplasm of exposed cells. These results demonstrate that cultured cells are capable of sequestering PrP106-126 and may indicate uptake pathways for PrP^Sc in various cell types. Toxicity of PrP106-126 may thus be mediated via a sequestration pathway that is not effective for this peptide in PrP-null cells.     

Induction of ligand-specific PrPC signaling in human neuronal cells

Cellular prion protein (PrP^C) has attracted considerable attention for its role in transmissible spongiform encephalopathies (TSEs). In spite of being a point of intense research effort critical questions still remain regarding the physiological function of PrP^C and how these functions may change with the conversion of the protein into the infectious and pathological conformation (PrP^Sc). While emerging evidence suggests PrP^C/Sc are involved in signal transduction there is little consensus on the signaling pathways associated with the normal and diseased states. The purported involvement of PrP^C in signal transduction, and the association of TSEs with neural pathology, makes kinome analysis of human neurons an interesting and appropriate model to characterize patterns of signal transduction following activation of PrP^C by two commonly employed experimental ligands; antibody-induced dimerization by 6H4 and the amino acids 106-126 PrP peptide fragment (PrP 106–126). Analysis of the induced kinome responses reveals distinct patterns of signaling activity following each treatment. Specifically, stimulation of human neurons with the 6H4 antibody results in alterations in mitogen activated protein kinase (MAPK) signaling pathways while the 106-126 peptide activates growth factor related signaling pathways including vascular endothelial growth factor (VEGF) signaling and the phosphoinositide-3 kinase (PI3K) pathway. These pathways were validated through independent functional assays. Collectively these results indicate that stimulation of PrP^C with distinct ligands, even within the same cell type, results in unique patterns of signaling. While this investigation highlights the apparent functional versatility of PrP^C as a signaling molecule and may offer insight into cellular mechanisms of TSE pathology it also emphasizes the potential dangers associated with attributing activation of specific intracellular events to particular receptors through artificial models of receptor activation.     

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.     

Molecular dynamics study of the fibril elongation of the prion protein fragment PrP106-126

The present paper aims at exploring the elongation of the PrP106-126 fibril under acid environments through molecular dynamics simulation. It shows that influenced by the edge strands of the fibril, single PrP106-126 peptide forms beta-sheet and becomes a new element of the fibril. Under acidic condition, single PrP106-126 fragment presents a much larger variety of conformations than it does under neural condition. However, acidic condition does not largely affect the stability of the PrP106-126 fibril. Consequently, the speed of the fibril elongation can be dramatically increased by lowering the pH value of the solution. The pH values are adjusted by either altering the protonation state of the residues or adding hydronium ions or hydroxyl ions.     

Induction of ligand-specific PrPC signaling in human neuronal cells

Cellular prion protein (PrP^C) has attracted considerable attention for its role in transmissible spongiform encephalopathies (TSEs). In spite of being a point of intense research effort critical questions still remain regarding the physiological function of PrP^C and how these functions may change with the conversion of the protein into the infectious and pathological conformation (PrP^Sc). While emerging evidence suggests PrP^C/Sc are involved in signal transduction there is little consensus on the signaling pathways associated with the normal and diseased states. The purported involvement of PrP^C in signal transduction, and the association of TSEs with neural pathology, makes kinome analysis of human neurons an interesting and appropriate model to characterize patterns of signal transduction following activation of PrP^C by two commonly employed experimental ligands; antibody-induced dimerization by 6H4 and the amino acids 106-126 PrP peptide fragment (PrP 106–126). Analysis of the induced kinome responses reveals distinct patterns of signaling activity following each treatment. Specifically, stimulation of human neurons with the 6H4 antibody results in alterations in mitogen activated protein kinase (MAPK) signaling pathways while the 106-126 peptide activates growth factor related signaling pathways including vascular endothelial growth factor (VEGF) signaling and the phosphoinositide-3 kinase (PI3K) pathway. These pathways were validated through independent functional assays. Collectively these results indicate that stimulation of PrP^C with distinct ligands, even within the same cell type, results in unique patterns of signaling. While this investigation highlights the apparent functional versatility of PrP^C as a signaling molecule and may offer insight into cellular mechanisms of TSE pathology it also emphasizes the potential dangers associated with attributing activation of specific intracellular events to particular receptors through artificial models of receptor activation.     

Effects of Lipid Composition and Phase on the Membrane Interaction of the Prion Peptide 106-126 Amide

Lipid rafts are specialized liquid-ordered (L_o) phases of the cell membrane that are enriched in sphingolipids and cholesterol (Chl), and surrounded by a liquid-disordered (L_d) phase enriched in glycerophospholipids. Lipid rafts are involved in the generation of pathological forms of proteins that are associated with neurodegenerative diseases. To investigate the effects of lipid composition and phase on the generation of pathological forms of proteins, we constructed an L_d-gel phase-separated 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)/sphingomyelin (from bovine brain (BSM))-supported lipid bilayer (SLB) and an L_d-L_o phase-separated POPC/BSM/Chl SLB. We used in situ time-lapse atomic force microscopy to study the interactions between these SLBs and the prion peptide K¹⁰⁶TNMKHMAGAAAAGAVVGGLG¹²⁶ (PrP106-126) amide, numbered according to the human prion-peptide sequence. Our results show that: 1), with the presence of BSM in the L_d phase, the PrP106-126 amide induces fully penetrated porations in the L_d phase of POPC/BSM SLB and POPC/BSM/Chl SLB; 2), with the presence of both BSM and Chl in the L_d phase, the PrP106-126 amide induces the disintegration of the L_d phase of POPC/BSM/Chl SLB; and 3), with the presence of both BSM and Chl in the L_o phase, PrP106-126 amide induces membrane thinning in the L_o phase of POPC/BSM/Chl SLB. These results provide comprehensive insight into the process by which the PrP106-126 amide interacts with lipid membranes.     

PrP106-126多肽诱导凋亡细胞中14-3-3β的降解

本研究将PrP106-126多肽和HeLa细胞共孵育4h和8h,采用Hoechst染色分析发现PrP106-126诱导凋亡细胞细胞核出现不同程度的染色质浓集,固缩及碎裂的细胞凋亡征象。Western blotting检测发现PrP106-126诱导细胞中的多聚ADP核糖聚合酶(poly ADP-ribose polymerase,PARP)降解,提示PrP106-126通过caspase3途径引起细胞凋亡现象。PrP106-126诱导的细胞中14-3-3β在不同孵育时间也出现降解,而Real-time PCR检测14-3-3β mRNA未发生变化。本研究证明PrP106-126通过caspase3诱导HeLa细胞凋亡,并可导致抗凋亡蛋白14-3-3β的降解而加速凋亡的形成。     

Prions,proteinase K and infectivity

It has been described that the breakdown of β-sheets in PrP^Sc by denaturation results in loss of infectivity and PK-sensitivity, suggesting a relationship between the structure and PK-resistance. It is also known that an important fraction of total PrP^Sc is PK-sensitive and can be isolated by the method we already described. Consequently, we decided to employ the PK-sensitive fraction of PrP^Sc as a potential and useful tool for structural studies. Thus, two essential questions were addressed in our recent article. First, the difference in the infectivity between the sensitive and resistant fractions and second, whether sensitive and resistant PrP^Sc shared the same conformation or were only different size multimers with the same basic conformation. Here we discuss our latest data in light of recent infectivity studies and their possible implications on the conformation of the prion.     

Structure of the Flexible Amino-Terminal Domain of Prion Protein Bound to a Sulfated Glycan

The intrinsically disordered amino-proximal domain of hamster prion protein (PrP) contains four copies of a highly conserved octapeptide sequence, PHGGGWGQ, that is flanked by two polycationic residue clusters. This N-terminal domain mediates the binding of sulfated glycans, which can profoundly influence the conversion of PrP to pathological forms and the progression of prion disease. To investigate the structural consequences of sulfated glycan binding, we performed multidimensional heteronuclear (¹H, ¹³C, ¹⁵N) NMR (nuclear magnetic resonance), circular dichroism (CD), and fluorescence studies on hamster PrP residues 23-106 (PrP 23-106) and fragments thereof when bound to pentosan polysulfate (PPS). While the majority of PrP 23-106 remain disordered upon PPS binding, the octarepeat region adopts a repeating loop-turn structure that we have determined by NMR. The β-like turns within the repeats are corroborated by CD data demonstrating that these turns are also present, although less pronounced, without PPS. Binding to PPS exposes a hydrophobic surface composed of aligned tryptophan side chains, the spacing and orientation of which are consistent with a self-association or ligand binding site. The unique tryptophan motif was probed by intrinsic tryptophan fluorescence, which displayed enhanced fluorescence of PrP 23-106 when bound to PPS, consistent with the alignment of tryptophan side chains. Chemical-shift mapping identified binding sites on PrP 23-106 for PPS, which include the octarepeat histidine and an N-terminal basic cluster previously linked to sulfated glycan binding. These data may in part explain how sulfated glycans modulate PrP conformational conversions and oligomerizations.