Binding of prion proteins to lipid membranes

A key molecular event in prion diseases is the conversion of the normal cellular form of the prion protein (PrPC) to an aberrant form known as the scrapie isoform, PrPSc. Under normal physiological conditions PrPC is attached to the outer leaflet of the plasma membrane via a GPI-anchor. It has been proposed that a direct interaction between PrP and lipid membranes could be involved in the conversion of PrPC to its disease-associated corrupted conformation, PrPSc. Recombinant PrP can be refolded into an alpha-helical structure, designated alpha-PrP isoform, or into beta-sheet-rich states, designated beta-PrP isoform. The current study investigates the binding of recombinant PrP isoforms to model lipid membranes using surface plasmon resonance spectroscopy. The binding of alpha- and beta-PrP to negatively charged lipid membranes of POPG, zwitterionic membranes of DPPC, and model raft membranes composed of DPPC, cholesterol, and sphingomyelin is compared at pH 7 and 5, to simulate the environment at the plasma membrane and within endosomes, respectively. It is found that PrP binds strongly to lipid membranes. The strength of the association of PrP with lipid membranes depends on the protein conformation and pH, and involves both hydrophobic and electrostatic lipid-protein interactions. Competition binding measurements established that the binding of alpha-PrP to lipid membranes follows a decreasing order of affinity to POPG>DPPC>rafts.     

Effect of chemical modifications of myelin basic protein on its interaction with lipid interfaces and cell fusion ability

Summary The ability of native and chemically modified myelin basic protein to induce fusion of chicken erythrocytes and to interact with lipids in monolayers at the air-water interface and liposomes was studied. Chemical modifications of myelin basic protein were performed by acetylation and succinylation: the positive charges of the native protein were blocked to an extent of about 90–95%.Cellular aggregation and fusion of erythrocytes into multinucleated cells was induced by the native myelin basic protein. This effect was diminished for both acetylated and succinylated myelin basic protein. Native myelin basic protein penetrated appreciably in sulphatide-containing lipid monolayers while lower penetration occurred in monolayers of neutral lipids. Contrary to this, both chemically modified myelin basic proteins did not show any selectivity to penetrate into interfaces of neutral or negatively charged lipids. The intrinsic fluorescence of the native and chemically modified myelin basic proteins upon interacting with liposomes constituted by dipalmitoylphosphatidycholine, glycosphingolipids, egg phosphatidic acid or dipalmitoylphosphatidyl glycerol was studied. The interaction with liposomes of anionic lipids is accompanied by a blue shift of the maximum of the native protein emission fluorescence spectrum from 346 nm to 335 nm; no shift was observed with liposomes containing neutral lipids. The acetylated and succinylated myelin basic proteins did not show changes of their emission spectra upon interacting with any of the lipids studied. The results obtained in monolayers and the fluorescence shifts indicate a lack of correlation between the ability of the modified proteins to penetrate lipid interfaces and the microenvironment sensed by the tryptophan-containing domain.Abbreviations MBP myelin basic protein - DPPC dipalmitoyl phosphatidylcholine - DPPG dipalmitoyl phosphatidylglycerol - PA phosphatidic acid     

Role of polysaccharide and lipid in lipopolysaccharide induced prion protein conversion

Conversion of native cellular prion protein (PrP^c) from an α-helical structure to a toxic and infectious β-sheet structure (PrP^Sc) is a critical step in the development of prion disease. There are some indications that the formation of PrP^Sc is preceded by a β-sheet rich PrP (PrP^β) form which is non-infectious, but is an intermediate in the formation of infectious PrP^Sc. Furthermore the presence of lipid cofactors is thought to be critical in the formation of both intermediate-PrP^β and lethal, infectious PrP^Sc. We previously discovered that the endotoxin, lipopolysaccharide (LPS), interacts with recombinant PrP^c and induces rapid conformational change to a β-sheet rich structure. This LPS induced PrP^β structure exhibits PrP^Sc-like features including proteinase K (PK) resistance and the capacity to form large oligomers and rod-like fibrils. LPS is a large, complex molecule with lipid, polysaccharide, 2-keto-3-deoxyoctonate (Kdo) and glucosamine components. To learn more about which LPS chemical constituents are critical for binding PrP^c and inducing β-sheet conversion we systematically investigated which chemical components of LPS either bind or induce PrP conversion to PrP^β. We analyzed this PrP conversion using resolution enhanced native acidic gel electrophoresis (RENAGE), tryptophan fluorescence, circular dichroism, electron microscopy and PK resistance. Our results indicate that a minimal version of LPS (called detoxified and partially de-acylated LPS or dLPS) containing a portion of the polysaccharide and a portion of the lipid component is sufficient for PrP conversion. Lipid components, alone, and saccharide components, alone, are insufficient for conversion.     

Disulfide bond as a structural determinant of prion protein membrane insertion

Conversion of the normal soluble form of prion protein, PrP (PrP^C), to proteinase K-resistant form (PrP^Sc) is a common molecular etiology of prion diseases. Proteinase K-resistance is attributed to a drastic conformational change from α-helix to β-sheet and subsequent fibril formation. Compelling evidence suggests that membranes play a role in the conformational conversion of PrP. However, biophysical mechanisms underlying the conformational changes of PrP and membrane binding are still elusive. Recently, we demonstrated that the putative transmembrane domain (TMD; residues 111–135) of Syrian hamster PrP penetrates into the membrane upon the reduction of the conserved disulfide bond of PrP. To understand the mechanism underlying the membrane insertion of the TMD, here we explored changes in conformation and membrane binding abilities of PrP using wild type and cysteine-free mutant. We show that the reduction of the disulfide bond of PrP removes motional restriction of the TMD, which might, in turn, expose the TMD into solvent. The released TMD then penetrates into the membrane. We suggest that the disulfide bond regulates the membrane binding mode of PrP by controlling the motional freedom of the TMD.     

Lipopolysaccharide induced conversion of recombinant prion protein

The conformational conversion of the cellular prion protein (PrP^C) to the β-rich infectious isoform PrP^Sc is considered a critical and central feature in prion pathology. Although PrP^Sc is the critical component of the infectious agent, as proposed in the “protein-only” prion hypothesis, cellular components have been identified as important cofactors in triggering and enhancing the conversion of PrP^C to proteinase K resistant PrP^Sc. A number of in vitro systems using various chemical and/or physical agents such as guanidine hydrochloride, urea, SDS, high temperature, and low pH, have been developed that cause PrP^C conversion, their amplification, and amyloid fibril formation often under non-physiological conditions. In our ongoing efforts to look for endogenous and exogenous chemical mediators that might initiate, influence, or result in the natural conversion of PrP^C to PrP^Sc, we discovered that lipopolysaccharide (LPS), a component of gram-negative bacterial membranes interacts with recombinant prion proteins and induces conversion to an isoform richer in β sheet at near physiological conditions as long as the LPS concentration remains above the critical micelle concentration (CMC). More significant was the LPS mediated conversion that was observed even at sub-molar ratios of LPS to recombinant ShPrP (90–232).     

Incorporation of the human erythrocyte sialoglycoprotein into recombined membranes containing cholesterol

Summary Glycophorin, the major sialoglycoprotein from the human erythrocyte membrane, has been isolated and recombined with phosphatidylcholine and cholesterol. Sucrose density gradient analysis of the recombinants shows that it is possible not only to recombine this protein with phospholipid, but also with phospholipid-cholesterol mixtures. Surprisingly, by the same analysis, it was possible to make a recombinant with cholesterol and glycophorin, only, in the absence of added phospholipid. The accessibility of the protein to trypsin was tested in each of these recombinants. In all the recombinants which contained either phospholipid, or phospholipid and cholesterol, the protein was protected from extensive hydrolysis. This is consistent with closed vesicles and incorporation of the protein into the recombinant membrane. Extensive hydrolysis of the protein occurred in the cholesterol-glycophorin recombinant indicating some differences in structure. Freeze-fracture electron microscopy of the phospholipid and the phospholipid-cholesterol recombinants showed mostly unilamellar vesicles, 1000 to 5000 Å in diameter. Intramembranous particles were observed on both fracture faces, and the fracture planes were those expected for phospholipid bilayers. The glycophorin-cholesterol recombinants also showed fracture planes consistent with bilayers, and revealed intramembranous particles. Pieces of membrane-like structures as well as apparent vesicular structures were observed. Finally in the recombinants of glycophorin with phospholipid and cholesterol, cholesterol is shown to reduce the population of the motionally restricted phospholipid headgroup environment, in proportion to the mole percent cholesterol content.     

Structural conservation of prion strain specificities in recombinant prion protein fibrils in real-time quaking-induced conversion

ABSTRACT

A major unsolved issue of prion biology is the existence of multiple strains with distinct phenotypes and this strain phenomenon is postulated to be associated with the conformational diversity of the abnormal prion protein (PrP^Sc). Real-time quaking-induced conversion (RT-QUIC) assay that uses Escherichia coli-derived recombinant prion protein (rPrP) for the sensitive detection of PrP^Sc results in the formation of rPrP-fibrils seeded with various strains. We demonstrated that there are differences in the secondary structures, especially in the β-sheets, and conformational stability between 2 rPrP-fibrils seeded with either Chandler or 22L strains in the first round of RT-QUIC. In particular, the differences in conformational properties of these 2 rPrP-fibrils were common to those of the original PrP^Sc. However, the strain specificities of rPrP-fibrils seen in the first round were lost in subsequent rounds. Instead, our findings suggest that nonspecific fibrils became the major species, probable owing to their selective growth advantage in the RT-QUIC. This study shows that at least some strain-specific conformational properties of the original PrP^Sc can be transmitted to rPrP-fibrils in vitro, but further conservation appears to require unknown cofactors or environmental conditions or both.     

Cellular prion protein: implications in seizures and epilepsy

1. Cellular prion (PrP^c) is a plasma membrane protein involved with copper uptake, protection against oxidative stress, cell adhesion, differentiation, signaling, and survival in the central nervous system2. Deletion of PrP^c gene (Prnp) in mice enhances sensitivity to seizures in vivo and neuronal excitability in vitro which can be related to: (i) disrupted Ca⁺²-activated K⁺ currents, with loss of I _HAP conductance in hippocampus; (ii) abnormal GABA-A inhibition in the hippocampus; (iii) mossy fiber reorganization in the hippocampus; (iv) changes in ectonucleotidases in both hippocampus and neocortex; and (v) higher levels of neocortical and subcortical oxidative stress. Moreover, postnatal Prnp knockout mice showed a significant reduction of after hyperpolarization potentials in hippocampal CA1 cells.3. Taken together, these findings suggest that loss of PrP^c function contributes to the hyperexcitable and synchronized activities underlying epileptic seizures generated in neocortex and hippocampus. Hence, the role of PrP^c on human symptomatic, cryptogenic or idiopathic epileptic syndromes deserves further investigation.     

朊蛋白的细胞生物学研究

朊蛋白病是人和牛羊等哺乳动物所患的致命性的神经系统变性疾病,它是由机体内正常的朊蛋白改变构象后所引起的疾病。本综述对朊蛋白在细胞生物学领域的认知和理解进行了归纳总结,阐述了正常和异常朊蛋白的翻译、表达、定位、裂解、转化等一系列过程,是对疾病本质的有益探索。     

Cooperative binding of dominant-negative prion protein to kringle domains

Conversion of the cellular prion protein (PrP(C)) to the pathogenic isoform (PrP(Sc)) is a major biochemical alteration in the progression of prion disease. This conversion process is thought to require interaction between PrP(C) and an as yet unidentified auxiliary factor, provisionally designated protein X. In searching for protein X, we screened a phage display cDNA expression library constructed from prion-infected neuroblastoma (ScN2a) cells and identified a kringle protein domain using full-length recombinant mouse PrP (recMoPrP(23-231), hereafter recMoPrP) expressing a dominant-negative mutation at codon 218 (recMoPrP(Q218K)). In vitro binding analysis using ELISA verified specific interaction of recMoPrP to kringle domains (K(1+2+3)) with higher binding by recMoPrP(Q218K) than by full-length recMoPrP without the mutation. This interaction was confirmed by competitive binding analysis, in which the addition of either a specific anti-kringle antibody or L-lysine abolished the interaction. Biochemical studies of the interactions between K(1+2+3) and various concentrations of both recMoPrP molecules demonstrated binding in a dose-dependent manner. A Hill plot analysis of the data indicates positive cooperative binding of both recMoPrP(Q218K) and recMoPrP to K(1+2+3) with stronger binding by recMoPrP(Q218K). Using full-length and an N-terminally truncated MoPrP(89-231), we demonstrate that N-terminal sequences enable PrP to bind strongly to K(1+2+3). Further characterization with truncated MoPrP(89-231) refolded in different conformations revealed that both alpha-helical and beta-sheet conformations bind to K(1+2+3). Our data demonstrate specific, high-affinity binding of a dominant-negative PrP as well as binding of other PrPs to K(1+2+3). The relevance of such interactions during prion pathogenesis remains to be established.     

Characterization of cell-surface prion protein relative to its recombinant analogue: insights from molecular dynamics simulations of diglycosylated, membrane-bound human prion protein

The prion protein (PrP) is responsible for several fatal neurodegenerative diseases via conversion from its normal to disease-related isoform. The recombinant form of the protein is typically studied to investigate the conversion process. This constructs lacks the co- and post-translational modifications present in vivo , there the protein has two N-linked glycans and is bound to the outer leaflet of the plasma membrane via a glycosylphosphatidylinositol (GPI) anchor. The inherent flexibility and heterogeneity of the glycans, the plasticity of the GPI anchor, and the localization of the protein in a membrane make experimental structural characterization of biological constructs of cellular prion protein (PrP^C) challenging. Yet this characterization is central in determining not only the suitability of recombinant (rec)-PrP^C as a model for biological forms of the protein but also the potential role of co- and post-translational modifications on the disease process. Here, we present molecular dynamics simulations of three human prion protein constructs: (i) a protein-only construct modeling the recombinant form, (ii) a diglycosylated and soluble construct, and (iii) a diglycosylated and GPI-anchored construct bound to a lipid bilayer. We found that glycosylation and membrane anchoring do not significantly alter the structure or dynamics of PrP^C, but they do appreciably modify the accessibility of the polypeptide surface PrP^C. In addition, the simulations of membrane-bound PrP^C revealed likely recognition domains for the disease-initiating PrP^C:PrP^Sc (infectious and/or misfolded form of the prion protein) binding event and a potential mechanism for the observed inefficiency of conversion associated with differentially glycosylated PrP species.     

The binding of prion proteins to serum components is affected by detergent extraction conditions

As many GPI anchored proteins, PrP(C) and its abnormal conformer PrP(Sc), are inserted into membrane microdomains known as rafts. Upon raft disruption, PrP(C) becomes soluble, while PrP(Sc) aggregates into insoluble structures. It was recently published that, as opposed to PrP(C), PrP(Sc), as well as its protease resistant core PrP27-30, can bind specifically to plasminogen and other serum components. These findings were suggested to have important physiological implications in transmissible spongiform encephalopathies (TSE) diagnosis and pathogenesis. In this work, we show that the binding of PrP(Sc) or PrP 27-30 to serum proteins occurs only at specific detergent combinations, in which disease associated PrPs are present in aggregated structures. At detergent conditions in which rafts are intact, it is actually PrP(C.) that binds to blood proteins, albeit not directly, but through neighboring rafts components. Our results therefore indicate that the binding of PrP(Sc) to blood components has no physiological relevance.     

Reversible aggregation of mouse prion protein derivatives with PrPSC-like structural properties

Three carbamylated derivatives of reduced mouse prion protein (mPrP) were isolated during the aborted oxidative folding in the presence of urea. These three prion protein derivatives (mPrP-a, mPrP-b, and mPrP-c) exist as monomer in the acidic solution (pH < 2.0) and exhibit prevalent random coil structure. However, they undergo rapid aggregation and transformation to a predominant -sheet structure upon exposure to ionic buffer with pH greater than 3.0. The stability of aggregates of mPrP conformers is in part dependent upon the time that they were allowed to develop. The nascent aggregates comprise a significant fraction of loosely packed mPrP monomers that can be dissociated by treatment with strong acidic solution. Matured aggregates acquired through prolonged sample incubation contain more tightly packed mPrP monomers that cannot be dissociated by strong acid but can be disaggregated by denaturant. The properties of reversible aggregation of mPrP-a, mPrP-b, and mPrP-c bear a striking resemblance to that observed with aggregates of hamster PrP^SC.     

Aggregation of cellular prion protein is initiated by proximity-induced dimerization

Prion diseases or transmissible spongiform encephalopathies (TSEs) are infectious and fatal neurodegenerative disorders in humans and animals. Pathological features of TSEs include the conversion of cellular prion protein (PrP(C)) into an altered disease-associated conformation generally designated PrP(Sc), abnormal deposition of PrP(Sc) aggregates, and spongiform degeneration of the brain. The molecular steps leading to PrP(C) aggregation are unknown. Here, we have utilized an inducible oligomerization strategy to test if, in the absence of any infectious prion particles, the encounter between PrP(C) molecules may trigger its aggregation in neuronal cells. A chimeric PrP(C) composed of one (Fv1) or two (Fv2) modified FK506-binding protein (Fv) fused with PrP(C) were created, and transfected in N2a cells. Similar to PrP(C), Fv1-PrP and Fv2-PrP were glycosylated, displayed normal localization, and anti-apoptotic function. When cells were treated with the dimeric Fv ligand AP20187, to induce dimerization (Fv1) or oligomerization (Fv2) of PrP(C), both dimerization and oligomerization of PrP(C) resulted in the de novo production, release and deposition of extracellular PrP aggregates. Aggregates were insoluble in non-ionic detergents and partially resistant to proteinase K. These findings demonstrate that homologous interactions between PrP(C) molecules may constitute a minimal and sufficient molecular event leading to PrP(C) aggregation and extracellular deposition.     

Mutant prion protein-mediated aggregation of normal prion protein in the endoplasmic reticulum: implications for prion propagation and neurotoxicity

Familial prion disorders are believed to result from spontaneous conversion of mutant prion protein (PrPM) to the pathogenic isoform (PrPSc). While most familial cases are heterozygous and thus express the normal (PrPC) and mutant alleles of PrP, the role of PrPC in the pathogenic process is unclear. Plaques from affected cases reveal a heterogeneous picture; in some cases only PrPM is detected, whereas in others both PrPC and PrPM are transformed to PrPSc. To understand if the coaggregation of PrPC is governed by PrP mutations or is a consequence of the cellular compartment of PrPM aggregation, we coexpressed PrPM and PrPC in neuroblastoma cells, the latter tagged with green fluorescent protein (PrPC-GFP) for differentiation. Two PrPM forms (PrP231T, PrP217R/231T) that aggregate spontaneously in the endoplasmic reticulum (ER) were generated for this analysis. We report that PrPC-GFP aggregates when coexpressed with PrP231T or PrP217R/231T, regardless of sequence homology between the interacting forms. Furthermore, intracellular aggregates of PrP231T induce the accumulation of a C-terminal fragment of PrP, most likely derived from a potentially neurotoxic transmembrane form of PrP (CtmPrP) in the ER. These findings have implications for prion pathogenesis in familial prion disorders, especially in cases where transport of PrPM from the ER is blocked by the cellular quality control.     

Differences between the prion protein and its homolog Doppel: a partially structured state with implications for scrapie formation

The key event in the pathogenesis of prion diseases is a conformational change in the prion protein (PrP). Models for conversion of PrP(C) into PrP(Sc) typically implicate an, as yet, unidentified intermediate. In an attempt to identify such an intermediate, we used native-state hydrogen exchange monitored with NMR. Although we were unable to detect an intermediate directly, we observed substantial protection above that expected based upon measurements of the global stability of PrP (>2 kcal mol(-1) super protection). This super protection implicates either structure in the denatured state or the presence of an intermediate. Similar experiments with Doppel, a homolog of PrP that does not form infectious prions, failed to demonstrate such super protection. This suggests that the partially structured state of PrP encompassing portions of the B and C helices, may be a significant factor in the ability of PrP to convert from PrP(C) to PrP(Sc).