DNA-induced partial unfolding of prion protein leads to its polymerisation to amyloid

The full-length mouse recombinant prion protein (23-231 amino acid residues) contains all of its structural elements viz. three alpha-helices and a short two-stranded antiparallel beta-sheet in its C-terminal fragment comprising 121-231 amino acid residues. The incubated mixture of this prion protein fragment and nucleic acid results in the formation of amyloid fibres evidenced from electron microscopy, birefringence and fluorescence of the fibre bound Congo Red and Thioflavin T dyes, respectively. The secondary structure of the amyloid formed in nucleic acid solution is similar to the in vivo isolated prion protein 27-30 amyloid but unlike in it, a hydrophobic milieu is absent in the 121-231 amyloid. Thermal denaturation study demonstrates a partial unfolding of the protein fragment in nucleic acid solution. We propose that nucleic acid catalyses unfolding of prion protein helix 1 followed by a nucleation-dependent polymerisation of the protein to amyloid.     

NCAM regulates the proliferation,apoptosis, autophagy,EMT, and migration of human melanoma cells via the Src/Akt/mTOR/cofilin signaling pathway

The neural cell adhesion molecule (NCAM) plays critical roles in multiple cellular processes in neural cells, mesenchymal stem cells, and various cancer cells. However, the effect and mechanism of NCAM in human melanoma cells are still unclear. In this study, we found that NCAM regulated the proliferation, apoptosis, autophagy, migration, and epithelial-to-mesenchymal transition of human melanoma cells by determining the biological behavior of NCAM knockdown A375 and M102 human melanoma cells. Further studies revealed that NCAM knockdown impaired the organization of actin cytoskeleton and reduced the phosphorylation of cofilin, an actin-cleaving protein. When cells were transfected with cofilin S3A (dephosphorylated cofilin), biological behavior similar to that of NCAM knockdown cells was observed. Research on the underlying molecular mechanism showed that NCAM knockdown suppressed activation of the Src/Akt/mTOR pathway. Specific inhibitors of Src and PI3K/Akt were employed to further verify the relationship between Src/Akt/mTOR signaling and cofilin, and the results showed that the phosphorylation level of cofilin decreased following inhibition of the Src/Akt/mTOR pathway. These results indicated that NCAM may regulate the proliferation, apoptosis, autophagy, migration, and epithelial-to-mesenchymal transition of human melanoma cells via the Src/Akt/mTOR/cofilin pathway-mediated dynamics of actin cytoskeleton.     

Nucleic acid and prion protein interaction produces spherical amyloids which can function in vivo as coats of spongiform encephalopathy agent

The infectious agent of transmissible spongiform encephalopathies (TSE) has been considered to be PrP(SC), a structural isoform of cellular prion protein PrP(C). PrP(SC) can exist as oligomers and/or as amyloid polymers. Nucleic acids induce structural conversion of recombinant prion protein PrP and PrP(C) to PrP(SC) form in solution and in vitro. Here, we report that nucleic acids, by interacting with PrP in solution, produce amyloid fibril and fibres of different morphologies, similar to those identified in the diseased brains. In addition, the same interaction produces polymer lattices and spherical amyloids of different dimensions (15-150 nm in diameters). The polymer lattices show apparent morphological similarity to the two-dimensional amyloid crystals obtained from linear amyloids isolated in vivo. The spherical amyloids structurally resemble "spherical particles" observed in natural spongiform encephalopathy (SE) and in scrapie-infected brains (TSE). We suggest that spherical amyloids, PrP(SC)-amylospheroids, are probable constituents of the coat of the spherical particles found in vivo and the latter can act as protective coats of the SE and TSE agents in vivo.     

神经细胞粘附分子结构特征和生理功能

神经细胞粘附分子是一类调节细胞与细胞、细胞与细胞外基质间粘附作用的膜表面糖蛋白,主要有NCAM-180、NCAM-140、NCAM-120三种形式,多与PSA结合在一起。在神经系统中,NCAM的表达具有时间和空间特异性,最主要的作用为调节神经系统的可塑性,这种作用可能是通过PSA-NCAM对AMPA的调节作用,主要是通过调节蛋白激酶的表达和细胞内Ca^2 浓度来实现的。     

The peculiar nature of unfolding of the human prion protein

Spontaneous conformational transition of the prion protein from an alpha-helical isoform to a beta-sheet-rich isoform underlies the pathogenesis of sporadic prion diseases. To study the rate-limiting steps of spontaneous conversion, the formation of amyloid fibrils by the recombinant human PrP C-terminal fragment spanning residues 90-231 (recPrP) was monitored in the presence of urea. The kinetics of spontaneous fibril formation displayed sigmoidal behavior involving a lag phase. The shortest lag phase was observed at partially denaturing conditions, close to the concentration of urea corresponding to the middle point of unfolding. This result indicates that unfolding intermediates may be important for the conversion. To test whether unfolding intermediates are formed, we employed size-exclusion chromatography and circular dichroism spectroscopy to monitor urea denaturation of recPrP. Both techniques showed a single sigmoidal transition with very similar thermodynamic parameters of denaturation and that the transition can be described by a simple equilibrium between folded and denatured states. Detailed analyses of data, however, revealed that the dimensions of both the native and denatured species gradually increases with urea. Expansion of the native species is also accompanied by an increase in efficiency of the energy transfer from a single Trp residue to 1-anilinonaphthalene-8-sulfonate dye as measured by fluorescence. These data illustrate that thermodynamic character of the native ensemble changes gradually with environmental conditions. Such behavior is consistent with the thermodynamically variable model, and may be linked to the ability of PrP to adopt distinct abnormal conformations under pathologic conditions.     

The protease-sensitive N-terminal polybasic region of prion protein modulates its conversion to the pathogenic prion conformer

Conversion of normal prion protein (PrP^C) to the pathogenic PrP^Sc conformer is central to prion diseases such as Creutzfeldt–Jakob disease and scrapie; however, the detailed mechanism of this conversion remains obscure. To investigate how the N-terminal polybasic region of PrP (NPR) influences the PrP^C-to-PrP^Sc conversion, we analyzed two PrP mutants: ΔN6 (deletion of all six amino acids in NPR) and Met4-1 (replacement of four positively charged amino acids in NPR with methionine). We found that ΔN6 and Met4-1 differentially impacted the binding of recombinant PrP (recPrP) to the negatively charged phospholipid 1-palmitoyl-2-oleoylphosphatidylglycerol, a nonprotein cofactor that facilitates PrP conversion. Both mutant recPrPs were able to form recombinant prion (recPrP^Sc) in vitro, but the convertibility was greatly reduced, with ΔN6 displaying the lowest convertibility. Prion infection assays in mammalian RK13 cells expressing WT or NPR-mutant PrPs confirmed these differences in convertibility, indicating that the NPR affects the conversion of both bacterially expressed recPrP and post-translationally modified PrP in eukaryotic cells. We also found that both WT and mutant recPrP^Sc conformers caused prion disease in WT mice with a 100% attack rate, but the incubation times and neuropathological changes caused by two recPrP^Sc mutants were significantly different from each other and from that of WT recPrP^Sc. Together, our results support that the NPR greatly influences PrP^C-to-PrP^Sc conversion, but it is not essential for the generation of PrP^Sc. Moreover, the significant differences between ΔN6 and Met4-1 suggest that not only charge but also the identity of amino acids in NPR is important to PrP conversion.     

Evidence for an association of prion protein and sphingolipid-mediated signaling

The physiological function of the cellular prion protein (PrP^c) is unclear. PrP^c associates with lipid rafts, highly glycolipid-rich membrane domains containing a large variety of signaling molecules, e.g., sphingolipids (SL). In this study, we investigated possible connections between PrP^c and sphingolipid-associated signaling pathways. Using PrP^c-wt and PrP^c-k.o. hippocampal cell lines and mouse brains we showed higher activity of neutral and acid sphingomyelinase (SMase) in PrP^c-k.o.-groups, while ceramide and sphingomyelin-levels were unchanged. Furthermore, despite lower basal expression levels of sphingosine kinase (SphK) in PrP^c-k.o.-groups, the levels of its metabolite sphingosine-1-phosphate were increased, whereas S1P₃-receptor expression was higher in PrP^c-wt-groups again. In addition, we detected enhanced activity of phospholipase D1, an enzyme that seems to be suitable to act as a connector between the S1P₃ receptor and continuative signaling. Finally, evidence for an impact on downstream signaling cascades, especially activation of the PI3K/Akt pathway, was found. In summary, our data suggest that PrP^c is involved in sphingolipid-associated signaling, modulating pathways that exert anti-apoptotic functions, hence indicating that PrP^c plays a role in neuroprotection.     

Cellular prion protein modulates the intracellular calcium response to hydrogen peroxide

The physiological function of the cellular prion protein (PrP(C)) is still under intense investigation. It has been suggested that PrP(C) has a protective role in neuronal cells, particularly against environmental stress caused by reactive oxygen species (ROS). Here we analysed the acute effect of a major ROS, hydrogen peroxide (H(2)O(2)), on intracellular calcium homeostasis in cultured cerebellar granule cells and immortalized hippocampal neuronal cells. Both neuronal cell culture models showed that the rise in intracellular calcium following application of H(2)O(2) was strongly dependent on the presence of PrP(C). Moreover, the N-terminal octapeptide repeats of PrP(C) were required for this effect, because neuronal cells expressing a PrP(C) lacking the N-terminus resembled the PrP(C)-deficient phenotype. Neurones deficient of fyn kinase, or pharmacological inhibition of fyn, also abrogated the calcium response to H(2)O(2) treatment, indicating that fyn activation is a critical step within the PrP(C) signalling cascade. Finally, we identified a possible role of this PrP(C) signalling pathway in the neuroprotective response of PrP(C) to oxidative stress. In conclusion, we put forward the hypothesis that PrP(C) functions as a sensor for H(2)O(2), thereby activating a protective signalling cascade involving fyn kinase that leads to calcium release from intracellular stores.     

The structural transition of the prion protein into its pathogenic conformation is induced by unmasking hydrophobic sites

A series of structural intermediates in the putative pathway from the cellular prion protein PrP(C) to the pathogenic form PrP(Sc) was established by systematic variation of low concentrations (<0.1%) of the detergent sodium dodecyl sulfate (SDS) or by the interaction with the bacterial chaperonin GroEL. Most extended studies were carried out with recombinant PrP (90-231) corresponding to the amino acid sequence of hamster prions PrP 27-30. Similar results were obtained with full-length recombinant PrP, hamster PrP 27-30 and PrP(C) isolated from transgenic, non-infected CHO cells. Varying the incubation conditions, i.e. the concentration of SDS, the GroEL and GroEL/ES, but always at neutral pH and room temperature, different conformations could be established. The conformations were characterized with respect to secondary structure as determined by CD spectroscopy and to molecular mass, as determined by fluorescence correlation spectroscopy and analytical ultracentrifugation: alpha-helical monomers, soluble alpha-helical dimers, soluble but beta-structured oligomers of a minimal size of 12-14 PrP molecules, and insoluble multimers were observed. A high activation barrier was found between the alpha-helical dimers and beta-structured oligomers. The numbers of SDS-molecules bound to PrP in different conformations were determined: Partially denatured, alpha-helical monomers bind 31 SDS molecules per PrP molecule, alpha-helical dimers 21, beta-structured oligomers 19-20, and beta-structured multimers show very strong binding of five SDS molecules per PrP molecule. Binding of only five molecules of SDS per molecule of PrP leads to fast formation of beta-structures followed by irreversible aggregation. It is discussed that strongest binding of SDS has an effect identical with or similar to the interaction with GroEL thereby inducing identical or very similar transitions. The interaction with GroEL/ES stabilizes the soluble, alpha-helical conformation. The structure and their stabilities and particularly the induction of transitions by interaction of hydrophobic sites of PrP are discussed in respect to their biological relevance.     

Binding of prion protein to lipid membranes and implications for prion conversion

The binding of the Syrian hamster prion protein, SHaPrP(90-231), to model lipid membranes was investigated by tryptophan fluorescence. Membranes composed of negatively charged or zwitterionic lipids, and raft-like membranes containing dipalmitoylphosphatidylcholine(1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), cholesterol and sphingomyelin, were investigated. It was found that SHaPrP(90-231) binds to negatively charged lipid membranes and raft-like membranes. Binding of PrP to negatively charged lipid membranes involves both electrostatic and hydrophobic lipid-protein interactions and results in partial insertion of PrP into the lipid bilayer. This membrane-inserted conformation of PrP is richer in beta-sheet structure and has a disruptive effect on the integrity of the lipid bilayer, leading to total release of vesicle contents. In contrast, the binding of PrP to raft-like membranes is driven by hydrophobic lipid-protein interactions and induces the formation of alpha-helical structure. This conformation of PrP with a high content of alpha-helix is formed only at pH 7 and does not destabilize the lipid bilayer. Our findings support the view that an interaction of PrP with lipid membranes could play a role in PrP conversion.     

Production of monoclonal antibodies to the prion protein and their characterization

Antibodies to the prion protein (PrP), particularly, monoclonal antibodies, are necessary tools in the diagnostics and study of prion diseases and potential means of their immunotherapy. For the production of monoclonal antibodies, BALB/c mice were immunized by a recombinant bovine PrP. Three stable hybridomas producing antibodies of IgM class were prepared. The antibodies were bound to PrP in a solid-phase enzyme immunoassay and immunoblotting. The epitope mapping accomplished with the use of synthetic peptides showed that an epitope located in region 25–36 of PrP corresponds to one antibody, and epitopes located in region 222–229, to the other two. The antibodies to fragment 222–229 purified by affinity chromatography recognized with a high specificity conglomerates of a pathogenic prion in the brain tissue of cows suffering from spongiform encephalopathy. Thus, in nontransgenic mice, PrP-specific monoclonal antibodies were produced, useful in studies and diagnostics of prion diseases.     

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).     

The cellular form of the prion protein guides the differentiation of human embryonic stem cells into neuron-, oligodendrocyte-, and astrocyte-committed lineages

Prion protein, PrP^C, is a glycoprotein that is expressed on the cell surface beginning with the early stages of embryonic stem cell differentiation. Previously, we showed that ectopic expression of PrP^C in human embryonic stem cells (hESCs) triggered differentiation toward endodermal, mesodermal, and ectodermal lineages, whereas silencing of PrP^C suppressed differentiation toward ectodermal but not endodermal or mesodermal lineages. Considering that PrP^C might be involved in controlling the balance between cells of different lineages, the current study was designed to test whether PrP^C controls differentiation of hESCs into cells of neuron-, oligodendrocyte-, and astrocyte-committed lineages. PrP^C was silenced in hESCs cultured under three sets of conditions that were previously shown to induce hESCs differentiation into predominantly neuron-, oligodendrocyte-, and astrocyte-committed lineages. We found that silencing of PrP^C suppressed differentiation toward all three lineages. Similar results were observed in all three protocols, arguing that the effect of PrP^C was independent of differentiation conditions employed. Moreover, switching PrP^C expression during a differentiation time course revealed that silencing PrP^C expression during the very initial stage that corresponds to embryonic bodies has a more significant impact than silencing at later stages of differentiation. The current work illustrates that PrP^C controls differentiation of hESCs toward neuron-, oligodendrocyte-, and astrocyte-committed lineages and is likely involved at the stage of uncommitted neural progenitor cells rather than lineage-committed neural progenitors.     

The double life of the ribosome: When its protein folding activity supports prion propagation

It is no longer necessary to demonstrate that ribosome is the central machinery of protein synthesis. But it is less known that it is also key player of the protein folding process through another conserved function: the protein folding activity of the ribosome (PFAR). This ribozyme activity, discovered more than 2 decades ago, depends upon the domain V of the large rRNA within the large subunit of the ribosome. Surprisingly, we discovered that anti-prion compounds are also potent PFAR inhibitors, highlighting an unexpected link between PFAR and prion propagation.

In this review, we discuss the ancestral origin of PFAR in the light of the ancient RNA world hypothesis. We also consider how this ribosomal activity fits into the landscape of cellular protein chaperones involved in the appearance and propagation of prions and other amyloids in mammals. Finally, we examine how drugs targeting the protein folding activity of the ribosome could be active against mammalian prion and other protein aggregation-based diseases, making PFAR a promising therapeutic target for various human protein misfolding diseases.     

Recombinant prion protein induces rapid polarization and development of synapses in embryonic rat hippocampal neurons in vitro

While a beta-sheet-rich form of the prion protein (PrPSc) causes neurodegeneration, the biological activity of its precursor, the cellular prion protein (PrPC), has been elusive. We have studied the effect of purified recombinant prion protein (recPrP) on rat fetal hippocampal neurons in culture. Overnight exposure to Syrian hamster or mouse recPrP, folded into an alpha-helical-rich conformation similar to that of PrPC, resulted in a 1.9-fold increase in neurons with a differentiated axon, a 13.5-fold increase in neurons with differentiated dendrites, a fivefold increase in axon length, and the formation of extensive neuronal circuitry. Formation of synaptic-like contacts was increased by a factor of 4.6 after exposure to recPrP for 7 days. Neither the N-terminal nor C-terminal domains of recPrP nor the PrP paralogue doppel (Dpl) enhanced the polarization of neurons. Inhibitors of protein kinase C (PKC) and of Src kinases, including p59Fyn, blocked the effect of recPrP on axon elongation, while inhibitors of phosphatidylinositol 3-kinase showed a partial inhibition, suggesting that signaling cascades involving these kinases are candidates for transduction of recPrP-mediated signals. The results predict that full-length PrPC functions as a growth factor involved in development of neuronal polarity.     

Identification of novel putative-binding proteins for cellular prion protein and a specific interaction with the STIP1 homology and U-Box-containing protein 1

ABSTRACT

Prion diseases involve the conversion of the endogenous cellular prion protein, PrP^C, into a misfolded infectious isoform, PrP^Sc. Several functions have been attributed to PrP^C, and its role has also been investigated in the olfactory system. PrP^C is expressed in both the olfactory bulb (OB) and olfactory epithelium (OE) and the nasal cavity is an important route of transmission of diseases caused by prions. Moreover, Prnp^?/? mice showed impaired behavior in olfactory tests. Given the high PrP^C expression in OE and its putative role in olfaction, we screened a mouse OE cDNA library to identify novel PrP^C-binding partners. Ten different putative PrP^C ligands were identified, which were involved in functions such as cellular proliferation and apoptosis, cytoskeleton and vesicle transport, ubiquitination of proteins, stress response, and other physiological processes. In vitro binding assays confirmed the interaction of PrP^C with STIP1 homology and U-Box containing protein 1 (Stub1) and are reported here for the first time. Stub1 is a co-chaperone with ubiquitin E3-ligase activity, which is associated with neurodegenerative diseases characterized by protein misfolding and aggregation. Physiological and pathological implications of PrP^C-Stub1 interaction are under investigation. The PrP^C-binding proteins identified here are not exclusive to the OE, suggesting that these interactions may occur in other tissues and play general biological roles. These data corroborate the proposal that PrP^C is part of a multiprotein complex that modulates several cellular functions and provide a platform for further studies on the physiological and pathological roles of prion protein.     

Cellular prion protein function in copper homeostasis and redox signalling at the synapse

The fundamental physiological function of native cellular prion (PrPC) remains unknown. Herein, the most salient observations as regards prion physiology are critically evaluated. These include: (i) the role of PrPC in copper homeostasis, particularly at the pre-synaptic membrane; (ii) involvement of PrPC in neuronal calcium disturbances; and (iii) the neuroprotective properties of PrPC in response to copper and oxidative stress. Ultimately, a tentative hypothesis of basic prion function is derived, namely that PrPC acts as a sensor for copper and/or free radical stimuli, thereby triggering intracellular calcium signals that finally translate into modulation of synaptic transmission and maintenance of neuronal integrity.     

Helix 3 is necessary and sufficient for prion protein's anti-Bax function

To identify the structural elements of the prion protein (PrP) necessary for its protective function against Bcl-2 associated protein X (Bax), we performed structure–function analyses of the anti-Bax function of cytosolic PrP (CyPrP) in MCF-7 cells. Deletions of 1, 2, or 3 N-terminal Bcl-2 homology domain 2-like octapeptide repeats (BORs), but not deletion of all four BORs, abolish CyPrPs anti-Bax function. Deletion of α-helix 3 (PrP23–199) or further C-terminal deletions of α-helix 1 and 2, and β-strand 1 and 2 (PrP23–172, PrP23–160, PrP23–143, and PrP23–127) eliminates CyPrPs protection against Bax-mediated cell death. The substitution of helix 3 amino acid residues K204, V210, and E219 by proline inhibits the anti-Bax function of CyPrP. The substitution of K204, but not V210 and E219, by alanine residues also prevents CyPrPs anti-Bax function. Expression of PrPs helix 3 displays anti-Bax activity in MCF-7 cells and in human neurons. Together, these results indicate that although the BOR domain has an influence on PrPs anti-Bax function, the helix 3 is necessary and sufficient for the anti-Bax function of CyPrP. Identification of helix 3 as the structural element for the anti-Bax function thus provides a molecular target to modulate PrPs anti-Bax function in cancer and neurodegeneration.