RESUMEN
There are two different prion conformations: (1) the cellular natural (PrPC) and (2) the scrapie (PrPSc), an infectious form that tends to aggregate under specific conditions. PrPC and PrPSc are widely different regarding secondary and tertiary structures. PrPSc contains more and longer ß-strands compared to PrPC. The lack of solved PrPSc structures precludes a proper understanding of the mechanisms related to the transition between cellular and scrapie forms, as well as the aggregation process. In order to investigate the conformational transition between PrPC and PrPSc, we applied MDeNM (molecular dynamics with excited normal modes), an enhanced sampling simulation technique that has been recently developed to probe large structural changes. These simulations yielded new structural rearrangements of the cellular prion that would have been difficult to obtain with standard MD simulations. We observed an increase in ß-sheet formation under low pH (≤ 4) and upon oligomerization, whose relevance was discussed on the basis of the energy landscape theory for protein folding. The characterization of intermediate structures corresponding to transition states allowed us to propose a conversion model from the cellular to the scrapie prion, which possibly ignites the fibril formation. This model can assist the design of new drugs to prevent neurological disorders related to the prion aggregation mechanism.
Asunto(s)
Simulación de Dinámica Molecular , Proteínas PrPC/química , Proteínas PrPSc/química , Agregado de Proteínas , Humanos , Concentración de Iones de Hidrógeno , Conformación Proteica en Lámina beta , Pliegue de ProteínaRESUMEN
Since the first description of prion diseases, great effort has been made toward comprehending this new paradigm in biology. Despite large advances in the field, many questions remain unanswered, especially concerning the conversion of PrP(C) into PrP(Sc). How this conformational transition evolves is a crucial problem that must be solved in order to attain further progress in therapeutics and prevention. Recent developments have indicated the requirement for partners of the prion protein in triggering the conversion. In the present review, we will explore the interaction of PrP with some of its most intriguing partners, such as sulfated glycans and nucleic acids. These molecules seem to play a dual role in prion biology and could be fundamental to explaining how prion diseases arise, as well as in the development of effective therapeutic approaches.
Asunto(s)
Glicosaminoglicanos/metabolismo , Ácidos Nucleicos/metabolismo , Enfermedades por Prión/metabolismo , Priones/metabolismo , Animales , Humanos , Modelos Moleculares , Proteínas PrPC/química , Proteínas PrPC/metabolismo , Proteínas PrPSc/química , Proteínas PrPSc/metabolismo , Priones/química , Unión Proteica , Conformación ProteicaRESUMEN
Conversion of the cellular prion protein (PrP(C)) into its altered conformation, PrP(Sc), is believed to be the major cause of prion diseases. Although PrP is the only identified agent for these diseases, there is increasing evidence that other molecules can modulate the conversion. We have found that interaction of PrP with double-stranded DNA leads to a protein with higher beta-sheet content and characteristics similar to those of PrP(Sc). RNA molecules can also interact with PrP and potentially modulate PrP(C) to PrP(Sc) conversion or even bind differentially to both PrP isoforms. Here, we investigated the interaction of recombinant murine PrP with synthetic RNA sequences and with total RNA extracted from cultured neuroblastoma cells (N2aRNA). We found that PrP interacts with N2aRNA with nanomolar affinity, aggregates upon this interaction, and forms species partially resistant to proteolysis. RNA does not bind to N-terminal deletion mutants of PrP, indicating that the N-terminal region is important for this process. Cell viability assays showed that only the N2aRNA extract induces PrP-RNA aggregates that can alter the homeostasis of cultured cells. Small RNAs bound to PrP give rise to nontoxic small oligomers. Nuclear magnetic resonance measurements of the PrP-RNA complex revealed structural changes in PrP, but most of its native fold is maintained. These results indicate that there is selectivity in the species generated by interaction with different molecules of RNA. The catalytic effect of RNA on the PrP(C)-->PrP(Sc) conversion depends on the RNA sequence, and small RNA molecules may exert a protective effect.
Asunto(s)
Neuroblastoma/química , Proteínas PrPC/química , Proteínas PrPSc/química , ARN Catalítico/química , ARN Neoplásico/química , Proteínas de Unión al ARN/química , Animales , Catálisis , Línea Celular Tumoral , Supervivencia Celular , ADN de Neoplasias/química , ADN de Neoplasias/genética , ADN de Neoplasias/metabolismo , Homeostasis , Espectroscopía de Resonancia Magnética , Ratones , Neuroblastoma/genética , Neuroblastoma/metabolismo , Proteínas PrPC/genética , Proteínas PrPC/metabolismo , Proteínas PrPSc/genética , Proteínas PrPSc/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , ARN Catalítico/genética , ARN Catalítico/metabolismo , ARN Neoplásico/genética , ARN Neoplásico/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismoRESUMEN
Crude brain homogenates of terminally diseased hamsters infected with the 263 K strain of scrapie (PrP Sc) were heated and/or pressurized at 800 MPa at 60 degrees C for different times (a few seconds or 5, 30, 120 min) in phosphate-buffered saline (PBS) of different pH and concentration. Prion proteins were analyzed on immunoblots for their proteinase K (PK) resistance, and in hamster bioassays for their infectivity. Samples pressurized under initially neutral conditions and containing native PrP Sc were negative on immunoblots after PK treatment, and a 6-7 log reduction of infectious units per gram was found when the samples were pressurized in PBS of pH 7.4 for 2 h. A pressure-induced change in the protein conformation of native PrP Sc may lead to less PK resistant and less infectious prions. However, opposite results were obtained after pressurizing native infectious prions at slightly acidic pH and in PBS of higher concentration. In this case an extensive fraction of native PrP Sc remained PK resistant after pressure treatment, indicating a protective effect possibly due to induced aggregation of prion proteins in such buffers.
Asunto(s)
Endopeptidasa K/química , Presión Hidrostática , Proteínas PrPSc/química , Animales , Encéfalo/metabolismo , Tampones (Química) , Fenómenos Químicos , Química Física , Cricetinae , Endopeptidasa K/metabolismo , Concentración de Iones de Hidrógeno , Proteínas PrPSc/metabolismo , Proteínas PrPSc/patogenicidad , Factores de TiempoRESUMEN
Crude brain homogenates of terminally diseased hamsters infected with the 263 K strain of scrapie (PrP Sc) were heated and/or pressurized at 800 MPa at 60°C for different times (a few seconds or 5, 30, 120 min) in phosphate-buffered saline (PBS) of different pH and concentration. Prion proteins were analyzed on immunoblots for their proteinase K (PK) resistance, and in hamster bioassays for their infectivity. Samples pressurized under initially neutral conditions and containing native PrP Sc were negative on immunoblots after PK treatment, and a 6-7 log reduction of infectious units per gram was found when the samples were pressurized in PBS of pH 7.4 for 2 h. A pressure-induced change in the protein conformation of native PrP Sc may lead to less PK resistant and less infectious prions. However, opposite results were obtained after pressurizing native infectious prions at slightly acidic pH and in PBS of higher concentration. In this case an extensive fraction of native PrP Sc remained PK resistant after pressure treatment, indicating a protective effect possibly due to induced aggregation of prion proteins in such buffers.
Asunto(s)
Animales , Cricetinae , Endopeptidasa K/química , Presión Hidrostática , Proteínas PrPSc/química , Tampones (Química) , Encéfalo/metabolismo , Química Física , Endopeptidasa K/metabolismo , Concentración de Iones de Hidrógeno , Proteínas PrPSc/metabolismo , Proteínas PrPSc/patogenicidad , Factores de TiempoRESUMEN
The main hypothesis for prion diseases proposes that the cellular protein (PrP(c)) can be altered into a misfolded, beta-sheet-rich isoform (PrP(Sc)). We describe here that host nucleic acid may catalyze the conversion between PrP(c) and PrP(Sc) isoforms, by reducing the protein mobility and by making the protein-protein interactions more likely. We summarize the findings, focusing in the biological relevance of the catalytic action of nucleic acid.
Asunto(s)
Ácidos Nucleicos , Proteínas PrPC , Conformación Proteica , ADN/química , ADN/metabolismo , Ácidos Nucleicos/química , Ácidos Nucleicos/metabolismo , Proteínas PrPC/química , Proteínas PrPC/metabolismo , Proteínas PrPSc/química , Proteínas PrPSc/metabolismo , Pliegue de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMEN
Prion diseases are associated with conformational conversion of the cellular prion protein, PrPC, into a misfolded form, PrPSc. We have investigated the equilibrium unfolding of the structured domain of recombinant murine prion protein, comprising residues 121-231 (mPrP-(121-231)). The equilibrium unfolding of mPrP-(121-231) by urea monitored by intrinsic fluorescence and circular dichroism (CD) spectroscopies indicated a two-state transition, without detectable folding intermediates. The fluorescent probe 4,4'-dianilino-1,1'-binaphthyl-5,5-disulfonic acid (bis-ANS) binds to native mPrP-(121-231), indicating exposure of hydrophobic domains on the protein surface. Increasing concentrations of urea (up to 4 M) caused the release of bound bis-ANS, whereas changes in intrinsic fluorescence and CD of mPrP took place only above 4 M urea. This indicates the existence of a partially unfolded conformation of mPrP, characterized by loss of bis-ANS binding and preservation of the overall structure of the protein, stabilized at low concentrations of urea. Hydrostatic pressure and low temperatures were also used to stabilize partially folded intermediates that are not detectable in the presence of chemical denaturants. Compression of mPrP to 3.5 kbar at 25 degrees C and pH 7 caused a slight decrease in intrinsic fluorescence emission and an 8-fold increase in bis-ANS fluorescence. Lowering the temperature to -9 degrees C under pressure reversed the decrease in intrinsic fluorescence and caused a marked (approximately 40-fold) increase in bis-ANS fluorescence. The increase in bis-ANS fluorescence at low temperatures was similar to that observed for mPrP at 1 atm at pH 4. These results suggest that pressure-assisted cold denaturation of mPrP stabilizes a partially folded intermediate that is qualitatively similar to the state obtained at acidic pH. Compression of mPrP in the presence of a subdenaturing concentration of urea stabilized another partially folded intermediate, and cold denaturation under these conditions led to complete unfolding of the protein. Possible implications of the existence of such partially folded intermediates in the folding of the prion protein and in the conversion to the PrPSc conformer are discussed.
Asunto(s)
Proteínas PrPC/química , Proteínas PrPSc/química , Animales , Dicroismo Circular , Relación Dosis-Respuesta a Droga , Concentración de Iones de Hidrógeno , Cinética , Ratones , Presión , Conformación Proteica , Desnaturalización Proteica , Pliegue de Proteína , Proteínas Recombinantes/química , Espectrometría de Fluorescencia , Temperatura , Termodinámica , Urea/farmacologíaRESUMEN
The main hypothesis for prion diseases proposes that the cellular protein (PrP(C)) can be altered into a misfolded, beta-sheet-rich isoform (PrP(Sc)), which in most cases undergoes aggregation. In an organism infected with PrP(Sc), PrP(C) is converted into the beta-sheet form, generating more PrP(Sc). We find that sequence-specific DNA binding to recombinant murine prion protein (mPrP-(23-231)) converts it from an alpha-helical conformation (cellular isoform) into a soluble, beta-sheet isoform similar to that found in the fibrillar state. The recombinant murine prion protein and prion domains bind with high affinity to DNA sequences. Several double-stranded DNA sequences in molar excess above 2:1 (pH 4.0) or 0.5:1 (pH 5.0) completely inhibit aggregation of prion peptides, as measured by light scattering, fluorescence, and circular dichroism spectroscopy. However, at a high concentration, fibers (or peptide aggregates) can rescue the peptide bound to the DNA, converting it to the aggregating form. Our results indicate that a macromolecular complex of prion-DNA may act as an intermediate for the formation of the growing fiber. We propose that host nucleic acid may modulate the delicate balance between the cellular and the misfolded conformations by reducing the protein mobility and by making the protein-protein interactions more likely. In our model, the infectious material would act as a seed to rescue the protein bound to nucleic acid. Accordingly, DNA would act on the one hand as a guardian of the Sc conformation, preventing its propagation, but on the other hand may catalyze Sc conversion and aggregation if a threshold level is exceeded.