RESUMO
Pulsed-field gradient (PFG) NMR is an important tool for characterization of biomolecules and supramolecular assemblies. However, for micrometer-sized objects, such as amyloid fibrils, these experiments become difficult to interpret because in addition to translational diffusion they are also sensitive to rotational diffusion. We have constructed a mathematical theory describing the outcome of PFG NMR experiments on rod-like fibrils. To test its validity, we have studied the fibrils formed by Sup35NM segment of the prion protein Sup35. The interpretation of the PFG NMR data in this system is fully consistent with the evidence from electron microscopy. Contrary to some previously expressed views, the signals originating from disordered regions in the fibrils can be readily differentiated from the similar signals representing small soluble species (e.g. proteolytic fragments). This paves the way for diffusion-sorted NMR experiments on complex amyloidogenic samples.
Assuntos
Amiloide/síntese química , Ressonância Magnética Nuclear Biomolecular , Proteínas Priônicas/síntese química , Amiloide/química , Difusão , Substâncias Macromoleculares/síntese química , Substâncias Macromoleculares/química , Proteínas Priônicas/química , RotaçãoRESUMO
Herein, we have successfully semi-synthesized a TDP-43 prion-like domain with Ser404 phosphorylation. We have demonstrated that Ser404 phosphorylation could accelerate the amyloid aggregation of the TDP-43 prion-like domain and aggravate its cytotoxicity.
Assuntos
Proteínas Amiloidogênicas/farmacologia , Proteínas de Ligação a DNA/farmacologia , Fragmentos de Peptídeos/farmacologia , Proteínas Priônicas/farmacologia , Serina/química , Proteínas Amiloidogênicas/síntese química , Proteínas Amiloidogênicas/metabolismo , Proteínas Amiloidogênicas/toxicidade , Animais , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/síntese química , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/toxicidade , Camundongos , Fragmentos de Peptídeos/síntese química , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/toxicidade , Fosforilação , Proteínas Priônicas/síntese química , Proteínas Priônicas/metabolismo , Proteínas Priônicas/toxicidade , Domínios Proteicos , Multimerização ProteicaRESUMO
Transmissible spongiform encephalopathy is associated with misfolding of prion protein (PrP) into an amyloid ß-rich aggregate. Previous studies have indicated that PrP interacts with Alzheimer's disease amyloid-ß peptide (Aß), but it remains elusive how this interaction impacts on the misfolding of PrP. This study presents the first inâ vitro evidence that Aß induces PrP-amyloid formation at submicromolar concentrations. Interestingly, systematic mutagenesis of PrP revealed that Aß requires no specific amino acid sequences in PrP, and induces the misfolding of other unrelated proteins (insulin and lysozyme) into amyloid fibrils in a manner analogous to PrP. This unanticipated nonspecific amyloidogenic effect of Aß indicates that this peptide might be involved in widespread protein aggregation, regardless of the amino acid sequences of target proteins, and exacerbate the pathology of many neurodegenerative diseases.
Assuntos
Peptídeos beta-Amiloides/química , Proteínas Priônicas/síntese química , Sequência de Aminoácidos , Humanos , Proteínas Priônicas/química , Agregados ProteicosRESUMO
Prions are suspected as pathogen of the fatal transmissible spongiform encephalopathies. Strategies to access homogenous prion protein (PrP) are required to fully comprehend the molecular mechanism of prion diseases. However, the polypeptide fragments from PrP show a high tendency to form aggregates, which is a gigantic obstacle of protein synthesis and purification. In this study, murine prion sequence 90 to 230 that is the core three-dimensional structure domain was constructed from three segments murine PrP (mPrP)(90-177), mPrP(178-212), and mPrP(213-230) by combining protein expression, chemical synthesis and chemical ligation. The protein sequence 90 to 177 was obtained from expression and finally converted into the polypeptide hydrazide by chemical activation of a cysteine in the tail. The other two polypeptide fragments of the C-terminal were obtained by chemical synthesis, which utilized the strategies of isopeptide and pseudoproline building blocks to complete the synthesis of such difficult sequences. The three segments were finally assembled by sequentially using native chemical ligation. This strategy will allow more straightforward access to homogeneously modified PrP variants. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.