The adaptive immunity of bacteria against foreign nucleic acids, mediated by CRISPR (clustered regularly interspaced short palindromic repeats), relies on the specific incorporation of short pieces of the invading foreign DNA into a special genomic locus, termed CRISPR array. The stored sequences (spacers) are subsequently used in the form of small RNAs (crRNAs) to interfere with the target nucleic acid. We explored the DNA-binding mechanism of the immunization protein Csn2 from the human pathogen Streptococcus agalactiae using different biochemical techniques, atomic force microscopic imaging and molecular dynamics simulations. The results demonstrate that the ring-shaped Csn2 tetramer binds DNA ends through its central hole and slides inward, likely by a screw motion along the helical path of the enclosed DNA. The presented data indicate an accessory function of Csn2 during integration of exogenous DNA by end-joining.
Aggregation of β-amyloid protein is a hallmark pathology of the neurodegenerative disorder Alzheimer's disease and proceeds from monomers to insoluble misfolded fibril forms via soluble and highly toxic oligomeric intermediates. Given the dual feature of being the most toxic form of the Aβ aggregate proteome and an early marker of pathogenesis, there is a need for sensitive methods that can be used to detect Aβ oligomers and investigate the dynamics of aggregation. Herein, we describe a method based on the application of an oligomer-sensitive fluorescent chemical probe pTP-TFE combined with the use of a QIAD (Quantitative determination of Interference with Aβ Aggregate Size Distribution) assay to correctly identify Aβ oligomers in high sensitivity. pTP-TFE was evaluated and compared to thioflavin T and pFTAA, the two most widely used amyloid fibril dyes, and shown to be the only probe capable of detecting significant differences across all oligomeric species of β-amyloid. Furthermore, by observing changes in pTP-TFE fluorescence emission over time, we could track the dynamics of oligomer populations and thereby obtain kinetic information on the Aβ42 dynamic aggregation model. Therefore, we have established a highly sensitive, readily available, and simple method for studying β-amyloid protein aggregation dynamics.
Today, only palliative therapies for Alzheimer's disease (AD) are available. Several lines of evidence suggest that the amyloid-β-peptide (Aβ) plays a central role in the pathogenesis of AD. Not only Aβ fibrils, but also small soluble Aβ oligomers in particular are suspected to be the major toxic species responsible for disease development and progression. We successfully tested Aβ binding D-peptides in AD transgenic mouse models regarding their therapeutic properties. Additionally, we used several biophysical and biochemical methods for the elucidation of the possible mechanism. The present study reports on in vitro and in vivo properties of the Aβ targeting D-enantiomeric amino acid peptide “D3”. We show that next to plaque load and inflammation reduction, oral application of the peptide improved the cognitive performance of AD transgenic mice. In addition, we provide in vitro data elucidating a novel potential mechanism underlying the observed in vivo activity of D3. The D-enantiomeric peptide D3 is able to modulate Aβ42 oligomerization in vitro. Regardless of its in vivo mechanism of action, D3 exerts therapeutically interesting activities.
Increasing evidence indicate oligomeric structures represent the most cytotoxic forms of misfolded proteins in neurodegenerative disorders. Hence there is interest in developing imaging probes that can distinguish these forms from fibrils. We have synthesised a luminescent conjugated oligopolythiophene (pTP-TFE) which is considered to bind to oligomer forms of beta-amyloid based on an aggregation assay. Hence it was of interest to characterise the binding profile of this compound in greater detail, to assess the relative binding to range of aggregated protein size, including oligomers. Compound pFTAA1 (another oligopolythiophene), pTP-TFE and Thioflavin T (ThT) were first used to monitor Aβ fibrillation conducted on a POLARstar Omega plated reader. Transmission Electron Microscopy (TEM) was used to characterize the morphology of both Aβ oligomers and fibrils species.To determine this profile of binding we also used the novel technique of QIAD (Quantitative determination of interference with Aβ aggregate size distribution) 2 which can purify and quantify different sizes of Aβ species - Aβ monomers, oligomers and fibrils. Subquently we applied QIAD on our compound, pTP-TFE, to separated and fractionated Aβ species. Fluorescent intensity values were measured after these three compounds mixed with all sizes of the Aβ aggregates. For fibrillation kinetics of Aβ aggregates, pTP-TFE reacted earlier than pFTAA, whereas ThT was the slowest. TEM micrographs showed small amount of smaller oligomeric species were present at 90 min while both pFTAA and ThT showed no fluorescence change. QIAD assay produced high amount of oligomers. Compared to binding fibrils Aβ protein (Fractions 10-14) pTP-TFE has higher fluorescence intensity towards the oligomeric fractions (Fraction 3-6) which indicates a greater binding to these forms.
In dieser Arbeit wurde die Charakterisierung und gezielte Eliminierung von toxischen Proteinaggregaten, am Beispiel der fur die Pathogenese der Alzheimer-Krankheit (AD) verantwortlichen Amyloid-β (Aβ) Oligomere, behandelt. Als Wirkstoffkandidaten dienten zunachst mit Phagen-Display bzw. mit Spiegelbild-Phagen-Display auf As-Bindung selektierte L- und D-Peptide (L3 und D3).
Eine Fraktionierung der mit D3 oder L3 inkubierten Aβ-Losungen mittels Dichtegradientenzentrifugation (DGZ) und nachfolgender Analyse durch SDS-PAGE zeigte, dass D3 in der Lage ist, in substochiometrischer Konzentration den Gehalt von Aβ-Oligomeren bestimmter Grose zu reduzieren. In aquimolarer Konzentration von D3 oder L3 werden As- Oligomere vollstandig eliminiert. Durch Fluoreszenzmarkierung des D3 mit FITC konnte gezeigt werden, dass die eliminierten Aβ-Oligomere mit D3 zu hochmolekularen Komplexen reagieren. Diese Komplexe sind nicht amyloid. Da D3 auch zu einer Verbesserung der kognitiven Fahigkeiten in einem AD-Mausmodell fuhrte, wird vermutet, dass seine Eigenschaft in vitro Aβ-Oligomere zu eliminieren, auch fur die beobachtete in vivo Wirksamkeit von Bedeutung ist.
Um diese Hypothese zu uberprufen und effizientere Wirkstoffe gegen Aβ-Oligomere und somit gegen AD zu entwickeln, wurden unterschiedliche D3-Derivate entworfen. Gleichzeitig wurde das auf der DGZ basierende Testsystem zu einem quantitativen Assay mit hoher Reproduzierbarkeit und Wiederfindungsrate fur das eingesetzte Aβ weiterentwickelt, mit dem der Einfluss der D3-Derivate auf die As-Aggregatgrosenverteilung untersucht werden konnte. Dazu wurde die auf einer DGZ basierende Fraktionierung der Aβ-Aggregate mit einer Umkehrphasen-HPLC-Analytik unter vollstandig denaturierenden Bedingungen kombiniert.
Mit diesem Testsystem wurden nun viele D3-Derivate auf die Verringerung von Aβ- Oligomeren getestet. Ein „Head to Tail“ Dimer des D3 (D3D3) erwies sich im Vergleich zu D3 als effektiver bezuglich dieser Eigenschaft, wodurch die Hypothese, dass D3D3 dank der hoheren Aviditat eine hohere Wirksamkeit zeigen sollte, bestatigt wurde. Die hohere Effektivitat von D3D3 gegenuber D3 lies sich ebenfalls im Tiermodell aufgrund verbesserter kognitiver und motorischer Fahigkeiten wiederfinden. Diese Ergebnisse legten nahe, dass mit dem entwickelten in vitro Testsystem die Wirksamkeit von Wirkstoffen, welche Aβ-Oligomere als Target haben, im Tiermodell vorhergesagt werden kann.
Mit diesem Verfahren gelang es weitere Derivate des D3, welche neben der Dimerisierung, gezielte Sequenzanderungen, auch Zyklisierungen und kovalente Kopplungen an andere Wirkstoffkandidaten erzeugt wurden, erfolgreich fur weitere Charakterisierungen im Tiermodell vorzuselektieren. Die durch Zugabe von D3 und seinen Derivaten eliminierten Aβ- Oligomere wurden biophysikalisch und biochemisch charakterisiert. So konnte gezeigt werden, dass diese Molekule fur humane Neuroblastom-Zellen toxisch sind, uberwiegend aus β- Faltblattern bestehen, sich jedoch strukturell von Fibrillen unterscheiden. Ein im Laufe dieser Arbeit etabliertes AFM-Abbildungsverfahren von Aβ-Oligomeren in Losung zeigte, dass diese Oligomere aus ca. 23 Monomer-Einheiten bestehen.
Alzheimer's disease (AD) is a progressive neurodegenerative disorder that affects millions of people worldwide. One AD hallmark is the aggregation of β-amyloid (Aβ) into soluble oligomers and insoluble fibrils. Several studies have reported that oligomers rather than fibrils are the most toxic species in AD progression. Aβ oligomers bind with high affinity to membrane-associated prion protein (PrP), leading to toxic signaling across the cell membrane, which makes the Aβ–PrP interaction an attractive therapeutic target. Here, probing this interaction in more detail, we found that both full-length, soluble human (hu) PrP(23–230) and huPrP(23–144), lacking the globular C-terminal domain, bind to Aβ oligomers to form large complexes above the megadalton size range. Following purification by sucrose density–gradient ultracentrifugation, the Aβ and huPrP contents in these heteroassemblies were quantified by reversed-phase HPLC. The Aβ:PrP molar ratio in these assemblies exhibited some limited variation depending on the molar ratio of the initial mixture. Specifically, a molar ratio of about four Aβ to one huPrP in the presence of an excess of huPrP(23–230) or huPrP(23–144) suggested that four Aβ units are required to form one huPrP-binding site. Of note, an Aβ-binding all-d-enantiomeric peptide, RD2D3, competed with huPrP for Aβ oligomers and interfered with Aβ–PrP heteroassembly in a concentration-dependent manner. Our results highlight the importance of multivalent epitopes on Aβ oligomers for Aβ–PrP interactions and have yielded an all-d-peptide–based, therapeutically promising agent that competes with PrP for these interactions. Alzheimer's disease (AD) is a progressive neurodegenerative disorder that affects millions of people worldwide. One AD hallmark is the aggregation of β-amyloid (Aβ) into soluble oligomers and insoluble fibrils. Several studies have reported that oligomers rather than fibrils are the most toxic species in AD progression. Aβ oligomers bind with high affinity to membrane-associated prion protein (PrP), leading to toxic signaling across the cell membrane, which makes the Aβ–PrP interaction an attractive therapeutic target. Here, probing this interaction in more detail, we found that both full-length, soluble human (hu) PrP(23–230) and huPrP(23–144), lacking the globular C-terminal domain, bind to Aβ oligomers to form large complexes above the megadalton size range. Following purification by sucrose density–gradient ultracentrifugation, the Aβ and huPrP contents in these heteroassemblies were quantified by reversed-phase HPLC. The Aβ:PrP molar ratio in these assemblies exhibited some limited variation depending on the molar ratio of the initial mixture. Specifically, a molar ratio of about four Aβ to one huPrP in the presence of an excess of huPrP(23–230) or huPrP(23–144) suggested that four Aβ units are required to form one huPrP-binding site. Of note, an Aβ-binding all-d-enantiomeric peptide, RD2D3, competed with huPrP for Aβ oligomers and interfered with Aβ–PrP heteroassembly in a concentration-dependent manner. Our results highlight the importance of multivalent epitopes on Aβ oligomers for Aβ–PrP interactions and have yielded an all-d-peptide–based, therapeutically promising agent that competes with PrP for these interactions.
The still elusive structural difference of non-infectious and infectious amyloid of the mammalian prion protein (PrP) is a major pending milestone in understanding protein-mediated infectivity in neurodegenerative diseases. Preparations of PrP-amyloid proven to be infectious have never been investigated with a high-resolution technique. All available models to date have been based on low-resolution data. Here, we establish protocols for the preparation of infectious samples of full-length recombinant (rec) PrP-amyloid in NMR-sufficient amounts by spontaneous fibrillation and seeded fibril growth from brain extract. We link biological and structural data of infectious recPrP-amyloid, derived from bioassays, atomic force microscopy, and solid-state NMR spectroscopy. Our data indicate a semi-mobile N-terminus, some residues with secondary chemical shifts typical of α-helical secondary structure in the middle part between ∼115 to ∼155, and a distinct β-sheet core C-terminal of residue ∼155. These findings are not in agreement with all current models for PrP-amyloid. We also provide evidence that samples seeded from brain extract may not differ in the overall arrangement of secondary structure elements, but rather in the flexibility of protein segments outside the β-core region. Taken together, our protocols provide an essential basis for the high-resolution characterization of non-infectious and infectious PrP-amyloid in the near future.
ABSTRACTThe still elusive structural difference of non-infectious and infectious amyloid of the mammalian prion protein (PrP) is a major pending milestone in understanding protein-mediated infectivity in neurodegenerative diseases. Preparations of PrP-amyloid proven to be infectious have never been investigated with a high-resolution technique. All available models to date have been based on low-resolution data. Here, we establish protocols for the preparation of infectious samples of full-length recombinant (rec) PrP−amyloid in NMR-sufficient amounts by spontaneous fibrillation and seeded fibril growth from brain extract. We link biological and structural data of infectious recPrP-amyloid, derived from bioassays, atomic force microscopy, and solid-state NMR spectroscopy. Our data indicate a semi-mobile N-terminus, some residues with secondary chemical shifts typical of α-helical secondary structure in the middle part between ∼115 to ∼155, and a distinct β-sheet core C-terminal of residue ∼155. These findings are not in agreement with all current models for PrP-amyloid. We also provide evidence that samples seeded from brain extract may not differ in the overall arrangement of secondary structure elements, but rather in the flexibility of protein segments outside the β-core region. Taken together, our protocols provide an essential basis for the high-resolution characterisation of non-infectious and infectious PrP-amyloid in the near future.
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