Amyloid-ß-Secondary Structure Distribution in Cerebrospinal Fluid and Blood Measured by an Immuno-Infrared-Sensor: A Biomarker Candidate for Alzheimer's Disease.

The misfolding of the Amyloid-beta (Aß) peptide into ß-sheet enriched conformations was proposed as an early event in Alzheimer's Disease (AD). Here, the Aß peptide secondary structure distribution in cerebrospinal fluid (CSF) and blood plasma of 141 patients was measured with an immuno-infrared-sensor. The sensor detected the amide I band, which reflects the overall secondary structure distribution of all Aß peptides extracted from the body fluid. We observed a significant downshift of the amide I band frequency of Aß peptides in Dementia Alzheimer type (DAT) patients, which indicated an overall shift to ß-sheet. The secondary structure distribution of all Aß peptides provides a better marker for DAT detection than a single Aß misfold or the concentration of a specific oligomer. The discrimination between DAT and disease control patients according to the amide I frequency was in excellent agreement with the clinical diagnosis (accuracy 90% for CSF and 84% for blood). The amide I band maximum above or below the decisive marker frequency appears as a novel spectral biomarker candidate of AD. Additionally, a preliminary proof-of-concept study indicated an amide I band shift below the marker band already in patients with mild cognitive impairment due to AD. The presented immuno-IR-sensor method represents a promising, simple, robust, and label-free diagnostic tool for CSF and blood analysis.

FTIR spectroscopy of biofluids revisited: an automated approach to spectral biomarker identification.

The extraction of disease specific information from Fourier transform infrared (FTIR) spectra of human body fluids demands the highest standards of accuracy and reproducibility of measurements because the expected spectral differences between healthy and diseased subjects are very small in relation to a large background absorbance of the whole sample. Here, we demonstrate that with the increased sensitivity of modern FTIR spectrometers, automatisation of sample preparation and modern bioinformatics, it is possible to identify and validate spectral biomarker candidates for distinguishing between urinary bladder cancer (UBC) and inflammation in suspected bladder cancer patients. The current dataset contains spectra of blood serum and plasma samples of 135 patients. All patients underwent cytology and pathological biopsy characterization to distinguish between patients without UBC (46) and confirmed UBC cases (89). A minimally invasive blood test could spare control patients a repeated cystoscopy including a transurethral biopsy, and three-day stationary hospitalisation. Blood serum, EDTA and citrate plasma were collected from each patient and processed following predefined strict standard operating procedures. Highly reproducible dry films were obtained by spotting sub-nanoliter biofluid droplets in defined patterns, which were compared and optimized. Particular attention was paid to the automatisation of sample preparation and spectral preprocessing to exclude errors by manual handling. Spectral biomarker candidates were identified from absorbance spectra and their 1(st) and 2(nd) derivative spectra using an advanced Random Forest (RF) approach. It turned out that the 2(nd) derivative spectra were most useful for classification. Repeat validation on 21% of the dataset not included in predictor training with Linear Discriminant Analysis (LDA) classifiers and Random Forests (RFs) yielded a sensitivity of 93 ± 10% and a specificity of 46 ± 18% for bladder cancer. The low specificity can be most likely attributed to the unbalanced and small number of control samples. Using this approach, spectral biomarker candidates in blood-derived biofluids were identified, which allow us to distinguish between cancer and inflammation, but the observed differences were tiny. Obviously, a much larger sample number has to be investigated to reliably validate such candidates.

Surface charge of polyoxometalates modulates polymerization of the scrapie prion protein.

Prions are composed solely of an alternatively folded isoform of the prion protein (PrP), designated PrP(Sc). N-terminally truncated PrP(Sc), denoted PrP 27-30, retains infectivity and polymerizes into rods with the ultrastructural and tinctorial properties of amyloid. We report here that some polyoxometalates (POMs) favor polymerization of PrP 27-30 into prion rods, whereas other POMs promote assembly of the protein into 2D crystals. Antibodies reacting with epitopes in denatured PrP 27-30 also bound to 2D crystals treated with 3 M urea. These same antibodies did not bind to either native PrP(Sc) or untreated 2D crystals. By using small, spherical POMs with Keggin-type structures, the central heteroatom was found to determine whether prion rods or 2D crystals were preferentially formed. An example of a Keggin-type POM with a phosphorous heteroatom is the phosphotungstate anion (PTA). Both PTA and a Keggin-type POM with a silicon heteratom have low-charge densities and favor formation of prion rods. In contrast, POMs with boron or hydrogen heteroatoms exhibiting higher negative charges encouraged 2D crystal formation. The 2D crystals of PrP 27-30 produced by selective precipitation with POMs were larger and more well ordered than those obtained by sucrose gradient centrifugation. Our findings argue that the negative charge of Keggin-type POMs determines the quaternary structure adopted by PrP 27-30. The mechanism by which POMs function in competing prion polymerization pathways--one favoring 2D crystals and the other, amyloid fibrils--remains to be established.

Natural and synthetic prion structure from X-ray fiber diffraction.

A conformational isoform of the mammalian prion protein (PrP(Sc)) is the sole component of the infectious pathogen that causes the prion diseases. We have obtained X-ray fiber diffraction patterns from infectious prions that show cross-beta diffraction: meridional intensity at 4.8 A resolution, indicating the presence of beta strands running approximately at right angles to the filament axis and characteristic of amyloid structure. Some of the patterns also indicated the presence of a repeating unit along the fiber axis, corresponding to four beta-strands. We found that recombinant (rec) PrP amyloid differs substantially from highly infectious brain-derived prions, both in structure as demonstrated by the diffraction data, and in heterogeneity as shown by electron microscopy. In addition to the strong 4.8 A meridional reflection, the recPrP amyloid diffraction is characterized by strong equatorial intensity at approximately 10.5 A, absent from brain-derived prions, and indicating the presence of stacked beta-sheets. Synthetic prions recovered from transgenic mice inoculated with recPrP amyloid displayed structural characteristics and homogeneity similar to those of naturally occurring prions. The relationship between the structural differences and prion infectivity is uncertain, but might be explained by any of several hypotheses: only a minority of recPrP amyloid possesses a replication-competent conformation, the majority of recPrP amyloid has to undergo a conformational maturation to acquire replication competency, or inhibitory forms of recPrP amyloid interfere with replication during the initial transmission.

Structural changes of membrane-anchored native PrP(C).

Misfolding and subsequent aggregation of endogenous proteins constitute essential steps in many human disorders, including Alzheimer and prion diseases. In most prion protein-folding studies, the posttranslational modifications, the lipid anchor in particular, were lacking. Here, we studied a fully posttranslationally modified cellular prion protein, carrying two N-glycosylations and the natural GPI anchor. We used time-resolved FTIR to study the prion protein secondary structure changes when binding to a raft-like lipid membrane via its GPI anchor. We observed that membrane anchoring above a threshold concentration induced refolding of the prion protein to intermolecular beta-sheets. Such transition is not observed in solution and is membrane specific. Excessive membrane anchoring, analyzed with molecular sensitivity, is thought to be a crucial event in the development of prion diseases.

Correction: Comparison of the molecular properties of retinitis pigmentosa P23H and N15S amino acid replacements in rhodopsin.

[This corrects the article DOI: 10.1371/journal.pone.0214639.].

Comparison of the molecular properties of retinitis pigmentosa P23H and N15S amino acid replacements in rhodopsin.

Mutations in the RHO gene encoding for the visual pigment protein, rhodopsin, are among the most common cause of autosomal dominant retinitis pigmentosa (ADRP). Previous studies of ADRP mutations in different domains of rhodopsin have indicated that changes that lead to more instability in rhodopsin structure are responsible for more severe disease in patients. Here, we further test this hypothesis by comparing side-by-side and therefore quantitatively two RHO mutations, N15S and P23H, both located in the N-terminal intradiscal domain. The in vitro biochemical properties of these two rhodopsin proteins, expressed in stably transfected tetracycline-inducible HEK293S cells, their UV-visible absorption, their Fourier transform infrared, circular dichroism and Metarhodopsin II fluorescence spectroscopy properties were characterized. As compared to the severely impaired P23H molecular function, N15S is only slightly defective in structure and stability. We propose that the molecular basis for these structural differences lies in the greater distance of the N15 residue as compared to P23 with respect to the predicted rhodopsin folding core. As described previously for WT rhodopsin, addition of the cytoplasmic allosteric modulator chlorin e6 stabilizes especially the P23H protein, suggesting that chlorin e6 may be generally beneficial in the rescue of those ADRP rhodopsin proteins whose stability is affected by amino acid replacement.

Prion protein alpha-to-beta transition monitored by time-resolved Fourier transform infrared spectroscopy.

The conformational change of the recombinant, murine prion protein (PrP) from an alpha-helical to a beta-sheet enriched state was monitored by time-resolved Fourier transform infrared (FT-IR) spectroscopy. The alpha-to-beta transition is induced by reduction of the single disulfide bond in PrP. This transition is believed to generate the scrapie form PrP(Sc), the supposed infectious agent of transmissible spongiform encephalopathies. We followed the kinetics of this conformational change using a novel method for amide I band analysis of the infrared (IR) spectra. The amide I analysis provides the secondary structure. The amide I decomposition was calibrated with the three dimensional structure of cellular PrP solved by nuclear magnetic resonance (NMR). The novel secondary structure analysis provides a root mean squared deviation (RMSD) of only 3% as compared to the NMR structure. Reduction of alpha-helical PrP caused the transient accumulation of a partially unfolded intermediate, followed by formation of a state with higher beta-sheet than alpha-helical structure contents. The novel approach allows us to now determine the secondary structure of the beta-sheet conformation. This was not determined by either NMR or X-ray. The experiments were performed in a double-sealed security cuvette developed for IR analysis of potentially infectious PrP samples outside the biosafety laboratory.

An infrared sensor analysing label-free the secondary structure of the Abeta peptide in presence of complex fluids.

The secondary structure change of the Abeta peptide to beta-sheet was proposed as an early event in Alzheimer's disease. The transition may be used for diagnostics of this disease in an early state. We present an Attenuated Total Reflection (ATR) sensor modified with a specific antibody to extract minute amounts of Abeta peptide out of a complex fluid. Thereby, the Abeta peptide secondary structure was determined in its physiological aqueous environment by FTIR-difference-spectroscopy. The presented results open the door for label-free Alzheimer diagnostics in cerebrospinal fluid or blood. It can be extended to further neurodegenerative diseases. An immunologic ATR-FTIR sensor for Abeta peptide secondary structure analysis in complex fluids is presented.

Mechanism of scrapie prion precipitation with phosphotungstate anions.

The phosphotungstate anion (PTA) is widely used to facilitate the precipitation of disease-causing prion protein (PrP(Sc)) from infected tissue for applications in structural studies and diagnostic approaches. However, the mechanism of this precipitation is not understood. In order to elucidate the nature of the PTA interaction with PrP(Sc) under physiological conditions, solutions of PTA were characterized by NMR spectroscopy at varying pH. At neutral pH, the parent [PW12O40](3-) ion decomposes to give a lacunary [PW11O39](7-) (PW11) complex and a single orthotungstate anion [WO4](2-) (WO4). To measure the efficacy of each component of PTA, increasing concentrations of PW11, WO4, and mixtures thereof were used to precipitate PrP(Sc) from brain homogenates of scrapie prion-infected mice. The amount of PrP(Sc) isolated, quantified by ELISA and immunoblotting, revealed that both PW11 and WO4 contribute to PrP(Sc) precipitation. Incubation with sarkosyl, PTA, or individual components of PTA resulted in separation of higher-density PrP aggregates from the neuronal lipid monosialotetrahexosylganglioside (GM1), as observed by sucrose gradient centrifugation. These experiments revealed that yield and purity of PrP(Sc) were greater with polyoxometalates (POMs), which substantially supported the separation of lipids from PrP(Sc) in the samples. Interaction of POMs and sarkosyl with brain homogenates promoted the formation of fibrillar PrP(Sc) aggregates prior to centrifugation, likely through the separation of lipids like GM1 from PrP(Sc). We propose that this separation of lipids from PrP is a major factor governing the facile precipitation of PrP(Sc) by PTA from tissue and might be optimized further for the detection of prions.

It's in your blood: spectral biomarker candidates for urinary bladder cancer from automated FTIR spectroscopy.

Blood samples of urinary bladder cancer (UBC) patients and patients with urinary tract infection were analysed with advanced automated high throughput Fourier transform infrared (HT-FTIR)-spectroscopy. Thin dried film samples were robotically prepared on multi-well titer plates (MTP) for absorbance measurements in transmission mode. Within the absorbance, 1st and 2nd derivative spectra of serum and two plasma preparations, discriminative patterns were identified and validated using bioinformatic tools. The optimal spectral resolution for data acquisition was determined. An accurate discrimination of the patient groups was achieved with three different independent spectral variable sets. The HT-FTIR blood test may support future clinical diagnostics.

Conformational diversity of wild-type Tau fibrils specified by templated conformation change.

Tauopathies are sporadic and genetic neurodegenerative diseases characterized by aggregation of the microtubule-associated protein Tau. Tau pathology occurs in over 20 phenotypically distinct neurodegenerative diseases, including Alzheimer disease and frontotemporal dementia. The molecular basis of this diversity among sporadic tauopathies is unknown, but distinct fibrillar wild-type (WT) Tau conformations could play a role. Using Fourier transform infrared spectroscopy, circular dichroism, and electron microscopy, we show that WT Tau fibrils and P301L/V337M Tau fibrils have distinct secondary structures, fragilities, and morphologies. Furthermore, P301L/V337M fibrillar seeds induce WT Tau monomer to form a novel fibrillar conformation, termed WT*, that is maintained over multiple seeding reactions. WT* has secondary structure, fragility, and morphology that are similar to P301L/V337M fibrils and distinct from WT fibrils. WT Tau is thus capable of conformational diversity that arises via templated conformation change, as has been described for amyloid beta, beta2-microglobulin, and prion proteins.

A unique sequence motif in the 54-kDa subunit of the chloroplast signal recognition particle mediates binding to the 43-kDa subunit.

Chloroplasts contain a novel type of signal recognition particle (cpSRP) that consists of two proteins, cpSRP54 and cpSRP43. cpSRP is involved in the post-translational targeting of the nuclear encoded light-harvesting chlorophyll-binding proteins (LHCPs) to the thylakoid membrane by forming a soluble cpSRP.LHCP transit complex in the stroma. Despite high sequence homology between chloroplast and cytosolic SRP54 proteins, the 54-kDa subunit of cpSRP is unique in its ability to bind cpSRP43. In this report, we identified a 10-amino acid long segment of cpSRP54 that forms the cpSRP43-binding site. This segment is located at position 530-539 close to the C terminus of cpSRP54. In addition, we demonstrate that arginine at position 537 is essential for binding cpSRP43 and that mutation of arginine 536 drastically reduced cpSRP43 binding. Mutations within the cpSRP43-binding site of cpSRP54 that reduced or completely abolished cpSRP complex formation also did inhibit transit complex formation and integration of LHCP into the thylakoid membrane, reflecting the importance of these residues for LHCP targeting. Alignment studies revealed that the cpSRP43-binding site is conserved in chloroplast SRP54 proteins and is not present in any SRP54 subunit of cytosolic SRPs.

De novo design of conformationally flexible transmembrane peptides driving membrane fusion.

Fusion of biological membranes is mediated by distinct integral membrane proteins, e.g., soluble N-ethylmaleimide-sensitive factor attachment protein receptors and viral fusion proteins. Previous work has indicated that the transmembrane segments (TMSs) of such integral membrane proteins play an important role in fusion. Furthermore, peptide mimics of the transmembrane part can drive the fusion of liposomes, and evidence had been obtained that fusogenicity depends on their conformational flexibility. To test this hypothesis, we present a series of unnatural TMSs that were designed de novo based on the structural properties of hydrophobic residues. We find that the fusogenicity of these peptides depends on the ratio of alpha-helix-promoting Leu and beta-sheet-promoting Val residues and is enhanced by helix-destabilizing Pro and Gly residues within their hydrophobic cores. The ability of these peptides to refold from an alpha-helical state to a beta-sheet conformation and backwards was determined under different conditions. Membrane fusogenic peptides with mixed Leu/Val sequences tend to switch more readily between different conformations than a nonfusogenic peptide with an oligo-Leu core. We propose that structural flexibility of these TMSs is a prerequisite of fusogenicity.