A pH-dependent conformational transition of Aβ peptide and physicochemical properties of the conformers in the glial cell

2002 ◽  
Vol 361 (3) ◽  
pp. 547-556 ◽  
Author(s):  
Yoichi MATSUNAGA ◽  
Nobuhiro SAITO ◽  
Akihiro FUJII ◽  
Junichi YOKOTANI ◽  
Tadakazu TAKAKURA ◽  
...  
Keyword(s):  
Glial Cell ◽  
Conformational Changes ◽  
Conformational Transition ◽  
Early Stage ◽  
Ph Dependence ◽  
Amyloid Β ◽  
Proteinase K ◽  
Aβ Peptide ◽  
Ph Dependent ◽  
Insight Into

In the present study we identified the epitopes of antibodies against amyloid β-(1–42)-peptide (Aβ1–42): 4G8 reacted with peptides corresponding to residues 17–21, 6F/3D reacted with peptides corresponding to residues 9–14, and anti 5-10 reacted with peptides corresponding to residues 5–10. The study also yielded some insight into the Aβ1–42 structures resulting from differences in pH. An ELISA study using monoclonal antibodies showed that pH-dependent conformational changes occur in the 6F/3D and 4G8 epitopes modified at pH 4.6, but not in the sequences recognized by anti 1-7 and anti 5-10. This was unique to Aβ1–40 and Aβ1–42 and did not occur with Aβ1–16 or Aβ17–42. The reactivity profile of 4G8 was not affected by blockage of histidine residues of pH-modified Aβ1–40 and Aβ1–42 with diethyl pyrocarbonate; however, the mutant [Gln11]Aβ1–40 abrogated the unique pH-dependence towards 4G8 observed with Aβ1–40. These findings suggest that these epitopes are cryptic at pH4.6, and that Glu11 is responsible for the changes. We suggest that the abnormal folding of 6F/3D epitope affected by pH masked the 4G8 epitope. A study of the binding of metal ions to Aβ1–42 suggested that Cu2+ and Zn2+ induced a conformational transition around the 6F/3D region at pH7.4, but did not affect the region when it was modified at pH4.6. However, Fe2+ had no effect, irrespective of pH. Aβ modified at pH 4.6 appeared to be relatively resistant to proteinase K compared with Aβs modified at pH7.4, and the former might be preferentially internalized and accumulated in a human glial cell. Our findings suggest the importance of microenvironmental changes, such as pH, in the early stage of formation of Aβ aggregates in the glial cell.

scholarly journals A pH-dependent conformational transition of Aβ peptide and physicochemical properties of the conformers in the glial cell

2002 ◽  
Vol 361 (3) ◽  
pp. 547 ◽  
Author(s):  
Yoichi MATSUNAGA ◽  
Nobuhiro SAITO ◽  
Akihiro FUJII ◽  
Junichi YOKOTANI ◽  
Tadakazu TAKAKURA ◽  
...  
Keyword(s):  
Physicochemical Properties ◽  
Glial Cell ◽  
Conformational Transition ◽  
Aβ Peptide ◽  
Ph Dependent

scholarly journals Cell-derived hexameric β-amyloid: a novel insight into composition, self-assembly and nucleating properties

2020 ◽  
Author(s):  
Devkee M Vadukul ◽  
Céline Vrancx ◽  
Pierre Burguet ◽  
Sabrina Contino ◽  
Nuria Suelves ◽  
...  
Keyword(s):  
Self Assembly ◽  
Critical Nucleus ◽  
Amyloid Fibril ◽  
Amyloid Β ◽  
Aβ Peptide ◽  
Amyloid A ◽  
Amyloid Fibril Formation ◽  
Secretase Activity ◽  
Β Amyloid ◽  
Insight Into

A key hallmark of Alzheimer's disease (AD) is the extracellular deposition of amyloid plaques composed primarily of the amyloidogenic amyloid-β (Aβ) peptide. The Aβ peptide is a product of sequential cleavage of the Amyloid Precursor Protein (APP), the first step of which gives rise to a C-terminal Fragment (C99). Cleavage of C99 by γ-secretase activity releases Aβ of several lengths and the Aβ42 isoform in particular has been identified as being neurotoxic. The misfolding of Aβ leads to subsequent amyloid fibril formation by nucleated polymerisation. This requires an initial and critical nucleus for self-assembly. Here, we identify and characterise the composition and self-assembly properties of cell-derived hexameric Aβ42 and show its nucleating properties which are dependent on the Aβ monomer availability. Identification of nucleating assemblies that contribute to self-assembly in this way may serve as therapeutic targets to prevent the formation of toxic oligomers.

scholarly journals Development of a quantitative and conformation-sensitive ATR-FTIR biosensor for Alzheimer's disease: The effect of deuteration on the detection of the Aβ peptide

2010 ◽  
Vol 24 (1-2) ◽  
pp. 61-66 ◽  
Author(s):  
Emilie Kleiren ◽  
Jean-Marie Ruysschaert ◽  
Erik Goormaghtigh ◽  
Vincent Raussens
Keyword(s):  
Alzheimer’S Disease ◽  
Alzheimer's Disease ◽  
Early Stage ◽  
Amyloid Β ◽  
Attenuated Total Reflection ◽  
Aβ Peptide ◽  
Deuterated Water ◽  
Germanium Crystal ◽  
Amyloid Oligomers ◽  
New Type

Alzheimer's disease (AD) is the most common form of dementia worldwide and represents a growing socio-economical issue. To date, no reliable diagnosis can be obtained at an early-stage of the disease, though it is now recognized that the aggregation of the amyloid β (Aβ) peptide is responsible for the onset of the disease. Recent studies have shown that soluble amyloid oligomers present in the physiological fluids were the most neurotoxic species and correlated best with the first signs of cognitive decline, which makes them good biomarkers in the development of a diagnostic tool.We describe here a new type of biosensor, based on attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy, that would be able to specifically detect and quantify the presence of the different forms of the Aβ peptide in solution. The principle of the detection relies on the recognition of the peptides by specific antibodies that were previously grafted on the surface of an ATR element, consisting of a functionalized germanium crystal. We show that the BIA-ATR technology is able to detect the presence of Aβ if incubated in deuterated water and that this step is crucial in the development of our conformation-sensitive biosensor for AD.

scholarly journals Monitoring phagocytic uptake of amyloid β into glial cell lysosomes in real time

2021 ◽  
Author(s):  
Priya Prakash ◽  
Krupal P. Jethava ◽  
Nils Korte ◽  
Pablo Izquierdo ◽  
Emilia Favuzzi ◽  
...  
Keyword(s):  
Real Time ◽  
Brain Tissue ◽  
Glial Cell ◽  
Amyloid Β ◽  
Tissue Sections ◽  
Health And Disease ◽  
Ph Dependent ◽  
Phagocytic Uptake

Glial cell phagocytosis of pH-dependent amyloid-β, AβpH, in live and fixed cultures, brain tissue sections, retina, cortex and in live animals useful for studying function in health and disease.

scholarly journals Local pH-dependent conformational changes leading to proteolytic susceptibility of cystatin C

1994 ◽  
Vol 302 (2) ◽  
pp. 411-416 ◽  
Author(s):  
P J Berti ◽  
A C Storer
Keyword(s):  
Ionic Strength ◽  
Conformational Change ◽  
Cystatin C ◽  
Conformational Changes ◽  
Ph Dependence ◽  
Significant Proportion ◽  
Cysteine Protease Inhibitor ◽  
Ionic Strength Dependence ◽  
Ph Dependent ◽  
Ph Range

Cystatin C, a cysteine protease inhibitor, was subject to hydrolysis at two sites when complexed with papain and in the presence of excess papain. A pH-dependent cleavage at His-86 increases Asp-87 was observed, as well as a pH-independent one at Gly-4 increases Lys-5. His-86 increases Asp-87 hydrolysis increased with decreasing pH and was characterized kinetically. It could be described by a single ionization with pKa = 3.4 +/- 0.2 and (kcat./Km)max. = 1.4 (+/- 0.4) x 10(4) M-1.s-1 at I = 0.3 M. C.d. spectroscopy, also at I = 0.3 M, demonstrated a conformational change with pKa = 3.2 +/- 0.2, indicating that the pH-dependence of hydrolysis was due to a conformational change in cystatin C. At I = 0.15 M, the pKa of the conformational change observed by c.d. shifted to 4.1 +/- 0.1. This indicates that at physiological ionic strength of 0.15 M, a significant proportion of cystatin C complexed with protease would be in a proteolytically labile conformation over the pH range 4.5 to 5, which is encountered in lysosomes. This may constitute a mechanism for clearing inappropriately localized cystatins. A pH-dependent conformational variability in this region of the inhibitor could explain the differences in the X-ray crystallographic and n.m.r. structures of the homologous chicken cystatin. The ionic-strength dependence of ionization indicates a hydrophobic stabilization of the ionizable group. The lack of pH-dependence of hydrolysis at Gly-4 increases Lys-5, with kcat./Km = 220 +/- 41 M-1.s-1 in the pH range 3.89 to 7.96 was unexpected in light of the normal, bell-shaped pH-dependence of papain-catalysed hydrolyses. This may reflect a different rate-limiting step of cystatin C hydrolysis.

scholarly journals Deletion of β-Strand and α-Helix Secondary Structure in Normal Prion Protein Inhibits Formation of Its Protease-Resistant Isoform

2001 ◽  
Vol 75 (21) ◽  
pp. 10024-10032 ◽  
Author(s):  
Ina Vorberg ◽  
Kaman Chan ◽  
Suzette A. Priola
Keyword(s):  
Secondary Structure ◽  
Conformational Changes ◽  
Cellular Localization ◽  
Conformational Transition ◽  
Neuroblastoma Cells ◽  
Proteinase K ◽  
Transmissible Spongiform Encephalopathies ◽  
Spongiform Encephalopathies ◽  
A Cell ◽  
Α Helix

ABSTRACT A fundamental event in the pathogenesis of transmissible spongiform encephalopathies (TSE) is the conversion of a normal, proteinase K-sensitive, host-encoded protein, PrP-sen, into its protease-resistant isoform, PrP-res. During the formation of PrP-res, PrP-sen undergoes conformational changes that involve an increase of β-sheet secondary structure. While previous studies in which PrP-sen deletion mutants were expressed in transgenic mice or scrapie-infected cell cultures have identified regions in PrP-sen that are important in the formation of PrP-res, the exact role of PrP-sen secondary structures in the conformational transition of PrP-sen to PrP-res has not yet been defined. We constructed PrP-sen mutants with deletions of the first β-strand, the second β-strand, or the first α-helix and tested whether these mutants could be converted to PrP-res in both scrapie-infected neuroblastoma cells (Sc+-MNB cells) and a cell-free conversion assay. Removal of the second β-strand or the first α-helix significantly altered both processing and the cellular localization of PrP-sen, while deletion of the first β-strand had no effect on these events. However, all of the mutants significantly inhibited the formation of PrP-res in Sc+-MNB cells and had a greatly reduced ability to form protease-resistant PrP in a cell-free assay system. Thus, our results demonstrate that deletion of the β-strands and the first α-helix of PrP-sen can fundamentally affect PrP-res formation and/or PrP-sen processing.

scholarly journals Cell-Derived Hexameric β-Amyloid: A Novel Insight Into Composition, Self-Assembly and Nucleating Properties

2020 ◽  
Author(s):  
Devkee Vadukul ◽  
Céline Vrancx ◽  
Pierre Burguet ◽  
Sabrina Contino ◽  
Nuria Suelves ◽  
...  
Keyword(s):  
Self Assembly ◽  
Critical Nucleus ◽  
Amyloid Fibril ◽  
Amyloid Β ◽  
Aβ Peptide ◽  
Amyloid A ◽  
Amyloid Fibril Formation ◽  
Secretase Activity ◽  
Β Amyloid ◽  
Insight Into

Abstract A key hallmark of Alzheimer’s disease (AD) is the extracellular deposition of amyloid plaques composed primarily of the amyloidogenic amyloid-β (Aβ) peptide. The Aβ peptide is a product of sequential cleavage of the Amyloid Precursor Protein (APP), the first step of which gives rise to a C-terminal Fragment (C99). Cleavage of C99 by γ-secretase activity releases Aβ of several lengths and the Aβ42 isoform in particular has been identified as being neurotoxic. The misfolding of Aβ leads to subsequent amyloid fibril formation by nucleated polymerisation. This requires an initial and critical nucleus for self-assembly. Here, we identify and characterise the composition and self-assembly properties of cell-derived hexameric Aβ42 and show its nucleating properties which are dependent on the Aβ monomer availability. Identification of nucleating assemblies that contribute to self-assembly in this way may serve as therapeutic targets to prevent the formation of toxic oligomers.

scholarly journals Simulation of pH-Dependent Conformational Transitions in Membrane Proteins: The CLC-ec1 Cl−/H+ Antiporter

Molecules ◽  
2021 ◽  
Vol 26 (22) ◽  
pp. 6956
Author(s):  
Ekaterina Kots ◽  
Derek M. Shore ◽  
Harel Weinstein
Keyword(s):  
Force Field ◽  
Conformational Changes ◽  
Intracellular Transport ◽  
Ph Dependence ◽  
Linear Interpolation ◽  
Acidic Ph ◽  
Gating Mechanism ◽  
Transporter Family ◽  
Ph Dependent ◽  
Dynamics Simulations

Intracellular transport of chloride by members of the CLC transporter family involves a coupled exchange between a Cl− anion and a proton (H+), which makes the transport function dependent on ambient pH. Transport activity peaks at pH 4.5 and stalls at neutral pH. However, a structure of the WT protein at acidic pH is not available, making it difficult to assess the global conformational rearrangements that support a pH-dependent gating mechanism. To enable modeling of the CLC-ec1 dimer at acidic pH, we have applied molecular dynamics simulations (MD) featuring a new force field modification scheme—termed an Equilibrium constant pH approach (ECpH). The ECpH method utilizes linear interpolation between the force field parameters of protonated and deprotonated states of titratable residues to achieve a representation of pH-dependence in a narrow range of physiological pH values. Simulations of the CLC-ec1 dimer at neutral and acidic pH comparing ECpH-MD to canonical MD, in which the pH-dependent protonation is represented by a binary scheme, substantiates the better agreement of the conformational changes and the final model with experimental data from NMR, cross-link and AFM studies, and reveals structural elements that support the gate-opening at pH 4.5, including the key glutamates Gluin and Gluex.

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