Helix Backbone Dynamics of the Alzheimer Amyloid Precursor Protein Transmembrane Domain - a γ-Secretase Substrate

Die Transmembrandomäne (TMD) des C99 Fragments, das Teil des Alzheimerschen Amyloid Vorläuferproteins ist, wird durch die γ-Sekretase sequentiell prozessiert. Die Abundanz an β-verzweigten Residuen in der Spaltregion gab Anlass zur These, dass lokales, transientes Auffalten der TMD-Helix Einfluss auf die Proteolyse hat. In dieser Arbeit wurde ihre Peptidrückgratdynamik mittels Deuterium-Wasserstoff-Austausch-Experimenten gekoppelt an die Elektrospray-Ionisation-Massen-spektrometrie von Modellpeptiden charakterisiert. Die N-terminale Glyzin-reiche Dimerregion ist sehr dynamisch. Die C-terminale Spaltregion ist moderat dynamisch und ist im Vergleich zu Nichtsubstrat-TMDs mit durchschnittlicher Residuenzusammensetzung nicht dynamischer, da ihre Threonine die Helix durch zusätzliche H-Brücken zum Rückgrat stabilisieren. Die Helixdynamik der freien C-Termini der Spaltfragmente erhöhte sich mit jeder weiteren Spaltstelle zum N-Terminus.The transmembrane domain (TMD) of the C99 fragment which is part of the Alzheimer’s Amyloid Precursor Protein is sequentially processed by the γ-secretase. The abundance of β-branched residues in the cleavage region gives the rationale that local, transient unfolding of the TMD helix might be important to proteolysis. In the present work, its backbone dynamics was characterized on model peptides with deuterium/hydrogen exchange experiments coupled to electrospray ionization mass spectrometry. The N-terminal glycine-rich dimerization domain is highly dynamic. The C-terminal cleavage region is moderately dynamic and is not more dynamic than a non-substrate TMD with average residue composition because its threonine residues stabilize the helix with additional H-bonds to the backbone. The helix dynamics of the free C-termini of the fragments was increased with each further cleavage site towards the N-terminus

Macroamphiphilic components of thermophilic actinomycetes: identification of lipoteichoic acid in thermobifida fusca

The cell envelopes of gram-positive bacteria contain structurally diverse membrane-anchored macroamphiphiles (lipoteichoic acids and lipoglycans) whose functions are poorly understood. Since regulation of membrane composition is an important feature of adaptation to life at higher temperatures, we have examined the nature of the macroamphiphiles present in the thermophilic actinomycetes Thermobifida fusca and Rubrobacter xylanophilus. Following hot-phenol-water extraction and purification by hydrophobic interaction chromatography, Western blotting with a monoclonal antibody against lipoteichoic acid strongly suggested the presence of a polyglycerophosphate lipoteichoic acid in T. fusca. This structure was confirmed by chemical and nuclear magnetic resonance analyses, which confirmed that the lipoteichoic acid is substituted with ?-glucosyl residues, in common with the teichoic acid of this organism. In contrast, several extraction methods failed to recover significant macroamphiphilic carbohydrate- or phosphate-containing material from R. xylanophilus, suggesting that this actinomycete most likely lacks a membrane-anchored macroamphiphile. The finding of a polyglycerophosphate lipoteichoic acid in T. fusca suggests that lipoteichoic acids may be more widely present in the cell envelopes of actinomycetes than was previously assumed. However, the apparent absence of macroamphiphiles in the cell envelope of R. xylanophilus is highly unusual and suggests that macroamphiphiles may not always be essential for cell envelope homeostasis in gram-positive bacteria

The Backbone Dynamics of the Amyloid Precursor Protein Transmembrane Helix Provides a Rationale for the Sequential Cleavage Mechanism of γ‑Secretase

The etiology of Alzheimer’s disease depends on the relative abundance of different amyloid-β (Aβ) peptide species. These peptides are produced by sequential proteolytic cleavage within the transmembrane helix of the 99 residue C-terminal fragment of the amyloid precursor protein (C99) by the intramembrane protease γ-secretase. Intramembrane proteolysis is thought to require local unfolding of the substrate helix, which has been proposed to be cleaved as a homodimer. Here, we investigated the backbone dynamics of the substrate helix. Amide exchange experiments of monomeric recombinant C99 and of synthetic transmembrane domain peptides reveal that the N-terminal Gly-rich homodimerization domain exchanges much faster than the C-terminal cleavage region. MD simulations corroborate the differential backbone dynamics, indicate a bending motion at a diglycine motif connecting dimerization and cleavage regions, and detect significantly different H-bond stabilities at the initial cleavage sites. Our results are consistent with the following hypotheses about cleavage of the substrate: First, the GlyGly hinge may precisely position the substrate within γ-secretase such that its catalytic center must start proteolysis at the known initial cleavage sites. Second, the ratio of cleavage products formed by subsequent sequential proteolysis could be influenced by differential extents of solvation and by the stabilities of H-bonds at alternate initial sites. Third, the flexibility of the Gly-rich domain may facilitate substrate movement within the enzyme during sequential proteolysis. Fourth, dimerization may affect substrate processing by decreasing the dynamics of the dimerization region and by increasing that of the C-terminal part of the cleavage region