Inhibition of Aß(1-40) fibril formation by cyclophilins.

Cyclophilins interact directly with the Alzheimer's disease peptide Aß (amyloid ß-peptide) and are therefore involved in the early stages of Alzheimer's disease. Aß binding to CypD (cyclophilin D) induces dysfunction of human mitochondria. We found that both CypD and CypA suppress in vitro fibril formation of Aß(1-40) at substoichiometric concentrations when present early in the aggregation process. The prototypic inhibitor CsA (cyclosporin A) of both cyclophilins as well as the new water-soluble MM258 derivative prevented this suppression. A SPOT peptide array approach and NMR titration experiments confirmed binding of Aß(1-40) to the catalytic site of CypD mainly via residues Lys(16)-Glu(22) The peptide Aß(16-20) representing this section showed submicromolar IC50 values for the peptidyl prolyl cis-trans isomerase activity of CypD and CypA and low-micromolar KD values in ITC experiments. Chemical cross-linking and NMR-detected hydrogen-deuterium exchange experiments revealed a shift in the populations of small Aß(1-40) oligomers towards the monomeric species, which we investigated in the present study as being the main process of prevention of Aß fibril formation by cyclophilins.

RNA single strands bind to a conserved surface of the major cold shock protein in crystals and solution.

Bacterial cold shock proteins (CSPs) regulate the cellular response to temperature downshift. Their general principle of function involves RNA chaperoning and transcriptional antitermination. Here we present two crystal structures of cold shock protein B from Bacillus subtilis (Bs-CspB) in complex with either a hexanucleotide (5'-UUUUUU-3') or heptanucleotide (5'-GUCUUUA-3') single-stranded RNA (ssRNA). Hydrogen bonds and stacking interactions between RNA bases and aromatic sidechains characterize individual binding subsites. Additional binding subsites which are not occupied by the ligand in the crystal structure were revealed by NMR spectroscopy in solution on Bs-CspB·RNA complexes. Binding studies demonstrate that Bs-CspB associates with ssDNA as well as ssRNA with moderate sequence specificity. Varying affinities of oligonucleotides are reflected mainly in changes of the dissociation rates. The generally lower binding affinity of ssRNA compared to its ssDNA analog is attributed solely to the substitution of thymine by uracil bases in RNA.

Monitoring fibril formation of the N-terminal domain of PABPN1 carrying an alanine repeat by tryptophan fluorescence and real-time NMR.

Intranuclear fibrils due to poly-alanine expansions in the N-terminal domain of the poly(A) binding protein PABPN1 correlate with the disease oculopharyngeal muscular dystrophy (OPMD). For monitoring fibril formation by fluorescence and real-time NMR spectroscopy, tryptophans were introduced either into the middle or C-terminal of the poly-alanine segment. The kinetics of fibril formation which were monitored by fluorescence spectroscopy were matched by real-time NMR kinetics. Our results show that fibril formation is concomitant with the burial of the tryptophans in the fibrillar core. Since no soluble pre-fibrillar intermediate(s) was detected, fibril formation of this domain may be regarded as a two state conversion from an unfolded soluble into folded insoluble species.

Hofmeister salts and potential therapeutic compounds accelerate in vitro fibril formation of the N-terminal domain of PABPN1 containing a disease-causing alanine extension.

The analysis of modulation of fibril formation helps to understand the mechanism of fibrillation processes besides opening routes for therapeutic intervention. Fibril formation was investigated with the N-terminal domain of the nuclear poly-A binding protein PABPN1, a protein in which mutation-based alanine extensions lead to the disease oculopharyngeal muscular dystrophy (OPMD). The disease is characterized by fibrillar inclusions consisting mainly of PABPN1. A systematic modulation of fibril formation kinetics was studied with trifluoroethanol, inorganic salts, low molecular weight organic substances, a poly-alanine peptide and anti-amyloidogenic compounds. Anions with salting out properties at high molar concentrations, poly-ethylene glycol and the poly-alanine peptide enhanced fibril formation rates. The effect of l-arginine on fibrillation rates depended on the counterion. Doxycycline and trehalose, compounds that have been found to mitigate OPMD symptoms in animal models, surprisingly accelerated fibril formation. Our results suggest that in the case of salts, primarily the salting out effects rather than electrostatic effects modulate fibril formation. The unexpected acceleration of fibril formation by trehalose and doxycycline questions the general view that these compounds per se impair fibril formation.