Large proteins have a great tendency to aggregate but a low propensity to form amyloid fibrils.

The assembly of soluble proteins into ordered fibrillar aggregates with cross-β structure is an essential event of many human diseases. The polypeptides undergoing aggregation are generally small in size. To explore if the small size is a primary determinant for the formation of amyloids under pathological conditions we have created two databases of proteins, forming amyloid-related and non-amyloid deposits in human diseases, respectively.

A causative link between the structure of aberrant protein oligomers and their toxicity.

The aberrant assembly of peptides and proteins into fibrillar aggregates proceeds through oligomeric intermediates that are thought to be the primary pathogenic species in many protein deposition diseases. We describe two types of oligomers formed by the HypF-N protein that are morphologically and tinctorially similar, as detected with atomic force microscopy and thioflavin T assays, though one is benign when added to cell cultures whereas the other is toxic. Structural investigation at a residue-specific level using site-directed labeling with pyrene indicated differences in the packing of the hydrophobic interactions between adjacent protein molecules in the oligomers.

Conformational properties of unfolded HypF-N.

We have measured the intramolecular diffusion rate between distant residues in the aggregation-prone protein HypF-N under various denaturing conditions. Using the method of cysteine quenching of the tryptophan triplet state, we find that intramolecular diffusion remains roughly constant at high concentrations of denaturant (2-6 M GdnHCl) and slows down at low concentrations of denaturant, but the decrease is not uniform throughout the chain. Extrapolation of these measurements to 0 M GdnHCl gives D approximately 10(-7) cm(2) s(-1), about 1 order of magnitude lower than unstructured peptides and at least 2 orders of magnitude higher than well-behaved proteins.

The folding process of acylphosphatase from Escherichia coli is remarkably accelerated by the presence of a disulfide bond.

The acylphosphatase from Escherichia coli (EcoAcP) is the first AcP so far studied with a disulfide bond. A mutational variant of the enzyme lacking the disulfide bond has been produced by substituting the two cysteine residues with alanine (EcoAcP mutational variant C5A/C49A, mutEcoAcP). The native states of the two protein variants are similar, as shown by far-UV and near-UV circular dichroism and dynamic light-scattering measurements.

The intrachain disulfide bridge is responsible of the unusual stability properties of novel acylphosphatase from Escherichia coli.

Acylphosphatase (AcP) activity in prokaryotes was classically attributed to some aspecific acid phosphatases. We identified an open reading frame for a putative AcP in the b0968 Escherichia coli gene and purified the recombinant enzyme after checking by RT-PCR that it was indeed expressed. EcoAcP has a predicted typical fold of the AcP family but displays a very low specific activity and a high structural stability differently from its mesophilic and similarly to its hyperthermophilic counterparts.

Nature and significance of the interactions between amyloid fibrils and biological polyelectrolytes.

Charged polyelectrolytes such as glycosaminoglycans and nucleic acids have frequently been found associated with the proteinaceous deposits in the tissues of patients with amyloid diseases. We have investigated the nature and generality of this phenomenon by studying the ability of different polyanions, including DNA, ATP, heparin, and heparan sulfate, to promote the aggregation of amyloidogenic proteins and to bind to the resulting aggregates. Preformed amyloid fibrils of human muscle acylphosphatase and human lysozyme, proteins with a net positive charge at physiological pH values, were found to bind tightly to the negatively charged DNA or ATP.

Glycine residues appear to be evolutionarily conserved for their ability to inhibit aggregation.

Six glycine residues of human muscle acylphosphatase (AcP) are evolutionarily conserved across the three domains of life. We have generated six variants of AcP, each having a glycine substituted by an alanine (G15A, G19A, G37A, G45A, G53A, and G69A). Three additional variants had Gly45 replaced by serine, glutamate, and arginine, respectively.

[Cefatrizine in dentistry. Clinical study].

The Authors evaluated the cefatrizine clinical effects on 50 patients with infectious diseases. The statistical analysis of results show a high efficacy and safety with no relevant side effects.