The identification of the factors that enable normally folded proteins to remain in their soluble and functional states is crucial for a comprehensive understanding of any biological system. We have determined a series of energy landscapes of the acylphosphatase from Drosophila melanogaster under a variety of conditions by combining NMR measurements with restrained molecular dynamics simulations. We thus analyzed the differences in the structures, dynamics, and energy surfaces of the protein in its soluble state or in situations where it aggregates through conformational states that have native-like structure, folding stability, and enzymatic activity. The study identifies the nature of the energy barriers that under normal physiological conditions prevent the protein ensemble from populating dangerous aggregation-prone states. We found that such states, although similar to the native conformation, have altered surface charge distribution, alternative topologies of the β-sheet region, and modified solvent exposure of hydrophobic surfaces and aggregation-prone regions of the sequence. The identified barriers allow the protein to undergo functional dynamics while remaining soluble and without a significant risk of misfolding and aggregation into nonfunctional and potentially toxic species.protein misfolding | free energy barriers | avoidance of protein aggregation | molecular simulations

In 5% (v/v) trifluoroethanol, pH 5.5, 25 degrees C one of the acylphosphatases from Drosophila melanogaster (AcPDro2) forms fibrillar aggregates that bind thioflavin T and Congo red and have an extensive beta-sheet structure, as revealed by circular dichroism. Atomic force microscopy indicates that the fibrils and their constituent protofilaments have diameters compatible with those of natural amyloid fibrils. Spectroscopic and biochemical investigation, carried out using near- and far-UV circular dichroism, intrinsic and 1-anilino-8-naphthalenesulfonic acid-derived fluorescence, dynamic light scattering, and enzymatic activity assays, shows that AcPDro2 has, before aggregation, a secondary structure content packing around aromatic and hydrophobic residues, hydrodynamic diameter, and catalytic activity indistinguishable from those of the native protein. The native protein was found to have the same conformational stability under native and aggregating conditions, as determined from urea-induced unfolding. The kinetic analysis supports models in which AcPDro2 aggregates initially without need to unfold and subsequently undergoes a conformational change into amyloid-like structures. Although fully or partially unfolded states have a higher propensity to aggregate, the residual aggregation potential that proteins maintain upon complete folding can be physiologically relevant and be directly involved in the pathogenesis of some protein deposition diseases.

Background: Psoriasis is a chronic hyperproliferative inflammatory skin disease, characterized by a generalized redox imbalance. Anti-tumor necrosis factor (TNF)-α therapy is widely used for the treatment of this disease, but its effect on blood redox status hasn't been explored. Objective: To investigate the effects of anti-TNF-α therapy on blood redox status in psoriatic patients. Methods: Twenty-nine psoriatic patients (PSO) were divided into two groups: one remained untreated (NRT) and to another the anti-TNF-α therapy was prescribed (TR). The levels of main oxidative stress markers and total antioxidant capacity (TAC) in plasma, levels of total reactive oxygen species (ROS) production, lipoperoxidation, TAC, glutathione content, and activity of NADPH oxidase in white blood cells (WBC) were evaluated in PSO, in NTR and TR after 6 months of the study. Results: Plasma levels of malondialdehyde (MDA) and protein carbonyl content (PCO), ROS production, lipoperoxidation, and glutathione content in WBC were increased, while TAC in both plasma and WBC was decreased in PSO with respect to controls. In the plasma of TR, levels of MDA and PCO were significantly lower with respect to PSO and NTR. The activity of NADPH oxidase was significantly increased in WBC of PSO and NTR but not in TR versus controls. Discussion: Our results represent novel data about the redox status of WBC in psoriatic patients. A significant redox-balancing effect of anti-TNF-α therapy, probably associated with the normalization of NADPH oxidase activity in WBC, was demonstrated.

The assembly of proteins into highly organized fibrillar aggregates is a key process in biology, biotechnology, and human disease. It has been shown that proteins retain a small, yet significant propensity to aggregate when they are folded into compact globular structures, and this may be physiologically relevant, particularly when considering that proteins spend most of their lifespan into such compact states. Proteins from the acylphosphatase-like structural family have been shown to aggregate via different mechanisms, with some members forming native-like aggregates as a first step of their aggregation process and others requiring unfolding as a first necessary step. Here we use the acylphosphatase from Sulfolobus solfataricus to show that assembly of folded protein molecules into native-like aggregates is prevented by single-point mutations that introduce structural protections within one of the most flexible region of the protein, the peripheral edge beta-strand 4. The resulting mutants do not form native-like aggregates, but can still form thioflavin T-binding and beta-structured oligomers, albeit more slowly than the wild-type protein. The kinetic data show that formation of the latter species proceeds via an alternative mechanism that is independent of the transient formation of native-like aggregates.

The acylphosphatases from Sulfolobus solfataricus and Drosophila melanogaster (Sso AcP and AcPDro2) were previously shown to form amyloid-like aggregates without the need to unfold initially. Inorganic phosphate (Pi), a competitive inhibitor binding specifically to the active site of these proteins, was found to stabilize, upon binding, the native state of AcPDro2 and to inhibit its conversion into amyloid-like fibrils. The inhibitory effect of Pi is suppressed only in a variant in which the Arg residue responsible for Pi binding is mutated. The study on Sso AcP shows that Pi retards both the formation of the initial nativelike oligomers and their subsequent conversion into protofibrils. Thus, stabilization of the native structure mediated by specific binding with small molecules can be an effective therapeutic strategy against protein deposition diseases that originate from initially folded proteins, independently of the structure of the protein, its aggregation pathway, and the particular aggregated species responsible for pathogenesis.