Copper-flavonoid family of complexes involved in alkaline phosphatase activation.

The flavonoid naringenin and a family of naringenin derivative Cu(II) complexes having phenanthroline-based second ligands were selected to study alkaline phosphatase activation. This enzyme plays a critical role in tissue formation, increasing the inorganic phosphate formation, favoring mineralization, and being essential to producing bone mineralization. The effects of those compounds on the function and structure of the enzyme were evaluated by kinetic measurements, fluorescence, FTIR, and UV-Vis spectroscopies. The results showed that naringenin did not affect alkaline phosphatase activity, having a value of the Michaelis-Menten-constant close to the enzyme (Km = 3.07 × 10-6). The binary complex, Cu(II)-naringenin, and the ternary complex Cu(II)-naringenin-phenanthroline behaved as an enzyme activator in all the concentrations range used in this study. Those complexes increased in c.a. 1.9% the catalytic efficiency concerning enzyme and naringenin. The ternary complex Cu(II)-naringenin-bathophenanthroline, provokes an activator mixed effect, dependent on the substrate concentrations. The different kinetic behavior can be correlated with different conformational changes observed under the interaction with ALP. Fluorescence experiments showed a raising of the binding constant with temperature. FTIR determinations showed that the complex with bathophenanthroline modifies the ALP structure but maintains the helical structure. The other copper complexes provoked a structural unfolding, decreasing the α-helix content. None of them affect the dephosphorylation enzyme ability. Even though the interactions and structural modifications on ALP are different, it is evident that the presence of copper favors enzymatic activity. The observed electrostatic interactions probably benefit the dissociation of the bound phosphate. The results suggest potential biological applications for the studied compounds.

Data regarding the sensibility to proteolysis of a natural apolipoprotein A-I mutant.

The article shows dataset of the proteolysis of a natural variant of apolipoprotein A-I (apoA-I) with a substitution of a leucine by and arginine in position 60 (L60R), in comparison with the protein with the native sequence (Wt). This information demonstrates the potential of in vitro partial proteolysis experiments as it may be applicable to different approaches in the biophysical field. We have analyzed by different electrophoresis techniques apoA-I variants, quantified the degree of proteolysis after staining and compared the proteolysis efficiency with the computed cleavage patterns. The data shown here clearly strengthen the usefulness of this approach to test protein flexibility, as it may be attained with enzymes which are not expected to modify in vivo this protein but have a well-known digestion pattern. In addition it is appropriate for evaluating protein catabolism, as it is exemplified here by the evidence with metalloproteinase 12 (MMP-12), which is a physiological protease that may elicit the pro-inflammatory processing of this variant within the lesions. We support the work "Structural analysis of a natural apolipoprotein A-I variant (L60R) associated with amyloidosis" (Gaddi, et al., 2020), gaining insights on protein folding from a characterization by proteolysis analysis [1].

Structural analysis of a natural apolipoprotein A-I variant (L60R) associated with amyloidosis.

The reason that determines the pathological deposition of human apolipoprotein A-I variants inducing organ failure has been under research since the early description of natural mutations in patients. To shed light into the events associated with protein aggregation, we studied the structural perturbations that may occur in the natural variant that shows a substitution of a Leucine by an Arginine in position 60 (L60R). Circular dichroism, intrinsic fluorescence measurements, and proteolysis analysis indicated that L60R was more unstable, more sensitive to cleavage and the N-terminus was more disorganized than the protein with the native sequence (Wt). A higher tendency to aggregate was also detected when L60R was incubated at physiological pH. In addition, the small structural rearrangement observed for the freshly folded variant led to the release of tumor necrosis factor-α and interleukin-1ß from a model of macrophages. However, the mutant preserved both its dimeric conformation and its lipid-binding capacity. Our results strongly suggest that the chronic disease may be a consequence of the native conformation loss which elicits the release of protein conformations that could be either cytotoxic or precursors of amyloid conformations.

Fibrillar conformation of an apolipoprotein A-I variant involved in amyloidosis and atherosclerosis.

BACKGROUND: Different protein conformations may be involved in the development of clinical manifestations associated with human amyloidosis. Although a fibrillar conformation is usually the signature of damage in the tissues of patients, it is not clear whether this species is per se the cause or the consequence of the disease. Hereditary amyloidosis due to variants of apolipoprotein A-I (apoA-I) with a substitution of a single amino acid is characterized by the presence of fibrillar protein within the lesions. Thus mutations result in increased protein aggregation. Here we set up to characterize the folding of a natural variant with a mutation leading to a deletion at position 107 (apoA-I Lys107-0). Patients carrying this variant show amyloidosis and severe atherosclerosis. METHODS: We oxidized this variant under controlled concentrations of hydrogen peroxide and analyzed the structure obtained after 30-day incubation by fluorescence, circular dichroism and microscopy approaches. Neutrophils activation was characterized by confocal microscopy. RESULTS: We obtained a high yield of well-defined stable fibrillar structures of apoA-I Lys107-0. In an in vitro neutrophils system, we were able to detect the induction of Neutrophils Extracellular Traps (NETs) when we incubated with oxidized apoA-I variants. This effect was exacerbated by the fibrillar structure of oxidized Lys 107-0. CONCLUSIONS: We conclude that a pro-inflammatory microenvironment could result in the formation of aggregation-prone species, which, in addition may induce a positive feed-back in the activation of an inflammatory response. GENERAL SIGNIFICANCE: These events may explain a close association between amyloidosis due to apoA-I Lys107-0 and atherosclerosis.