Prevention of amyloid β peptide (Aβ) deposition via facilitation of Aβ binding to its natural depot, human serum albumin (HSA), is a promising approach to preclude Alzheimer’s disease (AD) onset and progression. Previously, we demonstrated the ability of natural HSA ligands, fatty acids, to improve the affinity of this protein to monomeric Aβ by a factor of 3 (BBRC, 510(2), 248–253). Using plasmon resonance spectroscopy, we show here that another HSA ligand related to AD pathogenesis, serotonin (SRO), increases the affinity of the Aβ monomer to HSA by a factor of 7/17 for Aβ₄₀/Aβ₄₂, respectively. Meanwhile, the structurally homologous SRO precursor, tryptophan (TRP), does not affect HSA’s affinity to monomeric Aβ, despite slowdown of the association and dissociation processes. Crosslinking with glutaraldehyde and dynamic light scattering experiments reveal that, compared with the TRP-induced effects, SRO binding causes more marked changes in the quaternary structure of HSA. Furthermore, molecular docking reveals distinct structural differences between SRO/TRP complexes with HSA. The disintegration of the serotonergic system during AD pathogenesis may contribute to Aβ release from HSA in the central nervous system due to impairment of the SRO-mediated Aβ trapping by HSA.

The deposition of amyloid-β peptide (Aβ) in the brain is a critical event in the progression of Alzheimer’s disease (AD). This Aβ deposition could be prevented by directed enhancement of Aβ binding to its natural depot, human serum albumin (HSA). Previously, we revealed that specific endogenous ligands of HSA improve its affinity to monomeric Aβ. We show here that an exogenous HSA ligand, ibuprofen (IBU), exerts the analogous effect. Plasmon resonance spectroscopy data evidence that a therapeutic IBU level increases HSA affinity to monomeric Aβ₄₀/Aβ₄₂ by a factor of 3–5. Using thioflavin T fluorescence assay and transmission electron microcopy, we show that IBU favors the suppression of Aβ₄₀ fibrillation by HSA. Molecular docking data indicate partial overlap between the IBU/Aβ₄₀-binding sites of HSA. The revealed enhancement of the HSA–Aβ interaction by IBU and the strengthened inhibition of Aβ fibrillation by HSA in the presence of IBU could contribute to the neuroprotective effects of the latter, previously observed in mouse and human studies of AD.

Strontium salts are used for treatment of osteoporosis and bone cancer, but their impact on calcium-mediated physiological processes remains obscure. To explore Sr²⁺ interference with Ca²⁺ binding to proteins of the EF-hand family, we studied Sr²⁺/Ca²⁺ interaction with a canonical EF-hand protein, α-parvalbumin (α-PA). Evaluation of the equilibrium metal association constants for the active Ca²⁺ binding sites of recombinant human α-PA (‘CD’ and ‘EF’ sites) from fluorimetric titration experiments and isothermal titration calorimetry data gave 4 × 10⁹ M⁻¹ and 4 × 10⁹ M⁻¹ for Ca²⁺, and 2 × 10⁷ M⁻¹ and 2 × 10⁶ M⁻¹ for Sr²⁺. Inactivation of the EF site by homologous substitution of the Ca²⁺-coordinating Glu in position 12 of the EF-loop by Gln decreased Ca²⁺/Sr²⁺ affinity of the protein by an order of magnitude, whereas the analogous inactivation of the CD site induced much deeper suppression of the Ca²⁺/Sr²⁺ affinity. These results suggest that Sr²⁺ and Ca²⁺ bind to CD/EF sites of α-PA and the Ca²⁺/Sr²⁺ binding are sequential processes with the CD site being occupied first. Spectrofluorimetric Sr²⁺ titration of the Ca²⁺-loaded α-PA revealed presence of secondary Sr²⁺ binding site(s) with an apparent equilibrium association constant of 4 × 10⁵ M⁻¹. Fourier-transform infrared spectroscopy data evidence that Ca²⁺/Sr²⁺-loaded forms of α-PA exhibit similar states of their COO⁻ groups. Near-UV circular dichroism (CD) data show that Ca²⁺/Sr²⁺ binding to α-PA induce similar changes in symmetry of microenvironment of its Phe residues. Far-UV CD experiments reveal that Ca²⁺/Sr²⁺ binding are accompanied by nearly identical changes in secondary structure of α-PA. Meanwhile, scanning calorimetry measurements show markedly lower Sr²⁺-induced increase in stability of tertiary structure of α-PA, compared to the Ca²⁺-induced effect. Theoretical modeling using Density Functional Theory computations with Polarizable Continuum Model calculations confirms that Ca²⁺-binding sites of α-PA are well protected against exchange of Ca²⁺ for Sr²⁺ regardless of coordination number of Sr²⁺, solvent exposure or rigidity of sites. The latter appears to be a key determinant of the Ca²⁺/Sr²⁺ selectivity. Overall, despite lowered affinity of α-PA to Sr²⁺, the latter competes with Ca²⁺ for the same EF-hands and induces similar structural rearrangements. The presence of a secondary Sr²⁺ binding site(s) could be a factor contributing to Sr²⁺ impact on the functional activity of proteins.

Autoimmune uveitis is a relapsing blind-causing ocular condition with complex pathogenesis that is not completely understood. There is a high demand for accurate animal models of experimental autoimmune uveitis (EAU) suitable for elucidating the molecular mechanisms of the disease and testing new therapeutic approaches. Here, we demonstrated that photoreceptor Ca²⁺/Zn²⁺-sensor protein recoverin is a uveoretinal antigen in albino rabbits provoking typical autoimmune chorioretinitis 2–4 weeks after immunization. The pathologic process in recoverin-induced EAU shared features with human disease and included lymphocytic infiltration of the retina, Dalen–Fuchs nodules and foci of subtotal or total retinal atrophy, manifested as a decrease in amplitude of the a-wave of the electroretinogram. In some cases, changes in the retinal vascular pattern and subretinal hemorrhages were also observed. These signs were accompanied by a gradual accumulation of serum antibodies against recoverin. Biochemical examination of the aqueous humor (AH) revealed typical characteristics of inflammation and oxidative stress, including increased levels of TNF-α and IL-6 and decreased levels of IL-10, as well as decreased total antioxidant activity, superoxide dismutase and glutathione peroxidase activities, and increased zinc concentration. Consistently, metabolomic and targeted lipidomic analysis of AH showed high lactate and low ascorbic acid levels in early EAU; increased levels of key pro-inflammatory signaling lipids such as PGE2, TXB2, 11-HETE and Lyso-PAF; and reduced levels of the anti-inflammatory fatty acid DHA in advanced stages of the disease. Uveitic AH became enriched with recoverin, confirming disruption of the blood–ocular barrier and photoreceptor damage. Notably, the application of mitochondria-targeted antioxidant therapy impeded EAU progression by maintaining local antioxidant activity and suppressing TNF-α, IL-6 and PGE2 signaling. Overall, our results demonstrate that recoverin-induced EAU in rabbits represents an accurate model of human autoimmune posterior uveitis and suggest new directions for its therapy that can be trialed using the developed model.

Vascular endothelial growth factor-A (VEGF-A), a secreted homodimeric glycoprotein, is a critical regulator of angiogenesis in normal and pathological states. The binding of heparin (HE) to VEGF₁₆₅ (the major form of VEGF-A) modulates the angiogenesis-related cascade, but the mechanism of the observed changes at the structural level is still insufficiently explored. In the present study, we examined the effect of HE on the structural and physicochemical properties of recombinant human VEGF₁₆₅ (rhVEGF₁₆₅). The HE binding results in an increase of hydrophobic surface exposure in rhVEGF₁₆₅ without changes in its secondary structure. Differential scanning calorimetry measurements for intact and HE-bound rhVEGF₁₆₅ reveals the absence of any pronounced thermally induced transitions in the protein in the temperature range from 20 to 100 °C. The apolar area increase during the heparin binding explains the pronounced HE-induced oligomerization/aggregation of rhVEGF₁₆₅, as studied by chemical glutaraldehyde cross-linking and dynamic light scattering. Molecular modeling and docking techniques were used to model the full structure of dimeric VEGF₁₆₅ and to reveal putative molecular mechanisms underlying the function of the VEGF₁₆₅/HE system. In general, the results obtained can be a basis for explaining the modulating effect of HE on the biological activity of VEGF-A.

An imbalance between production and excretion of amyloid β peptide (Aβ) in the brain tissues of Alzheimer’s disease (AD) patients leads to Aβ accumulation and the formation of noxious Aβ oligomers/plaques. A promising approach to AD prevention is the reduction of free Aβ levels by directed enhancement of Aβ binding to its natural depot, human serum albumin (HSA). We previously demonstrated the ability of specific low-molecular-weight ligands (LMWLs) in HSA to improve its affinity for Aβ. Here we develop this approach through a bioinformatic search for the clinically approved AD-related LMWLs in HSA, followed by classification of the candidates according to the predicted location of their binding sites on the HSA surface, ranking of the candidates, and selective experimental validation of their impact on HSA affinity for Aβ. The top 100 candidate LMWLs were classified into five clusters. The specific representatives of the different clusters exhibit dramatically different behavior, with 3- to 13-fold changes in equilibrium dissociation constants for the HSA–Aβ40 interaction: prednisone favors HSA–Aβ interaction, mefenamic acid shows the opposite effect, and levothyroxine exhibits bidirectional effects. Overall, the LMWLs in HSA chosen here provide a basis for drug repurposing for AD prevention, and for the search of medications promoting AD progression.