In Search for Low-Molecular-Weight Ligands of Human Serum Albumin That Affect Its Affinity for Monomeric Amyloid ß Peptide.

An imbalance between production and excretion of amyloid ß peptide (Aß) in the brain tissues of Alzheimer's disease (AD) patients leads to Aß accumulation and the formation of noxious Aß oligomers/plaques. A promising approach to AD prevention is the reduction of free Aß levels by directed enhancement of Aß 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 Aß. 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 Aß. 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-Aß40 interaction: prednisone favors HSA-Aß 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.

Recognition of granulocyte-macrophage colony-stimulating factor by specific S100 proteins.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a pleiotropic myelopoietic growth factor and proinflammatory cytokine, clinically used for multiple indications and serving as a promising target for treatment of many disorders, including cancer, multiple sclerosis, rheumatoid arthritis, psoriasis, asthma, COVID-19. We have previously shown that dimeric Ca2+-bound forms of S100A6 and S100P proteins, members of the multifunctional S100 protein family, are specific to GM-CSF. To probe selectivity of these interactions, the affinity of recombinant human GM-CSF to dimeric Ca2+-loaded forms of 18 recombinant human S100 proteins was studied by surface plasmon resonance spectroscopy. Of them, only S100A4 protein specifically binds to GM-CSF with equilibrium dissociation constant, Kd, values of 0.3-2 µM, as confirmed by intrinsic fluorescence and chemical crosslinking data. Calcium removal prevents S100A4 binding to GM-CSF, whereas monomerization of S100A4/A6/P proteins disrupts S100A4/A6 interaction with GM-CSF and induces a slight decrease in S100P affinity for GM-CSF. Structural modelling indicates the presence in the GM-CSF molecule of a conserved S100A4/A6/P-binding site, consisting of the residues from its termini, helices I and III, some of which are involved in the interaction with GM-CSF receptors. The predicted involvement of the 'hinge' region and F89 residue of S100P in GM-CSF recognition was confirmed by mutagenesis. Examination of S100A4/A6/P ability to affect GM-CSF signaling showed that S100A4/A6 inhibit GM-CSF-induced suppression of viability of monocytic THP-1 cells. The ability of the S100 proteins to modulate GM-CSF activity is relevant to progression of various neoplasms and other diseases, according to bioinformatics analysis. The direct regulation of GM-CSF signaling by extracellular forms of the S100 proteins should be taken into account in the clinical use of GM-CSF and development of the therapeutic interventions targeting GM-CSF or its receptors.

Interaction of S100A6 Protein with the Four-Helical Cytokines.

S100 is a family of over 20 structurally homologous, but functionally diverse regulatory (calcium/zinc)-binding proteins of vertebrates. The involvement of S100 proteins in numerous vital (patho)physiological processes is mediated by their interaction with various (intra/extra)cellular protein partners, including cell surface receptors. Furthermore, recent studies have revealed the ability of specific S100 proteins to modulate cell signaling via direct interaction with cytokines. Previously, we revealed the binding of ca. 71% of the four-helical cytokines via the S100P protein, due to the presence in its molecule of a cytokine-binding site overlapping with the binding site for the S100P receptor. Here, we show that another S100 protein, S100A6 (that has a pairwise sequence identity with S100P of 35%), specifically binds numerous four-helical cytokines. We have studied the affinity of the recombinant forms of 35 human four-helical cytokines from all structural families of this fold to Ca2+-loaded recombinant human S100A6, using surface plasmon resonance spectroscopy. S100A6 recognizes 26 of the cytokines from all families of this fold, with equilibrium dissociation constants from 0.3 nM to 12 µM. Overall, S100A6 interacts with ca. 73% of the four-helical cytokines studied to date, with a selectivity equivalent to that for the S100P protein, with the differences limited to the binding of interleukin-2 and oncostatin M. The molecular docking study evidences the presence in the S100A6 molecule of a cytokine-binding site, analogous to that found in S100P. The findings argue the presence in some of the promiscuous members of the S100 family of a site specific to a wide range of four-helical cytokines. This unique feature of the S100 proteins potentially allows them to modulate the activity of the numerous four-helical cytokines in the disorders accompanied by an excessive release of the cytokines.

Specific S100 Proteins Bind Tumor Necrosis Factor and Inhibit Its Activity.

Tumor necrosis factor (TNF) inhibitors (anti-TNFs) represent a cornerstone of the treatment of various immune-mediated inflammatory diseases and are among the most commercially successful therapeutic agents. Knowledge of TNF binding partners is critical for identification of the factors able to affect clinical efficacy of the anti-TNFs. Here, we report that among eighteen representatives of the multifunctional S100 protein family, only S100A11, S100A12 and S100A13 interact with the soluble form of TNF (sTNF) in vitro. The lowest equilibrium dissociation constants (Kd) for the complexes with monomeric sTNF determined using surface plasmon resonance spectroscopy range from 2 nM to 28 nM. The apparent Kd values for the complexes of multimeric sTNF with S100A11/A12 estimated from fluorimetric titrations are 0.1-0.3 µM. S100A12/A13 suppress the cytotoxic activity of sTNF against Huh-7 cells, as evidenced by the MTT assay. Structural modeling indicates that the sTNF-S100 interactions may interfere with the sTNF recognition by the therapeutic anti-TNFs. Bioinformatics analysis reveals dysregulation of TNF and S100A11/A12/A13 in numerous disorders. Overall, we have shown a novel potential regulatory role of the extracellular forms of specific S100 proteins that may affect the efficacy of anti-TNF treatment in various diseases.

Ibuprofen Favors Binding of Amyloid-ß Peptide to Its Depot, Serum Albumin.

The deposition of amyloid-ß peptide (Aß) in the brain is a critical event in the progression of Alzheimer's disease (AD). This Aß deposition could be prevented by directed enhancement of Aß binding to its natural depot, human serum albumin (HSA). Previously, we revealed that specific endogenous ligands of HSA improve its affinity to monomeric Aß. 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 Aß40/Aß42 by a factor of 3-5. Using thioflavin T fluorescence assay and transmission electron microcopy, we show that IBU favors the suppression of Aß40 fibrillation by HSA. Molecular docking data indicate partial overlap between the IBU/Aß40-binding sites of HSA. The revealed enhancement of the HSA-Aß interaction by IBU and the strengthened inhibition of Aß 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.

Erythropoietin Interacts with Specific S100 Proteins.

Erythropoietin (EPO) is a clinically significant four-helical cytokine, exhibiting erythropoietic, cytoprotective, immunomodulatory, and cancer-promoting activities. Despite vast knowledge on its signaling pathways and physiological effects, extracellular factors regulating EPO activity remain underexplored. Here we show by surface plasmon resonance spectroscopy, that among eighteen members of Ca2+-binding proteins of the S100 protein family studied, only S100A2, S100A6 and S100P proteins specifically recognize EPO with equilibrium dissociation constants ranging from 81 nM to 0.5 µM. The interactions occur exclusively under calcium excess. Bioinformatics analysis showed that the EPO-S100 interactions could be relevant to progression of neoplastic diseases, including cancer, and other diseases. The detailed knowledge of distinct physiological effects of the EPO-S100 interactions could favor development of more efficient clinical implications of EPO. Summing up our data with previous findings, we conclude that S100 proteins are potentially able to directly affect functional activities of specific members of all families of four-helical cytokines, and cytokines of other structural superfamilies.

Specific cytokines of interleukin-6 family interact with S100 proteins.

Cytokines of interleukin-6 (IL-6) family are important signaling proteins involved in various physiological and pathological processes. Earlier, we described interactions between IL-11 and S100P/B proteins from the family of S100 proteins engaged in the pathogenesis of numerous diseases. We probed here interactions between seven IL-6 family cytokines (IL-6, IL-11, OSM, LIF, CNTF, CT-1, and CLCF1) and fourteen S100 proteins (S100A1/A4/A6/A7/A8/A9/A10/A11/A12/A13/A14/A15/B/P). Surface plasmon resonance spectroscopy revealed formation of calcium-dependent complexes between IL-11, OSM, CNTF, CT-1, and CLCF1 and distinct subsets of S100A1/A6/B/P proteins with equilibrium dissociation constants of 19 nM - 12 µM. The existence of a network of interactions between Ca2+-loaded S100 proteins and IL-6 family cytokines suggest regulation of these cytokines by the extracellular forms of S100 proteins.

Serotonin Promotes Serum Albumin Interaction with the Monomeric Amyloid ß Peptide.

Prevention of amyloid ß peptide (Aß) deposition via facilitation of Aß 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 Aß 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 Aß monomer to HSA by a factor of 7/17 for Aß40/Aß42, respectively. Meanwhile, the structurally homologous SRO precursor, tryptophan (TRP), does not affect HSA's affinity to monomeric Aß, 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 Aß release from HSA in the central nervous system due to impairment of the SRO-mediated Aß trapping by HSA.

Interferon Beta Activity Is Modulated via Binding of Specific S100 Proteins.

Interferon-ß (IFN-ß) is a pleiotropic cytokine used for therapy of multiple sclerosis, which is also effective in suppression of viral and bacterial infections and cancer. Recently, we reported a highly specific interaction between IFN-ß and S100P lowering IFN-ß cytotoxicity to cancer cells (Int J Biol Macromol. 2020; 143: 633-639). S100P is a member of large family of multifunctional Ca2+-binding proteins with cytokine-like activities. To probe selectivity of IFN-ß-S100 interaction with respect to S100 proteins, we used surface plasmon resonance spectroscopy, chemical crosslinking, and crystal violet assay. Among the thirteen S100 proteins studied S100A1, S100A4, and S100A6 proteins exhibit strictly Ca2+-dependent binding to IFN-ß with equilibrium dissociation constants, Kd, of 0.04-1.5 µM for their Ca2+-bound homodimeric forms. Calcium depletion abolishes the S100-IFN-ß interactions. Monomerization of S100A1/A4/A6 decreases Kd values down to 0.11-1.0 nM. Interferon-α is unable of binding to the S100 proteins studied. S100A1/A4 proteins inhibit IFN-ß-induced suppression of MCF-7 cells viability. The revealed direct influence of specific S100 proteins on IFN-ß activity uncovers a novel regulatory role of particular S100 proteins, and opens up novel approaches to enhancement of therapeutic efficacy of IFN-ß.

A Novel Approach to Bacterial Expression and Purification of Myristoylated Forms of Neuronal Calcium Sensor Proteins.

N-terminal myristoylation is a common co-and post-translational modification of numerous eukaryotic and viral proteins, which affects their interaction with lipids and partner proteins, thereby modulating various cellular processes. Among those are neuronal calcium sensor (NCS) proteins, mediating transduction of calcium signals in a wide range of regulatory cascades, including reception, neurotransmission, neuronal growth and survival. The details of NCSs functioning are of special interest due to their involvement in the progression of ophthalmological and neurodegenerative diseases and their role in cancer. The well-established procedures for preparation of native-like myristoylated forms of recombinant NCSs via their bacterial co-expression with N-myristoyl transferase from Saccharomyces cerevisiae often yield a mixture of the myristoylated and non-myristoylated forms. Here, we report a novel approach to preparation of several NCSs, including recoverin, GCAP1, GCAP2, neurocalcin δ and NCS-1, ensuring their nearly complete N-myristoylation. The optimized bacterial expression and myristoylation of the NCSs is followed by a set of procedures for separation of their myristoylated and non-myristoylated forms using a combination of hydrophobic interaction chromatography steps. We demonstrate that the refolded and further purified myristoylated NCS-1 maintains its Са2+-binding ability and stability of tertiary structure. The developed approach is generally suited for preparation of other myristoylated proteins.

Effect of Cu2+ and Zn2+ ions on human serum albumin interaction with plasma unsaturated fatty acids.

Human serum albumin (HSA) serves as a depot and carrier of multiple unrelated ligands including several participants of the pathogenesis of Alzheimer's disease (AD), such as amyloid ß peptide (Aß), Zn2+/Cu2+ ions, docosahexaenoic (DHA), linoleic (LA), and oleic (OA) acids. To explore the interplay between HSA interaction with Zn2+/Cu2+ and the plasma unsaturated fatty acids (DHA, LA, OA, and arachidonic acid (ArA)), we have studied the metal dependence of the fatty acid (FA) binding capacity of HSA (nmax) and structural consequences of the HSA-FA interactions. HSA loading with Zn2+ decreases nmax value by 0.3-1.5, while its saturation with Cu2+ causes the FA-dependent nmax changes by up to 0.9. The Cu2+-induced decline in nmax value for DHA is due to conformational rearrangements in HSA molecule. In other cases, the changes in nmax are attributed to steric hindarance/facilitation of the HSA-FA interaction because of the protein multimerization/monomerization, as confirmed by chemical crosslinking. The surface hydrophobicity of HSA is Cu2+-, Zn2+-, and FA-dependent and decreases upon the FA binding, according to bis-ANS fluorescence data. Overall, Zn2+ or Cu2+ binding selectively affect HSA interaction with the FAs studied, in part due to changes in quaternary structure of the protein.

The Use of Human, Bovine, and Camel Milk Albumins in Anticancer Complexes with Oleic Acid.

Oleic acid (OA) is a monounsaturated fatty acid that upon binding to milk proteins, such as α-lactalbumin and lactoferrin, forms potent complexes, which exert selective anti-tumor activity against malignant cells but are nontoxic for healthy normal cells. We showed that the interaction of OA with albumins isolated from human, bovine, and camel milk results in the formation of complexes with high antitumor activity against Caco-2, HepG-2, PC-3, and MCF-7 tumor cells. The antitumor effect of the complexes is mostly due to the action of oleic acid, similar to the case of OA complexes with other proteins. Viability of tumor cells is inhibited by the albumin-OA complexes in a dose dependent manner, as evaluated by the MTT assay. Strong induction of apoptosis in tumor cells after their treatment with the complexes was monitored by flow cytometry, cell cycle analysis, nuclear staining, and DNA fragmentation methods. The complex of camel albumin with OA displayed the most pronounced anti-tumor effects in comparison with the complexes of OA with human and bovine albumins. Therefore, these results suggest that albumins have the potential to be used as efficient and low cost means of tumor treatment.

Intrinsically Disordered Regions in Serum Albumin: What Are They For?

Serum albumin is a major plasma protein in mammalian blood. The importance of this protein lies in its roles in both bioregulation and transport phenomena. Serum albumin binds various metal ions and participates in the transport and storage of fatty acids, bilirubin, steroids amino acids, and many other ligands, usually with regions of hydrophobic surface. Although the primary role of serum albumin is to transport various ligand, its versatile binding capacities and high concentration mean that it can assume a number of additional functions. The major goal of this article is to show how intrinsic disorder is encoded in the amino acid sequence of serum albumin, and how intrinsic disorder is related to functions of this important serum protein.

Calcium-dependent interaction of monomeric S100P protein with serum albumin.

S100 proteins are multifunctional (intra/extra)cellular mostly dimeric calcium-binding proteins engaged into numerous diseases. We have found that monomeric recombinant human S100P protein interacts with intact human serum albumin (HSA) in excess of calcium ions with equilibrium dissociation constant of 25-50nM, as evidenced by surface plasmon resonance spectroscopy and fluorescent titration by HSA of S100P labelled by fluorescein isothiocyanate. Calcium removal or S100P dimerization abolish the S100P-HSA interaction. The interaction is selective, since S100P does not bind bovine serum albumin and monomeric human S100B lacks interaction with HSA. In vitro glycation of HSA disables its binding to S100P. The revealed selective and highly specific conformation-dependent interaction between S100P and HSA shows that functional properties of monomeric and dimeric forms of S100 proteins are different, and raises concerns on validity of cell-based assays and animal models used for studies of (patho)physiological roles of extracellular S100 proteins.

Modulation of linoleic acid-binding properties of human serum albumin by divalent metal cations.

Human serum albumin (HSA) is an abundant multiligand carrier protein, linked to progression of Alzheimer's disease (AD). Blood HSA serves as a depot of amyloid ß (Aß) peptide. Aß peptide-buffering properties of HSA depend on interaction with its ligands. Some of the ligands, namely, linoleic acid (LA), zinc and copper ions are involved into AD progression. To clarify the interplay between LA and metal ion binding to HSA, the dependence of LA binding to HSA on Zn2+, Cu2+, Mg2+ and Ca2+ levels and structural consequences of these interactions have been explored. Seven LA molecules are bound per HSA molecule in the absence of the metal ions. Zn2+ binding to HSA causes a loss of one bound LA molecule, while the other metals studied exert an opposite effect (1-2 extra LA molecules are bound). In most cases, the observed effects are not related to the metal-induced changes in HSA quaternary structure. However, the Zn2+-induced decline in LA capacity of HSA could be due to accumulation of multimeric HSA forms. Opposite to Ca2+/Mg2+-binding, Zn2+ or Cu2+ association with HSA induces marked changes in its hydrophobic surface. Overall, the divalent metal ions modulate LA capacity and affinity of HSA to a different extent. LA- and Ca2+-binding to HSA synergistically support each other. Zn2+ and Cu2+ induce more pronounced changes in hydrophobic surface and quaternary structure of HSA and its LA capacity. A misbalanced metabolism of these ions in AD could modify interactions of HSA with LA, other fatty acids and hydrophobic substances, associated with AD.