Phytosterols disaggregate bovine serum albumin under the glycation conditions through interacting with its glycation sites and altering its secondary structure elements

Highlights

• The disaggregation effects of 3 structurally different PS on glycated-BSA were studied.

• PS dose-dependently and structurally disaggregated BSA under the glycation conditions.

• PS altered the secondary structures, particles size, and spectra of glycated-BSA aggregates.

• PS interacted with some of the lysyl and arginine glycation sites of BSA.

• γ-oryzanol highly inhibited the glycation reactions since their unique structure.

Abstract

Discovering small molecules with protein-disaggregation effects is recently needed. For the first time, we intensely studied the anti-amyloidogenic effects of 3 structurally different phytosterols (PS), namely stigmasterol, β-sitosterol, and γ-oryzanol, on bovine serum albumin (BSA) under aggregations-promoting conditions using multispectral, microstructure, and molecular docking methods. Results found that PS dose- and structure- dependently inhibited BSA-aggregations under the glycation conditions through separating BSA-peak size, quenching Tryptophan-intensity, altering BSA-hydrophobicity, and microstructural declining the aggregates of glycated-BSA. Throughout the underlying mechanism beyond its disaggregation effects, PS reformed cross-β-sheet structure, SDS-PAGE-bands, and XRD-peaks of glycated-BSA aggregates. Most importantly, PS were found to bind with some lysyl and arginine glycation sites of BSA, specifically Lys114, Lys116, Lys136, Lys431, Arg427, and Arg185, via Hydrogen-bonding with their –OH-groups and pi-pi interactions of their steroid core. Taken together, the current results unleash that PS could restrict BSA-aggregations under the glycation conditions and their subsequent changes, which can assist in the design of reasonable therapeutics.

Introduction

Protein aggregations are certainly considered as one of the most exciting topics in biochemistry disciplines. The pathway of protein folding is distributed with stochastic-misfolding faults which often chief to forming aggregates-prone partly unfolded states, self-assembling to finally form protein-aggregates [1]. Such aggregates are specific kinds of insoluble structured protein-bonds which hold a basic core of overlapped β-sheet and able to interact with precise dyes such as Thioflavin-T and Congo red [2]. Most importantly, protein misfolding, aggregations, and therefore aggregate-depositions are nowadays of key interest due to their association with several human diseases [3], either neuropathic (Alzheimer, Parkinson, and so on) and/or non-neuropathic (diabetes type-II, and so on) [4]. Since these disorders touch millions of people every year, declining and/or mitigating aggregation is a public health priority.

There are several key steps involved in the destabilization of protein’s native conformations [5]. Most importantly, much attention has been dedicated to the function acted by glycation reactions of proteins in motivating aggregations. Throughout glycations, reducing sugars are covalently and nonenzymatically interacted with protein amino groups, namely lysine, arginine, and their N-terminus [6]. Glycation-induced aggregations, including proteins' β-amyloid, prions, and transthyretin, are found often glycated, showing the direct relation between amyloidosis and protein glycations [7]. The glycations are slowly accumulated in vivo leading to cellular modifications, different protein biologically dysfunctions [8], AGEs-accumulation [9], and toxic/misfolded aggregated-polypeptides [10].

Therefore, we hypothesized that inhibiting the protein glycation could also lead to inhibit the protein aggregations. Serum Albumin and its counterpart proteins, especially bovine serum protein (BSA), are highly soluble proteins rich in helical structure, common carrier, and convenient as a real model for scheming anti-amyloidogenic medications. BSA is a stable globular protein (66.4 k_D) with 583 amino acid residues. BSA has two tryptophan residues, namely Trp134 and Trp213, that possess intrinsic fluorescence, where Trp134 is placed on the surface of the molecule, and Trp213 is situated within a hydrophobic binding pocket of the protein. BSA also includes 3 homologous domains (I, II, and III), which are divided by 17 disulfide bonds into 9 loops (L1-L9) and each domain is composed of 2 subdomains (IA and IB, IIA and IIB, IIIA and IIIB) [11], [12], [13], [14], [15]. Experimental biochemical assays are commonly used to determine albumin concentration because the amount of this macromolecule has a significant effect on the biological systems [16], [17].

Great efforts have been done to identify potential additives, namely synthetic peptides, polyphenols, vitamins, and nucleotides that either inhibit or reverse protein aggregations [18], [19]. Herein, and for the first time we are reporting the inhibitory effects of structurally different phytosterols (PS) on thermally induced BSA-aggregations under the glycation conditions. In our recent findings, we found that PS inhibited the activity of glycation-related enzymes, resulting in reducing the glycemic ratio from the origin [20]. Additionally, we also investigated the ability of PS to delay AGEs-formations in BSA-glucose model in the primary and secondary steps of glycations through noncovalently binding with some of the lysyl glycation sites of BSA-structure and trapping dicarbonyls intermediates [21].

Therefore, we aimed to explore the effect of 3 structurally different PS, including stigmasterol (SS), β-sitosterol (βS), and γ-oryzanol (γO), on the in vitro aggregations of glycated-BSA. We used therefore multidimensional methods, including fluorescence spectroscopy, thioflavin-T (Th-T), 8-anilinonaphthalene-1-sulfonic acid (ANS), Congo red binding (CR), dynamic light scattering (DLS), transmission (TEM) and scanning (SEM) electron microscopies, sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), circular dichroism (CD), X-ray diffractograms (XRD), and molecular docking assays. The current results could concrete the platform for further efforts on scheming of appropriate molecules towards aggregations-associated disorders.