Molecular Interaction of Protein-Pigment C-Phycocyanin with Bovine Serum Albumin in a Gomphosis Structure Inhibiting Amyloid Formation

International Journal of Molecular Sciences

Article Molecular Interaction of Protein-Pigment C-Phycocyanin with Bovine Serum Albumin in a Gomphosis Structure Inhibiting Amyloid Formation

Yi-Cong Luo and Pu Jing *

Shanghai Food Safety and Engineering Technology Research Center, Bor S. Luh Food Safety Research Center, Key Lab of Urban Agriculture (South), School of Agriculture & Biology, Shanghai Jiao Tong University, Shanghai 200240, China; [email protected] * Correspondence: [email protected]

Received: 29 August 2020; Accepted: 20 October 2020; Published: 2 November 2020

Abstract: Accumulation of amyloid fibrils in organisms accompanies many diseases. Natural extracts offer an alternative strategy to control the process with potentially fewer side effects. In this study, the inhibition of C-phycocyanin from Spirulina sp. on amyloid formation of bovine serum albumin (BSA) during a 21-day incubation was investigated using fluorescence and circular dichroism (CD), and mechanisms were explored via kinetic fitting and molecular docking. C-phycocyanin (0–50 µg/mL) hindered the amyloid formation process of BSA with increased half-lives (12.43–17.73 days) based on fluorescence intensity. A kinetic model was built and showed that the k1 decreased from 1.820 10 2 d 1 to 2.62 10 3 d 1 with the increase of C-phycocyanin, while k showed no changes, × − − × − − 2 indicating that the inhibition of BSA fibrillation by C-phycocyanin occurred in a spontaneous process instead of self-catalyzed one. CD results show that C-phycocyanin inhibited conformational conversion (α-helices and β-sheets) of BSA from day 6 to day 18. Molecular docking suggested that C-phycocyanin may hinder BSA fibrillation by hydrogen-bonding > 6 of 27 α-helices of BSA in a gomphosis-like structure, but the unblocked BSA α-helices might follow the self-catalytic process subsequently.

Keywords: C-phycocyanin; blue food pigment; secondary interaction; fibrillation; non-covalent interaction

1. Introduction The process of aggregation and fibril formation called amyloid formation is widely found in proteins of organisms with unknown physiological functions. Accumulation of amyloid fibrils in organisms accompanies many diseases, such as Alzheimer’s, Parkinson’s, diabetes, prion diseases, etc. [1,2]. The mechanism of amyloid formation is still debated. Many different precursor proteins are involved in the fibrillation process; protein fibrillation is often accompanied by a decrease of α-helices and an increase of β-sheets [3]. Amyloid deposits have been under investigation in a number of pathological states, which then cause irreversible damage to the tissue [4], such as amyloid-β(Aβ) plaque deposits with neurofibrillary tangles containing tau proteins, which are the key pathognomonic manifestations of Alzheimer’s disease [5]. A range of small molecules (e.g., peptides and polyphenols) have been identified as impeding fibril formation. Peptides inhibitors or monomer binding-protein have been designed to block self-association and protein aggregation as a potential therapeutic target [6,7], although numerous phytochemicals such as polyphenols have been investigated to remodel mature amyloid fibrils to non-toxic aggregates [8,9]. A previous study showed that oral administration of the cyanobacteria Spriulina appeared to mediate effects. Spirulina exhibited a neuroprotective role against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine

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SpriulinaInt. J. Mol. Sci.appeared2020, 21, 8207to mediate effects. Spirulina exhibited a neuroprotective role against 1-methyl-4-2 of 11 phenyl-1,2,3,6-tetrahydropyridine neurotoxicity in a mice model of Parkinson’s disease. Spirulina may prevent memory dysfunction, possibly by lessening Aβ protein accumulation, reducing oxidativeneurotoxicity damage in a and mice mainly model augmenting of Parkinson’s the disease. catalase Spirulinaactivity inmay senescen preventce-accelerated memory dysfunction, mice [10]. Thepossibly pigment by lessening compound, Aβ protein namely accumulation, C-phycocyanin, reducing has been oxidative implicated damage in andprotec mainlytive benefits augmenting [11,12]. the C-phycocyanincatalase activity inaffected senescence-accelerated astrocytes-mediated mice [neuroprotection10]. The pigment against compound, oxidative namely brain C-phycocyanin, injury in astrocytehas been tissue implicated models in [11]. protective More recently, benefits C-phycocyanin [11,12]. C-phycocyanin from Spirulina affected was astrocytes-mediated found to inhibit α- synucleinneuroprotection and amyloid- againstβ oxidativefibril formation brain injury[13]. Molecular in astrocyte docking tissue studies models [14] [11]. showed More that recently, the phycocyaninC-phycocyanin interacted from Spirulina with thewas enzyme found β to-secretase, inhibit α -synucleinwhich catalyzes and amyloid- the proteolysisβ fibril of formation the amyloid [13]. precursorMolecular protein docking to studies form plaques. [14] showed that the phycocyanin interacted with the enzyme β-secretase, whichC-phycocyanin catalyzes the proteolysisis a homologous of the amyloid dimer precursor containing protein two to formidentical plaques. peptides and three chromophores,C-phycocyanin and is apractically homologous recognized dimer containing as a monomer, two identical which peptides can self-assemble and three chromophores,into trimer or hexamerand is practically in a solution recognized based on as concentration a monomer, [15]. which C-phycocyanin can self-assemble (Figure into 1a) trimer from Spirulina or hexamer sp. is in an a ediblesolution pigment based onprotein, concentration composing [15]. of C-phycocyanin α and β subunits. (Figure1 a)Three from tetrapyrroleSpirulina sp. chromophoreis an edible phycocyanobilinspigment protein, composing (Figure 1b) of areα andcovalentlyβ subunits. attached Three to tetrapyrrole the αβ monomer chromophore through phycocyanobilins three conserved cysteine(Figure1 b)residues are covalently [16]. attachedAdditionally, to the αβa monomervariety of through secondary three conservedinteractions cysteine occur residues between [16]. phycocyanobilinAdditionally, a variety and chromopeptide. of secondary interactions occur between phycocyanobilin and chromopeptide.

(b)

(a) (b)

Figure 1. StructureStructure of C-phycocyanin ((aa)) andand phycocyanobilin phycocyanobilin (yellow) (yellow) with with its its secondary secondary interaction interaction of ofamino amino acid acid residues residues on the onα thesubunit α subunit (b). The (bα). andTheβ αsubunits and β subunits are colored are in colored pink and in blue, pink respectively. and blue, respectively. Bovine serum albumin (BSA) is a helical protein containing 583 amino acids, and is the most abundantBovine protein serum in albumin the circulatory (BSA) systemis a helical that isprotein involved containing in the transport 583 amino of drug acids, molecules. and is the BSA most can abundantspontaneously protein form in the fibers circulatory at pH = system7.4, with that the is increaseinvolved of inβ the-sheets transport [17]. of Therefore, drug molecules. BSA is oftenBSA canused spontaneously as a model of proteinform fibers fibrillation at pH = [ 187.4,–20 with]. In the this increase study, weof β aimed-sheets to [17]. investigate Therefore, the BSA inhibition is often of usedC-phycocyanin as a model fromof proteinSpirulina fibrillation sp. on the[18–20]. fibrillation In this ofstudy, BSA. we This aimed was to evaluated investigate using the fluorescenceinhibition of C-phycocyaninand circular dichroism from Spirulina analyses sp. andon the to fibrillation study the mechanismof BSA. This of was the evaluated secondary using interaction fluorescence using andmolecular circular docking. dichroism analyses and to study the mechanism of the secondary interaction using molecular docking. 2. Results 2. Results 2.1. Inhibitory Effect of C-phycocyanin on Formation of Amyloid Fibrils 2.1. InhibitoryFluorometric Effect and of C-phycocya CD assaysnin were on Formation applied to of evaluate Amyloid theFibrils process of amyloid formation [21,22]. The precipitates of insoluble protein fiber started being visually notable until day 18 and plenty were Fluorometric and CD assays were applied to evaluate the process of amyloid formation [21,22]. generated on day 21. The precipitates of insoluble protein fiber started being visually notable until day 18 and plenty were generated2.2. Fluorescence on day Intensity 21. and Kinetic Fitting Results

2.2. FluorescenceThe fluorescence Intensity intensity and Kinetic was Fitting positively Results associated with protein amyloid formation due to the characteristics of fluorescence, and showed in the linear range at low concentrations of BSA The fluorescence intensity was positively associated with protein amyloid formation due to the <~260 µM[23], where 5 µM of BSA was applied in the experiment. Figure2 shows that the fluorescence characteristics of fluorescence, and showed in the linear range at low concentrations of BSA <~260 intensity as an index of the degree of inhibitory effect on the amyloid formation of BSA was negatively µM [23], where 5 µM of BSA was applied in the experiment. Figure 2 shows that the fluorescence associated with an increasing dosage of C-phycocyanin (0.5–50 µg/mL), suggesting a potential

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intensity as an index of the degree of inhibitory effect on the amyloid formation of BSA was Int. J. Mol. Sci. 2020, 21, 8207 3 of 11 negatively associated with an increasing dosage of C-phycocyanin (0.5–50 µg/mL), suggesting a potential interaction between BSA and C-phycocyanin. Considering these measurements contained interactiontime as a variable, between BSArepeated and C-phycocyanin.measures analysis Considering of variance these was measurementsselected for statistical contained analysis. time as The a variable,statistical repeated data with measures the same analysis dosage of of variance C-phycocya was selectednin were for defined statistical as a analysis.group. Results The statistical of intergroup data witheffect the analysis same dosage suggested of C-phycocyanin the effect of different were defined dosages as a of group. C-phycocyanin Results of intergroupon the amyloid effect formation analysis suggestedof BSA fibrosis the eff ectwas of significantly different dosages different of C-phycocyanin(F = 11.24, p = 0.0003 on the < amyloid 0.001). In formation the meantime, of BSA due fibrosis to the wasresult significantly of sphericity different test ( Fbeing= 11.24, 5.72p =×0.0003 10−8 << 0.05,0.001). Greenhouse–Geisser In the meantime, due correction to the result was of sphericityemployed. testMultivariate being 5.72 analysis10 8 < of0.05, variance Greenhouse–Geisser revealed an extremely correction significant was employed. main effect Multivariate of time (F analysis = 1496.4, of p × − variance= 5.691 × revealed 10−30 < 0.001, an extremely Greenhouse–Geisser significant main corrected). effect of Howe time (ver,F = the1496.4, fluorescencep = 5.691 intensity10 30 < in0.001 each, × − Greenhouse–Geissertreatment gradually corrected).increased during However, a 21-day the fluorescenceincubation indicated intensity that in eachC-phycocyanin treatment was gradually unable increasedto completely during terminate a 21-day incubationthe transformation indicated from that C-phycocyaninBSA to amyloid. was unable to completely terminate the transformation from BSA to amyloid.

FigureFigure 2. 2.Inhibitory Inhibitory eeffectﬀect ofof C-phycocyanin on amyloi amyloidd formation formation using using fluorometric ﬂuorometric assay assay for for a 21- a 21-dayday duration. duration.

TableTable1 shows1 shows the the kinetic kinetic fitting fitting results results of of fluorescence fluorescence analysis, analysis, whereas whereas the the kinetic kinetic fitting fitting plots plots 3 (Figures(Figures S1–S6) S1–S6) are are presented presented in thein the Supplementary Supplementary Materials. Data. All All ‘m ‘m’’ valuesvalues (3.6(3.6 ×10 10)3) inin treatment treatment × groupsgroups (0.5–50 (0.5–50µ gµg/mL)/mL) were were fixed fixed so so that that the the results result coulds could definitely definitely be be comparable. comparable. The The coe coefficientsfficients ofof k 1k1and and k k2 2were were designed designed for for two two types types of of amyloid amyloid formation: formation: k k1 1represented represented the the process process that that BSA BSA formsforms amyloid amyloid spontaneously, spontaneously, whereas whereas k2 kwas2 was for for the the self-catalyzed self-catalyzed amyloid amyloid formation. formation. The The half-life half-life (t(t1/21/2)) values values of of amyloid amyloid formation formation were were calculated calculated and and increased increased obviously obviously from from 12.43–17.73 12.43–17.73 days days withwith the the concentration concentration of of C-phycocyanin C-phycocyanin from from 0–50 0–50µ gµg/mL,/mL, indicating indicating that that C-phycocyanin C-phycocyanin exhibited exhibited 2 3 remarkableremarkable inhibitory inhibitory eeffffect.ect. The The variation variation of of k k1 1(~10(~10−2 to− 10to−3 10) was− ) wassignificant significant compared compared to k2 (~10 to k−25), 5 (~10suggesting− ), suggesting that k1 that was k 1thewas prominent the prominent coefficient coeffi cientof the of ki thenetics kinetics in the in theinhibitory inhibitory process process of ofC- C-phycocyaninphycocyanin on on amyloid amyloid formation. formation.

TableTable 1. 1.Kinetic Kinetic ﬁtting fitting result result of of amyloid amyloid formation. formation.

C-phycocyaninC-phycocyanin 2 Half-LifeHalf-Life k1 k1 kk22 RR2 (µg(µg/mL)/mL) (t(t1/1/22,, Days)Days) 0.0 0.0 1.820 1.82010 × 102 −2 3.14± 3.14 10× 103−3 4.198 4.198 ×10 105−5 ± 2.682.68 × 10−55 0.99310.9931 12.43 12.43 × − ± × − × − ± × − 0.5 0.5 1.445 1.44510 × 102 −2 2.07± 2.07 10× 103−3 4.003 4.003 ×10 105−5 ± 4.284.28 × 10−66 0.99450.9945 14.41 14.41 × − ± × − × − ± × − 2.5 2.5 1.256 1.25610 × 102 −2 3.42± 3.42 10× 103−3 4.894 4.894 ×10 105−5 ± 8.838.83 × 10−66 0.98040.9804 13.89 13.89 × − ± × − × − ± × − 5.0 5.0 1.247 1.24710 × 102 −2 1.77± 1.77 10× 104−4 4.444 4.444 ×10 105−5 ± 4.634.63 × 10−66 0.99390.9939 14.69 14.69 × − ± × − × − ± × − 25.025.0 5.610 5.61010 × 103 −3 9.42± 9.42 10× 104−4 5.930 5.930 ×10 105−5 ± 5.045.04 × 10−66 0.99280.9928 16.45 16.45 × − ± × − × − ± × − 50.050.0 2.620 2.62010 × 103 −3 3.03± 3.03 10× 104−4 7.012 7.012 ×10 105−5 ± 3.343.34 × 10−66 0.99690.9969 17.73 17.73 × − ± × − × − ± × −

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Int. J. Mol. Sci. 2020, 21, x 4 of 11 2.3. CD Spectra 2.3. CD SpectraCD spectra were analyzed and the ratios of secondary structures were calculated to determine the process of BSA amyloid formation in Figure3 according to the references [ 3,24]. Figure3a,b showed that CDBSA spectra alone changed were byanalyzed a decrease and of α the-helices ratios and of an secondary increase of β structures-sheets during were a 21-day calculated incubation, to determine the processwhich wasof BSA noted amyloid as a typical formation structural in change Figure in 3 fibrillation according formation to References [25]. Similar [3] and statistical [24]. Figure 3a,b showsanalysis that BSA as above alone was changed selected by for a the decrease difference of of αα-helices-helix, β-sheet and an and increaseβ-Turn between of β-sheets two groups. during a 21-day The insignificant variation of the secondary structures including ‘β-Turn’ (F = 0.002, p = 0.964 > 0.05) incubation, which was noted as a typical structural change in fibrillation formation [25]. The was observed during the incubation in Figure3c, indicating that it might not be involved in fibrillation insignificantformation. variation The formation of the of secondary ‘Random coil’ structures increased withincluding days, suggesting ‘β-Turn’ that was it mayobserved play an during the incubationimportant in Figure role in amyloid 3c, indicating formation. that Results it might of intergroup not be eff ectinvolved analysis in suggested the fibrillation that the eff ectformation. of The formationC-phycocyanin of ‘Random on the coil’ amyloid increased formation with of BSA days, fibrosis suggesting was significantly that diit ffmayerent (playFα-helix an= important1208.18, role in 6 pα = 4.088 10 < 0.001 and Fβ = 17.66, pβ = 0.014 < 0.05). Thus, the addition of amyloid-helix formation.× The− addition of C-phycocyanin-sheet -sheet in Figure 3a,b diminished the decrease of α- α β helicesC-phycocyanin and the increase in Figure of β3a,b-sheets diminished in BSA the from decrease day of6 to-helices day 18, and compared the increase to ofBSA-sheets alone, in suggesting BSA from day 6 to day 18, compared to the BSA alone, suggesting that C-phycocyanin interrupted that C-phycocyaninthe transformation interrupted and finally prolonged the transformation the amyloid formation and finally of BSA. prolonged Meanwhile, the C-phycocyanin amyloid formation of BSA. Meanwhile,notably affected C-phycocyanin the formation of ‘Randomnotably coil’affected in Figure the3 d.formation of ‘Random coil’ in Figure 3d.

Figure 3. Variation on the secondary structure ((a), α-helix; (b), β-sheet; (c), β-Turn; (d), random coil) Figureof 3. BSA Variation complexes on with the C-phycocyaninsecondary structure (50 µg/mL) ((a calculated), α-helix; using (b), theβ-sheet; CDSSTR (c), software β-Turn; during (d), random a coil) of BSA21-day complexes incubation. with C-phycocyanin (50 µg/mL) calculated using the CDSSTR software during a

21-day2.4. Molecular incubation. Docking

CD spectraThe C-phycocyanin were analyzed molecules and practically the ratios recognized of secondary as a monomer structures (PDB id: were 2vjr) calculated containing two to determine non-separable homologous units, and a self-assembled trimer or hexamer (PDB id: 1phn) in water [15] the processwere ﬁrst of consideredBSA amyloid to dock formation with BSA (PDB in Figure id: 4f5s) 3 inaccording this study. to The the monomer reference showed [3]. much Figure more 3a,b showed that BSAinteraction alone with changed BSA than by others a decrease and was ﬁnallyof α-helices selected asand the C-phycocyaninan increase molecularof β-sheets module during for a 21-day incubation,docking which analysis. was The optimizednoted as molecular a typical docking structural model ischange shown inin Figure fibrillation4, where C-phycocyaninformation [25]. Similar statistical analysis as above was selected for the difference of α-helix, β-sheet and β-Turn between two groups. The insignificant variation of the secondary structures including ‘β-Turn’ (F = 0.002, p = 0.964 > 0.05) was observed during the incubation in Figure 3c, indicating that it might not be involved in fibrillation formation. The formation of ‘Random coil’ increased with days, suggesting that it may play an important role in amyloid formation. Results of intergroup effect analysis suggested that the effect of C-phycocyanin on the amyloid formation of BSA fibrosis was significantly different (Fα-helix = 1208.18, pα-helix = 4.088 × 10−6 < 0.001 and Fβ-sheet = 17.66, pβ-sheet = 0.014 < 0.05). Thus, the addition of C- phycocyanin in Figure 3a,b diminished the decrease of α-helices and the increase of β-sheets in BSA from day 6 to day 18, compared to the BSA alone, suggesting that C-phycocyanin interrupted the transformation and finally prolonged the amyloid formation of BSA. Meanwhile, C-phycocyanin notably affected the formation of ‘Random coil’ in Figure 3d.

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2.4. Molecular Docking The C-phycocyanin molecules practically recognized as a monomer (PDB id: 2vjr) containing two non-separable homologous units, and a self-assembled trimer or hexamer (PDB id: 1phn) in Int.water J. Mol. [15] Sci. 2020 were, 21 first, 8207 considered to dock with BSA (PDB id: 4f5s) in this study. The monomer showed5 of 11 much more interaction with BSA than others and was finally selected as the C-phycocyanin molecular module for docking analysis. The optimized molecular docking model is shown in Figure 4, where interacted with BSA by binding to ~6 of 27 α-helices in BSA in a U-shaped cavity, likely a type of C-phycocyanin interacted with BSA by binding to ~6 of 27 α-helices in BSA in a U-shaped cavity, gomphosis structure where C-phycocyanin from bottom ﬁxed BSA. likely a type of gomphosis structure where C-phycocyanin from bottom fixed BSA.

FigureFigure 4. 4.Interaction Interaction of of BSA BSA (PDB (PDB id: id: 4f5s) 4f5s) and and C-phycocyanin C-phycocyanin (PDB (PDB id: id: 2vjr) 2vjr) using using molecular molecular docking. docking. SolventSolvent molecules molecules were were removed removed before before molecular molecular docking. docking. The The green green chain chain is BSA.is BSA.α and α andβ subunits β subunits ofof C-phycocyanin C-phycocyanin are are colored colored in in pinkpink and blue, blue, re respectively,spectively, whereas whereas phycocyanobilin phycocyanobilin is yellow. is yellow. Red Redarrows arrows represent represent the the BSA BSA α-helicesα-helices blocked blocked by by C-phycocyanin. C-phycocyanin.

FourFour characteristic characteristic areas, areas, A-D, A-D, in in Figure Figure4 were4 were defined defined in orderin order to exploreto explore the the secondary secondary interactions.interactions. In In these these areas, areas, a largea large amount amount of of hydrogen hydrogen bonds bonds was was found found and and most most hydrogen hydrogen bonds bonds + + possessedpossessed a bonda bond length length of of 2.0–3.5 2.0–3.5 Å. Å. Some Some groups, groups, like like peptide peptide linkage linkage and and –NH −NH2 or2 –NHor −NH3 on3 on the the chainchain side side of aminoof amino acid acid residues, residues, were were good good hydrogen hydrogen bond bond donors, donors, while while carbonyl carbonyl and carboxyl and carboxyl may performmay perform as good as hydrogen good hydrog bonden acceptors. bond acceptors. The α subunit The α subunit of C-phycocyanin of C-phycocyanin played played a key role a key with role numerouswith numerous X-H Y andX-H⋅⋅⋅ X-HY andπ hydrogenX-H⋅⋅⋅π hydrogen bonds, suggesting bonds, suggesting the significance the significance of cofactors of in cofactors the docking in the ··· ··· model.docking Figure model.5a shows Figure that 5a Lys-556 shows andthat Lys-573Lys-556 of and BSA Lys-573 in area of A wereBSA blockedin area A by were the residues blocked from by the theresiduesβ subunit from of C-phycocyaninthe β subunit of viaC-phycocyanin hydrogen bonding. via hydrogen Similarly, bonding. Lys-520 Similarly, and Lys-396 Lys-520 in area and B Lys-396 were blockedin area in B Figurewere blocked5b. The in hydrogen Figure 5b. bonds The shownhydrogen in areas bonds A shown and B contributedin areas A and to the B contributed increase in theto the boundaryincrease areain the between boundary BSA area and between C-phycocyanin, BSA and C-phycocyanin, and then allowed and two thenα-helices allowed from two BSAα-helices to enter from intoBSA the to U-shaped enter into cavity the of U-shaped the C-phycocyanin cavity of vertically.the C-phycocyanin Additionally, vertically. many hydrophilic Additionally, residues many (53.4%hydrophilic in total) residues were 25, (53.4% 21 and in 16total) residues were from25, 21 BSA, and 16α and residuesβ subunits from BSA, in the α cavity,and β subunits respectively, in the andcavity, contributed respectively, to the hydrophilicityand contributed of to the the cavity. hydrop Distanceshilicity of among the cavity. helices Distances in the cavity among were helices about in 7–11the Åcavity in Figure were6 abouta. As 7–11 a result Å in of Figure hydrophilicity 6a. As a result and spacesof hydrophilicity of the cavity, and water spaces (diameter: of the cavity, ~ 4 water Å) was(diameter: able to fill ~ 4 in Å) and was potential able to fill was in able and to potential build up was cross-bridge able to build linkages up cross-bridge among polypeptides linkages among via hydrogenpolypeptides bonding via (Figurehydrogen6b), bonding so as to access(Figure extra 6b), solvationso as to access energy. extra solvation energy.

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Figure 5. Secondary interaction of areas A (a), B (b), D (c) and C (d,e) in docking models. The green FigureFigure 5. 5. SecondarySecondary interaction interaction of of areas areas A A ( (aa),), B B ( (bb),), D D ( (cc)) and and C C ( (dd,,ee)) in in docking docking models. models. The The green green chain is BSA, whereas the pink and blue ones are α and β subunits of C-phycocyanin, respectively. chainchain is is BSA, BSA, whereas whereas the the pink pink and and blue blue ones ones are are αα andand ββ subunitssubunits of of C-phycoc C-phycocyanin,yanin, respectively. respectively. Area C containing phycocyanobilin (yellow structure) is viewed from different angles (d,e). Red AreaArea C containingcontaining phycocyanobilinphycocyanobilin (yellow (yellow structure) structure) is viewedis viewed from from diﬀerent different angles angles (d,e). ( Redd,e). atoms Red atoms are oxygen and blue atoms are nitrogen. Hydrogen bonds are shown in yellow dotted lines. atoms are oxygen and blue atoms are nitrogen. Hydrogen bonds are shown in yellow dotted lines. areUnit oxygen of length and is blue Å. atoms are nitrogen. Hydrogen bonds are shown in yellow dotted lines. Unit of Unitlength of islength Å. is Å.

Figure 6. Hydrophilic cavity (a) in areas A and B and a possible example in area A (b). Some distances were measured and are shown in brown lines. The example is in the triangle with side lengths of 11.4, Figure 6. Hydrophilic cavity (a) in areas A and B and a possible example in area A (b). Some distances Figure9.2 and 6. 10.6Hydrophilic Å. The gray cavity atoms (a) are in areashydrogen A and atoms B and in athe possible left panel. example The green in area chain A (isb). BSA. Some The distances pink werewereand measured measured blue chains and and are are are α andshown shown β subunits in in brow brown ofn lines.C-phycoc lines. The Theyanin, example example respectively. is is in in the the triangle triangle with with side side lengths lengths of of 11.4, 11.4, 9.29.2 and and 10.6 10.6 Å. Å. The The gray gray atoms atoms are are hydrogen hydrogen atoms atoms in in the the left left panel. panel. The The green green chain chain is is BSA. BSA. The The pink pink andandAlmost blue blue chains chains all of are aretested αα andand models ββ subunitssubunits contai of ofned C-phycoc C-phycocyanin, similaryanin, interactions respectively. respectively. shown in areas C and D, indicating that they were the core part of the docking models and provided the most stabilizing energy. Area C in AlmostFigureAlmost 5d,e all all ofshows of tested tested mainly models models secondary contai containedned interactions similar similar interactions interactionswithin the α shownsubunit shown in inand areas areas phycocyanobilin C C and and D, D, indicating indicating of C- thatthatphycocyanin. they they were were the theThe core core Arg-185, part part of of Lys-439, the the docking docking and modelArg-435 modelss and andfrom provided provided BSA were the the blocked most most stabilizing stabilizing by C-phycocyanin energy. energy. Area Area via C C inin Figurehydrogen Figure 5d,e5d,e bonding. shows shows mainly mainly secondary secondary interactions interactions within within the the α subunitα subunit and and phycocyanobilin phycocyanobilin of C- of phycocyanin. The Arg-185, Lys-439, and Arg-435 from BSA were blocked by C-phycocyanin via hydrogen bonding.

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C-phycocyanin. The Arg-185, Lys-439, and Arg-435 from BSA were blocked by C-phycocyanin via hydrogenInt. J. Mol. Sci. bonding. 2020, 21, x 7 of 11

3. Discussion A kinetic model was established to study the spontaneousspontaneous and self-catalytic process in BSA fibrillation,fibrillation, as as well well as as to to evaluate evaluate the the inhibitory inhibitory effect effect on onamyloid amyloid format formationion by C-phycocyanin. by C-phycocyanin. The Themodel model was simplified was simplified with a withhypothesis a hypothesis that the thatBSA theamyloid BSA is amyloid a monomer is a and monomer does not and exhibit does notmultiple exhibit molecular multiple action, molecular since action, numerous since dimer–aggregate numerous dimer–aggregate and aggregate–aggregate and aggregate–aggregate interactions interactionsoccurred in differential occurred in equations, differential resulting equations, in un resultinglikely solved in unlikely multiple solved variables multiple [26].variables However, [26 all]. However,factors including all factors the spontaneous including thetransformation spontaneous from transformation BSA to amyloid from [27], BSA the to polymerization- amyloid [27], thedependent polymerization-dependent induction of amyloid induction to the process of amyloid of BSA to the fibrillation process of [28], BSA the fibrillation mutual [28aggregation], the mutual of aggregationpolymerization-dependent of polymerization-dependent amyloids [29] and amyloids the po [lymer29] and solubility the polymer [30] may solubility need [to30 be] may considered need to becomprehensively considered comprehensively for an accurate for kinetic an accurate model in kinetic further model studies. in further studies. C-phycocyanin (0~50 µg/mL)µg/mL) hindered the amyloid formation process of BSA with increased half-lives (12.43–17.73 days) based on fluorescencefluorescence intensity and aaffectedffected spontaneous process (k1),), but notnot the the self-catalysis self-catalysis process process (k2) based(k2) based on the on kinetic the model.kinetic CDmodel. spectra CD showed spectra that showed C-phycocyanin that C- inhibitedphycocyanin conformational inhibited conf conversionormational of BSAconversion by decreasing of BSA αby-helices decreasing and increasing α-helices βand-sheets increasing from day β- 6sheets to day from 18, whichday 6 to was day consistent 18, which with was the consistent fluorescence with study.the fluorescence study. Molecule docking analyses showed that C-phycocya C-phycocyaninnin appeared to interact with one side of BSA in a type of gomphosis structure, and didifferedffered from most of thethe smallsmall inhibitorsinhibitors that combined and stabilized BSA from the inside [[31,32].31,32]. Since α-helix was the essential structure for spontaneous transformation in amyloid formation [33], [33], C-phycoc C-phycocyaninyanin might might hinder hinder the the process of fibrillation, fibrillation, likely by decelerating thethe spontaneousspontaneous transformation transformation in in amyloid amyloid formation. formation. However, However, BSA BSA still still had had a largea large number number of freeof freeα-helices α-helices and and followed followed the spontaneousthe spontaneous process process to form to amyloids,form amyloids, which which might catalyzemight catalyze others intoothers fibrillation into fibrillation in the self-catalytic in the self-catalytic process process (k2) in the (k2) kinetic in the model.kinetic Thatmodel. may That explain may whyexplain k2 waswhy little k2 was affected little byaffected C-phycocyanin. by C-phycocyanin. In other words, In other k1 declinedwords, k because1 declined some becauseα-helices some were α- blocked,helices were whereas blocked, k2 showed whereas no k significant2 showed no changes significant because changes the free becauseα-helices the mightfree α-helices be the major might part be involvedthe major in part the self-catalyticinvolved in the process. self-catalytic The mechanism process. of The C-phycocyanin mechanism inhibitingof C-phycocyanin amyloid inhibiting formation wasamyloid schemed formation in Figure was7 .schemed in Figure 7.

Figure 7. Mechanisms of phycocyanin inhibition on amyloid formation. Figure 7. Mechanisms of phycocyanin inhibition on amyloid formation. Hydrogen bonds in areas A and B allowed two α-helices from BSA to enter into a U-shaped cavity of theHydrogen C-phycocyanin bonds vertically.in areas A Areas and CB andallowed D, as two the α core-helices part offrom the BSA docking to enter models, into provided a U-shaped the mostcavity stabilizing of the C-phycocyanin energy. The mechanismvertically. Areas of the C inhibition and D, as of C-phycocyaninthe core part of on the amyloid docking formation models, isprovided likely to the inhibit most the stabilizing spontaneous energy. transformation The mechanis stepm of (k the1) ofinhibition BSA by stabilizingof C-phycocyanin at least on 6 α amyloid-helices, whereasformation k2 isshowed likely to no inhibit signiﬁcant the spontaneous changes because transformation the unblocked stepα (k-helices1) of BSA might by stabilizing exactly be theat least major 6 partα-helices, involved whereas in the k self-catalytic2 showed no process.significant Additionally, changes because alkaline the amino unblocked acids, α i.e.,-helices lysine might and arginine, exactly canbe the participate major part in ainvolved glycation in reaction the self-catalytic [34], which proc playsess. the Additionally, role in stimulating alkaline amyloid amino acids, aggregation i.e., lysine [35]. and arginine, can participate in a glycation reaction [34], which plays the role in stimulating amyloid aggregation [35]. As a result of the docking model, around 37.5% lysines and 47.6% arginines of BSA have been blocked, and thus potentially rendered the glycation process, which is consistent with the previous study of quercetin on BSA glycation [36].

Int. J. Mol. Sci. 2020, 21, 8207 8 of 11

As a result of the docking model, around 37.5% lysines and 47.6% arginines of BSA have been blocked, and thus potentially rendered the glycation process, which is consistent with the previous study of quercetin on BSA glycation [36].

4. Materials and Methods

4.1. Materials C-phycocyanin (from Spirulina sp.) was purchased from Sigma-Aldrich (Shanghai, China) with a purity of 99% determined by HPLC. Albumin from Bovine Serum (BSA) with a purity of 96% and ThT (thioﬂavine T) were purchased from Aladdin Bio-Chem Technology (Shanghai, China). All other chemicals used were of analytical reagent grade.

4.2. Inhibitory Effect of BSA on Amyloid Formation In Vitro The inhibitory assay was designed according to the previous method [37] using Tecan Infinite M200 Pro microplate reader (Männedorf, Switzerland). The final volume of the reaction system was 1000 µL, containing BSA (0.1 mM, 50 µL) and various volumes of C-phycocyanin (1 mg/mL). The solvent was phosphate buffer (0.1 mM, pH 7.4). Samples were incubated in Eppendorf tubes at 25 ◦C for 21 days and were detected by fluorometric assay with ThT (0.02 mM, 100 µL) according to previous research [38]. ThT is able to combine with β-sheets to produce strong fluorescence (λex = 440 nm, λem = 484 nm)[39]. The interference of C-phycocyanin on fluorescence has been eliminated; in fact, no fluorescence was detected for the C-phycocyanin at λex = 440 nm. A system including ThT (0.02 mM, 100 µL) with phosphate buffer (0.1 mM, pH 7.4, 900 µL) was used to eliminate the interference of the solvent.

4.3. Kinetic Analysis A kinetic model was established to study the spontaneous and self-catalytic process in BSA ﬁbrillation, as well as to evaluate the inhibitory eﬀect on amyloid formation by C-phycocyanin. In this kinetic model, A represents BSA and B represents amyloid. The kinetic constant of the spontaneous process is k1, and the kinetic constant of the self-catalytic process is k2.

k A 1 B → k A + B 2 2B → A diﬀerential equation was presented via this model.

d[B] = k [A] + k [A][B] (1) dt 1 2 With a hypothesis that the total amount of BSA and amyloid is constant ([A] + [B] = m), the diﬀerential equation was calculated and then came to an implicit expression.

! 1 k2[B] + k 1 k t = ln 1 ln 1 (2) k1 + mk2 m [B] − k1 + mk2 m − 4.4. Circular Dichroism Analysis Far-UV CD spectra (190–260 nm) were recorded on a JASCO J-815 spectropolarimeter (Jasco Inc., Easton, MD, USA). The scan speed was 100 nm/min with 1 nm bandwidth. The BSA (0.1 mM, 50 µL) alone or together with C-phycocyanin (1 mg/mL, 10 µL) was measured using a quartz cell of 1 mm path length. For the best signal to noise ratio (SNR), each absorbance curve was smoothed automatically. Base lining and analysis were done using Jasco J-720 software. In addition, CD spectra Int. J. Mol. Sci. 2020, 21, 8207 9 of 11 of C-phycocyanin in experimental condition were determined, yet there was no signal due to low concentration of C-phycocyanin in this study.

4.5. Molecular Docking Z-Dock Server 3.0.2 [40,41] and its score function [42] were used for molecular docking. Protein preparation was carried out by adding hydrogens, removing water molecules. All possible binding patterns between two proteins via translation and rotation were studied, and used an energy-based scoring function to evaluate each potential pattern. The crystal structure in the protein data bank was applied as monomer (PDB id: 2vjr), trimer (PDB id: 1phn) and hexamer (PDB id: 1phn) of C-phycocyanin for molecular docking with BSA (PDB id: 4f5s). The docking data were rendered and screened using PyMOL Viewer [43].

4.6. Data Analysis All the experiments were conducted in triplicate. CD data were analyzed using CDpro software to characterize the variation of the secondary structure of BSA during amyloid formation. Three algorithms including SELCON3, CONTIN/LL and CDSSTR were compared [24]. Among them, CDSSTR was selected for better results while SP-37A (SOLUBLE) was used as the reference protein. Curve ﬁtting for the kinetic model was done using Origin 2017 64-bit software. Data are expressed as the mean ± standard deviation.

Supplementary Materials: The following are available online at http://www.mdpi.com/1422-0067/21/21/8207/s1. Figure S1: CD data of BSA without the existence of C-phycocyanin; Figure S2: CD data of BSA with the existence of C-phycocyanin.; Figure S3: Two models between BSA and monomer of C-phycocyanin with less interactions; Figure S4: One example model between BSA and trimer of C-phycocyanin; Figure S5: One example model between BSA and hexamer of C-phycocyanin. Author Contributions: Conceptualization, Y.-C.L. and P.J.; methodology, Y.-C.L.; formal analysis, Y.-C.L.; investigation, Y.-C.L.; writing—original draft preparation, Y.-C.L.; writing—review and editing, P.J.; supervision, P.J.; project administration, P.J.; funding acquisition, P.J. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by National Natural Science Foundation of China (Grant No. 21978169) and the Shanghai Food Safety and Engineering Technology Research Center (Grant No. 19DZ2284200). Conﬂicts of Interest: The authors declare no conﬂict of interest.

Abbreviations

BSA Bovine serum albumin CD Circular dichroism ThT Thioﬂavine T

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